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Find video protocols related to scientific articles indexed in Pubmed.
Extensive crosstalk between O-GlcNAcylation and phosphorylation regulates cytokinesis.
Sci Signal
PUBLISHED: 01-14-2010
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Like phosphorylation, the addition of O-linked beta-N-acetylglucosamine (O-GlcNAcylation) is a ubiquitous, reversible process that modifies serine and threonine residues on nuclear and cytoplasmic proteins. Overexpression of the enzyme that adds O-GlcNAc to target proteins, O-GlcNAc transferase (OGT), perturbs cytokinesis and promotes polyploidy, but the molecular targets of OGT that are important for its cell cycle functions are unknown. Here, we identify 141 previously unknown O-GlcNAc sites on proteins that function in spindle assembly and cytokinesis. Many of these O-GlcNAcylation sites are either identical to known phosphorylation sites or in close proximity to them. Furthermore, we found that O-GlcNAcylation altered the phosphorylation of key proteins associated with the mitotic spindle and midbody. Forced overexpression of OGT increased the inhibitory phosphorylation of cyclin-dependent kinase 1 (CDK1) and reduced the phosphorylation of CDK1 target proteins. The increased phosphorylation of CDK1 is explained by increased activation of its upstream kinase, MYT1, and by a concomitant reduction in the transcript for the CDK1 phosphatase, CDC25C. OGT overexpression also caused a reduction in both messenger RNA expression and protein abundance of Polo-like kinase 1, which is upstream of both MYT1 and CDC25C. The data not only illustrate the crosstalk between O-GlcNAcylation and phosphorylation of proteins that are regulators of crucial signaling pathways but also uncover a mechanism for the role of O-GlcNAcylation in regulation of cell division.
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Regulation of calcium/calmodulin-dependent kinase IV by O-GlcNAc modification.
J. Biol. Chem.
PUBLISHED: 06-08-2009
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Similar to phosphorylation, GlcNAcylation (the addition of O-GlcNAc to Ser(Thr) residues on polypeptides) is an abundant, dynamic, and inducible post-translational modification. GlcNAcylated proteins are crucial in regulating virtually all cellular processes, including signaling, cell cycle, and transcription. Here we show that calcium/calmodulin-dependent kinase IV (CaMKIV) is highly GlcNAcylated in vivo. In addition, we show that upon activation of HEK293 cells, hemagglutinin-tagged CaMKIV GlcNAcylation rapidly decreases, in a manner directly opposing its phosphorylation at Thr-200. Correspondingly, there is an increase in CaMKIV interaction with O-GlcNAcase during CaMKIV activation. Furthermore, we identify at least five sites of GlcNAcylation on CaMKIV. Using site-directed mutagenesis, we determine that the GlcNAcylation sites located in the active site of CaMKIV can modulate its phosphorylation at Thr-200 and its activity toward cAMP-response element-binding transcription factor. Our results strongly indicate that the O-GlcNAc modification participates in the regulation of CaMKIV activation and function, possibly coordinating nutritional signals with the immune and nervous systems. This is the first example of an O-GlcNAc/phosphate cycle involving O-GlcNAc transferase/kinase cross-talk.
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O-GlcNAcylation of kinases.
Biochem. Biophys. Res. Commun.
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Recent evidence indicates that site-specific crosstalk between O-GlcNAcylation and phosphorylation and the O-GlcNAcylation of kinases play an important role in regulating cell signaling. However, relatively few kinases have been analyzed for O-GlcNAcylation. Here, we identify additional kinases that are substrates for O-GlcNAcylation using an in vitro OGT assay on a functional kinase array. Forty-two kinases were O-GlcNAcylated in vitro, representing 39% of the kinases on the array. In addition, we confirmed the in vivo O-GlcNAcylation of three identified kinases. Our results suggest that O-GlcNAcylation may directly regulate a substantial number of kinases and illustrates the increasingly complex relationship between O-GlcNAcylation and phosphorylation in cellular signaling.
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Samsung Salmonella Detection Kit. AOAC Performance Tested Method(SM) 021203.
J AOAC Int
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Salmonella, one of the most common causes of foodborne illness, is a significant public health concern worldwide. There is a need in the food industry for methods that are simple, rapid, and sensitive for the detection of foodborne pathogens. In this study, the Samsung Salmonella Detection Kit, a real-time PCR assay for the detection of Salmonella, was evaluated according to the current AOAC guidelines. The validation consisted of lot-to-lot consistency, stability, robustness, and inclusivity/exclusivity studies, as well as a method comparison of 10 different food matrixes. In the validation, the Samsung Salmonella Detection Kit was used in conjunction with the Applied Biosystems StepOnePlus PCR system and the Samsung Food Testing Software for the detection of Salmonella species. The performance of the assays was compared to the U.S. Department of Agriculture/Food Safety and Inspection Service-Microbiology Laboratory Guidebook (USDA/FSIS-MLG) 4.05: Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg, and Catfish and the and U.S. Food and Drug Administration/Bacteriological Analytical Manual (FDA/BAM) Chapter 5 Salmonella reference methods. The validation was conducted using an unpaired study design for detection of Salmonella spp. in raw ground beef, raw pork, raw ground pork, raw chicken wings, raw salmon, alfalfa sprouts, pasteurized orange juice, peanut butter, pasteurized whole milk, and shell eggs. The Samsung Salmonella Detection Kit demonstrated lot-to-lot consistency among three independent lots as well as ruggedness with minor modifications to changes in enrichment incubation time, enrichment incubation temperature, and DNA sample volume for PCR reaction. Stability was observed for 13 months at -20 degrees C and 3 months at 5 degrees C. For the inclusivity/exclusivity study, the Samsung Salmonella Detection Kit correctly identified 147 Salmonella species isolates out of 147 isolates tested from each of three different enrichment broths (a total of 441 isolates detected), and correctly excluded all 31 nontarget strains analyzed. For the method comparison, statistical analysis was conducted according to the Mantel-Haenszel Chi-square formula for unpaired test portions, and there was no significant difference in the number of positive samples detected between the Samsung Salmonella Detection Kit and the USDA/FSIS-MLG and FDA/BAM reference methods for all 10 food matrixes.
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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.