Gender differences in right ventricular function in patients with non-ischaemic cardiomyopathy

Aim

To evaluate sex-related differences in right ventricular (RV) function, assessed with cardiac magnetic resonance imaging, in patients with stable non-ischaemic dilated cardiomyopathy.

Methods

Prospective multicentre study. We included 71 patients (38 men) and 14 healthy volunteers.

Results

Mean age was 60.9 ± 12.2 years. Men presented higher levels of haemoglobin and white blood cell counts than women, and performed better in cardiopulmonary stress testing. A total of 24 patients (12 women) presented severe left ventricular (LV) systolic dysfunction, 32 (13 female) moderate and 15 (8 women) mild LV systolic dysfunction. In the group with severe LV systolic dysfunction, average right ventricular ejection fraction (RVEF) was normal in women (52 ± 4 %), whereas it was reduced in men (39 ± 3 %) p = 0.035. Only one woman (8 %) had severe RV systolic dysfunction (RVEF < 35 %) compared with 6 men (50 %) p < 0.001. In patients with moderate and mild LV dysfunction , the mean RVEF was normal in both men and women. In the 14 healthy volunteers, the lowest value of RVEF was 48 % and mean RVEF was normal in women (56 ± 2 %) and in men (51 ±  1 %), p = 0.08.

Conclusions

In patients with dilated cardiomyopathy, RV systolic dysfunction is found mainly in male patients with severe LV systolic dysfunction.     

Discovery, synthesis, and structure activity of a highly selective alpha7 nicotinic acetylcholine receptor antagonist

Nicotinic acetylcholine receptors (nAChRs) that contain an alpha7 subunit are widely distributed in neuronal and nonneuronal tissue. These receptors are implicated in the release of neurotransmitters such as glutamate and in functions ranging from thought processing to inflammation. Currently available ligands for alpha7 nAChRs have substantial affinity for one or more other nAChR subtypes, including those with an alpha1, alpha3, alpha6, and/or alpha9 subunit. An alpha-conotoxin gene was cloned from Conus arenatus. Predicted peptides were synthesized and found to potently block alpha3-, alpha6-, and alpha7-containing nAChRs. Structure-activity information regarding conotoxins from distantly related Conus species was employed to modify the C. arenatus derived toxin into a novel, highly selective alpha7 nAChR antagonist. This ligand, alpha-CtxArIB[V11L,V16D], has low nanomolar affinity for rat alpha7 homomers expressed in Xenopus laevis oocytes, and antagonism is slowly reversible. Kinetic analysis provided insight into the mechanism of antagonism. alpha-CtxArIB interacts with five ligand binding sites per alpha7 receptor, and occupation of a single site is sufficient to block function. The peptide was also shown to be highly selective in competition binding assays in rat brain membranes. alpha-CtxArIB[V11L,V16D] is the most selective ligand yet reported for alpha7 nAChRs.     

AlphaC-conotoxin PrXA: a new family of nicotinic acetylcholine receptor antagonists

We have purified a novel paralytic peptide with 32 AA and a single disulfide bond from the venom of Conus parius, a fish-hunting species. The peptide has the following sequence: TYGIYDAKPOFSCAGLRGGCVLPONLROKFKE-NH2, where O is 4-trans-hydroxyproline. The peptide, designated alphaC-conotoxin PrXA (alphaC-PrXA), is the defining member of a new, structurally distinct family of Conus peptides. The peptide is a competitive nAChR antagonist; all previously characterized conotoxins that competitively antagonize nAChRs are structurally and genetically unrelated. (Most belong to the alpha- and alphaA-conotoxin families.) When administered to mice and fish in vivo, alphaC-PrXA caused paralysis and death. In electrophysiological assays, alphaC-PrXA potently antagonized mouse muscle nicotinic acetylcholine receptors (nAChRs), with IC50 values of 1.8 and 3.0 nM for the adult (alpha1beta1 epsilondelta subunits) and fetal (alpha1beta1 gammadelta subunits) muscle nAChR subtypes, respectively. When tested on a variety of ligand-gated and voltage-gated ion channels, alphaC-PrXA proved to be a highly specific inhibitor of the neuromuscular nAChR. The peptide competes with alpha-bungarotoxin for binding at the alpha/delta and alpha/gamma subunit interfaces of the nAChR, with higher affinity for the alpha/delta subunit interface. AlphaC-PrXA is strikingly different from the many conopeptides shown to be nicotinic antagonists; it is most similar in its general biochemical features to the snake toxins known as Waglerins.     

Structure and sodium channel activity of an excitatory I1-superfamily conotoxin

Conotoxin iota-RXIA, from the fish-hunting species Conus radiatus, is a member of the recently characterized I1-superfamily, which contains eight cysteine residues arranged in a -C-C-CC-CC-C-C- pattern. iota-RXIA (formerly designated r11a) is one of three characterized I1 peptides in which the third last residue is posttranslationally isomerized to the d configuration. Naturally occurring iota-RXIA with d-Phe44 is significantly more active as an excitotoxin than the l-Phe analogue both in vitro and in vivo. We have determined the solution structures of both forms by NMR spectroscopy, the first for an I1-superfamily member. The disulfide connectivities were determined from structure calculations and confirmed chemically as 5-19, 12-22, 18-27, and 21-38, suggesting that iota-RXIA has an ICK structural motif with one additional disulfide (21-38). Indeed, apart from the first few residues, the structure is well defined up to around residue 35 and does adopt an ICK structure. The C-terminal region, including Phe44, is disordered. Comparison of the d-Phe44 and l-Phe44 forms indicates that the switch from one enantiomer to the other has very little effect on the structure, even though it is clearly important for receptor interaction based on activity data. Finally, we identify the target of iota-RXIA as a voltage-gated sodium channel; iota-RXIA is an agonist, shifting the voltage dependence of activation of mouse NaV1.6 expressed in Xenopus oocytes to more hyperpolarized potentials. Thus, there is a convergence of structure and function in iota-RXIA, as its disulfide pairing and structure resemble those of funnel web spider toxins that also target sodium channels.     

Phytochemical Composition and Antioxidant Activities of the Essential Oil and Extracts of Satureja subspicata Vis. Growing in Bosnia and Herzegovina

The phytochemical composition and the antioxidant activities of the essential oil, as well as methanol and hot water extracts of endemic Satureja subspicata Vis . growing in Bosnia and Herzegovina (BiH), were described. β‐Caryophyllene, cis‐β‐ocimene, and α‐pinene, identified by GC/MS and GC‐FID, were the dominant oil components. The major compound of both of extracts, identified by HPLC‐DAD, was rosmarinic acid. The analyzed essential oil showed moderate antioxidant activity. In this first report on the extracts of S. subspicata growing in BiH, the obtained results showed a high content of rosmarinic acid, as well as considerable amount of total phenols and flavonoids. Compared to the hot water extract, the methanol extract exhibits higher antioxidant potential, measured by DPPH and FRAP assay (IC₅₀ = 0.45 g/l and 1879.43 equiv. Fe²⁺ μm ), while the hot water extract showed higher potential in inhibition of linoleic acid oxidation (51.7% and 61.5% for 1 and 10 g/l). A good antioxidant potential of the tested extracts indicates their potential use as antioxidants, particularly for lipid protection, and partly explains the justification of the use of this plant in traditional medicine of BiH.     

Deep Tissue Injury in Development of Pressure Ulcers: A Decrease of Inflammasome Activation and Changes in Human Skin Morphology in Response to Aging and Mechanical Load

Molecular mechanisms leading to pressure ulcer development are scarce in spite of high mortality of patients. Development of pressure ulcers that is initially observed as deep tissue injury is multifactorial. We postulate that biomechanical forces and inflammasome activation, together with ischemia and aging, may play a role in pressure ulcer development. To test this we used a newly-developed bio-mechanical model in which ischemic young and aged human skin was subjected to a constant physiological compressive stress (load) of 300 kPa (determined by pressure plate analyses of a person in a reclining position) for 0.5–4 hours. Collagen orientation was assessed using polarized light, whereas inflammasome proteins were quantified by immunoblotting. Loaded skin showed marked changes in morphology and NLRP3 inflammasome protein expression. Sub-epidermal separations and altered orientation of collagen fibers were observed in aged skin at earlier time points. Aged skin showed significant decreases in the levels of NLRP3 inflammasome proteins. Loading did not alter NLRP3 inflammasome proteins expression in aged skin, whereas it significantly increased their levels in young skin. We conclude that aging contributes to rapid morphological changes and decrease in inflammasome proteins in response to tissue damage, suggesting that a decline in the innate inflammatory response in elderly skin could contribute to pressure ulcer pathogenesis. Observed morphological changes suggest that tissue damage upon loading may not be entirely preventable. Furthermore, newly developed model described here may be very useful in understanding the mechanisms of deep tissue injury that may lead towards development of pressure ulcers.     

DNA synthesis in Escherichia coli in the presence of cyanide

The addition of cyanide to an exponentially growing culture of Escherichia coli causes an immediate, but spontaneously reversible, inhibition of DNA synthesis. The small amount of DNA which is synthesized appears to be the product of the chromosomal replication fork. However, a substantial number of single-strand interruptions persist in the newly synthesized DNA, so that covalent linkage to the preformed DNA does not occur for many minutes. Since the presence of cyanide causes a shift from DPN to DPNH, it is suggested that the DPN-linked joining activity in these cells is inhibited because of the lowered DPN concentration.     

Characterization of alkaline proteases from a novel alkali-tolerant bacterium Bacillus patagoniensis

Summary The extracellular proteolytic activity produced by a moderately alkaliphilic bacterium, Bacillus patagoniensis PAT 05^T, was characterized. This strain, grown in a highly alkaline and saline medium, produced important levels of proteolytic activity. SDS-PAGE and zymogram analyses revealed two proteolytic active bands. Through isoelectricfocusing (IEF)-zymogram, an active band with alkaline pI and two slighter active bands with acid pI values were detected. The alkaline active enzyme in the IEF was purified and characterized. It showed a molecular mass of 29.4 kDa and its pI value was >‰10.3. Proteolytic activity of the culture supernatant showed an optimal temperature of approximately 60 °C and a plateau of maximum activity between pH 9.0 and 12.0. Such activity was not affected by H₂O₂ (10% v/v), 1,10-phenanthroline (10 mM), Triton X-100 (1% v/v) and Tween 20 (1% v/v), under the assay conditions. More than 80% of the activity was retained in 10 mM EDTA, 73% in 1 % (w/v) SDS and 63% in 2 M NaCl. The enzyme was inhibited by PMSF, indicating serine-protease activity. The proteolytic activity of the crude supernatant was thermosensitive with a half-life of 2.3 min at 70 °C, while high activity was detected at moderate temperatures. Considering PAT 05^T proteolytic activity characteristics, such as high optimum pH, high stability and residual activity in presence of oxidant, surfactant and chelating agents, this strain could be a potential source of enzymes for use as additives in detergent formulations or in the leather industry.     

Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

A major proportion of extracellular RNAs (exRNAs) do not copurify with extracellular vesicles (EVs) and remain in ultracentrifugation supernatants of cell-conditioned medium or mammalian blood serum. However, little is known about exRNAs beyond EVs. We have previously shown that the composition of the nonvesicular exRNA fraction is highly biased toward specific tRNA-derived fragments capable of forming RNase-protecting dimers. To solve the problem of stability in exRNA analysis, we developed a method based on sequencing the size exclusion chromatography (SEC) fractions of nonvesicular extracellular samples treated with RNase inhibitors (RI). This method revealed dramatic compositional changes in exRNA population when enzymatic RNA degradation was inhibited. We demonstrated the presence of ribosomes and full-length tRNAs in cell-conditioned medium of a variety of mammalian cell lines. Their fragmentation generates some small RNAs that are highly resistant to degradation. The extracellular biogenesis of some of the most abundant exRNAs demonstrates that extracellular abundance is not a reliable input to estimate RNA secretion rates. Finally, we showed that chromatographic fractions containing extracellular ribosomes are probably not silent from an immunological perspective and could possibly be decoded as damage-associated molecular patterns.