Characterization of transcriptional and posttranscriptional properties of native and cultured phenotypically modulated vascular smooth muscle cells

Various in vitro models are used for studying phenotypic modulation of vascular smooth muscle cells (VSMCs) and the established culture of vascular smooth muscle cells (cVSMCs) is most often used for this purpose. On the other hand, vascular interstitial cells (VICs) are native phenotypically modulated VSMCs present in blood vessels under normal physiological conditions. The aim of this work has been to compare the difference in expression of a number of VSMC-specific markers, which are commonly used for the characterisation of phenotypic modulation of VSMCs, between freshly dispersed VSMCs, VICs and cVSMCs from rat abdominal aorta. Our experiments show that VICs are present in the rat aorta and express markers of VSMCs. Both VICs and cVSMCs display the presence of sparse individual stress fibres enriched in alpha smooth muscle actin (αSM-actin), whereas in VSMCs, this protein is more densely packed. Compared with contractile VSMCs, both VICs and cVSMCs display decreased expression of VSMC-specific markers such as smoothelin, myosin light chain kinase and SM22α; however, the expression of two major cytoskeletal and contractile proteins (smooth muscle myosin heavy chain and αSM-actin) was downregulated in cVSMCs but not in VICs compared with contractile VSMCs. These results suggest different mechanisms for the phenotypic modulation of cVSMCs and VICs. VICs might therefore represent a novel convenient model for studying molecular mechanisms that govern the phenotypic modulation of VSMCs.     

Spermatozoa production by triploid males in the New Zealand freshwater snail Potamopyrgus antipodarum

Asexual lineages derived from dioecious taxa are typically assumed to be all female. Even so, asexual females from a variety of animal taxa occasionally produce males. The existence of these males sets the stage for potential gene flow across asexual lineages as well as between sexual and asexual lineages. A recent study showed that asexual triploid female Potamopyrgus antipodarum, a New Zealand freshwater snail often used as a model to study sexual reproduction, occasionally produce triploid male offspring. Here, we show that these triploid male P. antipodarum (1) have testes that produce morphologically normal sperm, (2) make larger sperm cells that contain more nuclear DNA than the sperm produced by diploid sexual males, and (3) produce sperm that range in DNA content from haploid to diploid, and are often aneuploid. Analysis of meiotic chromosomes of triploid males showed that aberrant pairing during prophase I probably accounts for the high variation in DNA content among sperm. These results indicate that triploid male P. antipodarum produce sperm, but the extent to which these sperm are able to fertilize female ova remains unclear. Our results also suggest that the general assumption of sterility in triploid males should be more closely examined in other species in which such males are occasionally produced. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 227–234.     

Asymmetric structure and domain binding interfaces of human tyrosyl‐tRNA synthetase studied by molecular dynamics simulations

Human tyrosyl‐tRNA synthetase (HsTyrRS) is composed of two structural modules: N‐terminal catalytic core and an EMAP II‐like C‐terminal domain. The structures of these modules are known, but no crystal structure of the full‐length HsTyrRS is currently available. An all‐atom model of the full‐length HsTyrRS was developed in this work. The structure, dynamics, and domain binding interfaces of HsTyrRS were investigated by extensive molecular dynamics (MD) simulations. Our data suggest that HsTyrRS in solution consists of a number of compact asymmetric conformations, which differ significantly by their rigidity, internal mobility, orientation of C‐terminal modules, and the strength of interdomain binding. Interfaces of domain binding obtained in MD simulations are in perfect agreement with our previous coarse‐grained hierarchical rotations technique simulations. Formation of the hydrogen bonds between R93 residue of the ELR cytokine motif and the residues A340 and E479 in the C‐module was observed. This observation supports the idea that the lack of cytokine activity in the full‐length HsTyrRS is explained by interactions between N‐modules and C‐modules, which block the ELR motif. Copyright © 2013 John Wiley & Sons, Ltd.     

S-nitrosoglutathione-induced toxicity in Drosophila melanogaster: Delayed pupation and induced mild oxidative/nitrosative stress in eclosed flies

The toxicity of the nitric oxide donor S-nitrosoglutathione (GSNO) was tested on the Drosophila melanogaster model system. Fly larvae were raised on food supplemented with GSNO at concentrations of 1.0, 1.5 or 4.0 mM. Food supplementation with GSNO caused a developmental delay in the flies. Biochemical analyses of oxidative stress markers and activities of antioxidant and associated enzymes were carried out on 2-day-old flies that emerged from control larvae and larvae fed on food supplemented with GSNO. Larval exposure to GSNO resulted in lower activities of aconitase in both sexes and also lower activities of catalase and isocitrate dehydrogenase in adult males relative to the control cohort. Larval treatment with GSNO resulted in higher carbonyl protein content and higher activities of glucose-6-phosphate dehydrogenase in males and higher activities of superoxide dismutase and glutathione-S-transferase in both sexes. Among the parameters tested, aconitase activity and developmental end points may be useful early indicators of toxicity caused by GSNO.     

Small-angle neutron and X-ray scattering analysis of the supramolecular organization of rhodopsin in photoreceptor membrane

The supramolecular organization of the visual pigment rhodopsin in the photoreceptor membrane remains contentious. Specifically, whether this G protein-coupled receptor functions as a monomer or dimer remains unknown, as does the presence or absence of ordered packing of rhodopsin molecules in the photoreceptor membrane. Completely opposite opinions have been expressed on both issues. Herein, using small-angle neutron and X-ray scattering approaches, we performed a comparative analysis of the structural characteristics of the photoreceptor membrane samples in buffer, both in the outer segment of photoreceptor cells, and in the free photoreceptor disks. The average distance between the centers of two neighboring rhodopsin molecules was found to be ~5.8 nm in both cases. The results indicate an unusually high packing density of rhodopsin molecules in the photoreceptor membrane, but molecules appear to be randomly distributed in the membrane without any regular ordering.     

A correlation between long-term in vitro dynamic calcification and abnormal flow patterns past bioprosthetic heart valves

In this paper, a long-term in vitro dynamic calcification of three porcine aortic heart valves was investigated using a combined approach that involved accelerated wear testing of the valves in the rapid calcification solution, hydrodynamic assessment of the progressive change of effective orifice area (EOA) along with the transaortic pressure gradient, and quantitative visualization of the flow. The motivation for this study was developing a standardized, economical, and feasible in vitro testing methodology for bioprosthetic heart valve calcification, which would address both the hydrodynamic performance of the valves as well as the subsequent changes in the flow field. The results revealed the failure of the test valves at 40 million cycles mark, associated with the critical decrease in the EOA, followed by the increase in the maximum value of viscous shear stress of up to 52%, compared to the values measured at the beginning of the study. The decrease in the EOA was subsequently followed by the rapid increase in the maximum streamwise velocity of the central orifice jet up to the value of about 2.8 m/s, compared to the initial value of 2 m/s, and to the value of 2.2 m/s in the case of a control valve along with progressive narrowing of the velocity profile for two test valves. The significance of the current work is in demonstrating a correlation between calcification of the aortic valve and spatial as well as the temporal development of abnormal flow features.     

Protein kinase C epsilon activation delays neuronal depolarization during cardiac arrest in the euthermic arctic ground squirrel

During the pre-hibernation season, arctic ground squirrels (AGS) can tolerate 8 min of asphyxial cardiac arrest (CA) without detectable brain pathology. Better understanding of the mechanisms regulating innate ischemia tolerance in AGS has the potential to facilitate the development of novel prophylactic agents to induce ischemic tolerance in patients at risk of stroke or CA. We hypothesized that neuroprotection in AGS involves robust maintenance of ion homeostasis similar to anoxia-tolerant turtles. Ion homeostasis was assessed by monitoring ischemic depolarization (ID) in cerebral cortex during CA in vivo and during oxygen glucose deprivation in vitro in acutely prepared hippocampal slices. In both models, the onset of ID was significantly delayed in AGS compared with rats. The epsilon protein kinase C (εPKC) is a key mediator of neuroprotection and inhibits both Na⁺/K⁺-ATPase and voltage-gated sodium channels, primary mediators of the collapse of ion homeostasis during ischemia. The selective peptide inhibitor of εPKC (εV1-2) shortened the time to ID in brain slices from AGS but not in rats despite evidence that εV1-2 decreased activation of εPKC in brain slices from both rats and AGS. These results support the hypothesis that εPKC activation delays the collapse of ion homeostasis during ischemia in AGS.     

Prostate Tissue Metal Levels and Prostate Cancer Recurrence in Smokers

Although smoking is not associated with prostate cancer risk overall, smoking is associated with prostate cancer recurrence and mortality. Increased cadmium (Cd) exposure from smoking may play a role in progression of the disease. In this study, inductively coupled plasma mass spectrometry was used to determine Cd, arsenic (As), lead (Pb), and zinc (Zn) levels in formalin-fixed paraffin embedded tumor and tumor-adjacent non-neoplastic tissue of never- and ever-smokers with prostate cancer. In smokers, metal levels were also evaluated with regard to biochemical and distant recurrence of disease. Smokers (N?=?25) had significantly higher Cd (median ppb, p?=?0.03) and lower Zn (p?=?0.002) in non-neoplastic tissue than never-smokers (N?=?21). Metal levels were not significantly different in tumor tissue of smokers and non-smokers. Among smokers, Cd level did not differ by recurrence status. However, the ratio of Cd ppb to Pb ppb was significantly higher in both tumor and adjacent tissue of cases with distant recurrence when compared with cases without distant recurrence (tumor tissue Cd/Pb, 6.36 vs. 1.19, p?=?0.009, adjacent non-neoplastic tissue Cd/Pb, 6.36 vs. 1.02, p?=?0.038). Tissue Zn levels were also higher in smokers with distant recurrence (tumor, p?=?0.039 and adjacent non-neoplastic, p?=?0.028). These initial findings suggest that prostate tissue metal levels may differ in smokers with and without recurrence. If these findings are confirmed in larger studies, additional work will be needed to determine whether variations in metal levels are drivers of disease progression or are simply passengers of the disease process.     

The enigmatic Crimean green lizard (Lacerta viridis magnifica) is extinct but not valid: Mitogenomics of a 120-year-old museum specimen reveals historical introduction

It has been proposed that Lacerta viridis magnifica Sobolevssky, 1930 represents an extinct species or subspecies of green lizard endemic to the southern Crimea. Using NGS protocols optimized for heavily degraded DNA, we sequenced the complete mitogenome of one of the originally formalin-preserved specimens collected in the late 19th century. A comparison with sequence data of other green lizards revealed that L. v. magnifica is a junior synonym of the northern subspecies of the western green lizard (L. b. bilineata Daudin, 1802), which occurs at least 1,500 km away, beyond the distribution ranges of other green lizards. In medieval times, a Genoese colony existed in the Crimean region where the extinct green lizards occurred. Until the early 20th century, close ties to Italy persisted, and locals of Genoese descent sent their children for education to Italy, where L. b. bilineata occurs. This suggests that the extinct Crimean green lizards have been introduced accidentally or intentionally from Italy. Our study exemplifies the value of historical formalin-preserved museum specimens for clarifying the status of questionable rare or extinct taxa.     

Competitive Mirror Image Phage Display Derived Peptide Modulates Amyloid Beta Aggregation and Toxicity

Alzheimer´s disease is the most prominent type of dementia and currently no causative treatment is available. According to recent studies, oligomeric species of the amyloid beta (Aβ) peptide appear to be the most toxic Aβ assemblies. Aβ monomers, however, may be not toxic per se and may even have a neuroprotective role. Here we describe a competitive mirror image phage display procedure that allowed us to identify preferentially Aβ_1–42 monomer binding and thereby stabilizing peptides, which destabilize and thereby eliminate toxic oligomer species. One of the peptides, called Mosd1 (monomer specific d-peptide 1), was characterized in more detail. Mosd1 abolished oligomers from a mixture of Aβ_1–42 species, reduced Aβ_1–42 toxicity in cell culture, and restored the physiological phenotype in neuronal cells stably transfected with the gene coding for human amyloid precursor protein.