The mitochondrial 60-kDa heat shock protein in marine invertebrates: biochemical purification and molecular characterization

Sessile marine invertebrates undergo constant direct exposure to the surrounding environmental conditions, including local and global environmental fluctuations that may lead to fatal protein damage. Induction of heat shock proteins (Hsps) constitutes an important defense mechanism that protects these organisms from deleterious stress conditions. In a previous study, we reported the immunological detection of a 60-kDa Hsp (Hsp60) in the sea anemone Anemonia viridis (formerly called Anemonia sulcata) and studied its expression under a variety of stress conditions. In the present study, we show that the sponge Tetilla sp. from tidal habitats with a highly variable temperature regime is characterized by an increased level of Hsp60. Moreover, we show the expression of Hsp60 in various species among Porifera and Cnidaria, suggesting a general importance of this protein among marine invertebrates. We further cloned the hsp60 gene from A viridis, using a combination of conventional protein isolation methods and screening of a complementary deoxyribonucleic acid library by polymerase chain reaction. The cloned sequence (1764 bp) encodes for a protein of 62.8 kDa (588 amino acids). The 62.8-kDa protein, which contains an amino terminal extension that may serve as a mitochondrial targeting signal, shares a significant identity with mitochondrial Hsp60s from several animals but less identity with Hsp60s from either bacteria or plants.     

Presence of normal human cell surface antigens in plasma of athymic mice bearing a human colon carcinoma and in normal human plasma

Summary The mixed haemadsorption (MHA) method was employed for detection of several normal antigenic components on the surface of human colon carcinoma cells (HT-29). The antigens were expressed by cells in monolayer cultures and in suspensions prepared by monolayer trypsinization, and by cells of tumours growing progressively in athymic mice. The plasma of such animals bearing medium sized and large, non-necrotic tumours contained all the antigens, as determined by the radial diffusion immune haemolysis method (RDIH); the plasma of animals with small or large heavily necrotic tumours did not contain detectable amounts of any of the determinants. The half-life of the determinants in the circulation as extracellular entities was ca. 20 h. The same antigens, and fibronectin, were found to be ubiquitously represented in normal human plasma. It is proposed that the presence of membrane antigens in plasma is the result of physiological shedding of cell surface constituents by living cells.     

A Single Disulfide Bond Disruption in the β3 Integrin Subunit Promotes Thiol/Disulfide Exchange,a Molecular Dynamics Study

The integrins are a family of membrane receptors that attach a cell to its surrounding and play a crucial function in cell signaling. The combination of internal and external stimuli alters a folded non-active state of these proteins to an extended active configuration. The β3 subunit of the platelet αIIbβ3 integrin is made of well-structured domains rich in disulfide bonds. During the activation process some of the disulfides are re-shuffled by a mechanism requiring partial reduction of some of these bonds; any disruption in this mechanism can lead to inherent blood clotting diseases. In the present study we employed Molecular Dynamics simulations for tracing the sequence of structural fluctuations initiated by a single cysteine mutation in the β3 subunit of the receptor. These simulations showed that in-silico protein mutants exhibit major conformational deformations leading to possible disulfide exchange reactions. We suggest that any mutation that prevents Cys560 from reacting with one of the Cys⁵⁶⁷–Cys⁵⁸¹ bonded pair, thus disrupting its ability to participate in a disulfide exchange reaction, will damage the activation mechanism of the integrin. This suggestion is in full agreement with previously published experiments. Furthermore, we suggest that rearrangement of disulfide bonds could be a part of a natural cascade of thiol/disulfide exchange reactions in the αIIbβ3 integrin, which are essential for the native activation process.     

Spectral Diversity and Regulation of Coral Fluorescence in a Mesophotic Reef Habitat in the Red Sea

The phenomenon of coral fluorescence in mesophotic reefs, although well described for shallow waters, remains largely unstudied. We found that representatives of many scleractinian species are brightly fluorescent at depths of 50–60 m at the Interuniversity Institute for Marine Sciences (IUI) reef in Eilat, Israel. Some of these fluorescent species have distribution maxima at mesophotic depths (40–100 m). Several individuals from these depths displayed yellow or orange-red fluorescence, the latter being essentially absent in corals from the shallowest parts of this reef. We demonstrate experimentally that in some cases the production of fluorescent pigments is independent of the exposure to light; while in others, the fluorescence signature is altered or lost when the animals are kept in darkness. Furthermore, we show that green-to-red photoconversion of fluorescent pigments mediated by short-wavelength light can occur also at depths where ultraviolet wavelengths are absent from the underwater light field. Intraspecific colour polymorphisms regarding the colour of the tissue fluorescence, common among shallow water corals, were also observed for mesophotic species. Our results suggest that fluorescent pigments in mesophotic reefs fulfil a distinct biological function and offer promising application potential for coral-reef monitoring and biomedical imaging.     

Antiviral activity of 3(2H)- and 6-chloro-3(2H)-isoflavenes against highly diverged, neurovirulent vaccine-derived, type2 poliovirus sewage isolates

Background

Substituted flavanoids interfere with uncoating of Enteroviruses including Sabin-2 polio vaccine strains. However flavanoid resistant and dependent, type-2 polio vaccine strains (minimally-diverged), emerged during in vitro infections. Between 1998–2009, highly-diverged (8 to >15%) type-2, aVDPV₂s, from two unrelated persistent infections were periodically isolated from Israeli sewage.

Aim

To determine whether highly evolved aVDPV₂s derived from persistent infections retained sensitivity to isoflavenes.

Methods

Sabin-2 and ten aVDPV₂ isolates from two independent Israeli sources were titered on HEp2C cells in the presence and absence of 3(2H)- Isoflavene and 6-chloro-3(2H)-Isoflavene. Neurovirulence of nine aVDPV₂s was measured in PVR-Tg-21 transgenic mice. Differences were related to unique amino acid substitutions within capsid proteins.

Principal Findings

The presence of either flavanoid inhibited viral titers of Sabin-2 and nine of ten aVDPV₂s by one to two log₁₀. The tenth aVDPV₂, which had unique amino acid substitution distant from the isoflavene-binding pocket but clustered at the three- and five-fold axies of symmetry between capsomeres, was unaffected by both flavanoids. Genotypic neurovirulence attenuation sites in the 5′UTR and VP1 reverted in all aVDPV₂s and all reacquired a full neurovirulent phenotype except one with amino acid substitutions flanking the VP1 site.

Conclusion

Both isoflavenes worked equally well against Sabin 2 and most of the highly-diverged, Israeli, aVDPV₂s isolates. Thus, functionality of the hydrophobic pocket may be unaffected by selective pressures exerted during persistent poliovirus infections. Amino acid substitutions at sites remote from the drug-binding pocket and adjacent to a neurovirulence attenuation site may influence flavanoid antiviral activity, and neurovirulence, respectively.     

Ascidian recruitment patterns on an artificial reef in Eilat (Red Sea)

Although ascidians are conspicuous members of the fouling community not much is known regarding their recruitment patterns in coral reefs. A 1-year study was carried out along the Red Sea coast of Israel to examine the effects of season and spatial distribution on ascidian recruitment to artificial marine structures. In general, autumn and spring were characterized by higher coverage with a significantly higher percentage of cover of Didemnum granulatum in autumn and higher numbers of Herdmania momus in spring. These species contributed the most to similarity between treatments consequently setting the pattern for each group (colonial and solitary). Halocynthia spinosa had significantly higher numbers during winter and Phallusia nigra was absent in spring and winter. H. momus showed a preference for horizontal surfaces. P. nigra and Ascidia cannelata showed a preference for floating units. It is concluded that the ascidian recruitment patterns are species-specific and vary between seasons, orientation and position on the substrata and in the water column.     

Heat-Stress and Light-Stress Induce Different Cellular Pathologies in the Symbiotic Dinoflagellate during Coral Bleaching

Coral bleaching is a significant contributor to the worldwide degradation of coral reefs and is indicative of the termination of symbiosis between the coral host and its symbiotic algae (dinoflagellate; Symbiodinium sp. complex), usually by expulsion or xenophagy (symbiophagy) of its dinoflagellates. Herein, we provide evidence that during the earliest stages of environmentally induced bleaching, heat stress and light stress generate distinctly different pathomorphological changes in the chloroplasts, while a combined heat- and light-stress exposure induces both pathomorphologies; suggesting that these stressors act on the dinoflagellate by different mechanisms. Within the first 48 hours of a heat stress (32°C) under low-light conditions, heat stress induced decomposition of thylakoid structures before observation of extensive oxidative damage; thus it is the disorganization of the thylakoids that creates the conditions allowing photo-oxidative-stress. Conversely, during the first 48 hours of a light stress (2007 µmoles m⁻² s⁻¹ PAR) at 25°C, condensation or fusion of multiple thylakoid lamellae occurred coincidently with levels of oxidative damage products, implying that photo-oxidative stress causes the structural membrane damage within the chloroplasts. Exposure to combined heat- and light-stresses induced both pathomorphologies, confirming that these stressors acted on the dinoflagellate via different mechanisms. Within 72 hours of exposure to heat and/or light stresses, homeostatic processes (e.g., heat-shock protein and anti-oxidant enzyme response) were evident in the remaining intact dinoflagellates, regardless of the initiating stressor. Understanding the sequence of events during bleaching when triggered by different environmental stressors is important for predicting both severity and consequences of coral bleaching.     

The Novel Mas agonist,CGEN-856S,Attenuates Isoproterenol-Induced Cardiac Remodeling and Myocardial Infarction Injury in Rats

CGEN-856S is a novel Mas agonist. Herein, we examined the effects of this peptide on isoproterenol (ISO)-induced cardiac remodeling and myocardial infarction (MI) injury. We also sought to determine whether CGEN-856S activates the underlying mechanisms related to Mas receptor activation. Heart hypertrophy and fibrosis were induced by ISO (2 mg·kg⁻¹·day⁻¹) in Wistar rats. After a 7-day treatment period with CGEN-856S (90 µg·kg⁻¹·day⁻¹) or vehicle, the cardiomyocyte diameter was evaluated in left ventricular sections stained with hematoxylin and eosin, and immunofluorescence labeling and quantitative confocal microscopy were used to quantify the deposition of type I and III collagen and fibronectin in the left ventricles. MI was induced by coronary artery ligation, and CGEN-856S (90 µg·kg⁻¹·day⁻¹) or saline was administered for 14 days. The Langendorff technique was used to evaluate cardiac function, and left ventricular sections were stained with Masson’s trichrome dye to quantify the infarct area. Using Chinese hamster ovary cells stably transfected with Mas cDNA, we evaluated whether CGEN-856S alters AKT and endothelial nitric oxide synthase (eNOS) phosphorylation. CGEN-856S reduced the degree of ISO-induced hypertrophy (13.91±0.17 µm vs. 12.41±0.16 µm in the ISO+CGEN-856S group). In addition, the Mas agonist attenuated the ISO-induced increase in collagen I, collagen III, and fibronectin deposition. CGEN-856S markedly attenuated the MI-induced decrease in systolic tension, as well as in +dT/dt and -dT/dt. Furthermore, CGEN-856S administration significantly decreased the infarct area (23.68±2.78% vs. 13.95±4.37% in the MI+CGEN-856S group). These effects likely involved the participation of AKT and NO, as CGEN-856S administration increased the levels of p-AKT and p-eNOS. Thus, our results indicate that CGEN-856S exerts cardioprotective effects on ISO-induced cardiac remodeling and MI-mediated heart failure in rats through a mechanism likely involving the eNOS/AKT pathway.     

Lower Paleolithic hominin ecology at the fringe of the desert: Faunal remains from Bizat Ruhama and Nahal Hesi, Northern Negev, Israel

The Southern Levant is a pivotal area for the study of hominin paleoecology during the Lower Paleolithic, because of its location on the out-of-Africa dispersal route and its significant ecological diversity. Important information has been gained by archaeofaunal studies, which usually reveal that exploitation of diverse Mediterranean environments with woodlands, marshes and lake margins, represents a dominant subsistence strategy for Lower Paleolithic hominins. Here, we present new taxonomic and taphonomic data from two sites in the southern coastal plain of the Southern Levant, at the fringe of the Negev Desert: Bizat Ruhama (Early Pleistocene) and Nahal Hesi (Middle Pleistocene). The sites preserve anthropogenic faunas, with the former signaling a marrow-exploitation strategy, perhaps related to scavenging from carnivore kills, and the latter showing evidence for primary access to fleshed ungulate carcasses. The species composition of these Northern Negev sites is unique for the Levantine Lower Paleolithic in that these sites lack typical woodland and riparian species, probably indicating an open, relatively uniform environment with patchy water sources and trees, much like this semiarid region today. Bizat Ruhama and Nahal Hesi are among the only Levantine Lower Paleolithic faunas associated with such a setting, thereby widening the known spectrum of environments exploited by hominins in the region. It is suggested that the two sites, coupled with the nearby Late Pleistocene evidence, reflect a largely stable semiarid environment on the northwestern fringe of the Negev Desert throughout much of the Pleistocene.     

Molecular dynamics simulations of palmitate entry into the hydrophobic pocket of the fatty acid binding protein

The entry of substrate into the active site is the first event in any enzymatic reaction. However, due to the short time interval between the encounter and the formation of the stable complex, the detailed steps are experimentally unobserved. In the present study, we report a molecular dynamics simulation of the encounter between palmitate molecule and the Toad Liver fatty acid binding protein, ending with the formation of a stable complex resemblance in structure of other proteins of this family. The forces operating on the system leading to the formation of the tight complex are discussed.