Solid phase synthesis of peptide hydroxamic acids on poly(ethylene glycol)‐based support

A novel resin designed for solid‐phase synthesis of peptide hydroxamic acids (PHA) combining the trityl linker with poly(ethylene glycol)‐based support, ChemMatrix® type, is described. The synthesis of PHA can be performed according to a standard protocol, providing products in excellent purity and reasonable yields. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

2-Methylwyosine,a Nucleoside With Restricted Anti Conformation in the East Region Enforced by Nucleobase Moiety Modification: Synthesis and Conformational Analysis by NMR and Molecular Dynamics

GRAPHICAL ABSTRACT

We synthesized a new 2-methyl derivative of wyosine using a multistep procedure starting from guanosine. We examined different synthetic paths and optimized the conditions for each step. Based on MD calculations and analysis of the ³ J _HH and J _C1′H1′ of the ribose moiety, we discovered that the sugar part adopted conformation specific for the East region rarely occurring in solution. This unusual conformational preference is probably due to steric repulsions between the methyl group at position 2 and the 5′-CH₂OH group. We observed that N-glycosidic bond stability weakened 14-fold upon the introduction of the methyl group in position 2 compared with wyosine.     

The Oxytricha trifallax Macronuclear Genome: A Complex Eukaryotic Genome with 16,000 Tiny Chromosomes

The macronuclear genome of the ciliate Oxytricha trifallax displays an extreme and unique eukaryotic genome architecture with extensive genomic variation. During sexual genome development, the expressed, somatic macronuclear genome is whittled down to the genic portion of a small fraction (∼5%) of its precursor “silent” germline micronuclear genome by a process of “unscrambling” and fragmentation. The tiny macronuclear “nanochromosomes” typically encode single, protein-coding genes (a small portion, 10%, encode 2–8 genes), have minimal noncoding regions, and are differentially amplified to an average of ∼2,000 copies. We report the high-quality genome assembly of ∼16,000 complete nanochromosomes (∼50 Mb haploid genome size) that vary from 469 bp to 66 kb long (mean ∼3.2 kb) and encode ∼18,500 genes. Alternative DNA fragmentation processes ∼10% of the nanochromosomes into multiple isoforms that usually encode complete genes. Nucleotide diversity in the macronucleus is very high (SNP heterozygosity is ∼4.0%), suggesting that Oxytricha trifallax may have one of the largest known effective population sizes of eukaryotes. Comparison to other ciliates with nonscrambled genomes and long macronuclear chromosomes (on the order of 100 kb) suggests several candidate proteins that could be involved in genome rearrangement, including domesticated MULE and IS1595-like DDE transposases. The assembly of the highly fragmented Oxytricha macronuclear genome is the first completed genome with such an unusual architecture. This genome sequence provides tantalizing glimpses into novel molecular biology and evolution. For example, Oxytricha maintains tens of millions of telomeres per cell and has also evolved an intriguing expansion of telomere end-binding proteins. In conjunction with the micronuclear genome in progress, the O. trifallax macronuclear genome will provide an invaluable resource for investigating programmed genome rearrangements, complementing studies of rearrangements arising during evolution and disease.     

Telocytes: new insight into the pathogenesis of gallstone disease

The major mechanisms of gallstone formation include biliary cholesterol hypersecretion, supersaturation and crystallization, mucus hypersecretion, gel formation and bile stasis. Gallbladder hypomotility seems to be a key event that triggers the precipitation of cholesterol microcrystals from supersaturated lithogenic bile. Telocytes, a new type of interstitial cells, have been recently identified in many organs, including gallbladder. Considering telocyte functions, it is presumed that these cells might be involved in the signalling processes. The purpose of this study was to correlate the quantity of telocytes in the gallbladder with the lithogenicity of bile. Gallbladder specimens were collected from 24 patients who underwent elective laparoscopic cholecystectomy for symptomatic gallstone disease. The control group consisted of 25 consecutive patients who received elective treatment for pancreatic head tumours. Telocytes were visualized in paraffin sections of gallbladders with double immunofluorescence using primary antibodies against c‐Kit (anti‐CD117) and anti‐mast cell tryptase. Cholesterol, phospholipid and bile acid levels were measured in gallbladder bile. The number of telocytes in the gallbladder wall was significantly lower in the study group than that in the control group (3.03 ± 1.43 versus 6.34 ± 1.66 cell/field of view in the muscularis propria, P < 0.001) and correlated with a significant increase in the cholesterol saturation index. The glycocholic and taurocholic acid levels were significantly elevated in the control subjects compared with the study group. The results suggest that bile composition may play an important role in the reduction in telocytes density in the gallbladder.     

Biosynthesis of GlcNAc-rich N- and O-glycans in the Golgi apparatus does not require the nucleotide sugar transporter SLC35A3

Nucleotide sugar transporters, encoded by the SLC35 gene family, deliver nucleotide sugars throughout the cell for various glycosyltransferase-catalyzed glycosylation reactions. GlcNAc, in the form of UDP-GlcNAc, and galactose, as UDP-Gal, are delivered into the Golgi apparatus by SLC35A3 and SLC35A2 transporters, respectively. However, although the UDP-Gal transporting activity of SLC35A2 has been clearly demonstrated, UDP-GlcNAc delivery by SLC35A3 is not fully understood. Therefore, we analyzed a panel of CHO, HEK293T, and HepG2 cell lines including WT cells, SLC35A2 knockouts, SLC35A3 knockouts, and double-knockout cells. Cells lacking SLC35A2 displayed significant changes in N- and O-glycan synthesis. However, in SLC35A3-knockout CHO cells, only limited changes were observed; GlcNAc was still incorporated into N-glycans, but complex type N-glycan branching was impaired, although UDP-GlcNAc transport into Golgi vesicles was not decreased. In SLC35A3-knockout HEK293T cells, UDP-GlcNAc transport was significantly decreased but not completely abolished. However, N-glycan branching was not impaired in these cells. In CHO and HEK293T cells, the effect of SLC35A3 deficiency on N-glycan branching was potentiated in the absence of SLC35A2. Moreover, in SLC35A3-knockout HEK293T and HepG2 cells, GlcNAc was still incorporated into O-glycans. However, in the case of HepG2 cells, no qualitative changes in N-glycans between WT and SLC35A3 knockout cells nor between SLC35A2 knockout and double-knockout cells were observed. These findings suggest that SLC35A3 may not be the primary UDP-GlcNAc transporter and/or different mechanisms of UDP-GlcNAc transport into the Golgi apparatus may exist.     

Stress-induced expression and structure of the putative gene hyp-1 for hypericin biosynthesis

The phenolic oxidative coupling protein (Hyp-1) with proposed activity in the biosynthesis of hypericin in Hypericum perforatum shares about 50 % sequence similarity with Bet.v.1-like/PR-10 proteins. In our previous study, we showed that this protein is not a limiting factor in hypericin biosynthesis. To ascertain the role of Hyp-1 in defense mechanisms, we have analyzed some structural features of the hyp-1 gene in 14 Hypericum species with different abilities to synthesise hypericin. We show that the hyp-1 gene possesses characteristics typical for genes encoding plant PR-10 proteins. The coding sequence of the hyp-1 gene is interrupted by a single 86- to 125-bp intron localised strictly in codon 62, which is a typical feature of the dicot PR-10 subfamily. The localisation of the intron is conserved in all 14 tested Hypericum species indicating a common evolutionary history with genes encoding PR-10 proteins. In addition, we report that the hyp-1 gene exhibits a similar response to stress conditions as the PR-10 proteins encoding genes. Following either wounding or infection by Agrobacterium tumefaciens, all analysed Hypericum species exhibited rapid and significant upregulation of hyp-1 gene expression; this was particularly observed in hypericin-producing species. On the other hand, in the presence of high levels of abscisic acid, different levels of gene expression were observed.     

The Relationships between Polymorphisms in Genes Encoding the Growth Factors TGF-β1, PDGFB,EGF, bFGF and VEGF-A and the Restenosis Process in Patients with Stable Coronary Artery Disease Treated with Bare Metal Stent

Background

Neointima forming after stent implantation consists of vascular smooth muscle cells (VSMCs) in 90%. Growth factors TGF-β1, PDGFB, EGF, bFGF and VEGF-A play an important role in VSMC proliferation and migration to the tunica intima after arterial wall injury. The aim of this paper was an analysis of functional polymorphisms in genes encoding TGF-β1, PDGFB, EGF, bFGF and VEGF-A in relation to in-stent restenosis (ISR).

Materials and Methods

265 patients with a stable coronary artery disease (SCAD) hospitalized in our center in the years 2007–2011 were included in the study. All patients underwent stent implantation at admission to the hospital and had another coronary angiography performed due to recurrence of the ailments or a positive result of the test assessing the coronary flow reserve. Angiographically significant ISR was defined as stenosis >50% in the stented coronary artery segment. The patients were divided into two groups–with angiographically significant ISR (n = 53) and without significant ISR (n = 212). Additionally, the assessment of late lumen loss (LLL) in vessel was performed. EGF rs4444903 polymorphism was genotyped using the PCR-RFLP method whilst rs1800470 (TGFB1), rs2285094 (PDGFB) rs308395 (bFGF) and rs699947 (VEGF-A) were determined using the TaqMan method.

Results

Angiographically significant ISR was significantly less frequently observed in the group of patients with the A/A genotype of rs1800470 polymorphism (TGFB1) versus patients with A/G and G/G genotypes. In the multivariable analysis, LLL was significantly lower in patients with the A/A genotype of rs1800470 (TGFB1) versus those with the A/G and G/G genotypes and higher in patients with the A/A genotype of the VEGF-A polymorphism versus the A/C and C/C genotypes. The C/C genotype of rs2285094 (PDGFB) was associated with greater LLL compared to C/T heterozygotes and T/T homozygotes.

Conclusions

The polymorphisms rs1800470, rs2285094 and rs6999447 of the TGFB1, PDGFB and VEGF-A genes, respectively, are associated with LLL in patients with SCAD treated by PCI with a metal stent implantation.