Characterization of the Erwinia carotovora pelB gene and its product pectate lyase.

The pelB gene encodes pectate lyase B, one of three pectate lyases identified in Erwinia carotovora EC. Pectate lyase B was purified from Escherichia coli containing the pelB gene on a recombinant plasmid. The activity of the protein was optimal at a pH of 8.3. The amino acid composition, N-terminal amino acid sequence, and C-terminal peptide sequence were determined and compared with the polypeptide sequence deduced from the DNA sequence of pelB. Purified pectate lyase B started at amino acid 23 of the predicted sequence, suggesting that a 22-amino-acid leader peptide had been removed. Pectate lyase B of E. carotovora EC and pectate lyase B of E. chrysanthemi EC16 contain 352 and 353 amino acids, respectively (N. T. Keen, S. Tanaki, W. Belser, D. Dahlbeck, and B. Staskawicz, J. Bacteriol. 168:595-606, 1986). The two proteins are 72% homologous on the basis of DNA sequence data, and 75% of the amino acids are identical.     

Synaptically activated increases in Ca2+ concentration in hippocampal CA1 pyramidal cells are primarily due to voltage-gated Ca2+ channels.

Changes in intracellular Ca2+ concentration ([Ca2+]i) in the soma and dendrites of hippocampal CA1 pyramidal neurons were measured using intracellularly injected fura-2. A large component of the [Ca2+]i elevation caused by high frequency stimulation of the Schaffer collaterals was correlated with the Na+ spikes triggered by the excitatory postsynaptic potentials (EPSPs). These spikes were generated in the soma and proximal dendrites and stimulated Ca2+ entry through voltage-gated Ca2+ channels. Suppressing spikes by hyperpolarizing the soma or by injecting QX-314 revealed a smaller nonspike component of Ca2+ entry. A substantial fraction of this component was mediated by the action of the EPSPs on voltage-gated Ca2+ channels, because it persisted in 2-amino-5-phosphonovaleric acid and because it was usually reduced when Ca2+ channel activity was suppressed by hyperpolarization. Ca2+ entry through the N-methyl-D-aspartate receptor channel could not be detected with certainty, perhaps because it was highly localized.     

HLA genotypes and HLA-linked genetic markers in Italian patients with classical 21-hydroxylase deficiency

Summary HLA genotype and HLA-linked marker data for 40 unrelated patients from central Italy and 2 unrelated patients from Sardinia with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21-OH-def) were analyzed. The results confirm that the HLA-linked 21-OH-def gene is associated with several different HLA determinants and complete HLA haplotypes, although the only determinant with significantly increased frequency was the complement C2 allele C2B. The HLA antigens B8 and DR3 were found in significantly decreased frequencies. The haplotype A3, Cw6, Bw47, BfF, DR7, which is exceptionally rare in the general population but which has been found in many other 21-OH-def patients from diverse geographical origins, was also found in one of the Italian patients. This and other HLA haplotype associations found among the Italian patients may represent mutations that have occurred on HLA haplotypes with genetic linkage disequilibrium or, alternatively, may represent mutations that have not yet had time to become randomly associated with different HLA complex determinants. The marked negative associations with B8 and DR3 could, however, result from an interaction between the gene products of the HLA complex and the 21-OH-def phenotype.     

C-protein from rabbit soleus (red) muscle.

A new form of skeletal-muscle C-protein has been isolated from rabbit soleus (red) muscle. This new form of C-protein has been purified to homogeneity by a procedure similar to that used to purify C-protein from white skeletal muscle. In soleus muscle, only this new form of C-protein could be detected, whereas in psoas (white) muscle, only the previously identified form of C-protein was detected. The content of C-protein in rabbit soleus muscle is comparable with that found in psoas muscle. Other rabbit skeletal muscles composed of a mixture of fibre types contained at least two forms of C-protein. C-Protein derived from red skeletal muscle bound to myosin isolated from either red or white tissue, with maximum binding occurring at a ratio of approximately 13 microgram of red C-protein/100 microgram of myosin. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate indicated that C-protein isolated from red skeletal muscle has a molecular weight approx. 7% greater than that of C-protein isolated from white skeletal muscle. The amino acid content of both forms of C-protein was similar but major differences in the mol % of isoleucine and threonine were found. Antiserum against C-protein from white rabbit skeletal muscle formed a single precipitin line with rabbit C-protein on double in agar. This antiserum did not form a precipitin line when diffused against red C-protein from rabbit skeletal muscle. Also, this antiserum bound specifically to the A-band region of myofibrils isolated from psoas (white) muscle, but it did not bind to myofibrils prepared from soleus (red) muscle.     

A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Accessibility of tyrosyl residues altered by formation of the histone 2A/2B complex

The availability of tyrosyl residues to surface iodination was analyzed for histone 2A (H2A), histone 2B (H2B), and the H2A/H2B complex. When H2A is free in solution (200 mM NaCl, pH 7.4) tyrosine-39 and one or both tyrosines-50 and -57 were readily iodinated. Tyrosines-83 and -121 of H2B were iodinated, both when the histone was free in solution and when it was associated with H2A, while tyrosines-37, -40, and -42 of H2B were not iodinated under either condition. When H2A and H2B were associated or covalently cross-linked, all tyrosyl residues of H2A were unavailable for iodination. We also found that the iodination of nondenatured H2A and H2B did not inhibit formation of the H2A/H2B complex. These results indicate that the amino-terminal regions of the hydrophobic portions of H2A and H2B undergo significant conformational changes upon formation of the H2A/H2B complex. These conformational shifts occur in the same region of the H2A/H2B complex that contains a contact site between H2A and H2B in the nucleosome, thus indicating an involvement of this region in chromatin assembly.     

Association between Gestational Weight Gain and Postpartum Diabetes: Evidence from a Community Based Large Cohort Study

We have investigated the prospective association between excess gestational weight gain (GWG) and development of diabetes by 21 years post-partum using a community-based large prospective cohort study in Brisbane, Australia. There were 3386 mothers for whom complete data were available on GWG, pre-pregnancy BMI and self-reported diabetes 21 years post-partum. We used The Institute of Medicine (IOM) definition to categorize GWG as inadequate, adequate and excessive. We found 839 (25.78%) mothers gained inadequate weight, 1,353 (39.96%) had adequate weight gain and 1,194 (35.26%) had gained excessive weight during pregnancy. At 21 years post-partum, 8.40% of mothers self-reported a diagnosis of diabetes made by their doctor. In the age adjusted model, we found mothers who gained excess weight during pregnancy were 1.47(1.11,1.94) times more likely to experience diabetes at 21 years post-partum compared to the mothers who gained adequate weight. This association was not explained by the potential confounders including maternal age, parity, education, race, smoking, TV watching and exercise. However, this association was mediated by the current BMI. There was no association for the women who had normal BMI before pregnancy and gained excess weight during pregnancy. The findings of this study suggest that women who gain excess weight during pregnancy are at greater risk of being diagnosed with diabetes in later life. This relationship is likely mediated through the pathway of post-partum weight-retention and obesity. This study adds evidence to the argument that excessive GWG during pregnancy for overweight mothers has long term maternal health implications.     

Palatability and feeding preferences of Uresiphita maorialis (Lepidoptera: Crambidae) for three Sophora species

In a three-hour bioassay, we tested the palatability and feeding preferences of Uresiphita maorialis (kōwhai moth) for Sophora tetraptera, Sophora microphylla and Sophora prostrata. Palatability tests showed no differences among the Sophora species. Feeding preferences, on the other hand, showed that S. tetraptera and S. microphylla leaves are preferred over S. prostrata leaves. Our results support our field observations in Wellington city parks and gardens showing that S. tetraptera and S. microphylla plants frequently have higher densities of larvae than S. prostrata.