This study investigated the effects of neuropeptide Y (NPY) on signaling proteins in C. elegans. NPY is a neurotransmitter that regulates anxiety in humans. The researchers extracted proteins from C. elegans exposed to NPY and analyzed them using ELISA. They found a statistically significant upregulation of Akt1 protein levels in the presence of NPY, but no significant effects on other proteins like p38 MAP kinase and Stat3. This suggests NPY may upregulate Akt1 through a CRE element in its gene. Further experiments are needed to validate the proposed signaling pathway and determine if NPY increases phospho-CREB levels.
It is safe to say that at least a 500% improvement in the overall antioxidant efficiency was seen during these preliminary in vitro tests due to ASEA exposure.
Gamma-irradiated Frozen Cells Validated for GPCR-mediated Kinase SignalingPerkinElmer, Inc.
The use of frozen cells for cell-based screening has become widely accepted within the drug discovery community. Separating cell production from the actual screening campaign not only increases your flexibility but also improves the data consistency as the cellular material can be controlled and validated before running the functional assay.
Personalized Medicine Opportunity Analysis - Team Neuropeptide - Stanford Ven...neuropeptide
Opportunity analysis project report by Team Neuropeptide, Stanford VentureLab class 2012. Our team is exploring business opportunities in the area of overlap of the fields of neurocomputation, genomics, and medicine. We are looking for successful ways to use neuromorphic algorithms for mining genomic databases and create opportunities for modern agile business models in more accessible and personalized healthcare.
It is safe to say that at least a 500% improvement in the overall antioxidant efficiency was seen during these preliminary in vitro tests due to ASEA exposure.
Gamma-irradiated Frozen Cells Validated for GPCR-mediated Kinase SignalingPerkinElmer, Inc.
The use of frozen cells for cell-based screening has become widely accepted within the drug discovery community. Separating cell production from the actual screening campaign not only increases your flexibility but also improves the data consistency as the cellular material can be controlled and validated before running the functional assay.
Personalized Medicine Opportunity Analysis - Team Neuropeptide - Stanford Ven...neuropeptide
Opportunity analysis project report by Team Neuropeptide, Stanford VentureLab class 2012. Our team is exploring business opportunities in the area of overlap of the fields of neurocomputation, genomics, and medicine. We are looking for successful ways to use neuromorphic algorithms for mining genomic databases and create opportunities for modern agile business models in more accessible and personalized healthcare.
Hplc method development for proteins and polypeptides ijsit 2.4.2IJSIT Editor
In the pharmaceutical field, there is considerable interest in the use of peptides and proteins for therapeutic
purposes. High performance liquid chromatography (HPLC) and its methods of complex peptide or protein
mixtures remains a general method of choice because of the resolution it provides. Unlike small organic
molecules whose chromatographic behavior is described by a finite partitioning equilibrium between the
stationary phase and the mobile phase, proteins and peptides typically do not exhibit such an effect. Instead,
they exhibit an adsorption phenomenon in which the polypeptide is adsorbed onto the stationary phase and
elutes only when the solvent strength of the mobile phase is sufficient to compete with the hydrophobic
forces keeping it there. For this reason, elution of peptides or proteins from reversed-phase supports is by
gradients of increasing solvent strength. There are other differences that one needs to be aware of in order to
develop HPLC methods for separations of proteins and peptides as efficiently as possible.
Dynamin I plays dual roles in the activity-dependent shift in exocytic mode i...Bryan Doreian
Under low stimulation, adrenal chromaffin cells release freely-soluble catecholamines through a restricted granule fusion pore while retaining the large neuropeptide-containing proteinacious granule core. Elevated activity causes dilation of the pore and release of all granule contents. Thus, physiological differential transmitter release is achieved through regulation of fusion pore dilation. We examined the mechanism for pore dilation utilizing a combined approach of peptide transfection, electrophysiology, electrochemistry and quantitative imaging techniques. We report that disruption of dynamin I function alters both fusion modes. Under low stimulation, interference with dynamin I does not affect granule fusion but blocks its re-internalization. In full collapse mode, disruption of dynamin I limits fusion pore dilation, but does not block membrane re-internalization. These data suggest that dynamin I is involved in both modes of exocytosis by regulating contraction or dilation of the fusion pore and thus contributes to activity-dependent differential transmitter release from the adrenal medulla.
Kinsenoside isolated from Anoectochilus formosanus suppresses LPS-Stimulated ...Cây thuốc Việt
In the present study, we reported that kinsenoside, a major component of Anoectochilus formosanus, inhibited inflammatory reactions in mouse peritoneal lavage macrophages and protects mice from endotoxin shock. In LPSstimulated mouse peritoneal lavage macrophages, kinsenoside inhibited the inflammatory mediators, such as nitric oxide, TNF-!, IL-1", monocyte chemoattractant protein 1, and macrophage migration inhibitory factor production. Furthermore, kinsenoside decreased the formation of a nuclear factor .BYDNA complex and nuclear p65 and p50 protein levels. Kinsenoside inhibited nuclear factor .B translocation through both I.B!-dependent and -independent pathway. In contrast, it stimulated anti-inflammatory cytokine IL-10 generation and enhanced the mRNA expression of IL-10 and suppressor of cytokine signaling 3 in the same cells induced by LPS. In an animal model, both pretreatment and posttreatment of kinsenoside increased the survival rate of ICR mice challenged by LPS (80 mg/kg, i.p.). Pretreatment with kinsenoside decreased serum levels of TNF-!, IL-1", IL-10, monocyte chemoattractant protein 1, and migration inhibitory factor at 1 h after sublethal dose of LPS (40 mg/kg, i.p.) in mice. In contrast, kinsenoside enhanced serum IL-10 level at 24 h after LPS injection in mice. In conclusion, kinsenoside inhibited the production of inflammatory mediators and enhanced antiinflammatory cytokine generation. Therefore, kinsenoside can alleviate acute inflammatory hazards.
Cell culture is the process by which prokaryotic, eukaryotic or plant cells are grown under controlled conditions. Mammalian cell culture technology has become a major field in modern biotechnology; mammalian cell culture refers to the cells of a mammalian, isolated from specific tissues (i.e. skin, liver, glands, etc.) and further cultivated and reproduced in an artificial medium. Cell culture technology is currently playing a major role in toxicity testing, cancer research, virology, genetic engineering, and gene therapy.
OBJECTIVE:
To observe the transfection of CHO and HEK cells with GFP
To observe the recombinant GFP using Western Blotting
To purify the transfected HEK and CHO cells using AKTA Pure Purification
Topical Delivery of Fenoprofen Proliposomes: Preparation, Evaluation and In V...inventionjournals
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.
Transcription factors of the nuclear factor κ B family are the paradigm for signaling dependent nuclear translocation and are ideally suited to analysis through image-based chemical genetic screening. The authors describe combining high-content image analysis with a compound screen to identify compounds affecting either nuclear import or export. Validation in silico and in vitro determined an EC50 for the nuclear export blocker leptomycin B of 2.4 ng/mL (4.4 nM). The method demonstrated high selectivity (Z′ >0.95), speed, and robustness in a screen of a compound collection. It identified the IκB protein kinase inhibitor BAY 11 7082 as an import inhibitor, the p38 mitogen-activated protein (MAP) kinase inhibitor PD98509 as an import enhancer, and phorbol ester as an export inhibitor. The results establish a robust method for identifying compounds regulating nucleocy- toplasmic import or export and also implicate MAP kinases in nuclear import of nuclear factor κ B
The successful of pregnancy in humans and rodents occur between the interaction maternal and fetal
interface, specially involving the participation of uNK cells. This interaction involved neo angiogenesis,
placentation and presence of mediators like nitric oxide. During the pregnancy the administration of LPS
in the dams can results in necrosis, preterm birth, IUGR, miscarriage or neurological problem. Once the
uNK cells are activated, they can produce vasodilators, like NO. So, the main purpose of this study was
to evaluate if LPS cause alteration in the uNK cells in pregnant mice and if the same behaviour can be
detected by NO in the blood. Also we evaluated the effect of LPS to cause neurological injuries. To do that
we used pregnant mice on gd 10th and those was treated with LPS for different times. Uterine samples
were collected at 0.5,1,2 and 6hr after LPS treated and processed for paraffin embedding and tissue
homogenate. The samples designated for paraffin embedding was performed the Dolichos biflorus (DBA)
lectin cytochemistry and anti-iNOS immunocytochemistry. The samples designated to tissue homogenates
were processed for SDS-PAGE and Western-blot using anti-iNOS and evaluate of NO concentration. We
found after 2h LPS exposure the mice showed fever and low capacity to explore different environment.
At the same time, we found increase in the nitrate/nitrito ratio in a dose dependent manner in the uterus
after 2h LPS exposure.
Commercial Application of Anoectochilus formosanus: Immunomodulating ActivitiesCây thuốc Việt
Anoectochilus formosanus is an important ethnomedicinal plant of Taiwan. We investigated the effect of oral administration of A. formosanus effective fraction (AFEF) on the innate immune response in mice. Male BALB/c mice were treated orally for 2 weeks with 500, 1000 and 1500 mg/kg of AFEF. Primary peritoneal macrophage harvest from mice that administered with AFEF (500 –1500 mg/kg) was directed to activate phagocytosis. AFEF significantly increased interferon-production from lymph node cells by ConA stimulation for 48 hours in AFEF (1500 mg/kg) treated group. AFEF might be the active fraction in activation of innate immunity.
1. RISE Program<br />NPY-Induced Kinase Activation in C. elegans Effects on Signaling Nodes<br />Ricardo Chiesa1, Francisco Fuster1, Jessica Díaz Rivera1<br />1Department of Biology, University of Puerto Rico, Cayey Campus<br />_____________________________________________________________________________________________________________________Abstract<br />Neuropeptide Y (NPY) acts as neurotransmitter by activating multiple receptors thus regulating a number of physiological functions. Studies demonstrated that receptor 1 has anxiolytic effects in human body. To further investigate the effect of NPY, we studied the reactions that the proteins had with the presence of NPY and the changes that NPY causes on GABA receptor. No statistically significant effect was observed in the proteins with the presence of NPY, just Akt1. These results suggests that NPY has an up regulation in Akt1 protein. <br />Introduction<br />Neuropeptides are small proteins that act on the cell's surface receptors. They are secreted by neurons for communication with other cells. They adhere to the receptors and trigger actions on the cell. Sometimes these actions lead to a whole network of action and reaction induced pathways. We are working with the effects of neuropeptide Y (NPY), to monitor the changes of a proposed pathway that is supposedly related to anxiolytic effects, specifically receptor Y1. NPY is a neurotransmitter found in the brain and the rest of the nervous system. It occurs naturally in the human body and it is proposed that it regulates many anxiety related conditions, like excessive food intake, memory, and even in learning.<br />The proteins will be extracted from the nematode Caenorhabditis elegans. The nematode is used because it produces many neurotransmitters that are comparable to the ones in the human nervous system. Even though it has a primitive nervous system it is found to be very useful because their hermaphroditic reproduction allows for a fast renewal supply of these animals, also their maturity span is between 3 to 4 days. The method used to extract these proteins in mostly with the integration of the technique of heat shock. The animal is exposed to a dramatic change of temperature, causing permeability of its membrane, basically expelling the contents of its cells which include proteins. The proposed pathway includes a chain of events that occur after NPY is introduced. Once NPY adheres to the receptor on the cell surface, it stimulates the activation of phospholipase C (PLC). The lipase breaks a phosphate groups from the cell's membrane, separating the phosphorus molecule from the hydrophobic region of the phospholipid. This leads to the release of Ca+ from the endoplasmic reticulum, which activates protein kinase C (PKC). PKC then carries the phosphate group to Hras, which becomes phosphorylated, or activated. This then leads to yet another chain reaction between proteins that pass down this phosphate group, becoming phosphorylated, until the process reaches RSK 90. This molecule now has the phosphate group and passes it to CREB, which phosphorylates the gene GABRA. This process turned the gene, on for the synthesis of GABA. GABA is then synthesized and attached to the cell membrane, waiting for a substrate to adhere to activate yet another pathway, linked to anxiolysis. Our primary objectives are: to monitor changes in signaling nodes with the presence of NPY, monitor the effect that NPY causes on GABA synthesis and validate the proposed pathway. We hypothesize that in the presence of NPY, there will be an up regulation of the GABRA gene and increase in the levels of proteins of the proposed pathway. To monitor these changes we used the ELISA assay. This technique helps us detect the presence of a specific protein. It uses a primary layer of antibodies that adhere to the protein, then a secondary layer that adheres to the primary antibody which then creates a union between the structures. Each protein that is detected with ELISA has a specific function: Akt1 is a critical regulator role in diverse cellular processes including growth, survival and the cell cycle, p38 MAP kinase participates in a signaling cascade controlling the cellular response to pro-inflammatory cytokinases and a variety of cellular stresses and Stat3 is an important signaling molecule for many cytokines and growth receptors.<br />-40514578<br />Methodology:<br />Cultivation of Caenorhabditis elegans:<br />This cultivation involves two major changes of medium. First these nematodes grew in a solid medium of agar, with a similarity to its normal environment. They had plenty of bacteria to feed on, this way there are in no stress. The bacteria they normally feed on is E. coli which was added to the medium.<br />The second medium in which they finished the process of cultivation is on a liquid medium. Here they have a liquid environment with many elements found on their normal aquatic environment in the roots of plants, including the bacteria to feed upon. <br />Protein extraction:<br />This process involved the use of centrifuging to push all contents down. After this is done, the supernatant in the liquid is removed to concentrate all the other contents left on the bottom. Then next step involves the repetition of the last step to concentrate even more the amount of proteins in a small pellet at the bottom of the tube. To preserve the proteins place them on water at room temperature for 15 min and later store at -80 degrees Celsius. <br />Protein Quantification BCA:<br />The BCA protein assay was based on a biuret reaction, which is the reduction of Cu2+ to Cu+ by proteins in an alkaline solution with concentration-dependent detection of the monovalent copper ions. Bicinchoninic acid is a chromogenic reagent that chelates the reduced copper, producing a purple complex with strong absorbance at 562 nm. <br />ELISA(Enzyme-linked Immunosorbent) Assay<br />We had to do a Reagent Preparation by preparing 1X Wash Buffer by diluting 20X Wash Buffer in equivalently purified water. Then we prepared the cell lysates by adding fresh media containing regulator for desired time. Removed media and rinse cells once with ice cold-PBS, to harvest cells under non-denaturing conditions. We removed the PBS and added 0.5 ml ice cold 1X Cell Lysis Buffer plus 1mM phenylmethylsulfony fluoride (PMSF) to each plate and then we put the plate to incubate for a period of 5 minutes. We scraped the cells to transfer to an appropriate tube. Microcentrifuging was using for 10 minutes at 4 degrees Celsius and transferred the supernatant (cell lysate) to a new tube to store at -80 degrees Celsius. Next step was the Test Procedure. We added 100 ul of Sample Diluent to a microcentrifuge tube. Transferred 100 ul of cell lysate into the tube and vortex for a few seconds. Then we added 100 ul of each diluted cell lysate to the appropriate well. The plate was sealed with tape and incubated it for 2 hours at 37 degrees Celsius. The tape was removed and all the wells were washed. We discarded plate contents into a receptacle and washed it 4 times with 1X Wash Buffer, 200 ul each time for each well. For each wash, strike plates on fresh towels hard enough to remove the residual solution in each well. We added 100 ul of Detection Antibody to each well, sealed with tape and incubated it for 1 hour at 37 degrees Celsius. The well wash procedure was repeated. The following step was to add 100 ul HRP-linked secondary antibody to each well, and again repeated wash wells procedure. <br />Then we added 100 ul of TMB Substrate to each well, sealed with tape and incubated the plate for a period of 30 minutes at 37 degrees Celsius. The last step was to add 100 ul of STOP Solution to each well and shake gently for a few seconds. The initial color reaction was blue, which changed to yellow with the addition of the STOP Solution. <br />Spectrophotometry<br />Place the 96 well plate in the spectrophotometer. Choose the two blanks and the experimental wells using “Microplate Manager 5.2.1 Program”. <br />Results:<br /> <br />-4058269<br />77416330529793221165211<br />-263053-194554<br />Discussion:<br />The levels of total Akt-1 were higher (statistical significance) in the +NPY protein extracts. However, phospho-Akt-1 levels were the same both in the control and experimental group. We have to corroborate if a CRE element is present in the Akt-1 gene. If future experiments show an increase in the levels of phospho-CREB, this could account for the increase in the levels of total Akt-1. Phospho-Stat-3 and phospho-p38 levels are the same both in control group (no NPY) and the experimental group (with NPY).<br />References:<br />[1] Domschke K, Dannlowski U, Hohoff C, et all. (2010). Neuropeptide Y (NPY) gene: Impact on emotional processing and treatment response in anxious depression. Eur Neuropsychopharmacol, 5, 301-9.<br />[2] Bacchi F, Mathe AA, Jimenez P, et. all. (2006). Anxiolytic-like effect of the selective Neuropeptide Y Y2 receptor antagonist BIIE0246 in the elevated plus-maze. Peptides,12, 3202-7.<br />[3] Geary, T. G., Kubiak, T. M. (2005). Neuropeptide G-protein couple receptors, their cognate ligands and behavior in Caernorhabditis elegans. TRENDS in Pharmacological Sciencies, 26, 56-58.<br />