Evidence for depletion of CASP5 Ala90Thr heterozygous genotype in aged subjects

Our previous studies, which included genotyping of multiple coding apoptotic gene polymorphisms, unexpectedly demonstrated a depletion of heterozygous CASP5 Ala90Thr (rs507879, c.268 G > A) genotypes in elderly subjects. Present investigation was aimed to validate this trend. An analysis of 510 subjects aged 75–103 years revealed 205 (40%) CASP5 Ala90Thr heterozygotes as compared to 254 (50%) expected from the minor allele frequency 0.470 (p = 0.000014). This deviation was not observed in 549 middle-aged (18–50 years) controls (270 (49%) heterozygotes observed vs. 274 (50%) expected; minor allele frequency 0.475; p = 0.743). Unfavorable significance of CASP5 heterozygous genotype may be explained by the role of the caspase-5 in inflammation-related processes. Almost all prior gene-longevity association studies focused on discrimination between “good” and “bad” gene variants. Here we present a distinct situation, where the combination of alternative alleles (i.e., heterozygosity) appears to be unfavorable as compared to the homozygous carriership of either gene variant.     

Electrophysiological characterization of neural stem/progenitor cells during in vitro differentiation: study with an immortalized neuroectodermal cell line

Despite the accumulating data on the molecular and cell biological characteristics of neural stem/progenitor cells, their electrophysiological properties are not well understood. In the present work, changes in the membrane properties and current profiles were investigated in the course of in vitro-induced neuron formation in NE-4C cells. Induction by retinoic acid resulted in neuronal differentiation of about 50% of cells. Voltage-dependent Na+ currents appeared early in neuronal commitment, often preceding any morphological changes. A-type K+ currents were detected only at the stage of network formation by neuronal processes. Flat, epithelial- like, nestin-expressing progenitors persisted beside differentiated neurons and astrocytes. Stem/progenitor cells were gap junction coupled and displayed large, symmetrical, voltage-independent currents. By the blocking of gap junction communication, voltage-independent conductance was significantly reduced, and delayed-rectifying K+ currents became detectable. Our data indicate that voltage-independent symmetrical currents and gap junction coupling are characteristic physiological features of neural stem and progenitor cells regardless of the developmental state of their cellular environment.     

Founder mutations in early-onset, familial and bilateral breast cancer patients from Russia

Previous studies indicate that founder mutations may play a noticeable role in breast cancer (BC) predisposition in Russia. Here we performed a systematic analysis of eight recurrent mutations in 302 BC cases (St.-Petersburg, Russia), which were selected due to the presence of clinical indicators of hereditary disease (bilaterality and/or early onset (≤40 years) and/or family history). BC-associated alleles were revealed in 46 (15.2%) women. BRCA1 5382insC mutation was detected in 29 (9.6%) patients, CHEK2 1100delC in 9 (3.0%), BRCA1 4153delA in 3 (1.0%), CHEK2 IVS2+1G>A in 2 (0.7%), and BRCA1 185delAG, BRCA2 6174delT and NBS1 657del5 in 1 (0.3%) patient each. No cases with BRCA1 300T>G (C61G) mutation was identified. The obtained data suggest that a significant fraction of hereditary BC cases in Russia can be diagnosed using only a limited number of simple PCR tests.     

Isolation and structures of erylosides from the Caribbean sponge Erylus formosus

Nine new triterpene glycosides, erylosides F1-F4 (1-4), M (5), N (6), O (7), P (8), and Q (9), along with previously known erylosides F (10) and H (11) were isolated from the sponge Erylus formosus collected from the Mexican Gulf (Puerto Morelos, Mexico). Structures of 1-4 were determined as the corresponding biosides having aglycons related to penasterol with additional oxidation patterns in their side chains. Erylosides 5-9 contain new variants of carbohydrate chains with three (5, 6), four (7), and six (8, 9) sugar units, respectively. Erylosides 5, 7, 8, and 6, 9 contain 14-carboxy-24-methylenelanost-8(9)-en-3beta-ol and penasterol as aglycons, respectively. In contrast with its epimer 2, the compound 3 induced the early apoptosis of Ehrlich carcinoma cells at a concentration of 100 microg/mL, while 1 and 10 activated the Ca2+ influx into mouse spleenocytes (130% of the control) at the same doses.     

Nonrandom distribution of oncogene amplifications in bilateral breast carcinomas: Possible role of host factors and survival bias

Amplification of HER2, C-MYC and CCND1 oncogenes is a hallmark of breast cancer (BC); however, its involvement in the bilateral form of this disease has not been investigated yet. In this study, 50 bilateral BC (biBC) pairs (100 tumors) and 72 control unilateral BC were examined using real-time PCR analysis of microdissected archival tissues. In biBC, the frequency of >3-fold oncogene amplification was 6/100 (6%) for HER2, 6/100 (6%) for C-MYC and 7/100 (7%) for CCND1. Altogether, 18/100 (18%) biBC tumors had increased gene dosage of at least one oncogene. Tumors forming synchronous biBC pairs had amplification in 11/46 cases (24%). In 3 of 8 patients with amplification-positive carcinomas, the amplification was detected in both neoplasms: 2 biBC had concordant activation of the same oncogene (HER2 and CCND1, respectively), and in the remaining case distinct oncogenes were affected (HER2 and C-MYC). In contrast, amplifications in metachronous biBC were strongly discordant: none of 27 first carcinomas carried this abnormality, while the frequency of amplification in second tumors (7/27; 26%) was similar to the one observed in unilateral BC (20/72; 28%). The trend toward concordance of oncogene amplification status in synchronous but not in metachronous biBC pairs can be explained by the nearly identical natural history of the disease in simultaneously arising tumors. The skewed pattern of amplifications in metachronous biBC might be attributed to their association with adverse BC prognosis; it appears that only patients with amplification-negative first BC have sufficient chances to survive until the development of the contralateral carcinoma.     

Non-imported conjunctivitis with detection of Chlamydia trachomatis

The authors describe conjunctivitis caused by Chlamydia trachomatis. The diagnosis was established by isolation on tissue cultures with a typical finding of intracytoplasmatic inclusions. Serum antibodies were slightly elevated, the complement binding reaction was 1:16, ELISA 1:64. From the clinical aspect trachoma was not involved. The patient was not in contact with foreigners from endemic areas. The disease was cured only after general administration of Tetracycline. The authors maintain that the diagnosis and prevention of ocular Chlamydia infections in adults and neonates should be extended.     

Electrochemical evidence that pyranopterin redox chemistry controls the catalysis of YedY,a mononuclear Mo enzyme

A long-standing contradiction in the field of mononuclear Mo enzyme research is that small-molecule chemistry on active-site mimic compounds predicts ligand participation in the electron transfer reactions, but biochemical measurements only suggest metal-centered catalytic electron transfer. With the simultaneous measurement of substrate turnover and reversible electron transfer that is provided by Fourier-transformed alternating-current voltammetry, we show that Escherichia coli YedY is a mononuclear Mo enzyme that reconciles this conflict. In YedY, addition of three protons and three electrons to the well-characterized “as-isolated” Mo(V) oxidation state is needed to initiate the catalytic reduction of either dimethyl sulfoxide or trimethylamine N-oxide. Based on comparison with earlier studies and our UV-vis redox titration data, we assign the reversible one-proton and one-electron reduction process centered around +174 mV vs. standard hydrogen electrode at pH 7 to a Mo(V)-to-Mo(IV) conversion but ascribe the two-proton and two-electron transition occurring at negative potential to the organic pyranopterin ligand system. We predict that a dihydro-to-tetrahydro transition is needed to generate the catalytically active state of the enzyme. This is a previously unidentified mechanism, suggested by the structural simplicity of YedY, a protein in which Mo is the only metal site.Most living species require a Mo enzyme (1), and apart from nitrogenase, all of these Mo-containing proteins are part of the large family of “mononuclear Mo” enzymes. The general ability of mononuclear Mo enzymes to catalyze two-electron oxygen atom transfer reactions has been attributed to the Mo(IV)/Mo(V)/Mo(VI) oxidation state cycling of the active site, and this mechanism is a common part of undergraduate syllabuses (1, 2). Escherichia coli YedY is a mononuclear Mo enzyme (3), and based on sequence homology, the majority of sequenced Gram-negative bacterial genomes encode a YedY-like protein (3–5). Uniquely for a mononuclear Mo enzyme, it has not been possible to form the YedY Mo(VI) state in experiments using ferricyanide as an oxidizing agent, and an unusually positive reduction potential for the Mo(V/IV) transition [+132 mV vs. standard hydrogen electrode (SHE) at pH 7] was determined from EPR experiments (6). Although the physiological substrate of YedY is unknown, a possible role in the reduction of reactive nitrogen species is suggested by experiments on the pathogen Campylobacter jejuni, where deletion of the Cj0379 YedY homolog generated a mutant that is deficient in chicken colonization and has a nitrosative stress phenotype (4). YedY catalysis can be assayed by measuring the two-electron reduction of either dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO) (3), but the inaccessibility of the YedY Mo(VI) means the enzyme mechanism does not proceed via the common two-electron Mo-redox cycle. In small-molecule analogs of mononuclear Mo enzymes, the pterin ligands are described as “noninnocent,” meaning that the redox processes could be ligand or metal based (7). This study explores the possibility that ligand-based redox chemistry plays a role in YedY catalysis.YedY has been structurally characterized via both X-ray crystallography and X-ray absorption spectroscopy (XAS) (3, 8, 9). In most mononuclear Mo enzymes, heme groups and iron sulfur clusters are found within the same protein as the Mo center, but the only metal site in YedY is Mo, making this enzyme a helpfully simple system for studying redox chemistry (Fig. 1) (1, 3). Within the active site, the X-ray structure was interpreted to show Mo(V) in a square pyramidal environment (3), identical to other members of the “sulfite oxidase” family of mononuclear Mo enzymes. In contrast, XAS has suggested a pseudooctahedral Mo center (8, 9) with an additional O (from Glu104) or N (from Asn45) axial ligand coordinating trans to the apical oxo group (Fig. 1) (8). The equatorial ligation is provided by one oxygen-containing ligand and three sulfur donor atoms, one provided by cysteine (Cys102) and two from the pyranopterin cofactor, which binds to the Mo in a bidentate fashion via the enedithiolate side chain (3).Open in a separate windowFig. 1.Structure of Escherichia coli YedY. (A and B) The protein structure, PDB ID code 1XDQ (3). (C) The active site in the as-isolated Mo(V) state containing the dihydro form of pyranopterin.A 2012 computational study provided evidence that two different oxidation states can be accessed by protein-bound pyranopterin ligands (10). Conformational analysis and electronic structure calculations were used to assign redox states to the pyranopterin ligands in all known mononuclear Mo enzyme structures (10). It was concluded that, although enzymes from the sulfite oxidase family (such as YedY) contain pyranopterin ligands in the “dihydro” form, the xanthine dehydrogenase family of enzymes contain the two-proton, two-electron more reduced “tetrahydro” form of the pyranopterin (10).Traditionally, redox potential measurements of enzymes have required substrate-free conditions to either permit a solution equilibrium to be established (spectroscopic redox titrations) or to prevent catalytic signals from masking the noncatalytic response (film electrochemistry). Fourier-transformed alternating-current voltammetry (FTacV) is a technique that offers the ability to measure catalytic chemical redox reactions and reversible electron transfer processes in a single experiment (11, 12). In the FTacV measurement, a large-amplitude sine wave of frequency f is superimposed on a linear voltage–time sweep (11, 13–15) and the resulting current–time response is measured and then Fourier transformed (FT) into the frequency domain to give a power spectrum of harmonic contributions at frequencies f, 2f, 3f, etc. Band selection of the individual harmonics followed by inverse FT resolves the data back into the time domain. The higher harmonic components only arise from fast, reversible redox reactions, devoid of catalysis and baseline contributions, but the aperiodic (dc) component (f = 0) gives the same catalytic information as a traditional dc cyclic voltammetry (dcV) experiment, and can therefore show catalytic turnover (13, 14, 16).In this study, we both discover previously unidentified mononuclear Mo enzyme redox chemistry as well as demonstrate the significant advantages of using FTacV to probe the mechanism of a redox active enzyme.