1) Abca12 null mice were generated that lack the Abca12 gene encoding an ABC transporter protein.
2) Abca12 null mice develop a thickened epidermis and die shortly after birth from dehydration, mimicking the human condition harlequin ichthyosis caused by ABCA12 mutations.
3) Lipid analysis found that Abca12 null mice have profoundly reduced levels of linoleic acid esters of hydroxyceramides in their skin, which are required for the skin permeability barrier, along with increased levels of their glucosylceramide precursors. This establishes that ABCA12 activity is required for generating essential skin lipids.
This study aimed to optimize the fabrication of fiber templated hydrogels containing conduits, improve seeding of human mesenchymal stem cells (hMSCs) onto the hydrogels, and differentiate the hMSCs into cardiomyocytes inside the hydrogels. Researchers fabricated poly(ethylene glycol) diacrylate hydrogels containing zein fiber conduits, seeded hMSCs at different densities, and attempted to induce cardiomyocyte differentiation over 16 days. Immunostaining showed some actin formation but no evidence of successful cardiomyocyte differentiation within the hydrogels or 2D controls. Further work is needed to optimize differentiation.
This study evaluated the use of adipose-derived stem cells (ADSCs) and neonatal articular chondrocytes (NChons) encapsulated in a methacrylated chondroitin sulfate hydrogel to catalyze cartilage formation. The results showed that the chondroitin sulfate hydrogel supported cell proliferation and deposition of cartilage markers like collagen and GAGs when ADSCs and NChons were co-cultured in the presence of TGF-β. Histological analysis and mechanical testing confirmed the formation of cartilage tissue only in the co-culture group with TGF-β. The study demonstrated the potential of a fully biodegradable chondroitin sulfate hydrogel for catalyzed cartilage
This document describes a study that investigated the interaction between nick-directed DNA loop repair and mismatch repair in human cells. The study constructed DNA substrates containing combinations of mismatches and loops. It was demonstrated that a nick 3' to a large loop can direct loop repair in human cell extracts in a bidirectional manner. However, when a mismatch was near a loop, the efficiency of nick-directed mismatch repair was reduced. This suggests interference between loop repair and mismatch repair when sites are adjacent to avoid double-stranded DNA breaks.
Homologous recombination (HR) is the exchange of genetic material between two similar or identical molecules of DNA. The document outlines the mechanism and molecular basis of HR, including key steps like double-strand break formation, strand invasion, and Holliday junction resolution. HR serves important biological roles like DNA repair and genetic diversity. It has practical applications in gene mapping, transgenics, and gene editing technologies. Precise genome editing using HR is becoming an alternative to traditional plant breeding for crop improvement.
This document presents the complete small subunit ribosomal RNA (SSU rRNA) sequences of Giardia ardeae, G. muris, G. duodenalis, and the diplomonad Hexamita. The sequences are compared to analyze phylogenetic relationships. The Giardia sequences range from 1432 to 1453 nucleotides in length, while Hexamita's is 1550 nucleotides. Secondary structure analysis reveals both typically eukaryotic and variable regions. A phylogenetic tree groups the Giardia species separately from Hexamita and Vairimorpha, suggesting they represent a separate kingdom within the domain Eucarya.
Loss of pollen-specific phospholipase NOT LIKE DAD triggers gynogenesis in maizePGS
This lecture was a part of Plant Genetics Seminars - PGS 2017/2018 at Assiut University. These seminars organized by Dr. Ahmed Sallam, Department of Genetics, Faculty of Agriculture, Assiut University
Abstract
Gynogenesis is an asexual mode of reproduction common to animals and plants, in which stimuli from the sperm cell trigger the development of the unfertilized egg cell into a haploid embryo. Fine mapping restricted a major maize QTL (quantitative trait locus) responsible for the aptitude of inducer lines to trigger gynogenesis to a zone containing a single gene NOT LIKE DAD (NLD) coding for a patatin-like phospholipase A. In all surveyed inducer lines, NLD carries a 4-bp insertion leading to a predicted truncated protein. This frameshift mutation is responsible for haploid induction because complementation with wild-type NLD abolishes the haploid induction capacity. Activity of the NLD promoter is restricted to mature pollen and pollen tube. The translational NLD::citrine fusion protein likely localizes to the sperm cell plasma membrane. In Arabidopsis roots, the truncated protein is no longer localized to the plasma membrane, contrary to the wild-type NLD protein. In conclusion, an intact pollen-specific phospholipase is required for successful sexual reproduction and its targeted disruption may allow establishing powerful haploid breeding tools in numerous crops.
1) Researchers isolated and characterized a novel bacteriophage (Horchata) that infects Mycobacterium smegmatis from a soil sample.
2) Transmission electron microscopy revealed that Horchata has an icosahedral head and long non-contractile tail, characteristic of the Siphoviridae morphology.
3) Genome sequencing and annotation showed that Horchata has a 67,863 bp genome with 30 genes of putative function and 71 genes of unknown function.
there are s many methods are used in diagnosis of human gene mutation which occur disorders ,here u get information about the diagnostic method for genetic mutation detection
This study aimed to optimize the fabrication of fiber templated hydrogels containing conduits, improve seeding of human mesenchymal stem cells (hMSCs) onto the hydrogels, and differentiate the hMSCs into cardiomyocytes inside the hydrogels. Researchers fabricated poly(ethylene glycol) diacrylate hydrogels containing zein fiber conduits, seeded hMSCs at different densities, and attempted to induce cardiomyocyte differentiation over 16 days. Immunostaining showed some actin formation but no evidence of successful cardiomyocyte differentiation within the hydrogels or 2D controls. Further work is needed to optimize differentiation.
This study evaluated the use of adipose-derived stem cells (ADSCs) and neonatal articular chondrocytes (NChons) encapsulated in a methacrylated chondroitin sulfate hydrogel to catalyze cartilage formation. The results showed that the chondroitin sulfate hydrogel supported cell proliferation and deposition of cartilage markers like collagen and GAGs when ADSCs and NChons were co-cultured in the presence of TGF-β. Histological analysis and mechanical testing confirmed the formation of cartilage tissue only in the co-culture group with TGF-β. The study demonstrated the potential of a fully biodegradable chondroitin sulfate hydrogel for catalyzed cartilage
This document describes a study that investigated the interaction between nick-directed DNA loop repair and mismatch repair in human cells. The study constructed DNA substrates containing combinations of mismatches and loops. It was demonstrated that a nick 3' to a large loop can direct loop repair in human cell extracts in a bidirectional manner. However, when a mismatch was near a loop, the efficiency of nick-directed mismatch repair was reduced. This suggests interference between loop repair and mismatch repair when sites are adjacent to avoid double-stranded DNA breaks.
Homologous recombination (HR) is the exchange of genetic material between two similar or identical molecules of DNA. The document outlines the mechanism and molecular basis of HR, including key steps like double-strand break formation, strand invasion, and Holliday junction resolution. HR serves important biological roles like DNA repair and genetic diversity. It has practical applications in gene mapping, transgenics, and gene editing technologies. Precise genome editing using HR is becoming an alternative to traditional plant breeding for crop improvement.
This document presents the complete small subunit ribosomal RNA (SSU rRNA) sequences of Giardia ardeae, G. muris, G. duodenalis, and the diplomonad Hexamita. The sequences are compared to analyze phylogenetic relationships. The Giardia sequences range from 1432 to 1453 nucleotides in length, while Hexamita's is 1550 nucleotides. Secondary structure analysis reveals both typically eukaryotic and variable regions. A phylogenetic tree groups the Giardia species separately from Hexamita and Vairimorpha, suggesting they represent a separate kingdom within the domain Eucarya.
Loss of pollen-specific phospholipase NOT LIKE DAD triggers gynogenesis in maizePGS
This lecture was a part of Plant Genetics Seminars - PGS 2017/2018 at Assiut University. These seminars organized by Dr. Ahmed Sallam, Department of Genetics, Faculty of Agriculture, Assiut University
Abstract
Gynogenesis is an asexual mode of reproduction common to animals and plants, in which stimuli from the sperm cell trigger the development of the unfertilized egg cell into a haploid embryo. Fine mapping restricted a major maize QTL (quantitative trait locus) responsible for the aptitude of inducer lines to trigger gynogenesis to a zone containing a single gene NOT LIKE DAD (NLD) coding for a patatin-like phospholipase A. In all surveyed inducer lines, NLD carries a 4-bp insertion leading to a predicted truncated protein. This frameshift mutation is responsible for haploid induction because complementation with wild-type NLD abolishes the haploid induction capacity. Activity of the NLD promoter is restricted to mature pollen and pollen tube. The translational NLD::citrine fusion protein likely localizes to the sperm cell plasma membrane. In Arabidopsis roots, the truncated protein is no longer localized to the plasma membrane, contrary to the wild-type NLD protein. In conclusion, an intact pollen-specific phospholipase is required for successful sexual reproduction and its targeted disruption may allow establishing powerful haploid breeding tools in numerous crops.
1) Researchers isolated and characterized a novel bacteriophage (Horchata) that infects Mycobacterium smegmatis from a soil sample.
2) Transmission electron microscopy revealed that Horchata has an icosahedral head and long non-contractile tail, characteristic of the Siphoviridae morphology.
3) Genome sequencing and annotation showed that Horchata has a 67,863 bp genome with 30 genes of putative function and 71 genes of unknown function.
there are s many methods are used in diagnosis of human gene mutation which occur disorders ,here u get information about the diagnostic method for genetic mutation detection
Biotechnology and gene expression profiling for mechanistic understanding of ...Hana Fayed
1) The study characterized aggregation of baby hamster kidney (BHK) cells cultivated in perfusion bioreactors by analyzing expression of 84 genes related to extracellular matrix and adhesion. 2) They found that genes THBS2 and NCAM1 were significantly up-regulated and down-regulated, respectively, in late-stage versus early-stage cultures of an aggregating cell line. 3) Comparing an aggregating versus non-aggregating cell line, they found that genes COL1A1, COL4A1, THBS2, and VCAN were up-regulated while NCAM1 and THBS1 were down-regulated in the aggregating line, indicating differences in extracellular matrix and adhesion pathways contribute to aggregation
Zinc finger nucleases (ZFNs) are engineered restriction enzymes
designed to target specific DNA sequences within the genome.
Assembly of zinc finger DNAbinding domain to a DNA-cleavage
domain.
Genetic stability relies on accurate DNA replication and repair mechanisms to correct approximately 1 million lesions per cell per day caused by various sources of DNA damage. If unrepaired, this damage can lead to mutations, genetic diseases like cancer, or cell death. The cell has multiple pathways to repair DNA damage, including base excision repair, nucleotide excision repair, mismatch repair, and homologous recombination. Homologous recombination is especially important for repairing double-stranded breaks using the undamaged sister chromatid as a template, and involves proteins such as RAD51, BRCA1, BRCA2, ATM, and the Fanconi anemia/BRCA pathway. Defects in DNA repair genes are
Discussion of latest work on simulating "evolve and resequence" experiments. Covers issues brought up by Burke et al.'s 2010 paper and how the simulations in Baldwin-Brown et al. (2014) address them.
Sperm DNA fragmentation can negatively impact fertility and pregnancy outcomes. DNA becomes vulnerable during spermatogenesis when protamine replaces histones in chromatin compaction. Tests can evaluate sperm DNA fragmentation levels, which are correlated with lower fertilization and implantation rates. Factors like oxidative stress, temperature, and infections can intrinsically or extrinsically damage sperm DNA. Repair mechanisms attempt to resolve DNA double-strand breaks, but extensive unrepaired damage may be incompatible with embryo development. Treatments aim to address underlying causes of DNA fragmentation or use testicular sperm or ICSI to bypass ejaculated sperm issues.
Gene therapy involves introducing genes into cells to treat disease. CRISPR is a gene editing technique using Cas9 protein and guide RNA. It allows DNA cutting and modification. Luxturna is a gene therapy for an inherited eye disease caused by RPE65 gene mutations. It uses AAV vector to insert a healthy RPE65 gene into retinal cells. In clinical trials, Luxturna improved patients' visual function and mobility in low light after 1 year as measured by a mobility test.
Genetic screening of CRISPR edited human-derived induced pluripotent stem cells was conducted to create a model of Costello syndrome. CRISPR was used to induce mutations in the HRAS gene of iPSCs. Of 12 samples screened, 2 were found to have a heterozygous mutation and 1 had a homozygous mutation, supporting that CRISPR can be used for gene editing in iPSCs. The mutated iPSCs will be differentiated into cardiomyocytes to model cardiac abnormalities in Costello syndrome.
The document provides an overview of DNA repair, including:
1) DNA is the only biological macromolecule that is repaired, as spontaneous and environmentally-induced damage occurs daily.
2) There are multiple pathways of DNA repair, including direct reversal, base excision repair, nucleotide excision repair, and double-strand break repair.
3) Defects in DNA repair can lead to genetic disorders and cancer, highlighting the importance of effective repair.
1. Researchers developed a new cell line called DEN-HSA derived from a spontaneous hemangiosarcoma in a dog.
2. Characterization showed DEN-HSA cells express markers of endothelial cells and form tube-like structures in vitro. They also secrete factors that strongly stimulate angiogenesis.
3. The DEN-HSA cell line maintains features of angiogenic endothelium and could be useful for studying novel therapies that target endothelial proliferation involved in diseases like cancer.
This thesis examines the role of BIGH3 C-terminus cleavage in MG63 osteosarcoma cells and the protection offered by bleed 8 antibody and extracellular matrix proteins. BIGH3 is an extracellular matrix protein whose C-terminal cleavage induces apoptosis. The student aims to determine if bleed 8 antibody and proteins like fibronectin, collagen, and decorin can prevent this cleavage. Cell culture, protein electrophoresis, immunoblotting, and densitometry are used to analyze interactions between BIGH3, bleed 8, and extracellular matrix proteins. Preliminary results suggest fibronectin and bleed 8 may offer some protection against BIGH3 C-terminal cleavage.
Cell cycle and DNA repair are essential processes in living cells. The cell cycle consists of interphase and the mitotic phase, and includes four main phases: G1, S, G2, and M phase. DNA is replicated in S phase and the cell divides in M phase. There are also checkpoint controls between phases to ensure DNA is properly replicated and repaired before the cell divides. DNA can become damaged through normal cellular processes, radiation, hydrolysis, oxidation and other environmental factors. Cells have several DNA repair mechanisms, including excision repair, photo reactivation, and mismatch repair to identify and correct DNA damage and prevent mutations. Unrepaired DNA damage can lead to diseases like xeroderma pigmentosum and at
DNA repair systems are essential for maintaining the integrity of genetic information. There are multiple pathways for repairing different types of DNA damage:
1) Mismatch repair corrects errors made during DNA replication by using methylation patterns to distinguish the template strand.
2) Base-excision repair involves DNA glycosylases that remove damaged bases, leaving abasic sites that are then repaired.
3) Nucleotide-excision repair removes larger distortions in the DNA double helix.
4) Direct repair mechanisms like photoreactivation can directly reverse some types of damage using light or chemical processes, without removing nucleotides. DNA repair pathways help prevent mutations and ensure fidelity of genetic information.
Mutations occur through endogenous and exogenous DNA damage and can be in the form of point mutations, frame shifts, or splicing errors. Mutation types include nonsense mutations which result in premature stop codons, missense mutations which code for different amino acids, and silent mutations which do not change the amino acid. Frameshift mutations change the reading frame by inserting or deleting nucleotides. Splicing errors can occur from mutations affecting splice sites or their specificity. Lethal mutations cause death while loss or gain of function mutations impact gene activity.
This document summarizes Richard Rodriguez's final lab report on analyzing the L34P mutation in the gap junction protein Cx31 using site-directed mutagenesis. Key findings include:
1) The L34P mutation is associated with Erythrokeratoderma vairabilis (EKV) skin disorder and causes the disease through a recessive inheritance pattern by preventing formation of normal gap junctions between keratinocytes.
2) Site-directed mutagenesis was used to introduce the L34P mutation into a Cx31 plasmid, which was then transformed into E. coli cells. Over 200 colonies grew on each transformation plate.
3) The mutated Cx31 plasmid will be used
Verifying the role of AID in Chronic Lymphocytic LeukemiaCharlotte Broadbent
This study aimed to verify the role of the enzyme activation-induced deaminase (AID) in chronic lymphocytic leukemia (CLL) using statistical bootstrapping methods. DNA sequences from CLL patients were analyzed before and after AID stimulation. Three tests found statistically significant evidence that AID is active in CLL: 1) more mutations in the variable region than constant region, 2) more mutations at AID hotspots than expected, and 3) fewer mutations at AID coldspots than expected. The results confirm AID's role in somatic hypermutation in CLL, furthering understanding of this disease.
1. MicroRNAs are small non-coding RNA molecules that regulate gene expression at the post-transcriptional level. They play important roles in skin biology and diseases.
2. Specific microRNAs like miR-203, miR-146a and miR-125b are involved in the pathogenesis of psoriasis by regulating inflammatory signaling pathways.
3. MicroRNAs also modulate processes like wound healing, angiogenesis, and skin cancer development by targeting genes involved in these processes. Dysregulation of microRNAs has been observed in melanoma and other skin malignancies.
DNA repair mechanisms are indispensable for cell maintenance. The most common repair mechanisms are base excision repair (BER) and nucleotide excision repair (NER). Deficiencies in repair can lead to diseases like cancer or myelodysplastic syndrome. Studies have found that some viruses like murine polyomavirus activate DNA damage repair proteins like ATR and MRE11 to promote viral replication. Understanding DNA repair is important for developing new treatments for diseases linked to repair deficiencies and viral infections.
DNA damage can result from metabolic processes, hydrolysis, or exogenous sources and can lead to various human diseases if left unrepaired. Defects in the cellular response to repair DNA damage can cause cancer, inflammation, cell aging, and neurodegeneration. Neurodegeneration involves the death of neurons and is associated with diseases like Huntington's, Parkinson's, and Alzheimer's. Neurons are post-mitotic cells that are susceptible to DNA damage from endogenous and exogenous agents. If the damage is not repaired, unrepaired DNA accumulates in neurons and can cause cell cycle reentry and apoptosis, leading to neurodegeneration. Chronic inflammation from infection or other sources can also cause DNA damage through reactive oxygen species
This document summarizes a study that investigated the complexes containing the RNA helicase DDX6, which is a key component of P-bodies involved in posttranscriptional regulation. The researchers identified DDX6 complexes using tandem affinity purification coupled with mass spectrometry. They found that DDX6 was present in three main complexes: the decapping complex, a CPEB-like complex, and an Ataxin2/Ataxin2L complex. Investigation of P-body assembly under various conditions identified three proteins required for assembly in all conditions: DDX6, 4E-T, and LSM14A. The results reveal that P-body assembly involves different pathways that nevertheless share these three key factors connecting
1) A digital microfluidic (DμF) platform was developed to automate the liquid handling steps for differentiating embryonic stem cells into cardiomyocytes and assaying the resulting tissue.
2) The DμF platform improved upon existing manual methods by automating embryoid body growth, differentiation medium exchanges, and non-invasive assays of beating cardiomyocyte tissue using electric fields.
3) Beating cardiomyocyte embryoid bodies were cultured on the DμF platform for up to 8 weeks and responses to known chronotropic agents were measured non-invasively through impedance changes, demonstrating the potential of the system for screening cardiotoxicity.
This study investigated how tumor secreted factors alter the responses of human endothelial cells (HUVECs) to matrix stiffness during capillary formation. The researchers used synthetic hydrogels of varying stiffness to culture HUVECs with or without tumor cells. They found that while HUVECs prefer a substrate of intermediate stiffness for optimal capillary formation in the absence of tumor factors, stimulation by tumor secreted factors disrupts the HUVECs' mechanosensitive behavior. Additionally, the anti-angiogenic drug vandetanib was less effective at reducing capillary formation when HUVECs were activated by tumor factors, particularly on stiffer gels. This suggests tumor activation alters the mechanosensitivity and drug responsiveness
Biotechnology and gene expression profiling for mechanistic understanding of ...Hana Fayed
1) The study characterized aggregation of baby hamster kidney (BHK) cells cultivated in perfusion bioreactors by analyzing expression of 84 genes related to extracellular matrix and adhesion. 2) They found that genes THBS2 and NCAM1 were significantly up-regulated and down-regulated, respectively, in late-stage versus early-stage cultures of an aggregating cell line. 3) Comparing an aggregating versus non-aggregating cell line, they found that genes COL1A1, COL4A1, THBS2, and VCAN were up-regulated while NCAM1 and THBS1 were down-regulated in the aggregating line, indicating differences in extracellular matrix and adhesion pathways contribute to aggregation
Zinc finger nucleases (ZFNs) are engineered restriction enzymes
designed to target specific DNA sequences within the genome.
Assembly of zinc finger DNAbinding domain to a DNA-cleavage
domain.
Genetic stability relies on accurate DNA replication and repair mechanisms to correct approximately 1 million lesions per cell per day caused by various sources of DNA damage. If unrepaired, this damage can lead to mutations, genetic diseases like cancer, or cell death. The cell has multiple pathways to repair DNA damage, including base excision repair, nucleotide excision repair, mismatch repair, and homologous recombination. Homologous recombination is especially important for repairing double-stranded breaks using the undamaged sister chromatid as a template, and involves proteins such as RAD51, BRCA1, BRCA2, ATM, and the Fanconi anemia/BRCA pathway. Defects in DNA repair genes are
Discussion of latest work on simulating "evolve and resequence" experiments. Covers issues brought up by Burke et al.'s 2010 paper and how the simulations in Baldwin-Brown et al. (2014) address them.
Sperm DNA fragmentation can negatively impact fertility and pregnancy outcomes. DNA becomes vulnerable during spermatogenesis when protamine replaces histones in chromatin compaction. Tests can evaluate sperm DNA fragmentation levels, which are correlated with lower fertilization and implantation rates. Factors like oxidative stress, temperature, and infections can intrinsically or extrinsically damage sperm DNA. Repair mechanisms attempt to resolve DNA double-strand breaks, but extensive unrepaired damage may be incompatible with embryo development. Treatments aim to address underlying causes of DNA fragmentation or use testicular sperm or ICSI to bypass ejaculated sperm issues.
Gene therapy involves introducing genes into cells to treat disease. CRISPR is a gene editing technique using Cas9 protein and guide RNA. It allows DNA cutting and modification. Luxturna is a gene therapy for an inherited eye disease caused by RPE65 gene mutations. It uses AAV vector to insert a healthy RPE65 gene into retinal cells. In clinical trials, Luxturna improved patients' visual function and mobility in low light after 1 year as measured by a mobility test.
Genetic screening of CRISPR edited human-derived induced pluripotent stem cells was conducted to create a model of Costello syndrome. CRISPR was used to induce mutations in the HRAS gene of iPSCs. Of 12 samples screened, 2 were found to have a heterozygous mutation and 1 had a homozygous mutation, supporting that CRISPR can be used for gene editing in iPSCs. The mutated iPSCs will be differentiated into cardiomyocytes to model cardiac abnormalities in Costello syndrome.
The document provides an overview of DNA repair, including:
1) DNA is the only biological macromolecule that is repaired, as spontaneous and environmentally-induced damage occurs daily.
2) There are multiple pathways of DNA repair, including direct reversal, base excision repair, nucleotide excision repair, and double-strand break repair.
3) Defects in DNA repair can lead to genetic disorders and cancer, highlighting the importance of effective repair.
1. Researchers developed a new cell line called DEN-HSA derived from a spontaneous hemangiosarcoma in a dog.
2. Characterization showed DEN-HSA cells express markers of endothelial cells and form tube-like structures in vitro. They also secrete factors that strongly stimulate angiogenesis.
3. The DEN-HSA cell line maintains features of angiogenic endothelium and could be useful for studying novel therapies that target endothelial proliferation involved in diseases like cancer.
This thesis examines the role of BIGH3 C-terminus cleavage in MG63 osteosarcoma cells and the protection offered by bleed 8 antibody and extracellular matrix proteins. BIGH3 is an extracellular matrix protein whose C-terminal cleavage induces apoptosis. The student aims to determine if bleed 8 antibody and proteins like fibronectin, collagen, and decorin can prevent this cleavage. Cell culture, protein electrophoresis, immunoblotting, and densitometry are used to analyze interactions between BIGH3, bleed 8, and extracellular matrix proteins. Preliminary results suggest fibronectin and bleed 8 may offer some protection against BIGH3 C-terminal cleavage.
Cell cycle and DNA repair are essential processes in living cells. The cell cycle consists of interphase and the mitotic phase, and includes four main phases: G1, S, G2, and M phase. DNA is replicated in S phase and the cell divides in M phase. There are also checkpoint controls between phases to ensure DNA is properly replicated and repaired before the cell divides. DNA can become damaged through normal cellular processes, radiation, hydrolysis, oxidation and other environmental factors. Cells have several DNA repair mechanisms, including excision repair, photo reactivation, and mismatch repair to identify and correct DNA damage and prevent mutations. Unrepaired DNA damage can lead to diseases like xeroderma pigmentosum and at
DNA repair systems are essential for maintaining the integrity of genetic information. There are multiple pathways for repairing different types of DNA damage:
1) Mismatch repair corrects errors made during DNA replication by using methylation patterns to distinguish the template strand.
2) Base-excision repair involves DNA glycosylases that remove damaged bases, leaving abasic sites that are then repaired.
3) Nucleotide-excision repair removes larger distortions in the DNA double helix.
4) Direct repair mechanisms like photoreactivation can directly reverse some types of damage using light or chemical processes, without removing nucleotides. DNA repair pathways help prevent mutations and ensure fidelity of genetic information.
Mutations occur through endogenous and exogenous DNA damage and can be in the form of point mutations, frame shifts, or splicing errors. Mutation types include nonsense mutations which result in premature stop codons, missense mutations which code for different amino acids, and silent mutations which do not change the amino acid. Frameshift mutations change the reading frame by inserting or deleting nucleotides. Splicing errors can occur from mutations affecting splice sites or their specificity. Lethal mutations cause death while loss or gain of function mutations impact gene activity.
This document summarizes Richard Rodriguez's final lab report on analyzing the L34P mutation in the gap junction protein Cx31 using site-directed mutagenesis. Key findings include:
1) The L34P mutation is associated with Erythrokeratoderma vairabilis (EKV) skin disorder and causes the disease through a recessive inheritance pattern by preventing formation of normal gap junctions between keratinocytes.
2) Site-directed mutagenesis was used to introduce the L34P mutation into a Cx31 plasmid, which was then transformed into E. coli cells. Over 200 colonies grew on each transformation plate.
3) The mutated Cx31 plasmid will be used
Verifying the role of AID in Chronic Lymphocytic LeukemiaCharlotte Broadbent
This study aimed to verify the role of the enzyme activation-induced deaminase (AID) in chronic lymphocytic leukemia (CLL) using statistical bootstrapping methods. DNA sequences from CLL patients were analyzed before and after AID stimulation. Three tests found statistically significant evidence that AID is active in CLL: 1) more mutations in the variable region than constant region, 2) more mutations at AID hotspots than expected, and 3) fewer mutations at AID coldspots than expected. The results confirm AID's role in somatic hypermutation in CLL, furthering understanding of this disease.
1. MicroRNAs are small non-coding RNA molecules that regulate gene expression at the post-transcriptional level. They play important roles in skin biology and diseases.
2. Specific microRNAs like miR-203, miR-146a and miR-125b are involved in the pathogenesis of psoriasis by regulating inflammatory signaling pathways.
3. MicroRNAs also modulate processes like wound healing, angiogenesis, and skin cancer development by targeting genes involved in these processes. Dysregulation of microRNAs has been observed in melanoma and other skin malignancies.
DNA repair mechanisms are indispensable for cell maintenance. The most common repair mechanisms are base excision repair (BER) and nucleotide excision repair (NER). Deficiencies in repair can lead to diseases like cancer or myelodysplastic syndrome. Studies have found that some viruses like murine polyomavirus activate DNA damage repair proteins like ATR and MRE11 to promote viral replication. Understanding DNA repair is important for developing new treatments for diseases linked to repair deficiencies and viral infections.
DNA damage can result from metabolic processes, hydrolysis, or exogenous sources and can lead to various human diseases if left unrepaired. Defects in the cellular response to repair DNA damage can cause cancer, inflammation, cell aging, and neurodegeneration. Neurodegeneration involves the death of neurons and is associated with diseases like Huntington's, Parkinson's, and Alzheimer's. Neurons are post-mitotic cells that are susceptible to DNA damage from endogenous and exogenous agents. If the damage is not repaired, unrepaired DNA accumulates in neurons and can cause cell cycle reentry and apoptosis, leading to neurodegeneration. Chronic inflammation from infection or other sources can also cause DNA damage through reactive oxygen species
This document summarizes a study that investigated the complexes containing the RNA helicase DDX6, which is a key component of P-bodies involved in posttranscriptional regulation. The researchers identified DDX6 complexes using tandem affinity purification coupled with mass spectrometry. They found that DDX6 was present in three main complexes: the decapping complex, a CPEB-like complex, and an Ataxin2/Ataxin2L complex. Investigation of P-body assembly under various conditions identified three proteins required for assembly in all conditions: DDX6, 4E-T, and LSM14A. The results reveal that P-body assembly involves different pathways that nevertheless share these three key factors connecting
1) A digital microfluidic (DμF) platform was developed to automate the liquid handling steps for differentiating embryonic stem cells into cardiomyocytes and assaying the resulting tissue.
2) The DμF platform improved upon existing manual methods by automating embryoid body growth, differentiation medium exchanges, and non-invasive assays of beating cardiomyocyte tissue using electric fields.
3) Beating cardiomyocyte embryoid bodies were cultured on the DμF platform for up to 8 weeks and responses to known chronotropic agents were measured non-invasively through impedance changes, demonstrating the potential of the system for screening cardiotoxicity.
This study investigated how tumor secreted factors alter the responses of human endothelial cells (HUVECs) to matrix stiffness during capillary formation. The researchers used synthetic hydrogels of varying stiffness to culture HUVECs with or without tumor cells. They found that while HUVECs prefer a substrate of intermediate stiffness for optimal capillary formation in the absence of tumor factors, stimulation by tumor secreted factors disrupts the HUVECs' mechanosensitive behavior. Additionally, the anti-angiogenic drug vandetanib was less effective at reducing capillary formation when HUVECs were activated by tumor factors, particularly on stiffer gels. This suggests tumor activation alters the mechanosensitivity and drug responsiveness
ABSTRACT- Coronary artery disease (CAD) is suspected as a leading cause of mortality in developed countries. Due
to cholesterol and fat deposit plaque is forming into the inner walls of the arteries of the heart, which leads to narrowing
of blood vessels of heart and reduce the blood flow rate into heart. Proprotein convertase subtilisin-like kexin type 9
(PCSK9) is one of the candidate gene that regulate lipoprotein retention pathway of CAD development. It is a newly
discovered serine protease that plays a key role in LDL-C homeostasis by mediating LDL receptor (LDLR). The LDL
receptor is breakdown through a post transcriptional mechanism and induces the production of very low-density
lipoprotein in the fasting state. The aim of this study was to investigate the frequency of single nucleotide
polymorphism (SNP) of PCSK9 gene of 155 CAD patients and 102 ages matched healthy controls. Serum lipids
including total cholesterol (TC), triglycerides (TG), HDL, LDL, and VLDL were analyzed. PCR-RFLP analysis was
carried out to genotype regions carrying Eam 1104I restriction site in the PCSK9. Gene considering significant
difference in serum TC, TG, HDL-C, LDL-C and VLDL-C levels (P<0.001, <0.0001) of patients and control samples.
In CAD patients, G allele frequency is less than A allele frequency. G allele is responsible for decreasing the
LDL: HDL ratio which shows evidence in having its protecting effect on the occurrence of CAD in West Bengal Population.
Key-words- CAD, PCSK9, SNP, Eam1104I, Polymorphism, West Bengal population
This document discusses using stem cells for diabetes treatment. It mentions:
1. Human embryonic stem cells, induced pluripotent stem cells, umbilical cord blood, and mesenchymal stromal cells can potentially be used for diabetes treatment.
2. Human embryonic stem cells can be differentiated into insulin-expressing cells through a stepwise process involving definitive endoderm and embryoid body formation under specific factors.
3. The goal is to generate enough functional insulin-producing beta cells to restore normal blood glucose levels in diabetics through cell-based therapies.
This document describes a study on expressing recombinant nanobodies in E. coli cells, extracting the proteins, and purifying them. E. coli WK6 cells were transformed with a plasmid containing the nanobody gene and expression was induced with IPTG. The cells were lysed and the proteins in the periplasmic space were extracted. Purification was done using immobilized metal affinity chromatography to bind the histidine-tagged nanobody, which was then eluted with imidazole buffer. SDS-PAGE and Western blot were used to analyze the expression and purity of the nanobody.
Cornea is the outermost anterior layer of the eye ball which
has unique optical and physio-mechanical properties to maintain
good vision. A stratified squamous epithelium is continued with the conjunctival epithelium in the limbal area and create a protective stratum on the ocular surface to defend internal structure from environmental threats
The red blood cell membrane consists of three layers - an outer glycocalyx, lipid bilayer in the middle, and inner phospholipid and cholesterol layer. The lipid bilayer contains phospholipids like phosphatidylcholine and sphingomyelin in the outer monolayer, and phosphatidylethanolamine, phosphatidylserine, and phosphoinositol in the inner monolayer. Transport proteins maintain this asymmetric distribution, which is important for cell integrity and survival. The membrane also contains proteins that contribute to cell structure, transport, adhesion, and signaling.
Objective: To investigate the changes in the retina due to deltamethrin toxicity and the process in cell inflammation and apoptosis.
Study Design: Sixteen Wistar albino rats were randomly divided into two groups as control (n=8) and deltamethrin (n=8) groups. Saline was given to the control group, and 0.5 mL of 5 mg/kg deltamethrin was given to the deltamethrin group for 14 days each. Blood was collected for biochemical analysis. Retinal tissue was processed for histological examination.
Results: Compared to the control group, MDA levels were high while GSH and CAT levels were low in the deltamethrin group. Histopathological analysis showed spaces between the pigment epithelium, irregularity in the delimiting membrane, degenerated ganglion, cone and bacillus cell, pyknotic nuclei, thinned inner limitation membrane, and thickened vascular wall. The control group showed FAS expression in the pigment layer limiting membranes, in the nuclei of many cone and bacillus cells, and ganglion cells in the control group sections. In the deltamethrin group, FAS expression was observed in the inner and outer limiting membranes of the pigment epithelium, cone and bacillus cells, and ganglion cell nuclei. In the control group, negative NOS expression in the pigment epithelium and outer limiting membranes, internal limitation membrane, and ganglion cells in the cone and bacillus cell nuclei were observed. In the deltamethrin group, NOS expression was positive in the pigment epithelium, cone and bacillus, and ganglion cell nuclei.
Conclusion: We suggest that deltamethrin toxicity induced apoptotic process due to increased inflammation in the retina and may cause visual impairment as a result of neural damage.
Keywords: deltamethrin, FAS, insecticides, NOS, nitric oxide synthase, retina
A family of acetylcholine-gated chloride channel subunits in Caenorhabditis e...Igor Putrenko
This document summarizes the cloning and characterization of acetylcholine-gated chloride channel subunits in Caenorhabditis elegans. The authors cloned four subunits, ACC-1, ACC-2, ACC-3, and ACC-4, which form a distinct clade of ligand-gated ion channel genes in C. elegans. They found that ACC-1 and ACC-2 form homomeric channels that are gated by acetylcholine but not nicotine, blocked by D-tubocurarine but not α-bungarotoxin, and conduct chloride ions. ACC-3 and ACC-4 may interact with ACC-1 and ACC-2. This study identifies the first molecular
This study examines the degradation pathway of the thiazide-sensitive NaCl cotransporter (NCC) in yeast and mammalian cells. The authors show that NCC is a substrate of endoplasmic reticulum-associated degradation (ERAD) in yeast. Using yeast strains with mutations in ERAD components, they identify the E3 ubiquitin ligase Hrd1 and the cytoplasmic Hsp70 chaperone Ssa1/Hsp70 as important for NCC ubiquitination and degradation. Expression of NCC in mammalian kidney cells reveals similar polyubiquitination and proteasome-dependent degradation. Cytoplasmic Hsp70 preferentially associates with immature glycosylated NCC, indicating its role
UPS in diabetic microvascular complicationsSaeed Aghdam
The document discusses the ubiquitin-proteasome system (UPS) and its role in the development of microvascular complications of diabetes, specifically diabetic retinopathy and diabetic nephropathy. The UPS regulates protein quality control and degradation in cells and is involved in processes like cell cycle regulation and signal transduction. Dysregulation of the UPS due to hyperglycemia and oxidative stress may contribute to retinal and kidney damage caused by diabetes. Studies have shown UPS activity is altered in the retina and kidney under diabetic conditions. Better understanding how diabetes affects UPS function in the eye could lead to new treatment strategies for diabetic retinopathy.
This document summarizes three research studies on skin and keratinocytes:
1) The first study found elevated expression of osteopontin splice variants in nonmelanoma skin cancers compared to normal skin and adult keratinocytes. It also found that human adult keratinocytes expressed basal or induced levels of only two osteopontin variants.
2) The second study generated a mouse model lacking the desmoglein 1 protein and found it led to perinatal lethality and impaired skin barrier formation. Desmoglein 1 appears essential for normal epidermal morphogenesis.
3) The third study used fluorescence polarization microscopy and magnetic tweezers to investigate protein organization and force transmission at
- Biological membranes are thin barriers that compartmentalize cells and organelles. They perform important functions including transport and signal transduction.
- Membranes are composed of a lipid bilayer along with embedded and peripheral proteins. The fluid bilayer is formed from phospholipids, cholesterol, and glycolipids.
- Transport across membranes can occur through passive diffusion, facilitated diffusion via carrier proteins and channels, or active transport with carriers that require energy.
1) The document describes a new DNA sequencing system that uses picolitre-sized reactors on a microfabricated chip to massively parallelize sequencing reactions and significantly increase throughput over previous methods.
2) Key aspects of the system include using emulsion PCR to amplify DNA fragments on beads in picolitre droplets and performing pyrosequencing reactions in the high-density array of picolitre wells on the chip.
3) The document demonstrates the capabilities of the system by sequencing the genome of Mycoplasma genitalium and assembling it de novo, achieving 96% coverage of the 580 kb genome in a single run at 99.96% accuracy.
Effectiveness Of The Treatment Before And After The...Olga Bautista
The document discusses cation exchange gel samples labeled IEX 19-22 that had the highest activity and were selected for analysis by gel electrophoresis. The gel banding patterns were the same across samples, allowing them to be combined for yield calculations. While dye ligand chromatography resulted in a lower purification than expected, it concentrated the protein of interest compared to earlier fractions as determined by activity assays.
This document discusses materials and methods used in a study involving the chemical fipronil and zinc. Twenty male albino rats were divided into four groups of five rats each: a control group, a zinc group that received zinc supplementation, a fipronil group exposed to the insecticide fipronil, and a combination group exposed to both zinc and fipronil. Biochemical assays were conducted to assess oxidative stress markers like superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase, glutathione, lipid peroxidation, and total protein in the rats. Chemicals used including fipronil and zinc sulfate were obtained from reputable suppliers. Kits for the biochemical assays were purchased from a diagnostic
This document summarizes a study investigating the epigenetic changes that occur during the transdifferentiation of pancreatic acinar cells (HPACs) to hepatocyte-like cells. The study found that DNA methylation in HPACs peaks at 24 hours after treatment with dexamethasone, and the cells display phenotypic changes associated with hepatocytes after 7 days. The study also examined the promoter sequence of the SGK1 gene, which is involved in the transdifferentiation process. The results suggest therapeutic potential for producing hepatocytes from pancreatic cells to treat liver disease in a more cost-effective manner than current alternatives.
Molecular Cloning of the Structural Gene for ExopolygalacturonateAlan Brooks
This document summarizes research on the cloning and characterization of a gene (pelX) from Erwinia chrysanthemi that encodes an exopolygalacturonate lyase (ExoPL). The pelX gene was cloned from a mutant strain lacking known pectate lyase genes. ExoPL was purified from a recombinant E. coli strain and characterized. A pelX mutant was constructed in E. chrysanthemi but retained pathogenicity, indicating ExoPL does not contribute to tissue maceration ability.
The document summarizes a study that investigated the cytotoxic effects and cell death mechanisms of the naturally occurring coumarin A/AA in various cancer cell lines and normal cells. The results showed that coumarin A/AA was cytotoxic to four cancer cell lines tested, including HeLa cells, and was significantly less toxic to normal peripheral blood cells. In HeLa cells, coumarin A/AA induced cell death through DNA fragmentation but not cell cycle disruption. It was found to induce the early release of the apoptosis-inducing factor AIF from mitochondria, suggesting a caspase-independent cell death pathway. This indicates coumarin A/AA may have potential applications in cancer treatment.
This thesis examines the mechanisms of apoptosis induced by hydrogen peroxide, staurosporine, and transforming growth factor-beta 1 in primary cultured thyroid cells. The author finds that apoptosis in these cells is caspase-3 dependent. Hydrogen peroxide causes mitochondrial aggregation and fragmentation without cytochrome c release, while staurosporine induces cytochrome c release from mitochondria. Selenium-substituted cells with high glutathione peroxidase activity are more resistant to hydrogen peroxide-induced apoptosis. The thyroid epithelial barrier is disrupted by these apoptotic stimuli by both caspase-dependent and independent mechanisms.
2. they develop a markedly thickened stratum corneum and
defects in the skin lipid barrier, as evidenced by electron
microscopy and functional assays of tissue water loss. To better
characterize the lipid barrier defect, an extensive analysis of the
lipid content of the skin of the ABCA12-deficient mice was
performed. As ceramides are the major lipid constituent of
lamellar sheets in the intercellular spaces of the stratum cor-neum,
attention was particularly focused on this class of lipids.
Using mass spectrometry, we were able to establish that a
marked decrease in multiple species of Ceramide 1 (also called
Cer(EOS) or esterified -hydroxy acid sphingosine) was pres-ent
in Abca12 null mice. This was associated with a significant
increase in the amount of precursor EOS-glucosyl ceramide in
the skin, suggesting that a loss of ABCA12 activity leads to a
failure to transport glucosylceramides to a location where
cleavage of the sugar moiety by glucocerebrosidase can occur.
These findings indicate that Abca12 null mice are an excellent
model of human harlequin ichthyosis, and they demonstrate
that ABCA12 activity is required for the generation of a specific
class of lipids that are essential to the formation of the epider-mal
permeability barrier.
EXPERIMENTAL PROCEDURES
Generation and Genotyping of Abca12 Null Mice—Mice that
were heterozygous for recombination at the Abca12 locus were
obtained through the National Institutes of Health Mutant
Mouse Resource Center, University of California, Davis. These
mice were generated by replacing exon 9 of Abca12 with a NeoR
cassette via homologous recombination. To confirm the geno-type
of these mice, Southern hybridization was conducted
using genomic DNA cleaved with the SpeI restriction endonu-clease
and a radiolabeled cDNA fragment that hybridizes to
intron 7/8 (nucleotides 4440–5010) of the Abca12 locus. This
probe detects a band of 15.4 kb in the wild-type locus and 8 kb
in the targeted gene. For PCR genotyping, three primer multi-plex
reactions were used (P1, common, 5-GGGGCGGTGAG-AAGTAAAGTAT-
3; P2, wild-type allele, 5-GGATTGGGA-AGACAATAGCAGG-
3; P3, targeted allele, 5-CTTGGCAG-AGTACATCTCAG-
3) that amplify a wild-type product of 356
bp and a targeted product of 271 bp. F2 heterozygous animals
(129SvEvBrd C57BL6/J) were intercrossed to produce
homozygous Abca12/ mice, as well as wild-type and het-erozygous
littermates. All animal procedures were approved by
the Massachusetts General Hospital Subcommittee on
Research Animal Care and were conducted in accordance with
the USDA Animal Welfare Act and the PHS Policy for the
Humane Care and Use of Laboratory Animals.
ABCA12 mRNA Quantitation—Total RNA was extracted
using the RNeasy Mini Kit (Qiagen) according to the manufac-turer’s
instructions (Qiagen). RNA concentration and quality
were assessed using the Agilent RNA Bioanalyzer. RNA (1 g)
was reversed transcribed using the SuperScript First-Strand
Synthesis System for RT-PCR (Invitrogen), and quantitative
real-time PCR reactions were performed on a Thermal cycler
(Thermo Fisher Scientific, Waltham, MA). Levels of -actin
mRNA were used to normalize ABCA12 mRNA levels. The
primer pairs were as follows: ABCA12, 5-AAGATGCTGAC-TGGAGACATAATTC-
3 and 5-GAAATACAAGTGCTCT-TCCACAGTT-
ABCA12 Maintains Skin Ceramide Linoleic Esters
3, which amplify exons 46 and 47 of the
Abca12 mRNA, and 5-CATACAAGCCACTGTTATCC-TCC-
3 and 5-CAGAGATTTCGTACCTCTCCTCA-3,
which amplify the -actin mRNA.
In Situ Hybridization—Automated in situ hybridization was
performed using the Discovery System (Ventana Medical Sys-tems,
Tucson, AZ). Sense and antisense probes that hybridize
to exons 46–51 of the Abca12 mRNA were generated using
digoxigenin-labeled UTP. The efficiency of transcription and
incorporation of digoxigenin-UTP to the riboprobes were eval-uated
by 5% acrylamide-urea gel electrophoresis and dot blot
on nylon membrane, respectively (DIG Nucleic Acid Detection
Kit, nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phos-phate,
Roche Molecular Biochemicals, Mannheim, Germany).
Slides were hybridized at 65 °C for 6 h, washed twice in 0.1SSC
buffer at 75 °C, each for 6 min. The detection was performed
using biotinylated anti-digoxigenin antibody (Biogenex, San
Ramon, CA) followed by streptavidin-alkaline-phosphatase
conjugate and visualized by nitro blue tetrazolium/5-bromo-4-
chloro-3-indolyl phosphate substrate reaction (Ventana
BlueMap Detection Kit, Ventana Medical Systems, Tucson,
AZ). Slides were counterstained by nuclear fast red (Vector
Laboratories, Inc., Burlingame, CA).
Immunoblotting Procedures and Generation of an ABCA12
Antibody—Mouse ABCA12 cDNA was amplified by RT-PCR
from adult C57/BL6 skin total RNA, and the sequence was ver-ified
on both strands by dideoxy sequencing. An anti-ABCA12
anti-serum was raised against the C-terminal 98 amino acids of
murine ABCA12 as we previously described for ABCA1 (23).
The specificity of the sera obtained was confirmed by immuno-blotting
of lysates from HEK293 cells transfected with the
cDNAs of ABCA12, ABCA7, or ABCA1, using the ABCA12
antiserum (diluted 1:1000) and pre-immune control serum
(diluted 1:1000). The antiserum recognized a band of290 kDa
only in lysates of cells transfected with the ABCA12 cDNA.
Equal amounts of skin lysates (20 g of total cell protein in a
lysis buffer composed of 20 mM Tris-HCl, pH 7.5, 150 mm
NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% deoxycholate, 0.1%
SDS, and 0.001% Sigma protease mixture) were electrophore-sed
on 4–12% or 6% SDS-PAGE gels and transferred onto
nitrocellulose membranes. Membranes were blocked overnight
at 4 °C in 1 PBS, 5% nonfat dry milk, and 1% bovine serum
albumin, and were probed with the anti-ABCA12 or anti-
ABCA1 antiserum, diluted 1:1000, at room temperature for 2 h.
Membranes were washed in 1PBS containing 0.1% Tween 20.
Antibody binding was detected using a horseradish peroxidase-conjugated
goat anti-rabbit antibody (Sigma) and enhanced
chemiluminescence (Amersham Biosciences). For analysis of
filaggrin solubility and processing, skin lysates were prepared
with the buffer as above, or with a denaturing thiocyanate
buffer (1 M NaSCn, 50mM HEPES, 10mM EDTA, 0.3mM ortho-phenanthroline,
20 g/ml phenylmethylsulfonyl fluoride, and
0.1% isopropanol) as previously described (24, 25). To enhance
extraction of cornified proteins using the thiocyanate buffer,
lysates were sonicated and snap frozen (2). Precipitated pro-teins
were solubilized in 8Murea before boiling in reducing SDS
loading buffer.
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3. Histological, Immunofluorescent, and Ultrastructural
Procedures—For hematoxylin and eosin (HE) histological
analysis, skin samples were fixed overnight (4% formaldehyde,
1 PBS, 4 °C), embedded in paraffin, and 5-m sections were
stained and imaged using a Nikon Eclipses 50i microscope. For
immunofluorescence staining, OCT-embedded skin was snap
frozen in liquid nitrogen-cooled isopentane, and sections (5
m) were fixed (acetone:methanol (1:1), 20 °C) and blocked
with Superblock (Thermo Scientific) (26). Slides were incu-bated
with primary antibodies for loricrin, filaggrin, K10, K14
(Covance), and Ki67b (Abcam Inc.), or with an equivalent
amount of non-immune rabbit IgG. They were washed three
times (0.1% Nonidet P-40, 1PBS) and primary antibody bind-ing
was detected with a CY3-labeled goat anti-rabbit IgG anti-body
(Santa Cruz Biotechnology, Santa Cruz, CA). Nuclei were
counterstained with Hoechst 33258 dye (1g/ml, 0.1% Nonidet
P-40, 1PBS) (Sigma-Aldrich), and slides were imaged using a
Nikon OPTIPHOT photomicroscope (Nikon Eclipse E 800).
For electron microscopy studies, skin taken from littermate
E18.5 embryos was prepared and imaged as described previ-ously
(27). In brief, dorsal skin was fixed overnight at 4 °C in 2%
glutaraldehyde, 2% paraformaldehyde, with 0.06% calcium
chloride in 0.1 M sodium cacodylate buffer (pH 7.3). Skin sam-ples
were post-fixed (0.2% ruthenium tetroxide, 0.5% potas-sium
ferrocyanide in 0.1 M sodium cacodylate, pH 6.8, room
temp in darkness, 30 min), dehydrated, and embedded in Epon-
812 resin (Electron Microscopy Sciences, Hatfield, PA). Ultra-thin
sections cut on a Reichert Ultracut E ultramicrotome were
collected onto Formvar-coated slot grids and examined
unstained or after double staining with uranyl acetate and lead
citrate using a JEOL 1011 transmission electron microscope
with an ATM digital camera at 80 kV. A survey of corneocyte
interstitial lamellae and granulocyte lamellar storage bodies in
the epidermis of littermate-paired E18.5 pups (Abca12/ and
Abca12/; n3) was carried out on 100 micrographs at mag-nifications
of 80,000–120,000.
In Utero BrdUrd Incorporation and Imaging—Pregnant mice
were injected intraperitoneally with 5-bromo-2-deoxyuridine
(BrdUrd, 100 g/g, Invitrogen) and sacrificed 1 h later as
described with the following modifications (28). Dorsal skin
from the labeled E18.5 embryos was embedded, sectioned, and
fixed as described above, and slides were stained with an anti-
BrdU antibody (Invitrogen). Nuclei were counterstained with
Hoechst 33258 dye (Sigma-Aldrich). IP Lab Spectrum software
(4.0) was used to quantify the area of BrdUrd-positive nuclei per
area of epidermal tissue (Scanalytics, Rockville, MD). Average
values derived from five images of each embryo sample were
compared (Abca12/, n 3 and Abca12/, n 4).
Skin Permeability Assays—Dye penetration assays were per-formed
with toluidine blue as previously described (29). In
brief, E18.5 embryos were dehydrated by incubations in 25%,
50%, 75%, and 100% methanol/PBS (1 min per incubation). The
embryos were washed in PBS and stained in 0.0125% toluidine
blue for 10 min. After staining, embryos were immediately
washed in PBS three times and photographed. Trans epidermal
water loss (TEWL) was measured in 1-cm2 skin sections using a
gravimetric assay as previously described (30).
Mass Spectrometry Lipid-profiling Procedures—Phospholip-ids
in skin lysates from individual E18.5 embryos were deter-mined
using an electrospray ionization-MS/MS approach as
reported previously (16). In brief, skin (5–10 mg) cleaned of
all subcutaneous tissue (Abca12/ and Abca12/ E18.5
embryos, n 5) was minced with scissors and vortex-homog-enized
with steel beads in 0.5 ml of 1 PBS (BioSpec, Bartles-ville,
OK). Homogenate was extracted with 2.5 ml of chloro-form/
methanol/distilled water (1:1:0.5, v/v), and two 0.5-ml
chloroform extractions. Combined organic phases were
wash with 0.5 ml of KCl (1 M, 1) and with 0.5 ml of dH20
(2) and dried with N2 gas. Samples were resuspended in 1
ml of chloroform, and phospholipid scans were performed as
described (16). To analyze ceramides and glucosylceramides,
additional scans were performed for precursors ofm/z 264 in
the positive mode (collision energy, 50 V) for GlcCers and
high mass Cers and neutral loss of m/z 316 in the negative
mode (collision energy, 50 V) for low mass Cers. Internal
standards for quantitating these sphingolipids were d18:1/
14:0Cer and d18:1/12:0GalCer.
Levels of free fatty acids in skin lysates from individual E18.5
embryos were determined by methyl esterification (22): sam-ples
were mixed with methanol:methylene chloride and acetyl
chloride, incubated for 1 h at 75 °C, cooled, and then 7% potas-sium
carbonate was added. Lipids were extracted with hexane,
washed with acetonitrile and injected (splitless) into an Agilent
6890 gas chromatography-MS (G2613A system, Agilent Tech-nologies,
Palo Alto, CA) equipped with a Supelcowax-10 col-umn
(30 m 0.25 mm 0.25 m film, Supelco, Bellafonte,
PA). The temperature program for the gas chromatography
oven was as follows: Initial temperature 150 °C for 2 min,
increased to 200 °C (10 °C/min), held at 200 °C for 4 min,
increased to 240 °C (5 °C/min), held at 240 °C for 3 min, and
finally increased to 270 °C (10 °C/min) and held at 270 °C for 5
min.Amodel 5973N mass-selective detector (Agilent Technol-ogies)
in scan mode and coupled with Enhanced Chemstation
G1701CA versionCsoftware was used to identify the generated
mass spectra by comparison to a spectral library created from
known fatty acids. The amount of each fatty acid species was
quantified relative to an internal standard of heptadecanoic
acid (17:0) added to the samples before extraction. Total cho-lesterol
in the skin was determined as previously described (31).
Statistical Analysis—All statistical analyses were per-formed
using Student’s t test with sample sizes indicated in
the figure legends for each comparison that was made. A p
value of 0.05 was considered statistically significant.
RESULTS
Homozygous Null Abca12/ Mutations in Mice Result in
Neonatal Lethality and Epidermal Hyperkeratosis—Thetarget-ing
strategy used to produce mice lacking ABCA12 is shown in
Fig. 1A. To confirm that the heterozygous mice we obtained
from the NIH Mutant Mouse Resource had the correctly rear-ranged
gene structure, we performed Southern blot analysis.
Appropriate homologous recombination leading to the dele-tion
of exon 9 was confirmed in E18.5 embryos derived from
Abca12/ heterozygous matings (Fig. 1B). One or both wild-type
15.4-kb SpeI fragments were replaced by an 8.0-kb band in
ABCA12 Maintains Skin Ceramide Linoleic Esters
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4. ABCA12 Maintains Skin Ceramide Linoleic Esters
FIGURE 1. Deletion of Abca12 exon 9 in mice blocks ABCA12 expression. A, exon 9 of the Abca12 locus (wt) was disrupted by homologous recombination.
The resulting targeted allele (tg) contains aNeoRcassette and truncates the 2596-amino acidABCA12open reading frame after amino acid 328. B, Southern and
PCR analysis of DNA from day 18.5 embryos demonstrated transmission of the targeted allele and generation of the null state. Position of the DNA probe and
PCR primers are indicated in the diagram. ABCA12 mRNA levels are significantly diminished in the Abca12/ mice as determined by quantitative RT-PCR (n
5, S.D., p 0.5) (C) and by in situ hybridization (D); scale bar, 20 m. E, Abca12/ mice lack ABCA12 protein as determined by immunoblots of skin lysates
using anti-ABCA12 C-terminal antibody. Serial probing of the blot for ABCA1, an ABCA12 homologue, demonstrated equal loading of the membrane, and no
compensatory change in ABCA1 expression.
animals heterozygous or homozygous, respectively, for the tar-geted
gene locus. Having established by Southern blot that the
desired homologous recombination at exon 9 of the Abca12
locus had occurred in parental strains, high throughput geno-typing
of mouse offspring was subsequently performed using an
assay that enabled all three potential genotypes to be deter-mined
in a single, multiplexed PCR reaction (Fig. 1B). To estab-lish
that the gene rearrangement led to loss of expression of
ABCA12, assays of both ABCA12 mRNA and protein levels
were performed. ABCA12 message levels in the skin were first
determined using reverse transcriptase-quantitative PCR
amplifications of exons (46 and 47) downstream of the deleted
exon 9. Fig. 1C demonstrates that, although wild-type animals
have readily detected levels of ABCA12 mRNA, null mice do
not. These data indicate that the targeted deletion of exon 9
results in the production of an mRNA that is either unstable or
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5. FIGURE 2. Loss of ABCA12 cause neonatal lethality and epidermal hyperkeratosis. A, Abca12/ day 18.5 embryos (E18.5) when kept hydrated are similar
in size and weight to Abca12/ littermates, but after exposure to air rapidly dry out and become encased in a collodion-like shell (B). The Abca12/ epidermis
displays a markedly thickened, hyperkeratotic epidermis as shown in hematoxylin eosin-stained skin sections; scale bar, 20 m (C) (sc stratum corneum,
sg stratum granulosum, ss stratum spinosum, and sb stratum basale).
poorly transcribed. These in vitro amplification results were
confirmed by an in situ hybridization assay using a probe
encompassing exons 46 to 51. ABCA12 mRNA was detected in
the skin of wild-type animals by the antisense probe, but the
same probe was unable to generate any signal of ABCA12
expression in animals homozygous for the targeted deletion
(Fig. 1D). Consistent with the RNA analyses, immunoblots of
proteins extracted from epidermis revealed no ABCA12 pro-tein
in null mice, but a band of the expected size was presented
in heterozygotes and wild-type mice. All three groups of mice
had similar levels of expression of the closely related ABCA1
transporter (Fig. 1E). In composite, these results indicate that
deletion of exon 9 of Abca12 prevents expression of ABCA12.
Abrogation of ABCA12 expression led to death in mice, as no
homozygous null animals survived the immediate postnatal
period. However, if day 18.5 embryos were genotyped, near
Mendelian frequencies of each genotype were obtained (wt:het:
null, 104:155:72). Larger genotyping studies will be needed to
determine whether Abca12/ animals have slightly reduced
viability in utero, as suggested by the genotyping results, but the
absence of the transporter is clearly compatible with embryonic
development to term. After birth, if the null animals are kept in
an ambient air environment, mortality is universal, indicating a
vital role for ABCA12 in survival ex utero.
To investigate the cause of death of Abca12/ mice, studies
were carried out on late term E18.5 embryos delivered by cesar-ean
section. When kept hydrated, the weight of Abca12/
embryos was indistinguishable from that of wild-type and het-erozygous
littermates (wt, 0.96 0.28 g, n 16; heterozygous,
1.06 0.26 g, n 22; null 1.02 0.21 g, n 20; p 0.44 wt
versus null) (Fig. 2A). However, when exposed to ambient tem-perature
and humidity, the Abca12/ pups rapidly dehy-drated
within a matter of minutes, with their skin contracting
and encasing them in a collodion-like shell (Fig. 2B). Consistent
with a specific defect in skin function, histological analysis
showed a markedly hyperkeratotic stratum corneum in the
Abca12/ mice (Fig. 2C). Further histological analysis did not
reveal gross pathologies of the other major organs, including
the lungs, which the Abca12/ neonates were able to aerate,
and which contain surfactant and type II pneumatocyte lamel-lar
bodies as determined by electron microscopic analysis (sup-plemental
Fig. S1).
Loss of the Interstitial Lamellar Barrier in the Abca12/
Skin Increases Trans-epidermal Water Loss—The markedly
thicken stratum corneum observed in the Abca12/ animals
and their rapid demise after birth suggested that the loss of
ABCA12 activity had disrupted their epidermal permeability
barrier. To test this hypothesis, E18.5 Abca12/embryos were
stained with toluidine blue, a dye that does not penetrate the
epidermis upon formation of the permeability barrier. As seen
in Fig. 3A, the null mouse uniformly takes up the dye, whereas
the wild-type littermate can effectively exclude it, consistent
with the latter’s ability to form an intact permeability barrier.
To investigate the bi-directional function of the permeability
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6. barrier, TEWL rates were measured. Skin samples from
Abca12/, Abca12/, and Abca12/ E18.5 embryos were
tested for TEWL rates using a gravimetric assay. Confirming
the results of the toluidine blue dye test, the TEWL assay
demonstrated significantly greater water loss through the
epidermis of Abca12/ skin compared with that taken from
wild-type or heterozygous mice (Fig. 3B). A slightly greater
rate of water loss was noted from the heterozygous skin rel-ative
to the homozygous wild-type samples, but this trend
did not reach statistical significance, consistent with the his-tological
observations showing no gross abnormality of the
skin of the E18.5 Abca12/ embryos.
Having confirmed that loss of ABCA12 function had dis-rupted
the epidermal permeability barrier, we then investigated
the ultrastructural conformation of the epidermis that is known
to be involved in the creation of the skin’s barrier function. We
were particularly interested in assessing whether the corneo-cytes
of the thickened Abca12/ stratum corneum lacked the
characteristic interstitial lamellar lipid barrier, as has been
reported in infants with the HI phenotype. As expected,
Abca12/ E18.5 embryos showed a clearly organized stratum
corneum with interstitial lamellar material, as well as lamellar
storage bodies in the stratum granulosum (Fig. 4, A–D). In con-trast,
their Abca12/ littermates demonstrated a stratum cor-neum
thickened 6-fold that lacked organized lamellar struc-tures
in the interstitial spaces (Fig. 4, E and F). The interstitial
spaces of the null skin were instead filled with a disorganized
multivesicular material (Fig. 4F). Similarly, in the stratum
granulosum of the Abca12/ epidermis there was no evidence
of intact lamellar storage bodies, although numerous multive-sicular
bodies were found, which are the presumed precursor of
the lamellar body (Fig. 4, G and H). In aggregate, these findings
demonstrate that loss of ABCA12 function in mice compro-mises
the structural integrity of the skin’s lamellar permeability
barrier, leading to an inability to maintain water homeostasis,
causing the demise of the newborn Abca12/ pups after expo-sure
to air.
Loss of ABCA12 Function Does Not Alter E18.5 Epidermal
Proliferation Indices but Is Associated with Altered Keratin 14
Expression, Filaggrin Solubility, and Parakeratosis—To further
explore the cause of the thickened stratum corneum of
Abca12/ skin, we investigated whether loss of ABCA12 func-tion
induced aberrant cellular proliferation of the epidermis.
Epidermal DNA synthesis of E18.5 embryos in utero was meas-ured
after injecting bromodeoxyuridine (BrdUrd) into the per-itoneum
of the pups’ mother. After 1 h of exposure, embryos
were sacrificed, and epidermal nuclei positive for BrdUrd were
detected in skin sections using an anti-BrdUrd antibody. As
shown in Fig. 5A, BrdUrd-positive cells were observed in the
basal layers of both Abca12/ and Abca12/, but increased
numbers of BrdUrd-positive cells were not found in the
Abca12/ basal layer, nor in the normally quiescent upper
cellular layers of the stratum spinosum and stratum granulo-sum
(supplemental Fig. S2). We considered whether the
thicken stratum corneum and/or loss of ABCA12 transport
activity may have inhibited access of BrdUrd to the proliferat-ing
cellular layers of the Abca12/ epidermis, thus giving an
underestimate of cell division. To address this possibility, we
assessed cellular proliferation by an alternative method,
directly staining for the proliferation marker Ki67. Consistent
with the BrdUrd incorporation assay, expression of Ki67 in
Abca12/ epidermis was not found to be increased over that
seen in littermate paired Abca12/ samples (Fig. 5B). Finally,
we stained for Keratin 6, which is expressed in epidermal kerat-inocytes
that hyperproliferate, but no Keratin 6 staining was
observed in the cellular layers of the Abca12/ epidermis
(data not shown). Considered together, our evidence in late
term Abca12/ embryos provided no evidence that aberrant
cellular proliferation rates were driving the expansion of the
stratum corneum.
Because an increase in proliferation markers was not
observed in the Abca12/ epidermis, we explored whether
loss of ABCA12 had altered the expression of the keratins and
cornified envelope proteins that are associated with the differ-entiation
and maturation of basal cells into granulocytes and
corneocytes. Keratin 14 expression, a marker of the basal layer,
was detected in the Abca12/ skin only in the lowest cellular
layer of the epidermis. Interestingly, in the Abca12/ epider-mis,
K14 expression was more expansive and appeared to per-sist
in first cells of the stratum spinosum (Fig. 5C). Expression of
FIGURE 3. Abca12/ mice fail to develop the epidermal permeability
barrier. A, littermate E18.5 embryos stained with toluidine blue indicates loss
of ABCA12 function increases inward permeability to this dye. B, loss of
ABCA12 function significantly increases outward trans-epidermal water loss
rates as measured in littermate E18.5 embryos using the gravimetric assay
(Abca12/, n 3; Abca12/, n 7; Abca12/, n 6; S.E.: p 0.11,
Abca12/ versus Abca12/; p 0.00005, Abca12/ versus Abca12/).
ABCA12 Maintains Skin Ceramide Linoleic Esters
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7. FIGURE 4. Loss of ABCA12 transport function induces hyperkeratosis of the stratum corneum (SC) and disrupts interstitial lamellar lipid structure.
A, electron micrograph of the Abca12/epidermis showing a normal SC (10,000). E, Abca12/epidermis showing massive hyperkeratosis of the SC (2000
magnification, which was required to capture the expanded SC). B, interstitial spaces of the Abca12/SC demonstrate the lipid lamellar structures required for
permeability barrier function (arrow, 80,000), which are absent and replaced by a disorganized multivesicular material in the interstitial spaces of the
Abca12/ SC (F, arrow, 80,000). C andD, lamellar storage bodies present in the Abca12/ stratum granulosum (C andD, 150,000). The Abca12/ stratum
granulosum lacked lamellar storage bodies but did contain numerous multivesicular bodies, the presumptive precursor of the lamellar body (G and H,
150,000).
Keratin 10, which marks cells in the suprabasal layers that have
initiated terminal differentiation, was detected in the spinosum
and granular layers and was found to be similar between the
wild-type and null samples (Fig. 5D). The late differentiation
markers loricrin and filaggrin, which are expressed by granulo-cytes
and are incorporated into the cornified envelope, were
present in the Abca12/ granular and stratum corneum lay-ers.
Compared with the staining of these same proteins in wild-type
skin, however, the distribution appeared more punctate,
particularly for filaggrin (Fig. 5, E and F). Because skin from HI
patients has been associated with a block in the processing of
profilaggrin to filaggrin (20), immunoblots were used to meas-ure
amounts of these products that were extractable with either
a mild detergent or with the strong denaturant, urea. Signifi-cantly,
less processed filaggrin was detected in the detergent
extracts of Abca12/ samples, whereas profilaggrin, K14,
involucrin, and-actin levels were similar (Fig. 6A). In contrast,
both profilaggrin and filaggrin levels were as high, if not higher,
in the null samples extracted with a strong denaturant (Fig. 6B).
These results suggest that loss of ABCA12 does not induce a
ABCA12 Maintains Skin Ceramide Linoleic Esters
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8. ABCA12 Maintains Skin Ceramide Linoleic Esters
FIGURE 5. Loss of ABCA12 function does not alter E18.5 epidermal cell proliferation indices. A, DNA
synthesis rates are similar in E18.5 Abca12/ and Abca12/ embryos as determined by in utero incorporation
of the thymidine analogue, BrdUrd. Shown are skin sections from littermate-paired embryos labeled for1hand
stained with an anti-BrdUrd antibody (green) and counterstained for nuclei with Hoechst 33258 (blue). B, stain-ing
for the Ki67 proliferation maker (magenta) is also similar in wild-type and null samples and is confined to the
basal layer. C, staining for the basal cell differentiation marker Keratin 14 (red) is expanded into the first cell layer
of the stratum spinosum in the Abca12/ epidermis. D, staining for Keratin 10 is similar between the geno-types
and is largely confined to the stratum spinosum and stratum granulosum. Staining for the cornified
envelope marker loricrin (E) and filaggrin (F) show a more granular distribution in the Abca12/ epidermis.
The basal epidermal layer is indicated by filled arrowheads, and open arrowheads denote parakeratotic nuclei in
the Abca12/ stratum corneum. Scale bar, 20m.
block in the processing of profilaggrin to filaggrin, although the
solubility characteristics of the protein are altered. Finally, it
was noted that the Abca12/ epidermis exhibited signs of
abnormal parakeratosis, because the uppermost layers of the
stratum corneum were found con-sistently
to stain positively for
nuclear material (open triangles
denote stained nuclei in the stra-tum
corneum, Fig. 5). Thus, in mice,
loss of ABCA12 function does not
block filaggrin processing, but it
does affect the extraction properties
of that protein from skin, and is
associated with a persistence of
nuclear material in the stratum
corneum.
Lipid Mass Spectrometry Profiling
of Abca12/ Skin Shows Loss of
Cer(EOS) and Accumulation of
GlucosylCer(EOS)—The ultrastruc-tural
analysis described above indi-cated
that ABCA12 function was
critical for maintaining the charac-teristic
lamellar appearance of the
interstitial spaces in the stratum
corneum. To form these structures
of the permeability barrier, a com-plex
mix of lipids are packaged into
lamellar storage bodies and subse-quently
extruded into the extracel-lular
space of the stratum corneum.
This mix of lipids includes choles-terol,
free fatty acids, phospholipids,
and ceramides. Ceramide composi-tion
and structure are particularly
important for the formation of the
permeability barrier and its lamellar
structure. The very hydrophobic
linoleic esters of -hydroxy long-chain
ceramides are thought to have
unique biophysical characteristics
that aid in formation of the lamellar
barrier. To gain further insight into
ABCA12 function, keeping in mind
its membership in a lipid trans-porter
family, we sought to charac-terize
the level of the various lipids
that form the lamellar barrier,
using a broad mass spectrometry
approach. Total cholesterol levels
in the skin of the Abca12/ mice
did not differ significantly from
that of littermate matched Abca12/
mice (supplemental Fig. S3). This
was also the case for total phos-phatidylcholine,
phosphatidyleth-anolamine,
phosphatidylserine,
sphingomyelin, and free fatty acids
(Table 1 and supplemental Figs. S4–S8). However, total cera-mide
levels were significantly reduced in the skin of Abca12/
mice (Table 1), and this reduction was most profound for levels
of the linoleic esters of-hydroxy long-chain ceramides (Fig. 7,
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9. ABCA12 Maintains Skin Ceramide Linoleic Esters
A and B). Because these ceramides are formed from glucoslyc-eramide
precursors (32), which are the substrates packaged
into the lamellar storage bodies in the epidermal granular layer
(33), we further analyzed levels of glucosyl Cer(EOS) precur-sors.
As opposed to the 90% drop in the levels of the Cer(EOS)
species, the corresponding glucosylCer(EOS) species were
increased by nearly 5-fold when ABCA12 function was lost (Fig.
7B). The Cer(EOS) and GlcCer(EOS) were identified by scan-ning
for the sphingoid base. To confirm their identities, prod-uct
ion analysis was performed. Collision-induced fragmenta-tion
in negative mode of the [M OAc] ions for each of the
long-chain ceramides shown in Fig. 7 (and for the most prom-inent
GlcCers (d18:1/50:2 and d18:1/52:3)), produced an m/z
279 fragment. This mass is consistent with a 18:2 anion, as
expected for a linoleic ester of the -hydroxy ceramide. Loss of
ABCA12 also affected the levels of un-esterifed Cer(OS) spe-cies,
but this effect was not as profound and varied relative to
the acyl chain length (supplemental Fig. S9). The Cer(NS) and
GlcCer(NS) species were largely unaffected by loss of ABCA12
(supplemental Figs. S9 and S10). Thus, a broad profiling of lip-ids
indicates ABCA12 transport function is critical for main-taining
skin linoleic esters of -hydroxy long-chain ceramides
and when this function is lost, glucosyl precursors of these cer-amides
accumulate.
DISCUSSION
In this study, we have explored the function of ABCA12 by
investigating homologous recombinant mice lacking its expres-sion.
Abca12/ mice developed to embryonic day 18.5 with
near Mendelian frequencies, but universally failed to survive ex
utero. The skin of these mice has a markedly expanded stratum
corneum composed of disorganized and poorly condensed cor-neocytes
that lack their characteristic interstitial lipid lamellae.
The loss of this lipid lamellae results in formation of a defective
water permeability barrier in the skin, leading to massive water
loss from the Abca12/ epidermis. A broad-scale lipidomic
analysis of Abca12/ skin detected a reduction in the levels of
linoleic esters of -hydroxy long-chain ceramides that was
associated with a corresponding increase in the glucosylated
precursors of those same lipids. These data indicate that
ABCA12 plays an essential role in the formation of the skin’s
permeability barrier via effects on a ceramide that plays a spe-cialized
role in generating that barrier.
In children and adults, mutations in ABCA12 have been
associated with disruption of the lamellar permeability barrier
and skin ichthyoses of variable severity. Initially, in 2003,
ABCA12 missense mutations were associated with lamellar
ichthyosis type 2, a less severe non-fatal scaling condition of the
skin (6). Subsequently, in 2005, two reports associated trunca-tion
and deletion mutations in ABCA12 with the more severe
and often fatal harlequin ichthyosis (3, 5). The HI phenotype is
characterized by a markedly thickened stratum corneum and
loss of interstitial lamellar structures between the corneocytes
of the expanded stratum corneum. The skin of the Abca12/
mice analyzed here recapitulated these features of HI. As esti-mated
by our electron micrographs, the Abca12/ stratum
corneum is expanded 5- to 6-fold, and the corneocytes within
this expanded SC lacked all evidence of interstitial lamellar
structures. Consistent with a loss of corneocyte interstitial
lamellae, the granulocyte layer of the Abca12/ epidermis
showed no evidence of the lamellar storage organelles that are
the presumptive source of the lipids that form the corneocyte
interstitial lamellae. However, we did observe numerous mul-tivesicular
bodies in the Abca12/granulocytes, which may
represent lamellar bodies precursors that are the source of the
disorganized multivesicular material observed in the interstitial
spaces of the expanded Abca12/stratum corneum. These
ultrastructural observations are entirely consistent with the
functional assays we performed that indicated a permeability
barrier failure, i.e. rapid dehydration of the null animals,
increased skin water loss by gravimetric assay, and an inability
to exclude the water soluble toluidine blue dye.
During the preparation of this report, Yanagi et al. (34) pub-lished
on-line an independent investigation of their own
Abca12/ mice. Although the skin histological and ultrastruc-tural
findings in their mice are similar to those we report, there
are several interesting differences in the two studies. Yanagi et
al. concluded that their Abca12/ mice failed to inflate their
lungs due to a deficiency in pulmonary surfactant and likely
FIGURE 6. Loss of ABCA12 function alters filaggrin solubility but not proc-essing.
A, immunoblot analysis of Keratin 14 (K14), involucrin (Inv), profilaggrin
(Profil), and filaggrin (Fil) extracted from the E18.5 skin using mild detergent
buffer. B, profilaggrin and filaggrin extracted from skin using denaturing buffer.
TABLE 1
Loss of ABCA12 function significantly reduces skin ceramide levels
Lipid ABCA12/ ABCA12/ p valuea
Ceramideb 0.67 0.28 0.35 0.17 0.04
Phosphatidylcholine 3.98 1.02 3.34 0.41 0.23
Phosphatidylethanolamine 1.31 0.62 1.09 0.14 0.46
Phosphatidylinositol 0.29 0.08 0.26 0.05 0.38
Phosphatidylserine 0.10 0.03 0.07 0.01 0.08
Sphingomyelin 0.74 0.21 0.66 0.08 0.43
Cholesterolc 1.74 0.15 1.68 0.15 0.51
Free fatty acid 16.2 3.6 20.4 0.5 0.27
a p values are derived from a two-tailed Student’s t-test (n 5, S.E.).
b Ceramide, phospholipid, and free fatty acid values are expressed as nanomoles/mg
of skin.
c Cholesterol values are expressed as micrograms/mg of skin tissue.
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10. ABCA12 Maintains Skin Ceramide Linoleic Esters
FIGURE 7. Loss ofABCA12transport function blocks Cer(EOS) formationandinduces a buildup of GlcCer(EOS).A, representative spectra showing the loss
of the Cer(EOS) peaks in the Abca12/ skin sample (peak at 1020.2 m/z derived from the standard mixture is shown for comparison). B, quantitation of the
Cer(EOS) and corresponding glucosyl Cer(EOS) species in the wild-type and null skin samples (n 5, S.E.; *, p 0.05).
died from respiratory failure.Wedid not find this to be the case
in the mice we produced, because they were able to expand and
aerate their lungs, as evidenced by histological exam and the
ability of Abca12/lungs to float in phosphate-buffered saline
(supplemental Fig. S1, A and B). Furthermore, electron micro-graphs
of Abca12/ lungs showed type II alveolar cells that
were able to form lamellar bodies, unlike the epidermal cells in
these animals, and secrete surfactant in a normal manner (sup-plemental
Fig. S1C). Finally, when we kept Abca12/ mice in a
highly hydrated environment, they could survive and breathe
for several hours after birth, a finding inconsistent with an
inability to aerate the lung due to the lack of surfactant produc-tion
(supplemental video). Mice that were not hydrated died
much more rapidly.
Further studies will be needed to reconcile these disparate
observations, because they could reflect differences in the
genetic disruptions engineered into the mice or some strain
variability. Although we have found no evidence for expression
of full-length ABCA12 protein in our null mice using either a
C-terminal antibody (Fig. 1E) or an N-terminal antibody (data
not shown), we cannot exclude the possibility that a truncated
transcript derived from the rearranged locus in the mice could
lead to some functional activity that accounts for the differ-ences
in lung phenotype seen. Although we cannot rule out a
role for ABCA12 in the lung, several observations make us cau-tious
about accepting a major role for ABCA12 in pulmonary
function. Previously, we reported the generation of mice lack-ing
ABCA3, and those animals died much more rapidly after
birth than did Abca12/ mice (16). The Abca3/ mice were
never able to inflate their lungs, a finding subsequently con-firmed
by several other groups, and they failed to form lung
lamellar bodies and secrete surfactant into the alveolar space
(15, 16, 35, 36). Furthermore, we found ABCA3 to be highly
expressed in the lung, whereas ABCA12 is not (Ref. 16 and data
not shown). Finally, our reading of the literature does not sug-gest
that immediate respiratory compromise is a common fea-
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11. ture of human harlequin ichthyosis, whereas children lacking
ABCA3 activity cannot breathe without assistance. For all of
these reasons, we believe that ABCA3 is the major surfactant
transporter in the lung, whereas ABCA12’s primary locus of
activity is the skin.
To gain insight into the primary function of ABCA12, we
hypothesized that it would play a role in lipid transport and
therefore influence lipid levels in the skin. To explore that
hypothesis, gas chromatography and electrospray ionization
mass spectrometry approaches were used to profile a broad
spectrum of skin lipids. Previously, we used this approach to
show a unique and specific role for ABCA3 in maintaining lung
levels of phosphatidylglycerol and short-chain phosphatidyl-choline
species, which are packaged in lamellar surfactant stor-age
organelles in the type II alveolar cells (16). In the current
study, we found that loss of ABCA12 transport function causes
a 90% reduction in the amount of linoleic esters of long-chain
-hydroxyceramides. Associated with this finding was an
5-fold increase in the levels of the glucosyl precursors of these
-hydroxyceramide esters. As studies in-glucocerebrosidase-deficient
fibroblasts have shown that cells can prevent signifi-cant
glucosylceramide accumulation by metabolizing these lip-ids
to other sphingolipid forms, it is perhaps not surprising that
the decline in ceramide mass we measured was not matched by
an equivalent mass increase in glucosylceramides (37).
The quantitative and specific molecular characterization of
lipid species affected by the loss of ABCA12 that we report in
this study are consistent with the qualitative, immunohisto-chemical
results reported by Yanagi et al. (34), who noted a loss
of ceramide staining in the stratum corneum of their mice, as
well as accumulation of glucosylceramide staining intracellu-larly.
Because glucosylceramides are packaged in the lamellar
bodies of skin granulocytes and subsequently converted by
-glucocerebrosidase to the corresponding non-glycated cera-mides
(38), it is tempting to speculate that ABCA12 does indeed
play a direct role in lipid transport involving these specialized
ceramides. Loss of this transport activity would thereby result
in the failure to extrude these glucosylated precursors into the
extracellular environment, where -glucocerebrosidase pro-cesses
them.
Whereas more direct experimental data are needed to estab-lish
the mechanism proposed above, several lines of evidence
support this hypothesis. As sphingomyelin and ceramide are
produced in the same biosynthetic pathway, but sphingomyelin
levels were unaffected by the loss of ABCA12 activity, our data
indicate that ABCA12 is not essential for generalized sphingo-lipid
synthesis. Similarly, we found that loss of ABCA12 activity
did not significantly affect skin-free fatty acids levels, including
that of linoleic acid, which indicates that the loss of the linoleic
ceramide esters was not secondary to insufficient cellular lino-leate
supplies. Although it is formally possible that ABCA12
could play a role in transporting -glucocerebrosidase rather
than its substrate lipids, studies in animals either lacking the
enzyme in the skin or treated with an enzyme inhibitor have
reported no disruption of the epidermal lamellar body structure
(38, 39). Additionally, loss of -glucocerebrosidase, or its acti-vator
Saposin-C caused a different spectrum of ceramide and
glucosylceramide alterations in the skin than those we report
here in the Abca12/ mice (40, 41). Thus, we believe that
current data are most consistent with a model that posits
ABCA12 functions as a lipid transporter that is required for the
transport of a specialized class of ceramides into the lamellar
body.
Our findings with ABCA12, in concert with studies of the
other ABCA family members, suggest a general paradigm for
the biological role of members of the ABCA transporter family.
This paradigm posits that each member of the ABCA family
will have a unique lipid substrate or spectrum of substrates that
it transports across a cellular membrane. This transport enables
the lipid to associate with an acceptor protein or enzyme that
resides on the distal side of the membrane that must be crossed.
We further suggest that each member of the transporter family
has evolved to enable transport of a specialized class of lipids
and that the expression of the transporter may be restricted in a
cell or tissue-specific fashion to enable a specialized function.
Supporting the latter hypothesis in the current study are our
findings that several classes of skin lipid levels shown to be
substrates for transport by other members of the ABCA family
were unaffected by the loss of ABCA12 (16).
How the loss of ABCA12 transport function provokes the
development of an extremely hyperkeratotic stratum corneum
is unclear. It had been proposed that in response to the loss of
the permeability barrier, the epidermis mounts a compensatory
cellular proliferation response in an effort to restore the barrier
(42). As analyzed by BrdUrd incorporation into epidermal ke-ratinocytes
and by expression of independent proliferation
markers in these cells, we found no evidence that loss of
ABCA12 levels induced cellular proliferation in the epidermis.
However, we did find evidence of altered K14 expression in the
basal layer, and of residual nuclear material in the thickened
stratum corneum. Additionally, the solubility of filaggrin in tis-sue
extracts was affected by loss of ABCA12 expression. How
these changes relate to a loss of ABCA12 function is unclear,
but they may be secondary to signals generated by the loss of the
skin ceramide barrier. The generation of ABCA12 null mice
should facilitate future investigations into these unanswered
questions.
In summary, our results indicate that ABCA12 plays a critical
role in the formation of the skin’s permeability barrier via an
effect on the generation of a highly specialized class of ceramide
esters. These findings raise the possibility that strategies
designed to increase the amount of these specific ceramides in
the skin could ameliorate defects in skin function in individuals
with harlequin ichthyosis and, potentially, other disorders of
the skin lipid permeability barrier.
Acknowledgments—We thank Dr. Dennis Brown (Program in Mem-brane
Biology) for electron microscopy support and Mary Roth for
mass spectrometry support.
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