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Find video protocols related to scientific articles indexed in Pubmed.
Specific catalysis of asparaginyl deamidation by carboxylic acids: kinetic, thermodynamic, and quantitative structure-property relationship analyses.
Mol. Pharm.
PUBLISHED: 03-27-2014
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Asparaginyl (Asn) deamidation could lead to altered potency, safety, and/or pharmacokinetics of therapeutic protein drugs. In this study, we investigated the effects of several different carboxylic acids on Asn deamidation rates using an IgG1 monoclonal antibody (mAb1*) and a model hexapeptide (peptide1) with the sequence YGKNGG. Thermodynamic analyses of the kinetics data revealed that higher deamidation rates are associated with predominantly more negative ?S and, to a lesser extent, more positive ?H. The observed differences in deamidation rates were attributed to the unique ability of each type of carboxylic acid to stabilize the energetically unfavorable transition-state conformations required for imide formation. Quantitative structure property relationship (QSPR) analysis using kinetic data demonstrated that molecular descriptors encoding for the geometric spatial distribution of atomic properties on various carboxylic acids are effective determinants for the deamidation reaction. Specifically, the number of O-O and O-H atom pairs on carboxyl and hydroxyl groups with interatomic distances of 4-5 Å on a carboxylic acid buffer appears to determine the rate of deamidation. Collectively, the results from structural and thermodynamic analyses indicate that carboxylic acids presumably form multiple hydrogen bonds and charge-charge interactions with the relevant deamidation site and provide alignment between the reactive atoms on the side chain and backbone. We propose that carboxylic acids catalyze deamidation by stabilizing a specific, energetically unfavorable transition-state conformation of l-asparaginyl intermediate II that readily facilitates bond formation between the ?-carbonyl carbon and the deprotonated backbone nitrogen for cyclic imide formation.
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Trehalose limits BSA aggregation in spray-dried formulations at high temperatures: implications in preparing polymer implants for long-term protein delivery.
J Pharm Sci
PUBLISHED: 02-04-2013
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Polymer implants are promising systems for sustained release applications but their utility for protein delivery has been hindered because of concerns over drug stability at elevated temperatures required for processing. Using bovine serum albumin (BSA) as a model, we have assessed whether proteins can be formulated for processing at elevated temperatures. Specifically, the effect of trehalose and histidine-HCl buffer on BSA stability in a spray-dried formulation has been investigated at temperatures ranging from 80°C to 110°C. When both the sugar and buffer are present, aggregation is suppressed even when exposed to 100°C, the extrusion temperature of poly(lactide-co-glycolide) (PLGA), a bioresorbable polymer. Estimation of aggregation rate constants (k) indicate that though both trehalose and histidine-HCl buffer contribute to BSA stability, the effect because of trehalose alone is more pronounced. BSA-loaded PLGA implants were prepared using hot-melt extrusion process and in vitro release was conducted in phosphate buffered saline at 37°C. Comparison of drug released from implants prepared using four different formulations confirmed that maximal release was achieved from the formulation in which BSA was least aggregated. These studies demonstrate that when trehalose and histidine-HCl buffer are included in spray-dried formulations, BSA stability is maintained both during processing at 100°C and long-term residence within implants.
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Nestin-Expressing Stem Cells Promote Nerve Growth in Long-Term 3-Dimensional Gelfoam®-Supported Histoculture.
PLoS ONE
PUBLISHED: 01-01-2013
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We have previously reported that hair follicles contain multipotent stem cells which express nestin. The nestin-expressing cells form the hair follicle sensory nerve. In vitro, the nestin-expressing hair follicle cells can differentiate into neurons, Schwann cells, and other cell types. In the present study, the sciatic nerve was excised from transgenic mice in which the nestin promoter drives green fluorescent protein (ND-GFP mice). The ND-GFP cells of the sciatic nerve were also found to be multipotent as the ND-GFP cells in the hair follicle. When the ND-GFP cells in the mouse sciatic nerve cultured on Gelfoam® and were imaged by confocal microscopy, they were observed forming fibers extending the nerve. The fibers consisted of ND-GFP-expressing spindle cells, which co-expressed the neuron marker ?-III tubulin, the immature Schwann-cell marker p75(NTR) and TrkB which is associated with neurons. The fibers also contain nestin-negative spherical cells expressing GFAP, a Schwann-cell marker. The ?-III tubulin-positive fibers had growth cones on their tips expressing F-actin, indicating they are growing axons. When the sciatic nerve from mice ubiquitously expressing red fluorescent protein (RFP) was co-cultured on Gelfoam® with the sciatic nerve from ND-GFP transgenic mice, the interaction of nerves was observed. Proliferating nestin-expressing cells in the injured sciatic nerve were also observed in vivo. Nestin-expressing cells were also observed in posterior nerves but not in the spinal cord itself, when placed in 3-D Gelfoam® culture. The results of the present report suggest a critical function of nestin-expressing cells in peripheral nerve growth and regeneration.
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Inhibition and eradication of human glioma with tumor-targeting Salmonella typhimurium in an orthotopic nude-mouse model.
Cell Cycle
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Malignant glioma tumors are the most common primary central nervous system tumors. Despite the multidisciplinary approach to treatment, prognosis remains poor. In this study, we demonstrated that the Salmonella typhimurium A1-R tumor-targeting strain can inhibit and eradicate human glioma in an orthotopic nude-mouse model. S. typhimurium A1-R was administered by injection through a craniotomy open-window or intravenously in nude mice. To establish the model, 2x10(5) U87-RFP human glioma cells were injected stereotactically into the mouse brain through the craniotomy open window. Two weeks after glioma-cell implantation, mice were treated with S. typhimurium A1-R [2x10(7) CFU/200 ?l intravenous injection (i.v.) or 1x10(6) CFU/1 ?l intracranial injection (i.c.)] once a week for 3 weeks. Brain tumors were observed by fluorescence imaging through the craniotomy open window over time. S. typhimurium A1-R, administered i.c., inhibited brain tumor growth 7.6-fold compared with untreated mice (p=0.009) and improved survival 73% (p=0.001). Two of ten mice appeared to have their tumors eradicated. Intravenous administration of S. typhimurium A1-R was not effective. The craniotomy open window enabled observation of tumor growth in the brain in real time in both treated and untreated mice. The results of the present study demonstrate that bacterial therapy of brain cancer is a novel, effective and safe treatment strategy in a highly treatment-resistance cancer.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.