Effects of omega-3 polyunsaturated fatty acids on cardiac sarcolemmal Na(+)/H(+) exchange

Myocardial ischemia-reperfusion activates the Na(+)/H(+) exchanger, which induces arrhythmias, cell damage, and eventually cell death. Inhibition of the exchanger reduces cell damage and lowers the incidence of arrhythmias after ischemia-reperfusion. The omega-3 polyunsaturated fatty acids (PUFAs) are also known to be cardioprotective and antiarrhythmic during ischemia-reperfusion challenge. Some of the action of PUFAs may occur via inhibition of the Na(+)/H(+) exchanger. The purpose of our study was to determine the capacity for selected PUFAs to alter cardiac sarcolemmal (SL) Na(+)/H(+) exchange. Cardiac membranes highly enriched in SL vesicles were exposed to 10-100 microM eicosapentanoic acid (EPA) or docosahexanoic acid (DHA). H(+)-dependent (22)Na(+) uptake was inhibited by 30-50% after treatment with > or =50 microM EPA or > or =25 microM DHA. This was a specific effect of these PUFAs, because 50 microM linoleic acid or linolenic acid had no significant effect on Na(+)/H(+) exchange. The SL vesicles did not exhibit an increase in passive Na(+) efflux after PUFA treatment. In conclusion, EPA and DHA can potently inhibit cardiac SL Na(+)/H(+) exchange at physiologically relevant concentrations. This may explain, in part, their known cardioprotective effects and antiarrhythmic actions during ischemia-reperfusion.     

Structure of arterivirus nsp4. The smallest chymotrypsin-like proteinase with an alpha/beta C-terminal extension and alternate conformations of the oxyanion hole

Arteriviruses are enveloped, positive-stranded RNA viruses and include pathogens of major economic concern to the swine- and horse-breeding industries. The arterivirus replicase gene encodes two large precursor polyproteins that are processed by the viral main proteinase nonstructural protein 4 (nsp4). The three-dimensional structure of the 21-kDa nsp4 from the arterivirus prototype equine arteritis virus has been determined to 2.0 A resolution. Nsp4 adopts the smallest known chymotrypsin-like fold with a canonical catalytic triad of Ser-120, His-39, and Asp-65, as well as a novel alpha/beta C-terminal extension domain that may play a role in mediating protein-protein interactions. In different copies of nsp4 in the asymmetric unit, the oxyanion hole adopts either a collapsed inactive conformation or the standard active conformation, which may be a novel way of regulating proteolytic activity.     

Expression of different mutant p53 transgenes in neuroblastoma cells leads to different cellular responses to genotoxic agents

The involvement of p53 as a determinant of chemosensitivity or radiosensitivity is not well understood and is complicated by numerous contradictory reports. Here we have addressed this issue using a series of isogenic clones derived from two neuroblastoma cell lines that express wild-type p53 genes, Nub7 and IMR32. Two different mutant p53 transgenes were used in an attempt to disrupt p53 function in the clones. Our findings indicate that the cellular response is dependent on the genotoxic agent used as well as on the specific p53 transgene used. Cellular radiosensitivity showed no association with apoptosis or with the ability of the cells to arrest in G1 after irradiation. An association was observed, however, between gamma-radiation sensitivity and DNA double-strand break rejoining activity.     

Protein acylation in the cardiac muscle like cell line,H9c2

Besides serving as oxidisable substrates, fatty acids (FA) are involved in co- and post-translational modification of proteins (protein acylation). Despite the high rate of fatty acid utilisation in the heart, information on protein acylation in cardiac muscle is scarce. To explore this subject in more detail, we used the H9c2 cell line as an experimental model. After incubation with ³H-palmitate or ³H-myristate, cells were lysed and proteins precipitated, followed by extensive delipidation. The delipidated proteins were subjected to SDS-PAGE and transferred to nitro-cellulose prior to autoradiography. In addition, TLC was used to separate the various lipid classes. The first aspect we addressed was the extent of protein acylation as a function of time, relative to fatty acid incorporation into various lipid classes. Cells were incubated for 30 min, 1 h and 2 h with 100 Ci palmitate (PA, 2.3 nmol) or 125 Ci myristate (MA, 2.5 nmol). Palmitoylation increased from 0.48 ± 0.25 to 1.25 ± 0.56 Ci/mg protein between 30 min to 2 h, while myristoylation increased from 0.25 ± 0.12 to 0.77 ± 0.36 Ci/mg protein. Furthermore, delipidated proteins subjected to autoradiography showed that a set of distinct proteins was labelled with ³H-palmitate. Incorporation into phospholipids (PL) increased from 40–60% of the total amount of radio-labelled PA or MA supplied between 30 min and 2 h. Only the FA pool differed between MA and PA, with a higher FA content present after incubations with MA. Second, we investigated palmitoylation and incorporation into cellular lipids as a function of the amount of PA applied. Palmitoylation showed saturation at high PA concentrations. The percentage incorporation of ³H-PA in the various lipids depended on the amount of PA added: a decline in the PL pool with a concomitant increase in the size of the diacylglycerol pool at high PA concentrations. Third, inhibition of palmitoylation by cerulenin and tunicamycin was investigated. While both were able to inhibit palmitoylation, cerulenin also inhibited the incorporation of PA into various lipid classes, indicating differences in inhibitory action.     

Helminths of the ocelot from southern Texas

In the USA, the ocelot (Leopardus pardalis) is a highly endangered felid found only in a few remaining vestiges of native thornshrub brushland in the Lower Rio Grande Valley (LRGV) of extreme southern Texas. From 1987-1998, carcasses of 15 adult ocelots that died of vehicular accidents or natural causes were examined for helminths. All cats had 1-8 (mean = 3) helminth species. All were infected with 1-101 (mean +/- SE = 32 +/- 7) Toxascaris leonina. Other helminths from these ocelots were Alaria marcianae, Brachylaima sp., Mesocestoides lineatus, Taenia rileyi, Oncicola canis, Dirofilaria immitis, Physaloptera rara, Ancylostoma tubaeformae, Cylicospirura chevreuxi, Vogeloides felis, and Metathelazia californica. Additionally, two cats had scarring of the aorta with lesions typical of those caused by Spriocerca lupi, although larval nematodes were not seen. A clinal variation in size of nearly three orders of magnitude was noted in the diplostomatid trematodes in the small intestine of one adult male ocelot. Despite the differences in size, all specimens appeared morphologically identical and were regarded as A. marcianae. Helminth prevalences and abundances, including those of potentially pathogenic species like D. immitis, were low. Although a single heartworm infection may have contributed to the death of one ocelot, helminth infections in general seemed to be of no great consequence to this endangered ocelot population. The helminth fauna of ocelots in the LRGV is reflective of that from wild felids in general; all have been reported previously from the bobcat (Lynx rufus) and mountain lion (Puma concolor) elsewhere in Texas.     

Pericentric heterochromatin becomes enriched with H2A.Z during early mammalian development

Determining how chromatin is remodelled during early development, when totipotent cells begin to differentiate into specific cell types, is essential to understand how epigenetic states are established. An important mechanism by which chromatin can be remodelled is the replacement of major histones with specific histone variants. During early mammalian development H2A.Z plays an essential, but unknown, function(s). We show here that undifferentiated mouse cells of the inner cell mass lack H2A.Z, but upon differentiation H2A.Z expression is switched on. Strikingly, H2A.Z is first targeted to pericentric hetero chromatin and then to other regions of the nucleus, but is excluded from the inactive X chromosome and the nucleolus. This targeted incorporation of H2A.Z could provide a critical signal to distinguish constitutive from facultative heterochromatin. In support of this model, we demonstrate that H2A.Z can directly interact with the pericentric heterochromatin binding protein INCENP. We propose that H2A.Z functions to establish a specialized pericentric domain by assembling an architecturally distinct chromatin structure and by recruiting specific nuclear proteins.     

Activities of Bacillus thuringiensis Insecticidal Crystal Proteins Cyt1Aa and Cyt2Aa against Three Species of Sheep Blowfly

The toxicity of Bacillus thuringiensis Cyt1Aa protein to sheep blowfly larvae depends on its solubilization and proteolytic activation. Cyt1Aa crystals were not toxic. Full-length and trypsin-digested Cyt1Aa proteins were toxic to larvae of three species of sheep blowfly. Neither full-length nor trypsin-digested Cyt2A soluble crystal proteins were toxic.