Heterogeneity in the β-subunit of translational initiation factor eIF-2 during brain development

We have detected by immunoblotting analysis of crude fractions from suckling and adult rat brain, resolved by two-dimensional isoelectric focusing-dodecyl sulfate polyacrylamide gel electrophoresis, the presence of two different forms of the subunit of polypeptide initiation factor 2 (eIF-2). These two forms differ in their apparent molecular weights and also in their isoelectric point values. Quantitation of both forms in the crude fractions shows that, the most basic form ₁ (pI: 6.1, 52 kDa), is present in higher levels of the salt wash ribosomal fractions obtained from both, suckling and adult animals, than in the postmicrosomal fraction corresponding to the same animals. The most acidic form, ₂ (pI: 5.9, 50 kDa), is present in the highest level in the postmicrosomal supernatant from adult animals. A close parallelism is found between ₁ levels and eIF-2 activity.Special issue dedicated to Dr. Santiago Grisolia.     

Age-dependent changes in brain protein synthesis in the rat

Brain protein synthesis was studied in vivo, in brain slices, and in cell-free systems in rats aged 1, 16, and 24 months. We observed a highly significant reduction in amino acid incorporation with advancing age. This reduction was observed in vivo, in slices, in postmitochondrial supernatant, microsomes, and membrane-bound polysomes. Free heavy polysomes showed no age-dependent decline but formed a smaller proportion of total ribosomes in older animals. These studies suggest that in the rat brain protein synthesis declines before senescence, possibly due to an impairment in the initiation process.     

Synthesis of 13,14-secotestosterone derivatives

A number of testosterone analogs with a 13,14-secosteroidal fragment have been prepared from (13S)-13-iodo-6beta-methoxy-3alpha, 5-cyclo-13,14-seco-5alpha-androstan-14,17-dione. The key steps involved stereoselective deiodination of the starting compound with triphenylphosphine and selective protection of the 17-keto group with trimethylsilylcyanide. Removal of iodine at C-13 proceeded with inversion of the configuration at C-13, which has been established by X-ray crystallography. 13,14-Secotestosterone analogues substituted and non-substituted at C-14 have been prepared. The obtained compounds containing flexible CD ring fragments are of great interest for comparative studies in biological tests together with testosterone and other steroids with a rigid tetracyclic skeleton.     

The synthesis of functionalized 13,14-seco-steroids via Grob fragmentation

A synthetic methodology for the synthesis of 13,14-seco-steroids with substituents at C-14 and C-17 is described. The approach involves Grob fragmentation of 14beta-hydroxy-17beta-tosylates, hydroboration-oxidation of the intermediate delta13(17)-olefin, and hydride reduction of the 14-ketone. An unambiguous structural assignment of (13R,14S,17S)-14,17-diacetoxy-3-methoxy-7alpha-methyl-13,14-secoestra-1,3,5(10)-triene was determined by X-ray analysis.     

The effect of food concentration and population density of mosquito larvae (<Emphasis Type="Italic">Anopheles stephensi</Emphasis>, Diptera,Culicidae) on their feeding behavior

The influence of food concentration and population density on the feeding behavior of Anopheles stephensi (Diptera, Culicidae) larvae has been shown experimentally. Lack of food resulted in larvae switching from sweeping the surface film to scraping the substrate off the bottom. The effect of the larval population density was more complex and depended on the food concentration. As a result, the ratio between the larvae keeping to the surface and to the bottom of the vessel was determined by interactions which led to their redistribution between the two trophic niches: the surface film and the bottom substrate. The traits observed ensure plasticity in the use of trophic resources within a water body.     

Characterization of the single-stranded DNA binding protein pV(VGJΦ) of VGJΦ phage from Vibrio cholerae

pV(VGJΦ), a single-stranded DNA binding protein of the vibriophage VGJΦ was subject to biochemical analysis. Here, we show that this protein has a general affinity for single-stranded DNA (ssDNA) as documented by Electrophoretic Mobility Shift Assay (EMSA). The apparent molecular weight of the monomer is about 12.7kDa as measured by HPLC-SEC. Moreover, isoelectrofocusing showed an isoelectric point for pV(VGJΦ) of 6.82 pH units. Size exclusion chromatography in 150mM NaCl, 50mM sodium phosphate buffer, pH 7.0 revealed a major protein species of 27.0kDa, suggesting homodimeric protein architecture. Furthermore, pV(VGJΦ) binds ssDNA at extreme temperatures and the complex was stable after extended incubation times. Upon frozen storage at -20°C for a year the protein retained its integrity, biological activity and oligomericity. On the other hand, bioinformatics analysis predicted that pV(VGJΦ) protein has a disordered C-terminal, which might be involved in its functional activity. All the aforementioned features make pV(VGJΦ) interesting for biotechnological applications.     

TLP01, an mshA mutant of Vibrio cholerae O139 as vaccine candidate against cholera

No commercially live vaccine against cholera caused by Vibrio cholerae O139 serogroup is available and it is currently needed. Virulent O139 strain CRC266 was genetically modified by firstly deleting multiple copies of the filamentous phage CTXφ, further tagging by insertion of the endoglucanase A coding gene from Clostridium thermocellum into the hemagglutinin/protease gene and finally deleting the mshA gene, just to improve the vaccine biosafety. One of the derived strains designated as TLP01 showed full attenuation and good colonizing capacity in the infant mouse cholera model, as well as highly immunogenic properties in the adult rabbit and rat models. Since TLP01 lacks MSHA fimbriae, it is refractory to infection with another filamentous phage VGJφ and therefore protected of acquiring CTXφ from a recombinant hybrid VGJφ/CTXφ. This strategy could reduce the possibilities of stable reversion to virulence out of the human gut. Furthermore, this vaccine strain was impaired to produce biofilms under certain culture conditions, which might have implications for the strain survival in natural settings contributing to vaccine biosafety as well. The above results has encouraged us to consider TLP01 as a live attenuated vaccine strain having an adequate performance in animal models, in terms of attenuation and immunogenicity, so that it fulfills the requirements to be evaluated in human volunteers.     

Isolation of Eukaryotic Initiation Factor 2 from Rat Brain

Eukaryotic initiation factor 2 (eIF-2) was isolated from salt-washed microsomes of 4-day-old rat brain which show a high rate of protein synthesis. A three-step purification scheme was employed, including heparin-Sepharose, phosphocellulose, and DEAE-cellulose column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated factor revealed three polypeptides with molecular weights of 43,000, 54,000, and 59,000 and 90% purity. The rat brain eIF-2 forms ternary complexes with [3H]methionyl-tRNAi and GTP. In terms of specific activity, the purification does not correspond to that revealed by electrophoretic analysis. During purification there is an apparent loss of additional factors that modulates the activity of eIF-2 and explains the high rate of activity of the crude fraction.     

Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) induces initiation factor 2 alpha phosphorylation and translation inhibition in PC12 cells

We have investigated the effect of the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) on protein synthesis rate and initiation factor 2 (eIF2) phosphorylation in PC12 cells differentiated with nerve growth factor. FCCP treatment induced a very rapid 2-fold increase in intracellular Ca(2+) concentration that was accompanied by a strong protein synthesis rate inhibition (68%). The translation inhibition correlated with an increased phosphorylation of the alpha subunit of eIF2 (eIF2 alpha) (25% vs. 7%, for FCCP-treated and control cells, respectively) and a 1.7-fold increase in the double-stranded RNA-dependent protein kinase activity. No changes in the PKR endoplasmic reticulum-related kinase or eIF2 alpha phosphatase were found. Translational regulation may play a significant role in the process triggered by mitochondrial calcium mobilization.     

Ischemia-induced phosphorylation of initiation factor 2 in differentiated PC12 cells: role for initiation factor 2 phosphatase

An in vitro model of ischemia was obtained by subjecting PC12 cells differentiated with nerve growth factor to a combination of glucose deprivation plus anoxia. Immediately after the ischemic period, the protein synthesis rate was significantly inhibited (80%) and western blots of cell extracts revealed a significant accumulation of phosphorylated eukaryotic initiation factor 2, alpha subunit, eIF2(alphaP) (42%). Upon recovery, eIF2(alphaP) levels returned to control values after 30 min, whereas protein synthesis was still partially inhibited (33%) and reached almost control values within 2 h. The activities of the mammalian eIF2alpha kinases, double-stranded RNA-activated protein kinase, mammalian GCN2 homologue, and endoplasmic reticulum-resident kinase, were determined. None of the eIF2alpha kinases studied showed increased activity in ischemic cells as compared with controls. Exposure of cells to cell-permeable inhibitors of protein phosphatases 1 and 2A, calyculin A or tautomycin, induced dose- and time-dependent accumulation of eIF2(alphaP), mimicking an ischemic effect. Protein phosphatase activity, as measured with [(32)P]phosphorylase a as a substrate, diminished during ischemia and returned to control levels upon 30-min recovery. In addition, the rate of eIF2(alphaP) dephosphorylation was significantly lower in ischemic cells, paralleling both the greatest translational inhibition and the highest eIF2(alphaP) levels. The endogenous phosphatase activity from control and ischemic extracts showed different sensitivity to inhibitor 2 and fostriecin in in vitro assays, inhibitor-2 effect in ischemic cells being lower than in control cells. Together these results indicate that an eIF2alpha phosphatase, probably protein phosphatase 1, is implicated in the ischemia-induced eIF2(alphaP) accumulation in PC12 cells.