New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein

Cytoplasmic Polyadenylation Element Binding proteins (CPEBs) are a family of polyadenylation factors interacting with 3’UTRs of mRNA and thereby regulating gene expression. Various functions of CPEBs in development, synaptic plasticity, and cellular senescence have been reported. Four CPEB family members of partially overlapping functions have been described to date, each containing a distinct alternatively spliced region. This region is highly conserved between CPEBs-2-4 and contains a putative phosphorylation consensus, overlapping with the exon seven of CPEB3. We previously found CPEBs-2-4 splice isoforms containing exon seven to be predominantly present in neurons, and the isoform expression pattern to be cell type-specific. Here, focusing on the alternatively spliced region of CPEB3, we determined that putative neuronal isoforms of CPEB3 are phosphorylated. Using a new phosphospecific antibody directed to the phosphorylation consensus we found Protein Kinase A and Calcium/Calmodulin-dependent Protein Kinase II to robustly phosphorylate CPEB3 in vitro and in primary hippocampal neurons. Interestingly, status epilepticus induced by systemic kainate injection in mice led to specific upregulation of the CPEB3 isoforms containing exon seven. Extensive analysis of CPEB3 phosphorylation in vitro revealed two other phosphorylation sites. In addition, we found plethora of potential kinases that might be targeting the alternatively spliced kinase consensus site of CPEB3. As this site is highly conserved between the CPEB family members, we suggest the existence of a splicing-based regulatory mechanism of CPEB function, and describe a robust phosphospecific antibody to study it in future.     

Ribosomal RNA gene sequences and hominoid phylogeny

Sequences totaling 3,500 bases from the 28S rRNA gene and from one of the ribosomal internal transcribed spacers (ITS1) have been determined for human, chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), and orangutan (Pongo pygmaeus). Analyses of the rRNA alignments show (1) a clustering of substitutions in the "variable regions" of the 28S gene, (2) a 1.5-3-fold increase in divergence in the transcribed spacer over that in the exon, and (3) that human and chimpanzee are the most closely related pair, in agreement with the results of Miyamoto et al., Sibley and Ahlquist, and Caccone and Powell.     

Critical thermal maxima of three species of Hawaiian estuarine fish: a comparative study*

Crticial thermal maxima (CTM) of three species of Hawaiian estuarine fishes were determined in relation to acclimation temperature and time of day. The species studied were Mugil cephalus L., Chelon engeli (Bleeker) and Kuhlia sandvicensis (Steindachner). Critical thermal maxima of all three species were significantly affected by acclimation temperature and time. In relation to acclimation temperature, Mugil cephalus and Chelon engeli have similar critical thermal maxima with Kuhlia sandvicensis having a critical thermal maxima about 1° C below that of the other two species studied. Mugil cephalus and Chelon engeli showed an increasing critical thermal maxima toward midday which decreased therafter. Critical thermal maximum of Kuhlia sandvicensis increased throughout the day.     

Evidence of two inapparent nonoccluded viral infections of Culex tarsalis

Culex tarsalis from a mosquito colony maintained at Bakersfield, California, were infected with two noninclusion cytoplasmic viruses. Both viruses were polyhedral with average diameters of ca. 56 nm and 33 nm for C. tarsalis virus 1 and 2 (CTV1 and CTV2), respectively. CTV1 particles had an average prevalence in the colony of 24% over a 2-year period and CTV2 had a prevalence of 5%.The viruses were first observed in negatively stained preparations of crushed heads using sodium phosphotungstate. Both viruses were seen in thin sectioned material from the salivary glands, fat body, and nervous tissue of infected mosquitoes.CTV1 was retained through six serial passages by needle inoculations of infected mosquito suspensions into virus-free C. tarsalis adults. An estimated total dilution of >;10¹⁸ indicated CTV1 had multiplied in C. tarsalis. The coincident appearance of CTV2 in some of the inoculated mosquitoes through the 5th passage suggested that this virus also multiplied on serial passage.CTV1 had a median incubation period of ca. 6.6 days in adult C. tarsalis kept at 25°C. Preliminary data indicated that the median survival period of male and female C. tarsalis injected with CTV1 was reduced 2 or 3 days over that of inoculated control insects when incubated at 25°C. Infection may cause a degeneration of salivary glands in adult females.CTV1 did not infect Culex pipiens and did not produce plaques in Vero or duck embryo cell cultures.     

Oxidation of hydrogen sulfide by Thiobacillus denitrificans: desulfurization of natural gas

It has been demonstrated that Thiobacillus denitrificans may be readily cultivated anaerobically in batch reactors on H(2)S (g) under sulfide-limiting conditions. Under these conditions sulfide concentrations in the culture medium were less than 1 muM, resulting in very low concentrations of H(2)S in the reactor outlet gas. The stoichiometry of the reaction was determined, and stable reactor operation was demonstrated at reactor loadings as high as 4-5 mmol H(2)S oxidized/h g biomass. Maximum loading was estimated at 5.4-7.6 mmol H(2)S/h g biomass under the conditions employed in this study. Indicators of reactor upset were determined and recovery from upset conditions demonstrated. Barotolerance of T. denitrificans to 12.5 MPa as well as a relative insensitivity to pressurization-depressurization cycles were also demonstrated. T. denitrificans was observed to be very sensitive to CH(3)SH but relatively tolerant of CS(2), COS, and CH(3)SCH(3).     

Release of [3H]noradrenaline by α1-adrenoceptor agonists

Mouse isolated vas deferens preincubated with [³-H]noradrenaline was superfused and the effect of ₁-adrenoceptor agonists was studied on the release of total radioactivity ([³H]noradrenaline +³H-metabolites) and [³H]noradrenaline. Reverse phase high pressure liquid chromatography (HPLC) combined with scintillation spectrometry was used to separate [³H]noradrenaline from its metabolites. Among the ₁-adrenoceptor agonists (1-phenylephrine, ST-587(2-(2-chloro-5-trifluoromethyl phenylimino)-imidazole), (–)-amidephrine, methoxamine, cirazoline and l-noradrenaline) studied l-phenylephrine, ST-587 and l-noradrenaline were capable of releasing³H-noradrenaline. The effect of noradrenaline was stereospecific. As determined by HPLC combined with scintillation spectrometry the release of total radioactivity in response to l-noradrenaline is mainly due to [³H]noradrenaline. It is suggested that l-noradrenaline, l-phenylephrine, and ST-587 in addition to their direct effect on different receptors they also have indirect action through the release of noradrenaline which might be partly involved in the pharmacological responses. The mechanisms whereby l-noradrenaline and l-phenylephrine release noradrenaline would appear to involve a saturable Ca-independent and a cocaine and temperature sensitive process. On the basis of our findings among the ₁-adrenoceptor agonist studied (–)-amidephrine, methoxamine and cirazoline is a better choice than l-phenylephrine or ST-587 for selective stimulation of postjunctional ₁-adrenoceptor, they do not release noradrenaline.     

Effect of Potassium on the Release of [3H]Noradrenaline from Rabbit and Human Pulmonary Artery

The release of [3H]noradrenaline ( [3H]NA) from rabbit and human isolated pulmonary artery has been measured. Removal of external potassium ions enhanced both the resting and stimulated release of [3H]NA from the strips. On adding K+ to tissues which had been suspended in K+-free Krebs solution, the release of [3H]NA was reduced in both stimulated and unstimulated tissues. Selective inhibition of presynaptic alpha 2-adrenoceptors by yohimbine significantly potentiated the release of [3H]NA evoked by stimulation in K+-free solution. The presynaptic inhibitory effect of NA was much less pronounced when the release was enhanced by the removal of external K+. Since the activity of NA, K-ATPase may be affected by removing K+ or by adding it to tissue previously kept in K+-free solution, the results may indicate involvement of the sodium pump in NA release.