This document summarizes a proteomic analysis of the excretory/secretory (ES) proteins of Toxoplasma gondii, the causative agent of toxoplasmosis. 34 proteins were identified from the ES proteins of the RH strain of T. gondii using LC/MS/MS analysis and spectral counting methods. The three most abundant proteins identified were GRA1, GRA7, and GRA2. A variety of other proteins were also identified, including micronemal proteins, rhoptry proteins, and dense granule proteins, which play important roles in T. gondii host cell invasion and survival within host cells. This analysis provides new insights into the ES proteome of T
Objective: To identify interstitial cells of Cajal (ICC) in the common bile duct of Kunming mice.
Study Design: Common bile ducts obtained from the Kunming mice were prepared for immunohistochemical investigations using the c-kit antibody. Immunoelectron microscopy was used to detect the expression of c-kit in the ICC of the common bile duct. Transmission electron microscopy showed ultrastructure of ICC in the murine bile duct. Reverse transcription–polymerase chain reaction (RT-PCR) and western blot were used to confirm the expression of mRNA specific for the c-kit gene and production of c-kit protein in the Kunming mice common bile duct.
Results: Immunohistochemistry revealed that ICC in the murine common bile duct are c-kit positive and the ICC are located in the tela submucosa and the tunica muscularis of the murine common bile duct and do not connect with each other. Immunoelectron microscopy confirmed the expression of Kit by ICC in the murine common bile duct. Transmission electron microscopy showed that ICC in the murine common bile duct have long processes, abundant mitochondria, plenty of smooth endoplasmic reticulum (sER), a lot of lysosomes, and dense bodies. The caveolae of ICC are distinctive. At the same time, RT-PCR indicated that the Kunming mice common bile duct expressed mRNA specific for the c-kit gene, and western blot analysis showed the evidence of production of c-kit protein in the Kunming mice common bile duct.
Conclusion: ICC are found in the Kunming mice common bile duct, which is likely to lead to the development of motility study of the common bile duct.
Keywords: common bile duct; electron microscopy; immuno-electron microscopy; interstitial cells of Cajal; intestines; smooth muscle; tyrosine kinase receptor (c-kit)
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
Enterocin 55 produced by non rabbit-derived strain Enterococcus faecium EF55 ...Agriculture Journal IJOEAR
— Ent55 is produced by poultry strain Enterococcus faecium EF55. It is substance which can be allotted to Class II enterocins; thermo-stable, small peptide. Because producer strain has shown beneficial effect in poultry and broiler rabbits as well, we decided to apply Ent55 in broiler rabbit husbandry. Ent55 showed antimicrobial activity in broiler rabbits by reduction of staphylococci, Clostridiae, pseudomonads and coliforms. Its beneficial effect was demonstrated by stimulation of phagocytic activity as well as by reduction of Eimeria spp. oocysts. GPx values were lower; it means, no oxidative stress was evoked. Moreover, it has not negative influence on growth performance and biochemical parameters. Our results indicated that enterocin produced by not-autochtonous strain can also have protective and beneficial effect in broiler rabbits.
Effective in vitro gene delivery to murine cancerous brain cells using carbon...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
Carbon nanotube (CNT) has been widely applied at molecular and cellular levels due to its exceptional properties. Studies based on conjugation of CNTs with biological molecules indicated that biological activity is preserved. Polyethylenimine (PEI) is explored in designing novel gene delivery vectors due to its ability to condense plasmid DNA through electrostatic attraction. In this study functionalization and grafting polyethylenimine onto the surface of carbon nanotube was used to improve the solubility and biocompatibility.
Materials and Methods:
The effect of molecular weight of polymer on final efficacy of vectors has been investigated using three different molecular weights of polymer. In this study no linker was used and both segments (PEI and CNT) were directly attached resulted in the synthesis of three different vectors. Synthesized vectors were tested for their ability to condense plasmid DNA and cellular toxicity using ethidium bromide and MTT assays. Size and Zeta potential of nanoparticles was determined using Malvern zeta sizer. Evaluation of transfection efficiency of vectors was carried out on N2A cell line by different methods including qualitative fluorescence imaging, flow cytometry and luciferase assay.
Results:
All three synthesized vectors bear positive surface charges with sizes in the range of 85-190 nm. More than 80 percent of treated cells were viable and in the case of V25 significant improvement in reducing cytotoxicity compared to unmodified polymer was observed. Obtained results indicated that vector containing PEI 1.8 kDa has the greatest improvement in terms of its transfection efficiency compared to unmodified polymer.
Conclusion:
Conjugation of PEI with carbon nanotube les to new vectors with lowered cytotoxicity and higher transfection efficiency. The highest transfection efficiency was obtained with the lowest molecular weight PEI.
Objective(s):
This study aimed to find the effects of silver nanoparticles (Ag-NPs) (40 nm) on skin wound healing in mice Mus musculus when innate immune system has been suppressed.
Materials and Methods:
A group of 50 BALB/c mice of about 8 weeks (weighting 24.2±3.0 g) were randomly divided into two groups: Ag-NPs and control group, each with 25 mice. Once a day at the same time, a volume of 50 microliters from the nanosilver solution (10ppm) was applied to the wound bed in the Ag-NPs group while in the untreated (control) group no nanosilver solution was used but the wound area was washed by a physiological solution. The experiment lasted for 14. Transforming growth factor beta (TGF-β), complement component C3, and two other immune system factors involving in inflammation, namely C-reactive protein (CRP) and rheumatoid factor (RF) in sera of both groups were assessed and then confirmed by complement CH50 level of the blood.
Results:
The results show that wound healing is a complex process involving coordinated interactions between diverse immunological and biological systems and that Ag-NPs significantly accelerated wound healing and reduce scar appearance through suppression of immune system as indicated by decreasing levels of all inflammatory factors measured in this study.
Conclusion:
Exposure of mice to Ag-NPs can result in significant changes in innate immune function at the molecular levels. The study improves our understanding of nanoparticle interaction with components of the immune system and suggests that Ag-NPs have strong anti-inflammatory effects on skin wound healing and reduce scarring.
Objective: To identify interstitial cells of Cajal (ICC) in the common bile duct of Kunming mice.
Study Design: Common bile ducts obtained from the Kunming mice were prepared for immunohistochemical investigations using the c-kit antibody. Immunoelectron microscopy was used to detect the expression of c-kit in the ICC of the common bile duct. Transmission electron microscopy showed ultrastructure of ICC in the murine bile duct. Reverse transcription–polymerase chain reaction (RT-PCR) and western blot were used to confirm the expression of mRNA specific for the c-kit gene and production of c-kit protein in the Kunming mice common bile duct.
Results: Immunohistochemistry revealed that ICC in the murine common bile duct are c-kit positive and the ICC are located in the tela submucosa and the tunica muscularis of the murine common bile duct and do not connect with each other. Immunoelectron microscopy confirmed the expression of Kit by ICC in the murine common bile duct. Transmission electron microscopy showed that ICC in the murine common bile duct have long processes, abundant mitochondria, plenty of smooth endoplasmic reticulum (sER), a lot of lysosomes, and dense bodies. The caveolae of ICC are distinctive. At the same time, RT-PCR indicated that the Kunming mice common bile duct expressed mRNA specific for the c-kit gene, and western blot analysis showed the evidence of production of c-kit protein in the Kunming mice common bile duct.
Conclusion: ICC are found in the Kunming mice common bile duct, which is likely to lead to the development of motility study of the common bile duct.
Keywords: common bile duct; electron microscopy; immuno-electron microscopy; interstitial cells of Cajal; intestines; smooth muscle; tyrosine kinase receptor (c-kit)
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
Enterocin 55 produced by non rabbit-derived strain Enterococcus faecium EF55 ...Agriculture Journal IJOEAR
— Ent55 is produced by poultry strain Enterococcus faecium EF55. It is substance which can be allotted to Class II enterocins; thermo-stable, small peptide. Because producer strain has shown beneficial effect in poultry and broiler rabbits as well, we decided to apply Ent55 in broiler rabbit husbandry. Ent55 showed antimicrobial activity in broiler rabbits by reduction of staphylococci, Clostridiae, pseudomonads and coliforms. Its beneficial effect was demonstrated by stimulation of phagocytic activity as well as by reduction of Eimeria spp. oocysts. GPx values were lower; it means, no oxidative stress was evoked. Moreover, it has not negative influence on growth performance and biochemical parameters. Our results indicated that enterocin produced by not-autochtonous strain can also have protective and beneficial effect in broiler rabbits.
Effective in vitro gene delivery to murine cancerous brain cells using carbon...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
Carbon nanotube (CNT) has been widely applied at molecular and cellular levels due to its exceptional properties. Studies based on conjugation of CNTs with biological molecules indicated that biological activity is preserved. Polyethylenimine (PEI) is explored in designing novel gene delivery vectors due to its ability to condense plasmid DNA through electrostatic attraction. In this study functionalization and grafting polyethylenimine onto the surface of carbon nanotube was used to improve the solubility and biocompatibility.
Materials and Methods:
The effect of molecular weight of polymer on final efficacy of vectors has been investigated using three different molecular weights of polymer. In this study no linker was used and both segments (PEI and CNT) were directly attached resulted in the synthesis of three different vectors. Synthesized vectors were tested for their ability to condense plasmid DNA and cellular toxicity using ethidium bromide and MTT assays. Size and Zeta potential of nanoparticles was determined using Malvern zeta sizer. Evaluation of transfection efficiency of vectors was carried out on N2A cell line by different methods including qualitative fluorescence imaging, flow cytometry and luciferase assay.
Results:
All three synthesized vectors bear positive surface charges with sizes in the range of 85-190 nm. More than 80 percent of treated cells were viable and in the case of V25 significant improvement in reducing cytotoxicity compared to unmodified polymer was observed. Obtained results indicated that vector containing PEI 1.8 kDa has the greatest improvement in terms of its transfection efficiency compared to unmodified polymer.
Conclusion:
Conjugation of PEI with carbon nanotube les to new vectors with lowered cytotoxicity and higher transfection efficiency. The highest transfection efficiency was obtained with the lowest molecular weight PEI.
Objective(s):
This study aimed to find the effects of silver nanoparticles (Ag-NPs) (40 nm) on skin wound healing in mice Mus musculus when innate immune system has been suppressed.
Materials and Methods:
A group of 50 BALB/c mice of about 8 weeks (weighting 24.2±3.0 g) were randomly divided into two groups: Ag-NPs and control group, each with 25 mice. Once a day at the same time, a volume of 50 microliters from the nanosilver solution (10ppm) was applied to the wound bed in the Ag-NPs group while in the untreated (control) group no nanosilver solution was used but the wound area was washed by a physiological solution. The experiment lasted for 14. Transforming growth factor beta (TGF-β), complement component C3, and two other immune system factors involving in inflammation, namely C-reactive protein (CRP) and rheumatoid factor (RF) in sera of both groups were assessed and then confirmed by complement CH50 level of the blood.
Results:
The results show that wound healing is a complex process involving coordinated interactions between diverse immunological and biological systems and that Ag-NPs significantly accelerated wound healing and reduce scar appearance through suppression of immune system as indicated by decreasing levels of all inflammatory factors measured in this study.
Conclusion:
Exposure of mice to Ag-NPs can result in significant changes in innate immune function at the molecular levels. The study improves our understanding of nanoparticle interaction with components of the immune system and suggests that Ag-NPs have strong anti-inflammatory effects on skin wound healing and reduce scarring.
Structure of a carotenoid gene cluster from Pantoea sp. strain C1B1YArunkumar K.R.
Structure of a carotenoid gene
cluster from Pantoea sp. strain C1B1Y and characterization of β-carotene hydroxylase (crtZ) gene
by functional complementation in Escherichia coli.
Src jbbr-20-120 Dr. ihsan edan abdulkareem alsaimary PROFESSOR IN MEDICAL M...dr.Ihsan alsaimary
Dr. ihsan edan abdulkareem alsaimary
PROFESSOR IN MEDICAL MICROBIOLOGY AND MOLECULAR IMMUNOLOGY
ihsanalsaimary@gmail.com
mobile : 009647801410838
university of basrah - college of medicine - basrah -IRAQ
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay.
Correction of the Secondary Immunodeficiency at Radiation Sickness with the H...IJSRP Journal
The influence of herbal remedies on immunological and hematological parameters in mice with radiation sickness was studied. It has been revealed that the supromed, pro-vision, biomyrin and biophthysetham correct the inhibited immune response to erythrocytes of the sheep under radiation exposure, and also the studied means promote the increase in the number of erythrocytes and leukocytes in the blood in mice with radiation sickness. Under the influence of biomyrin, the number of blood leukocytes increases by 1.25 times, providas by 1.33 times, supercedes by 1.42 times and biofetizoetham by 1.47 times.
Synthesis, antiviral and cytotoxicity activities of N-Sulphonamidomethyl benz...SriramNagarajan15
A series of novel N-sulphonamido methyl benztriazole derivatives had been synthesized by combining benztriazole, formaldehyde and sulphonamides. Structure of synthesized compounds was elucidated by spectral analysis. Synthesized compounds were evaluated for in-vitro antiviral activity against HIV, HSV and Vaccinia viruses in cell culture. N-Sulphonamido methyl benzotriazole (BT-SN) inhibits Herpes Simplex Virus (HSV) -2 and Vaccinia virus at 34 µg/ml, respectively. HSV-1 at the concentration of 45 µg/ml. The minimum cytotoxic concentration was found to be more than 100µg/ml. So these compounds are suitable for designing newer derivatives and molecular modifications in them may help in optimizing antiviral activity.
Objective(s):
This study aimed to address the gold nanoparticle(GNP)-dose and exposure duration effect on the kidney function of rats: in vivo.
Materials and Methods:
A total of 32 healthy male Wistar rats were used in this study. Animals were randomly divided into groups, three GNP-treated groups and control group. Group 1, 2 and 3 received. /5 cc of solution containing 5, 10,100 ppm Au via IP injection for 7 successive days, respectively. The control group was treated with 0.5% normal saline. Several biochemical parameters such as BUN (blood urea nitrogen), creatine and uric acid were evaluated at various time points (7 and 14 days). After 14 days, the tissue of kidney was collected and investigated.
Results:
There was no significant difference between the control and the intervention group regarding the amount of creatine-BUN and uric acid. The amount of creatine-BUN and uric acid showed increase in all the groups [except group1 (creatine) and group 2 (uric acid)] in the 7 and 14 days after intervention compared to the control group, but this difference was not significant. Results of histopatological tissue kidney showed: in group 1 and 3, complete destruction of the proximal tubules and distal cortical, in group 2, almost complete destruction of proximal tubules and distal.
Conclusions:
The induced histological alterations might be an indication of injured renal tubules due to GNPs toxicity that become unable to deal with the accumulated residues resulting from metabolic and structural disturbances caused by these NPs
Evaluation of infection course in mice induced by L. major in presence of pos...Nanomedicine Journal (NMJ)
Abstract:
An inoculation of virulent Leishmania major is known as leishmanization (LZ) which is proven to be the most effective control measure against Cutaneous Leishmaniasis (CL). However, using LZ is restricted due to various side effects such as uncontrolled lesion development. In the present research, the efficacy of cationic nanoliposomes containing CpG oligodeoxynucleotides (CpG ODN) as an improved adjuvant delivery system was studied to diminish the lesion development and infection course of L. major after inoculation into the mice. BALB/c mice were inoculated subcutaneously (SC) with L. major plus empty DSPC, DSPC (CpG ODN), DSPC (Non CpG ODN), empty DMPC, DMPC (CpG ODN), DMPC (Non CpG ODN) or HEPES buffer. The results showed that group of mice received DMPC (CpG ODN) nanoliposomes developed a significantly smaller lesion and showed minimum number of L. major in the spleen and draining lymph nodes. In addition, using DMPC (CpG ODN) liposomes resulted in a Th1 type of immune response with a preponderance of IgG2a isotype which is concurrent with the production of DMPC (CpG) induced IFN-γ in the spleen of the mice. Taken together, the results suggested that immune modulation using DMPC (CpG ODN) nanoliposomes might be a practical approach to improve the safety of LZ
Keywords:
DMPC (CpG ODN) nanoliposomes; CpG ODN; L. major; Leishmanization; Immune response
Genotoxicity of Goji Berry (Lyciumbarbarum) In Vivo Mammalian Cellsinventionjournals
Lyciumbarbarum (Gojji berry) belongs to family Salonaceae which is found in China and Himalayan. This herb is used to prevent various diseases and in medical treatments as an alternative medicine being widely used for its antioxidant and revitalizing potential effects. In recent years, Gojji has become increasingly popular in Europe and North America as a "superfruit" and dietary supplement. The belief that herbal products do not bring any risk to health, is part of popular culture. However the term "natural" assigned to many products cannot assure no health risk. The aim of this study was to evaluate the possible genotoxic effects of aqueous extract of Lyciumbarbarum (Gojji berry) by micronucleus test and comet assay. Thirty Rattus norvegicus were divided into three equal groups: 1) experimental group, submitted to Gojji berry (200mg/kg orally); 2) positive control group (cyclophosphamide), and; 3) negative control group (distilled water). Micronucleus Tests were done by smear method of bone marrow cells performed after 48h for acute, and 72h for chronic exposure. The comet assay was performed on peripheral blood taken from the tail of each animal 4h, and 24h after intervention. Cytotoxicity was assessed by observing the DNA damage measuring the percentage of DNA in the tail (% DNA- measurement of the proportion of the total DNA present in the tail) and the tail moment (TM-tail length times the percentage of DNA in the tail), calculated by 100 nucleoids per animal and the presence of micronuclei in 2,000 polychromatic erythrocytes per animal. Analysis of variance (ANOVA) followed by Tukey test at 5% significance was used comparing the results. The data showed no significant difference in the frequency of DNA damage and the number of micronuclei between the experimental group and the negative control group. The results also suggest that the aqueous extract of Lyciumbarbarum (Gojji berry) at the dose of 200 mg/kg showed no genotoxic effect, which could, to a certain point, justifies its use.
Structure of a carotenoid gene cluster from Pantoea sp. strain C1B1YArunkumar K.R.
Structure of a carotenoid gene
cluster from Pantoea sp. strain C1B1Y and characterization of β-carotene hydroxylase (crtZ) gene
by functional complementation in Escherichia coli.
Src jbbr-20-120 Dr. ihsan edan abdulkareem alsaimary PROFESSOR IN MEDICAL M...dr.Ihsan alsaimary
Dr. ihsan edan abdulkareem alsaimary
PROFESSOR IN MEDICAL MICROBIOLOGY AND MOLECULAR IMMUNOLOGY
ihsanalsaimary@gmail.com
mobile : 009647801410838
university of basrah - college of medicine - basrah -IRAQ
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay.
Correction of the Secondary Immunodeficiency at Radiation Sickness with the H...IJSRP Journal
The influence of herbal remedies on immunological and hematological parameters in mice with radiation sickness was studied. It has been revealed that the supromed, pro-vision, biomyrin and biophthysetham correct the inhibited immune response to erythrocytes of the sheep under radiation exposure, and also the studied means promote the increase in the number of erythrocytes and leukocytes in the blood in mice with radiation sickness. Under the influence of biomyrin, the number of blood leukocytes increases by 1.25 times, providas by 1.33 times, supercedes by 1.42 times and biofetizoetham by 1.47 times.
Synthesis, antiviral and cytotoxicity activities of N-Sulphonamidomethyl benz...SriramNagarajan15
A series of novel N-sulphonamido methyl benztriazole derivatives had been synthesized by combining benztriazole, formaldehyde and sulphonamides. Structure of synthesized compounds was elucidated by spectral analysis. Synthesized compounds were evaluated for in-vitro antiviral activity against HIV, HSV and Vaccinia viruses in cell culture. N-Sulphonamido methyl benzotriazole (BT-SN) inhibits Herpes Simplex Virus (HSV) -2 and Vaccinia virus at 34 µg/ml, respectively. HSV-1 at the concentration of 45 µg/ml. The minimum cytotoxic concentration was found to be more than 100µg/ml. So these compounds are suitable for designing newer derivatives and molecular modifications in them may help in optimizing antiviral activity.
Objective(s):
This study aimed to address the gold nanoparticle(GNP)-dose and exposure duration effect on the kidney function of rats: in vivo.
Materials and Methods:
A total of 32 healthy male Wistar rats were used in this study. Animals were randomly divided into groups, three GNP-treated groups and control group. Group 1, 2 and 3 received. /5 cc of solution containing 5, 10,100 ppm Au via IP injection for 7 successive days, respectively. The control group was treated with 0.5% normal saline. Several biochemical parameters such as BUN (blood urea nitrogen), creatine and uric acid were evaluated at various time points (7 and 14 days). After 14 days, the tissue of kidney was collected and investigated.
Results:
There was no significant difference between the control and the intervention group regarding the amount of creatine-BUN and uric acid. The amount of creatine-BUN and uric acid showed increase in all the groups [except group1 (creatine) and group 2 (uric acid)] in the 7 and 14 days after intervention compared to the control group, but this difference was not significant. Results of histopatological tissue kidney showed: in group 1 and 3, complete destruction of the proximal tubules and distal cortical, in group 2, almost complete destruction of proximal tubules and distal.
Conclusions:
The induced histological alterations might be an indication of injured renal tubules due to GNPs toxicity that become unable to deal with the accumulated residues resulting from metabolic and structural disturbances caused by these NPs
Evaluation of infection course in mice induced by L. major in presence of pos...Nanomedicine Journal (NMJ)
Abstract:
An inoculation of virulent Leishmania major is known as leishmanization (LZ) which is proven to be the most effective control measure against Cutaneous Leishmaniasis (CL). However, using LZ is restricted due to various side effects such as uncontrolled lesion development. In the present research, the efficacy of cationic nanoliposomes containing CpG oligodeoxynucleotides (CpG ODN) as an improved adjuvant delivery system was studied to diminish the lesion development and infection course of L. major after inoculation into the mice. BALB/c mice were inoculated subcutaneously (SC) with L. major plus empty DSPC, DSPC (CpG ODN), DSPC (Non CpG ODN), empty DMPC, DMPC (CpG ODN), DMPC (Non CpG ODN) or HEPES buffer. The results showed that group of mice received DMPC (CpG ODN) nanoliposomes developed a significantly smaller lesion and showed minimum number of L. major in the spleen and draining lymph nodes. In addition, using DMPC (CpG ODN) liposomes resulted in a Th1 type of immune response with a preponderance of IgG2a isotype which is concurrent with the production of DMPC (CpG) induced IFN-γ in the spleen of the mice. Taken together, the results suggested that immune modulation using DMPC (CpG ODN) nanoliposomes might be a practical approach to improve the safety of LZ
Keywords:
DMPC (CpG ODN) nanoliposomes; CpG ODN; L. major; Leishmanization; Immune response
Genotoxicity of Goji Berry (Lyciumbarbarum) In Vivo Mammalian Cellsinventionjournals
Lyciumbarbarum (Gojji berry) belongs to family Salonaceae which is found in China and Himalayan. This herb is used to prevent various diseases and in medical treatments as an alternative medicine being widely used for its antioxidant and revitalizing potential effects. In recent years, Gojji has become increasingly popular in Europe and North America as a "superfruit" and dietary supplement. The belief that herbal products do not bring any risk to health, is part of popular culture. However the term "natural" assigned to many products cannot assure no health risk. The aim of this study was to evaluate the possible genotoxic effects of aqueous extract of Lyciumbarbarum (Gojji berry) by micronucleus test and comet assay. Thirty Rattus norvegicus were divided into three equal groups: 1) experimental group, submitted to Gojji berry (200mg/kg orally); 2) positive control group (cyclophosphamide), and; 3) negative control group (distilled water). Micronucleus Tests were done by smear method of bone marrow cells performed after 48h for acute, and 72h for chronic exposure. The comet assay was performed on peripheral blood taken from the tail of each animal 4h, and 24h after intervention. Cytotoxicity was assessed by observing the DNA damage measuring the percentage of DNA in the tail (% DNA- measurement of the proportion of the total DNA present in the tail) and the tail moment (TM-tail length times the percentage of DNA in the tail), calculated by 100 nucleoids per animal and the presence of micronuclei in 2,000 polychromatic erythrocytes per animal. Analysis of variance (ANOVA) followed by Tukey test at 5% significance was used comparing the results. The data showed no significant difference in the frequency of DNA damage and the number of micronuclei between the experimental group and the negative control group. The results also suggest that the aqueous extract of Lyciumbarbarum (Gojji berry) at the dose of 200 mg/kg showed no genotoxic effect, which could, to a certain point, justifies its use.
The Future of Brand Marketing = In-App Mobile VideoInMobi
In-app mobile video usage is exploding as highlighted in the latest App Annie Market Index report. Nearly half of mobile video viewers can only (or mostly only) be reached in-app according to IAB. The recent SMOX MMA study with Coca-Cola, Walmart & Mastercard showed that mobile video can outperform cable television. Learn all about in-app mobile video in terms of vertical video, completion rates, viewability, true full-screen formats, rich interactive features, programmatic buying, and more.
Speaker:
Anne Frisbie
Senior Vice President, InMobi Exchange and Global Alliances
InMobi
Development of cell culture samples for drug screening with bone marrow stem ...Apollo Hospitals
Side population (SP) refers to a group of cells, which is capable to efflux Hoechst 33342, a DNA-binding dye. SP cells exist both in normal and tumor tissues. SP abundance is used as an indicator for disease prognosis and drug screening in some studies. Our study concentrates on the factors influencing SP analysis.
Determination and comparison rate of expression markers of osteoblast derived...IJERD Editor
Nowadays high accident rates, fractures leading to permanent bone disorders and the impossibility of bone transplant have made scientists to look for new methods of repairing injured bones. Considering the application of stem cells in bone tissue engineering, there exists the necessity to investigate various culture methods and suitable fields and scaffolds. Thus, we decided to induce adipose-derived stem cells into osteoblast cells in two systems of pellet culture and monolayer and compare osteogenic markers. Methods: Stem cells have been separated via mechanical and enzymatic methods and cultured in monolayer and pellet culture models with osteogenic medium. Then, RNA was separated from differentiated cells, complementary DNA (cDNA) was synthesized and amplified. Polymerase chain reaction (PCR) product was transferred to electrophoresis gel. The intensity of the bands was measured by Image-J software and analyzed by SPSS.
Applying new techniques to blood assays in cats has enabled researchers to reduce the amount of blood needed nutrition study sample collection studies by 80%, with concomitant benefits for animal welfare. Presented at the Waltham InternationaI Nutrition Science Symposium, October 2016, Chicago
Colletotrichum causes anthracnose in crops around the world producing postharvest losses up to 60%. There are a great variety of Colletotrichum strains isolated from mango orchards. Thus, it is important to characterize their pathogenicity, as well as to perform a correct identification, in order to implement good strategies to eradicate the produced disease. The aim of this work is to identify Colletotrichum spp. and to determine the production of Pectate Lyase (PL) as a virulence factor in the pathogenicity process. Macroscopic characteristics of isolated colony vary from grey to salmon, sometimes showing luxuriant orange conidial masses with grey or white bottom. Conidia vary from 10.39 to 14.83 × 2.75 to 3.40 μm corresponding to C. gloeosporioides or C. acutatum according to Sutton. Growth rates vary from 0.1948 to 0.2239 day-1. The pectate lyase activity was induced by mango cells (240.81 VS 398U/L). According to CgInt and ITS4 PCR amplification M2V and SA correspond to C. gloeosporioides.
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
1. Analysis of the ES Proteins of T. gondii Bull. Korean Chem. Soc. 2014, Vol. 35, No. 10 3071
http://dx.doi.org/10.5012/bkcs.2014.35.10.3071
Comprehensive Proteome Analysis of the Excretory/Secretory Proteins
of Toxoplasma gondii
Won-Kyu Lee, Hye-Jin Ahn,†
Je-Hyun Baek,‡
Chong-Heon Lee,#
Yeon Gyu Yu,*
and Ho-Woo Nam†,*
Department of Chemistry, College of Natural Sciences, Kookmin University, Seoul 136-702, Korea
*
E-mail: ygyu@kookmin.ac.kr
†
Department of Parasitology and the Catholic Institute of Parasitic Diseases, College of Medicine,
Catholic University of Korea, Seoul 137-701, Korea. *
E-mail: howoo@catholic.ac.kr
‡
Center of Biomedical Mass Spectrometry (CBMS), Diatech Korea Co., Ltd., Seoul 138-826, Korea
#
Department of Oral Pathology, College of Dentistry, Dankook University, Cheonan 330-714, Korea
Received July 5, 2014, Accepted July 26, 2014
Proteomic analyses of the excretory/secretory proteins from the RH strain of Toxoplasma gondii have been
performed to understand their functions in the host-parasite interaction. A total of 34 proteins were identified
from LC/MS/MS analysis and their abundance was estimated by spectral counting methods. Among them, 8
species of micronemal proteins (MICs), 2 species of rhoptry proteins (ROPs), and 6 species of dense granular
proteins (GRAs) were confirmed. Besides these, 18 species of protein were newly identified, and their cellular
functions were estimated from sequence analysis. The three most abundant of the 34 identified extractor/
secretory proteins−GRA1, GRA7 and GRA2−were confirmed to be highly expressed in T. gondii using the
spectral count method. This phenomenon is another demonstration of the importance of GRA proteins for the
penetration and survival of T. gondii.
Key Words : Excretory/secretory protein, Toxoplasma gondii, Micronemal protein, Rhoptry protein, Dense
granule protein
Introduction
Toxoplasma gondii is a member of Apicomplexa phylum
and is the causative agent of toxoplasmosis.1
It is a widely
distributed protozoan parasite that infects various warm-
blooded animals, including humans. Decoster et al. (1988)
first described several excretory and secretory antigens
(ESAs) of T. gondii in the sera of toxoplasmosis patients.2
ESA have been studied with regard to cell-mediated
immunity,3-6
cell biology,7-10
and biochemical process.11,12
Most ESAs of T. gondii are released from highly specialized
secretory organelles such as micronemes, rhoptries, and
dense granules, which are involved in the penetrating pro-
cess and the intracellular interactions within the parasito-
phorous vacuole (PV). The micronemes secrete a collection
of adhesion proteins, termed microneme proteins (MICs)
that mediate host cell entry.13-15
Rhoptry proteins (ROPs)
and lipids form the junction moving during invasion,16
eventually resulting in a parasitophorous vacuole that en-
ables the efficient procurement of nutrients and the evasion
of host immune defenses.17,18
Dense granule proteins (GRAs)
appear to facilitate the formation of specialized tubules that
enable nutrient acquisition by the parasite, and some of these
proteins, most notably GRA7, are also secreted into host
cells.19
ROPs are also injected into the host cell, resulting in
the extensive modification of host gene expression and
signaling pathways.20
Eui-Sun Son and Ho woo Nam (2001),
attempted to profile the production, subcellular localization,
and exhaustion of excretory/secretory proteins (ESPs). This
demonstrates that they play an essential role in providing the
appropriate environment for the entry of the parasite into
host cells.21
A couple of proteins have been identified in ESP
including a 42 kD protease,22
GRA3 and GRA10.23
In a
recent report on ESP, manipulation of the host PI3K/Akt
signaling pathway and Nox4 gene expression is influenced
by ESP, and this mechanism is involved in T. gondii survival
and proliferation.24
Using two dimensional electrophoresis (2DE) and multi-
dimensional protein identification technology, approximate-
ly 100 spots of ESPs related to host cell invasion could be
identified. Many proteins were present in multiple spots,
consistent with the presence of post-translational modifi-
cations.25,26
In a similar study, A23187 used to stimulate
calcium mediated ESP from T. gondii. As a result, total of
213 protein spots were identified by 2DE.27
Purified micro-
neme proteins, which play in long or short range interactions
during parasite attachment and entry, have also been analyz-
ed using proteomic techniques.28,29
Although there has been
a substantial amount of T. gondii proteomics research, an
extensive study on the identification of ESPs has not been
reported.
In this study, we performed proteomic analysis of ESPs in
the RH strain of T. gondii, identified 34 proteins, and analyz-
ed their abundance.
Experimental
Chemicals. Acrylamide and bis-acrylamide cocktail solu-
2. 3072 Bull. Korean Chem. Soc. 2014, Vol. 35, No. 10 Won-Kyu Lee et al.
tions were obtained from Bio-Rad (USA). The chemicals 3-
[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
(CHAPS), and dithiothreitol (DTT) were purchased from
GE Healthcare (USA). Urea, thiourea, iodoacetamide, and
the other reagents for the polyacrylamide gel preparation
were from Sigma (USA). All other chemicals were acquired
from standard sources and were of molecular biology grade
or higher.
Preparation of ESP. The RH strain of T. gondii was
maintained by peritoneal passages in Balb/c mice. Tachy-
zoites were purified by centrifugation over 40% Percoll (GE
Healthcare, USA) in PBS solution.21
Purified tachyzoites
(3 × 108
) were incubated at 37 °C for 1 h under mild agitation
in 1.0 mL Hank’s balanced salt solution (Gibco BRL, USA).
After centrifugation for 5 min at 6,000 rpm, the supernatant
containing ESPs was recovered.
Western Blot Analysis. ESPs were separated in 12%
SDS-PAGE gels and transferred onto nitrocellulose sheets
(NC, Schlleicher and Shuell, Keene, USA). NC papers block-
ed by 5% skim milk in PBS/0.05% Tween-20 were incubated
with total anti-T. gondii polyclonal antibody diluted 1:1,000,
and then with 1:3,000 diluted HRP-conjugated goat anti-
mouse IgG antibody (Cappel, Costa Mesa, USA). They were
soaked in enhanced chemiluminescence (ECL) solution
(Intron, Korea) for 1 min and exposed to an X-ray film for
visualization (Konica, Japan).
Enzymatic In-gel Digestion. The proteins separated by
SDS-PAGE were excised from the gel, and the gel pieces
containing protein were destained with 50% acetonitrile
(ACN) containing 50 mM NH4HCO3 and vortexed until
CBB was completely removed. These gel pieces were then
dehydrated in 100% acetonitrile and Vacuum-dried for 20
min in CentriVap®
DNA Centrifugal concentrator. For the
digestion, gel pieces were reduced using 10 mM DTT in 50
mM NH4HCO3 for 45 min at 56 °C, followed by alkylation
by 55 mM iodoacetamide in 50 mM NH4HCO3 for 30 min in
dark. Finally, each gel pieces were treated with 12.5 ng/µL
sequencing grade modified trypsin (Promega, USA) in 50
mM NH4HCO3 buffer (pH 7.8) at 37 °C for overnight.
Following digestion, tryptic peptides were extracted with
5% formic acid in 50% ACN solution at room temperature
for 20 min. The supernatants were collected and dried by
CentriVap®
DNA Centrifugal concentrator. The samples
were purified and concentrated in 0.1% formic acid using
StageTip C18 (Thermo, German) before MS analysis.
Liquid Chromatography and Mass Spectrometric
Analysis. The tryptic peptides were loaded onto a fused
silica microcapillary column (12 cm × 75 µm) packed with
C18 reversed phase resin (5 µm, 100 Å). LC separation was
conducted under a linear gradient as follows: a 3-40%
solvent B (0.1% formic acid in 100% ACN) gradient, with a
flow rate of 400 nL/min for 60 min. The column was direct-
ly connected to an LTQ linear ion-trap mass spectrometer
(Finnigan, USA) equipped with a nano-electrospray ion
source. The electrospray voltage was set at 1.80 kV, and the
threshold for switching from MS to MS/MS was 2000. The
normalized collision energy for MS/MS was 35% of the
main radio frequency amplitude (RF) and the duration of
activation was 30 ms. All spectra were acquired in data-
dependent scan mode. Each full MS scan was followed by
five consecutive MS/MS scans corresponding to the five
most intense peaks in the full MS scan. Repeat count of
peaks for dynamic exclusion was 1, and its repeat duration
was 30 sec. The dynamic exclusion duration was set to 180
sec and the width of exclusion mass was ± 1.5 Da. The list
size of dynamic exclusions was 50.
Database Searching. All MS/MS samples were analyzed
using SEQUEST (Thermo Fisher Scientific, USA; version
v.27, rev. 11). SEQUEST was searched with a fragment ion
mass tolerance of 1.00 Da and a parent ion tolerance of 3.0
Da. Carbamidomethylation of cysteine was specified in
SEQUEST as a fixed modification. Oxidation of methionine
was specified in SEQUEST as a variable modification.
Scaffold (version Scaffold_4.2, Proteome Software Inc.,
USA) was used to validate MS/MS based peptide and protein
identifications (Peptide/Protein FDR 1%) which contained
at least 2 identified peptides. SEQUEST was used to search
the TgondiiRH_ORFs_Anotated_GRA_ROP_MIC_Uniprot
_Mouse_Contaminants.fasta database (248,268 entries includ-
ing decoy sequences) assuming that the peptide fragments
were resulted from trypsin digestion. The T. gondii RH
protein database was reconstructed with TgondiiRH_ORFs
fasta (from http://toxodb.org/toxo/showApplication.do),
GRA/ROP/MIC.fasta, and mouse.fasta from UniportKB
(http://www.uniprot.org). The protein abundance and rank
estimation were defined by the previously described Fabb
index.30,31
The putative function of proteins, which were not listed in
UniProtKB, was further analyzed using Pfam (http://pfam.
Figure 1. Western blot analysis of the ESPs of the Toxoplasma
gondii (RH) strain. The ESP bands on nitrocellulose membrane
were reacted with a reference serum from a mouse infected with
RH tachyzoites. ESP bands were detected by anti T. gondii
polyclonal antibody. M, size marker; lane 1 and 2, two-batch
samples of ESP; lane 3, Total T. gondii lysate.
3. Analysis of the ES Proteins of T. gondii Bull. Korean Chem. Soc. 2014, Vol. 35, No. 10 3073
xfam.org) and gene ontology databases (http://www.ebi.ac.
uk/ego/).
Criteria for Protein Identification. Scaffold (version
Scaffold_4.3.2, Proteome Software Inc., USA) was used to
validate MS/MS based peptide and protein identifications.
Peptide identifications were accepted if they could be
established at greater than 81.0% probability to achieve a
false discovery rate (FDR) less than 1.0% by the Scaffold
Local FDR algorithm. Protein identifications were accepted
if they could be established at greater than 98.0% probability
to achieve an FDR less than 1.0% and contained at least 2
identified peptides. Protein probabilities were assigned by
the Protein Prophet algorithm.32
Proteins that contained
similar peptides and could not be differentiated based on
MS/MS analysis alone were grouped to satisfy the principles
of parsimony.
Results and Discussion
Western Blot Analysis. When detected using an anti
T. gondii polyclonal antibody, about 20 bands appeared in
the total ESP from T. gondii (Fig. 1). We confirmed proper
separation of ESP using western blot analysis results
compared with a reference.21
The molecular mass of 15 ESP
were estimated as 110, 97, 86, 80, 70, 60, 54, 42, 40, 36, 30,
28, 26, 22, and 19 kD, which were compatible with the
previously described ESA of T. gondii,3,4
with minor differ-
ences. The protein samples identified by western blot analysis
were stored at −80 °C until analyzed.
Identification of ESP by LC/MS/MS Analysis. The
ESPs were digested by trypsin and the resulting peptides
were analyzed by LC/MS/MS. A total of 314 proteins were
identified by MS analysis. Among them, 280 proteins were
Table 1. The list of identified proteins found in ESP of Toxoplasma gondii
Rank
(by Abundance)
Identified Proteins
(34/314)
Molecular
Weight (kDa)
# of Unique
Peptides (1st)
# of Unique
Peptides (2nd)
# of Total Spectra
(merged)
# of Estimated Protein
Abundance
1 GRA1 20 10 11 275.48 232.75
2 GRA7 26 10 10 152.12 98.86
3 GRA2 20 6 6 108.61 91.77
4 TgESP-1 36 9 15 155.91 73.18
5 MIC2 83 19 19 265.67 54.09
6 MIC5 20 6 4 52.59 44.43
7 TgESP-2 13 1 3 29.67 38.57
8 TgESP-3 10 1 2 20.49 34.63
9 TgESP-4 15 4 4 28.44 32.04
10 TgESP-5 36 2 9 66.01 30.99
11 MIC10 23 5 4 34.18 25.11
12 TgESP-6 20 3 3 27.16 22.95
13 TgESP-7 64 13 13 82.66 21.83
14 TgESP-8 16 1 2 20.45 21.59
15 GRA5 13 3 4 14.21 18.47
16 MIC17A 39 9 6 42.10 18.24
17 TgESP-9 19 0 5 20.12 17.89
18 MIC11 22 4 3 20.87 16.03
19 GRA6 24 2 4 21.30 15.00
20 TgESP-10 17 2 3 14.23 14.15
21 MIC1 42 5 6 35.11 14.12
22 MIC6 37 4 3 25.02 11.43
23 GRA4 36 0 2 20.12 9.44
24 TgESP-11 13 1 2 7.12 9.25
25 TgESP-12 75 4 5 33.42 7.53
26 TgESP-13 14 1 2 5.43 6.56
27 TgESP-14 14 0 2 3.77 4.55
28 TgESP-15 16 0 2 3.77 3.98
29 TgESP-16 35 1 2 5.43 2.62
30 TgESP-17 27 2 1 3.75 2.35
31 ROP15 34 0 3 3.77 1.87
32 ROP2 475 7 9 42.20 1.50
33 MIC8 75 2 1 5.41 1.22
34 TgESP-18 28 2 0 1.66 1.00
4. 3074 Bull. Korean Chem. Soc. 2014, Vol. 35, No. 10 Won-Kyu Lee et al.
identified as mouse proteins and only 34 proteins were
confirmed to be derived from T. gondii. The reason was that
the ESP were obtained from the abdominal cavity of mice.
Mice proteins outnumbered T. gondii by far, therefore ESP
identification was difficult, relatively. The 34 identified
proteins are listed in Table 1 along with protein name, mole-
cular weight, number of unique peptides, number of total
spectra, number of estimated protein copy and rank by
abundance. Among them, 16 proteins registered in the
UniProtKB were listed in Table 2 along with protein name,
alternative name, UniProtKB ID, function by Gene Ontology
database, and reference. A total of 18 species of protein,
including TgESP-1, were newly identified in this study. The
amino acid sequences of these 18 proteins were described in
Support Information 1.
Among the identified proteins, proteins related to the host-
parasite interaction were of particular interest. All compo-
nents of ESP are known to be released from highly speci-
alized secretory organelles of T. gondii, micronemes (Micro-
nemal protein, MIC), rhoptries (Rhoptry protein, ROP), and
dense granules (Dense granule protein, GRA), which func-
tion in the penetrating process and during subsequent intra-
cellular interactions within the parasitophorous vacuole (PV).
From these proteins, 8 species of MIC protein, 2 species of
ROP protein, and 6 species of GRA protein were identified
(Table 2). The Gene Ontology database functions of these
proteins are antigen presenting in the extracellular region,
the factors of transport vesicles, and pathogenesis. The ROP2
protein has a kinase domain, and it is believed to be involved
in protein phosphorylation.
The frequencies of the 34 proteins from T. gondii were
further analyzed using the spectral count method and the
abundances of the proteins are listed in Table 1. GRA1 is the
most abundant protein followed by GRA7 and GRA2.
Interestingly, the top 3 abundant proteins are all GRA pro-
teins. The GRA1 protein is considerably more abundant than
the other 33 kinds of identified proteins, and is more than
double the abundance of GRA7 or GRA2. GRA1 has been
reported to be useful as a diagnostic marker for chronic
toxoplasmosis and vaccine development.33
In the case of
GRA7, the T. gondii actively recruits host microtubules,
resulting in the selective attraction of endo-lysosomes to the
PV. GRA7 acts like a lasso that sequesters host endocytic
organelles in the vacuolar space.19
The expression of GRA1
and GRA7 is much higher than the other proteins, and the
function of these proteins shows their considerable impor-
tance for interactions with the host system. Some of these
GRAs have been shown to be targeted to the parasitophor-
ous vacuolar space, the parasitophorous vacuole membrane
or the intravacuolar tubular network where they play signi-
ficant roles in the biogenesis and modification of the para-
sitophorous vacuole where it interfaces with the host cell.33
TgESP-1, the 4th
most abundant protein out of our identified
ESPs, it has a similar sequence to elongation factor 1-alpha,
suggesting it is involved in protein synthesis. We can infer
that ESPs, including TgESP-1, are involved in the synthesis
Table 2. The list of reported protein on UniProtKB
Name Alternative name UniProt ID Function Reference
GRA1 Major antigen p24 P13403 Extracellular region Cesbron-Delauw et al. (1989)35
GRA7 29 kDa excretory dense
granule protein
O00933 Integral component of membrane
Transport vesicle
Hans-Georg (1998)36
GRA2 28 kDa antigen P13404 Symbiont-containing vacuole Mercier et al. (1989)37
MIC2 O00816 None Wan et al. (1997)38
MIC5 V4ZHQ9 None Saouros et al. (2012)39
MIC10 20 kDa excretory-secretory antigen B9PRE4 None Hoff et al. (2001)40
GRA5 p21 Q07828 Integral component of membrane
Transport vesicle
Lecordier et al. (1993)41
MIC17A V4Z8E3 Blood coagulation
Proteolysis
Extracellular region
MIC11 B9PUW8 None Harper et al. (2004)42
GRA6 Antigen p32 Q27003 Integral component of membrane
Transport vesicle
Lecordier et al. (1995)43
MIC1 O00834 Cell adhesion
Pathogenesis
Fourmaux et al. (1996)44
MIC6 Q9XYH7 Cell adhesion
Pathogenesis
Reiss et al. (2001)45
GRA4 Antigen H11 Q27002 Integral component of membrane
Transport vesicle
Mevelec et al. (1992)46
ROP15 A4GWX6 None
ROP2 Q06AK3 ATP binding
Protein kinase activity
Qiu et al. (2009)47
MIC8 D8UY25 None Kessler et al. (2008)48
5. Analysis of the ES Proteins of T. gondii Bull. Korean Chem. Soc. 2014, Vol. 35, No. 10 3075
or modification of host proteins. GRA proteins are potent
antigens that can induce strong parasite-directed T-cell and
B-cell responses.34
Because of the abundance of their expre-
ssion, the proteins GRA1, GRA2, GRA7 and TgESP-1 deserve
to be considered as promising candidates for vaccine develop-
ment.
Functional Estimation of Unknown Proteins. The cellular
function of 18 newly identified proteins were not listed in
the UniProtKB database (Table 3). The functions of these
proteins were estimated from their structural homologues
using the Pfam database, UniProt database, and Gene Onto-
logy database. The putative functions indicated that TgESP-
1 and TgESP-4 were involved in protein synthesis; TgESP-
2, TgESP-3, TgESP-5 and TgESP-10 were involved in the
cytoskeleton; TgESP-7 was involved in blood coagulation
and proteolysis; TgESP-9 was involved in the oxydoreduc-
tation reaction; TgESP-11 was involved in carbohydrate
metabolism; TgESP-12, TgESP-14, and TgESP-15 were involv-
ed in nucleotide synthesis; and TgESP-13 was involved in
protein translation. TgESP-6 and TgESP-8 have a domain of
the Kazal-type serine protease inhibitor, but the function of
this domain is not recorded in the GO database. TgESP-16
has a domain common to Cytochrome C and Quinol oxidase
polypeptide I, but the function of this domain is not recorded
in the GO database. TgESP-17 and TgESP-18 do not have a
known structural domain. TgESP-1 and TgESP-4 are struc-
turally similar to elongation factor 1-alpha, TgESP-2 and
TgESP-3 are structurally similar to profilin, TgESP-6 and
TgESP-8 are structurally similar to the tachyzoite serine
proteinase inhibitor, and TgESP-5 and TgESP-10 are struc-
turally similar to actin. The function of these 18 kinds of
proteins, inferred from the structural information, indicates
that they are involved in the cytoskeleton, protein synthesis,
proteolysis and translation. It can be considered that ESPs
from T. gondii, including these 18 proteins, are actively
involved in protein metabolism.
Table 3. The list of novel reported proteins
Name Location Domain Similar protein Putative function
TgESP-1 TGRH_chrIa:1271142-1272140(−) Elongation factor Tu GTP binding
domain
Elongation factor Tu domain 2
Elongation factor Tu C-terminal domain
Elongation factor 1-alpha Protein biosynthesis
TgESP-2 TGRH_chrIa:634885-635256(−) None Putative profilin Actin cytoskeleton
organization
TgESP-3 TGRH_chrIa:634582-634851(−) None Inflammatory profilin Actin cytoskeleton
organization
TgESP-4 TGRH_chrIa:1272355-1272774(−) Elongation factor Tu GTP binding
domain
Elongation factor 1-alpha Protein biosynthesis
TgESP-5 TGRH_chrIb:1038884-1039867(+) Actin Actin ACT1 Cytoskeleton
TgESP-6 TGRH_chrIb:754143-754679(−) Kazal-type serine protease inhibitor
domain
Tachyzoite serine
proteinase inhibitor
None
TgESP-7 TGRH_chrIb:571277-573040(+) PAN domain Microneme protein MIC4 Blood coagulation
Proteolysis
TgESP-8 TGRH_chrIb:752726-753154(−) Kazal-type serine protease inhibitor
domain
Tachyzoite serine
proteinase inhibitor
None
TgESP-9 TGRH_chrIa:743195-743719(+) Thioredoxin Thioredoxin Oxidoreductase
TgESP-10 TGRH_chrIb:1040267-1040731(+) Actin Actin ACT1 Cytoskeleton
TgESP-11 TGRH_chrIa:827883-828242(+) Glucose-6-phosphate dehydrogenase,
C-terminal domain
Glucose-6-phosphate
1-dehydrogenase
Carbohydrate
metabolism
TgESP-12 TGRH_chrIb:1811657-1813663(+) RNA recognition motif-containing
protein
RNA recognition motif
WW domain
Nucleotide binding
TgESP-13 TGRH_chrIb:1231496-1231858(−) Ribosomal protein S28e Ribosomal protein RPS28 Translation
TgESP-14 TGRH_chrIa:1939363-1939734(−) Nucleoside diphosphate kinase Nucleoside diphosphate
kinase, putative
CGU/TP biosynthetic
process
TgESP-15 TGRH_chrIb:1813125-1813553(+) RNA recognition motif. RNA recognition motif-
containing protein
Nucleotide binding
TgESP-16 TGRH_chrIb:1686978-1687934(−) Cytochrome C and Quinol oxidase
polypeptide I
Hyaluronan/mRNA-bind-
ing family protein
None
TgESP-17 TGRH_chrIb:74822-75550(−) None None None
TgESP-18 TGRH_chrIa:757447-758244(+) None Sporozoite protein with an
altered thrombospondin
repeat SPATR
None
6. 3076 Bull. Korean Chem. Soc. 2014, Vol. 35, No. 10 Won-Kyu Lee et al.
Conclusion
We have analyzed ESPs from T. gondii using LC/MS/MS
analysis techniques and estimated their abundance using
spectral counting methods. A total of 314 proteins were
identified, and among those there were 34 proteins identified
that are ESPs of T. gondii and 18 of those proteins are novel
and reported in this study for the first time. The putative
functions of the 18 proteins indicate they are involved in
protein metabolism, oxidoreductation, carbohydrate meta-
bolism, and nucleotide binding and protein translation. The
34 proteins identified are listed by abundance, and among
the top 3 most abundant proteins are GRA proteins. These
quantitative results revealed that GRA proteins are important
for the penetration and survival of T. gondii. In addition,
these high abundant proteins deserve to be considered as
promising candidates for anti-parasite drug or vaccine develop-
ment.
Acknowledgments. This research was supported by MSIP
& PAL, XFEL project, Korea (2014-SB-7) and the Basic
Science Research Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Edu-
cation, Science, and Technology (2012-R1A1A2-002612
and 2013-R1A1A2-062683).
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