Combined Use of a Solid-Phase Hexapeptide Ligand Library with Liquid Chromatography and Two-Dimensional Difference Gel Electrophoresis for Intact Plasma Proteomics

The intact plasma proteome is of great interest in biomarker studies because intact proteins reflect posttranslational protein processing such as phosphorylation that may correspond to disease status. We examined the utility of a solid-phase hexapeptide ligand library in combination with conventional plasma proteomics modalities for comprehensive profiling of intact plasma proteins. Plasma proteins were sequentially fractionated using depletion columns for albumin and immunoglobulin, and separated using an anion-exchange column. Proteins in each fraction were treated with a solid-phase hexapeptide ligand library and compared to those without treatment. Two-dimensional difference gel electrophoresis demonstrated an increased number of protein spots in the treated samples. Mass spectrometric studies of these protein spots with unique intensity in the treated samples resulted in the identification of high- and medium-abundance proteins. Our results demonstrated the possible utility of a solid-phase hexapeptide ligand library to reveal greater number of intact plasma proteins. The characteristics of proteins with unique affinity to the library remain to be clarified by more extensive mass spectrometric protein identification, and optimized protocols should be established for large-scale plasma biomarker studies

The Application of a Three-Step Proteome Analysis for Identification of New Biomarkers of Pancreatic Cancer

We searched for novel tumor markers of pancreatic cancer by three-step serum proteome analysis. Twelve serum abundant proteins were depleted using immunoaffinity columns followed by fractionation by reverse-phase high-performance liquid chromatography. Proteins in each fraction were separated by two-dimensional gel electrophoresis. Then the gel was stained by Coomassie Brilliant Blue. Protein spots in which the expression levels were significantly different between cancer and normal control were identified by LC-MS/MS. One hundred and two spots were upregulated, and 84 spots were downregulated in serum samples obtained from patients with pancreatic cancers, and 58 proteins were identified by mass spectrometry. These candidate proteins were validated using western blot analysis and enzyme-linked immunosorbent assay (ELISA). As a result of these validation process, we could confirm that the serum levels of apolipoprotein A-IV, vitamin D-binding protein, plasma retinol-binding protein 4, and tetranectin were significantly decreased in patients with pancreatic cancer

The C/EBP Site in the Feline Immunodeficiency Virus (FIV) Long Terminal Repeat (LTR) Is Necessary for Its Efficient Replication and Is Also Involved in the Inhibition of FIV LTR-Directed Gene Expression by Pseudorabies Virus ICP4

AbstractWe investigated effects of site-specific mutation of the putative C/EBP binding site in the feline immunodeficiency virus (FIV) long terminal repeat (LTR) on the basal promoter activity in Crandell feline kidney (CRFK) cells and on replication efficiency in CRFK cells and a T-lymphoblastoid cell line, MYA-1 cells. Mutation of the C/EBP site reduced the basal promoter activity in CRFK cells and prevented efficient FIV replication in both CRFK and MYA-1 cells. Gel-mobility-shift assay using nuclear extracts from CRFK and MYA-1 cells revealed that the nuclear factor(s) actually binds to the C/EBP site, but there was a clear difference in the binding patterns to the C/EBP site between CRFK and MYA-1 cell nuclear proteins. Furthermore, we demonstrated that the C/EBP site is necessary for inhibition of FIV LTR-directed gene expression by pseudorabies virus (PRV) ICP4. The C/EBP site is sufficient to confer inhibitory effect by PRV ICP4 on heterologous promoters. These data suggest that the C/EBP site in the FIV LTR is important for the positive regulation of FIV gene expression and replication and is also required for the negative regulation of FIV gene expression by PRV ICP4

Phylogenetic analysis of the long terminal repeat of feline immunodeficiency viruses from Japan, Argentina and Australia

The nucleotide sequences of the long terminal repeat of five Japanese, five Argentine and three Australian isolates of feline immunodeficiency virus (FIV) were determined and compared with those of isolates previously described. The results revealed that the Japanese isolates were found to cluster with nucleotide sequence similarity of 95.6%–99.4%. The Australian isolates also clustered with nucleotide sequence similarity of 97.2%–99.4%. The Argentine isolates formed two groups; the LP9 isolate is closely related to the Japanese isolates, whereas the LP1, LP3, LP20 and LP24 isolates are distant from both the Japanese and Australian isolates. From these results, FIV can be divided into three groups, namely: (I) the Californian, Australian and British isolates; (II) the Japanese isolates and one Argentine LP9 isolate; (III) the other Argentine isolates.Facultad de Ciencias Veterinaria

Belediye Müzesi

Taha Toros Arşivi, Dosya No: 114-Müzelerİstanbul Kalkınma Ajansı (TR10/14/YEN/0033) İstanbul Development Agency (TR10/14/YEN/0033

Involvement of SIK3 in Glucose and Lipid Homeostasis in Mice

Salt-inducible kinase 3 (SIK3), an AMP-activated protein kinase-related kinase, is induced in the murine liver after the consumption of a diet rich in fat, sucrose, and cholesterol. To examine whether SIK3 can modulate glucose and lipid metabolism in the liver, we analyzed phenotypes of SIK3-deficent mice. Sik3−/− mice have a malnourished the phenotype (i.e., lipodystrophy, hypolipidemia, hypoglycemia, and hyper-insulin sensitivity) accompanied by cholestasis and cholelithiasis. The hypoglycemic and hyper-insulin-sensitive phenotypes may be due to reduced energy storage, which is represented by the low expression levels of mRNA for components of the fatty acid synthesis pathways in the liver. The biliary disorders in Sik3−/− mice are associated with the dysregulation of gene expression programs that respond to nutritional stresses and are probably regulated by nuclear receptors. Retinoic acid plays a role in cholesterol and bile acid homeostasis, wheras ALDH1a which produces retinoic acid, is expressed at low levels in Sik3−/− mice. Lipid metabolism disorders in Sik3−/− mice are ameliorated by the treatment with 9-cis-retinoic acid. In conclusion, SIK3 is a novel energy regulator that modulates cholesterol and bile acid metabolism by coupling with retinoid metabolism, and may alter the size of energy storage in mice

Cohesin-dependent globules and heterochromatin shape 3D genome architecture in S. pombe

Eukaryotic genomes are folded into three-dimensional structures, such as self-associating topological domains, the borders of which are enriched in cohesin and CCCTC-binding factor (CTCF) required for long-range interactions1-7. How local chromatin interactions govern higher-order folding of chromatin fibers and the function of cohesin in this process remain poorly understood. Here we perform genome-wide chromatin conformation capture (Hi-C) analysis8 to explore the high-resolution organization of the Schizosaccharomyces pombe genome, which despite its small size exhibits fundamental features found in other eukaryotes9. Our analyses of wild type and mutant strains reveal key elements of chromosome architecture and genome organization. On chromosome arms, small regions of chromatin locally interact to form “globules”. This feature requires a function of cohesin distinct from its role in sister chromatid cohesion. Cohesin is enriched at globule boundaries and its loss causes disruption of local globule structures and global chromosome territories. By contrast, heterochromatin, which loads cohesin at specific sites including pericentromeric and subtelomeric domains9-11, is dispensable for globule formation but nevertheless affects genome organization. We show that heterochromatin mediates chromatin fiber compaction at centromeres and promotes prominent interarm interactions within centromere-proximal regions, providing structural constraints crucial for proper genome organization. Loss of heterochromatin relaxes constraints on chromosomes, causing an increase in intra- and inter-chromosomal interactions. Together, our analyses uncover fundamental genome folding principles that drive higher-order chromosome organization crucial for coordinating nuclear functions