Visualization of ribonucleotide reductase catalytic oxidation establishes thioredoxins as its major reductants in yeast

Thioredoxins and/or glutaredoxins assist ribonucleotide reductase, and other such enzymes that require disulfide bond reduction during their catalytic cycle. In Saccharomyces cerevisiae, the presence of either pathway is essential but which of these pathways operates in ribonucleotide reductase reduction and how this function contributes to the pathways' essential nature have not been definitively established. We have identified two in vivo redox forms of the S. cerevisiae ribonucleotide reductase R1 subunit, which correspond to catalytically reduced or oxidized enzymes. Cells lacking thioredoxins, which exhibit an elongated S phase, accumulate R1 in its oxidized form and also contain significantly decreased deoxyribonucleotide levels during the S phase. Overexpressing R1 in these cells increases both the amount of the R1 reduced form and the concentrations of deoxyribonucleotides and accelerates DNA replication. These results establish thioredoxins as the major RNR reducing system in yeast and indicate that impaired RNR reduction accounts for the S phase defects of thioredoxin-deficient cells.     

Characterization of EN-1078D, a poorly differentiated human endometrial carcinoma cell line: a novel tool to study endometrial invasion in vitro

Background  

To date, tools to study metastasis in endometrial cancers are insufficiently developed. The aim of this study was to characterize the cell line EN-1078D, a new endometrial carcinoma cell line derived from a metastasis to the ovary.     

In situ conformation of spider silk proteins in the intact major ampullate gland and in solution

To understand the spinning process of dragline silk by spiders, the protein conformation before spinning has to be determined. Raman confocal spectromicroscopy has been used to study the conformation of the proteins in situ in the intact abdominal major ampullate gland of Nephila clavipes and Araneus diadematus spiders. The spectra obtained are typical of natively unfolded proteins and are very similar to that of a mixture of recombinant silk proteins. Vibrational circular dichroism reveals that the conformation is composed of random and polyproline II (PPII) segments with some alpha-helices. The alpha-helices seem to be located in the C-terminal part whereas the repetitive sequence is unfolded. The PPII structure can significantly contribute to the efficiency of the spinning process in nature.     

Naphthalene/quinoline amides and sulfonylureas as potent and selective antagonists of the EP4 receptor

Two new series of EP₄ antagonists based on naphthalene/quinoline scaffolds have been identified as part of our on-going efforts to develop treatments for inflammatory pain. One series contains an acidic sulfonylurea pharmacophore, whereas the other is a neutral amide. Both series show subnanomolar intrinsic binding potency towards the EP₄ receptor, and excellent selectivity towards other prostanoid receptors. While the amide series generally displays poor pharmacokinetic parameters, the sulfonylureas exhibit greatly improved profile. MF-592, the optimal compound from the sulfonylurea series, has a desirable overall preclinical profile that suggests it is suitable for further development.     

The identification of substituted benzothiophene derivatives as PGE(2) subtype 4 receptor antagonists: From acid to non-acid

We disclose herein our preliminary SAR study on the identification of substituted benzothiophene derivatives as PGE₂ subtype 4 receptor antagonists. A potent EP₄ antagonist 6a (K_i = 1.4 nM with 10% HSA) was identified. Furthermore, we found that an acidic group was not essential for the EP₄ antagonizing activity in the series and neutral replacements were identified. This opens a new direction for future EP₄ antagonist design.     

Sensory experience differentially modulates the mRNA expression of the polysialyltransferases ST8SiaII and ST8SiaIV in postnatal mouse visual cortex

Polysialic acid (PSA) is a unique carbohydrate composed of a linear homopolymer of α-2,8 linked sialic acid, and is mainly attached to the fifth immunoglobulin-like domain of the neural cell adhesion molecule (NCAM) in vertebrate neural system. In the brain, PSA is exclusively synthesized by the two polysialyltransferases ST8SiaII (also known as STX) and ST8SiaIV (also known as PST). By modulating adhesive property of NCAM, PSA plays a critical role in several neural development processes such as cell migration, neurite outgrowth, axon pathfinding, synaptogenesis and activity-dependent plasticity. The expression of PSA is temporally and spatially regulated during neural development and a tight regulation of PSA expression is essential to its biological function. In mouse visual cortex, PSA is downregulated following eye opening and its decrease allows the maturation of GABAergic synapses and the opening of the critical period for ocular dominance plasticity. Relatively little is known about how PSA levels are regulated by sensory experience and neuronal activity. Here, we demonstrate that while both ST8SiaII and ST8SiaIV mRNA levels decrease around the time of eye opening in mouse visual cortex, only ST8SiaII mRNA level reduction is regulated by sensory experience. Using an organotypic culture system from mouse visual cortex, we further show that ST8SiaII gene expression is regulated by spiking activity and NMDA-mediated excitation. Further, we show that both ST8SiaII and ST8SiaIV mRNA levels are positively regulated by PKC-mediated signaling. Therefore, sensory experience-dependent ST8SiaII gene expression regulates PSA levels in postnatal visual cortex, thus acting as molecular link between visual activity and PSA expression.     

Antiretroviral Therapy Uptake,Attrition, Adherence and Outcomes among HIV-Infected Female Sex Workers: A Systematic Review and Meta-Analysis

Purpose

We aimed to characterize the antiretroviral therapy (ART) cascade among female sex workers (FSWs) globally.

Methods

We systematically searched PubMed, Embase and MEDLINE in March 2014 to identify studies reporting on ART uptake, attrition, adherence, and outcomes (viral suppression or CD4 count improvements) among HIV-infected FSWs globally. When possible, available estimates were pooled using random effects meta-analyses (with heterogeneity assessed using Cochran''s Q test and I² statistic).

Results

39 studies, reporting on 21 different FSW study populations in Asia, Africa, North America, South America, and Central America and the Caribbean, were included. Current ART use among HIV-infected FSWs was 38% (95% CI: 29%–48%, I² = 96%, 15 studies), and estimates were similar between high-, and low- and middle-income countries. Ever ART use among HIV-infected FSWs was greater in high-income countries (80%; 95% CI: 48%–94%, I² = 70%, 2 studies) compared to low- and middle-income countries (36%; 95% CI: 7%–81%, I² = 99%, 3 studies). Loss to follow-up after ART initiation was 6% (95% CI: 3%–11%, I² = 0%, 3 studies) and death after ART initiation was 6% (95% CI: 3%–11%, I² = 0%, 3 studies). The fraction adherent to ≥95% of prescribed pills was 76% (95% CI: 68%–83%, I² = 36%, 4 studies), and 57% (95% CI: 46%–68%, I² = 82%, 4 studies) of FSWs on ART were virally suppressed. Median gains in CD4 count after 6 to 36 months on ART, ranged between 103 and 241 cells/mm³ (4 studies).

Conclusions

Despite global increases in ART coverage, there is a concerning lack of published data on HIV treatment for FSWs. Available data suggest that FSWs can achieve levels of ART uptake, retention, adherence, and treatment response comparable to that seen among women in the general population, but these data are from only a few research settings. More routine programme data on HIV treatment among FSWs across settings should be collected and disseminated.     

Effect of organic and inorganic acids on concentrated chitosan solutions and gels

Rheological properties of concentrated chitosan aqueous solutions and gels in the presence of different organic and inorganic acids were investigated. Viscosities of the solutions increased with polymer concentration and degree of ionization. Strong gels were obtained at pH around 2 with oxalic, phosphoric and sulfuric acids. Gelation was favored by simple and short chain length acids and was governed by ionic interactions. The gels could be distinguished from solutions by the frequency independence of their dynamic moduli and their high apparent activation energy for flow.     

Oligomerization of the alpha and beta isoforms of the thromboxane A2 receptor: relevance to receptor signaling and endocytosis

Thromboxane A(2) (TXA(2)) is a potent mediator of inflammation, vasoconstriction and oxidative stress. The TXA(2) receptor (TP) is a G protein-coupled receptor (GPCR) that is expressed as two alternatively spliced isoforms, alpha (343 residues) and beta (407 residues) that share the first 328 residues. For many years GPCRs were assumed to exist and function as monomeric species, but increasing evidence suggests that a dimer is the minimal functional unit of GPCRs. In the present report, using co-immunoprecipitation of differentially tagged TP expressed in HEK293 cells, we demonstrate that TPalpha and TPbeta form homo- and hetero-oligomers. Immunoblotting of lysates from human platelets with an anti-TP specific antibody revealed the presence of endogenously expressed TP oligomers. We show that TP oligomerization is an agonist-independent process highly affected by the reducing agent dithiothreitol suggesting the involvement of disulfide bonds in TP oligomerization. Over-expression of G protein-coupled receptor kinases and arrestins did not modulate the extent of receptor dimerization/oligomerization. Co-expression of two TP signaling-deficient mutants, R60L and E2402R, resulted in rescuing of receptor signal transduction suggesting that dimers/oligomers constitute the functional units of this receptor. Interestingly, TPalpha which does not undergo constitutive or agonist-induced endocytosis on its own was subjected to both types of endocytosis when co-expressed with TPbeta, indicating that TPalpha can display intracellular trafficking when complexed through hetero-oligomerization with TPbeta.     

New antitubulin derivatives in the combretastatin A4 series: synthesis and biological evaluation

Two series of combretastatin A4 derivatives (acrylamide=carboxamide and carbamate) were synthesized in order to improve the water solubility and stabilize the cis-configuration of the double bond. Their cytotoxic effects were evaluated against MCF-7, KB-3-1 and IGROV human cancer cell lines, as well as their inhibitory activity on tubulin polymerization. Results were compared to those of carboxamide 1, chosen as reference. Potent inhibitions were observed on both tests in the carboxamide series, particularly for compound 4d bearing a fluorine group in replacement of the 3-hydroxyl of CA4. In contrast, most of the carbamates were either inactive or displayed only moderate cytotoxicities. Interestingly, a submicromolar IC(50) was measured on MCF-7 cells for 6g, although this compound was totally devoid of antitubulin activity.