Delayed cardioprotection is associated with the sub-cellular relocalisation of ventricular protein kinase Cɛ, but not p42/44MAPK

Molecular and Cellular Biochemistry - Both noradrenaline administration to rats and rapid cardiac pacing in dogs induces delayed protection of the heart against ischaemia-induced ventricular...     

Utility of the white gene in estimating phylogenetic relationships among mosquitoes (Diptera: Culicidae)

The utility of a nuclear protein-coding gene for reconstructing phylogenetic relationships within the family Culicidae was explored. Relationships among 13 species representing three subfamilies and nine genera of Culicidae were analyzed using a 762-bp fragment of coding sequence from the eye color gene, white. Outgroups for the study were two species from the sister group Chaoboridae. Sequences were determined from clone PCR products amplified from genomic DNA, and aligned following conceptual intron splicing and amino acid translation. Third codon positions were characterized by high levels of divergence and biased nucleotide composition, the intensity and direction of which varied among taxa. Equal weighting of all characters resulted in parsimony and neighboring-joining trees at odds with the generally accepted phylogenetic hypothesis based on morphology and rDNA sequences. The application of differential weighting schemes recovered the traditional hypothesis, in which the subfamily Anophelinae formed the basal clade. The subfamily Toxorhynchitinae occupied an intermediate position, and was a sister group to the subfamily Culicinae. Within Culicinae, the genera Sabethes and Tripteroides formed an ancestral clade, while the Culex-Deinocerites and Aedes- Haemagogus clades occupied increasingly derived positions in the molecular phylogeny. An intron present in the Culicinae- Toxorhynchitinae lineage and one outgroup taxon was absent in the basal Anophelinae lineage and the second outgroup taxon, suggesting that intron insertions or deletions may not always be reliable systematic characters.      

Development of the macronuclear anlage in the ciliate Chilodonella uncinata

The development of the macronuclear anlage in C. uncinata occurs in three steps. During stage I, distinct Feulgen-positive elements are visible in the anlage. Along with the increase in the volume of the anlage, the DNA content of the latter increases from 2 C to about 32 C, indicating four cycles of replication. Simultaneously, the number of Feulgen-positive elements also increases, suggesting that these bodies represent the individual chromosomes and that after each duplication cycle, the daughter chromosomes fall apart. During stage II, the anlage increases greatly in volume and shows a diffused Feulgen stain, in which no structured elements are discernible. During stage III, small Feulgen-positive granules reappear inside the anlage, and gradually become bigger as well as more numerous; at the same time the anlage as a whole starts condensing and becomes more and more densely staining, until it attains the appearance of a vegetative macronucleus about to divide. During stages II and III, the DNA content of the macronuclear anlage goes on increasing, until it nearly reaches the G₂ value of the vegetative macronucleus, which is about 128 C. The problem as to whether there is an elimination of some DNA in between stages I and II has however not yet been resolved and needs further study.     

Immunolocalization of a nuclear protein bound to the sphere organelle during oogenesis and embryogenesis inPleurodeles waltl

Summary The distribution of a nuclear antigen ofPleurodeles waltl oocytes, recognized by the monoclonal antibody B24/1, has been studied during oogenesis and early embryonic development. In stage I oocytes the antigen was localized in the nucleoplasm and on two atypical structures of lampbrush chromosomes, the spheres (S) and the mass (M). The immunostaining increased as the oocyte developed. In stage VI oocytes, the nucleoplasm and spheres showed intense staining. At this stage, the nucleoplasm often contained free spheres which were also labelled. The staining of M diminished during oogenesis, as did its size. Immunoblots of nuclear proteins of oocytes at different stages confirmed that there was an accumulation of this protein during oogenesis. During embryonic development, the nuclei of all the cells of blastula and gastrula were labelled by this antibody: there was no embryonic regionalization. Starting from the neurula stage, the staining progressively disappeared from the nuclei of ectodermal and mesodermal cells. In the tailbud stage, only the endodermal cell nuclei showed faint staining. Immunoblots of proteins from embryos of different stages showed that the quantity of this protein was constant until the young gastrula stage and then decreased progressively; in the young tailbud stage, this protein was practically absent. B24/1 is the first described protein of the sphere. This protein is accumulated in the oocyte nucleus and behaves like a maternal polypeptide, shifting early in the nuclei during embryonic development. Thus, B24/1 probably has a function required from the early developmental stages, perhaps in relation with small nuclear ribonucleoproteins.     

Treatment of intact hepatocytes with either the phorbol ester TPA or glucagon elicits the phosphorylation and functional inactivation of the inhibitory guanine nucleotide regulatory protein Gi

The antiserum AS7 can specifically immunoprecipitate alpha-Gi from membrane extracts as well as from a mixture of purified alpha-Gi and alpha-Go as ascertained using [32P]ADP-ribosylated G-proteins. Using this antiserum to immunoprecipitate alpha-Gi from hepatocytes labelled with 32P it was evident that alpha-Gi was phosphorylated under basal (resting) conditions. Challenge of hepatocytes with the tumour promoting phorbol ester TPA, however, elicited a marked enhancement of the phosphorylation state of alpha-Gi. This was accompanied by the loss of inhibitory effect of Gi on adenylate cyclase, as judged by the inability of low concentrations of p[NH]ppG to inhibit forskolin-stimulated adenylate cyclase activity. Such actions were mimicked by treatment of hepatocytes with either glucagon or TH-glucagon, an analogue of glucagon which is incapable of activating adenylate cyclase and elevating intracellular cyclic AMP concentrations. Pre-treatment of hepatocytes with either glucagon, TPA or insulin did not affect the ability of pertussis toxin to cause the NAD+-dependent, [32P]ADP-ribosylation of alpha-Gi in membrane fractions isolated from such pre-treated hepatocytes. We suggest that protein kinase C can elicit the phosphorylation and functional inactivation of alpha-Gi in intact hepatocytes. As pertussis toxin only causes the ADP-ribosylation of the holomeric form of Gi, it may be that phosphorylation leaves alpha-Gi in its holomeric state.     

Insulin stimulates the tyrosyl phosphorylation and activation of the 52 kDa peripheral plasma-membrane cyclic AMP phosphodiesterase in intact hepatocytes.

The 52 kDa subunit of the peripheral-plasma-membrane insulin-stimulated high-affinity cyclic AMP phosphodiesterase can be specifically detected by the antibody PM1 by Western-blotting procedures and also can be immunoprecipitated from a hepatocyte extract. PM1-mediated immunoprecipitation from hepatocyte extracts showed that insulin treatment of intact 32P-labelled hepatocytes caused the rapid phosphorylation of the peripheral-plasma-membrane cyclic AMP phosphodiesterase. Phosphoamino acid analysis and the use of a phosphotyrosine-specific antibody indicated that phosphorylation occurred on tyrosyl residue(s) of this phosphodiesterase. Prior treatment of hepatocytes with glucagon (10 nM) completely blocked the insulin-mediated tyrosyl phosphorylation of this 52 kDa protein, as detected with both the PM1 and the anti-phosphotyrosine antibodies. Treatment of hepatocytes with glucagon alone did not increase the phosphorylation state of the peripheral-plasma-membrane cyclic AMP phosphodiesterase. The specific anti-phosphotyrosine antibody also detected the insulin-stimulated phosphorylation of proteins of 180 kDa, 95 kDa and 39 kDa. Prior treatment of hepatocytes with glucagon decreased the ability of insulin to phosphorylate the 180 kDa and 39 kDa species, but not the 95 kDa species.     

Inseminatory behavior of Philophthalmus megalurus and Philophthalmus hegeneri in concurrent infections of chicks

Sperm labeled with ³H-thymidine and³H-tyrosine detected by autoradiography were used to determine inseminatory patterns in concurrent infections of Philophthalmus megalurus and Philophthalmus hegeneri. In infections of one labeled P. megalurus and 1 to 3 unlabeled P. hegeneri self-insemination but not cross-insemination was detected. When P. hegeneri was labeled and transplanted with 1 to 3 unlabeled P. megalurus no insemination was found. Transplantation of one labeled P. megalurus with various numbers of unlabeled adults of both species resulted in exclusive cross-insemination ofP. megalurus. When one labeled P. hegeneri was in the presence of unlabeled adults of both species, only cross-insemination of P. hegeneri was detected. Thus no insemination between species was found precluding hybrid production. This may be due to a separation of feeding sites within the microenvironment of the conjunctival sac of the chick's eye. Both species carried on normal inseminatory behavior in the presence of the other species. Recovery rates of both species from concurrent infections were not greatly different than those from single species infections. However, the recovery rate forP. megalurus (61·2 %) was much greater than for P. hegeneri (44·7 %).