Analysis of the gene of Vibrio hollisae encoding the hemolysin similar to the thermostable direct hemolysin of Vibrio parahaemolyticus

The gene (designated as Vh-tdh) of Vibrio hollisae 9041 encoding a hemolysin similar to the thermostable direct hemolysin (TDH) of V. parahaemolyticus contained a 567-base-pair open reading frame (ORF), which was 93.3-93.5% homologous to those of the tdh genes of V. parahaemolyticus, V. cholerae non-01, and V. mimicus encoding TDH or similar hemolysins. Comparative analysis of the nucleotide sequence containing the Vh-tdh ORF with published nucleotide and amino acid sequences suggested that the Vh-tdh gene and other tdh genes diverged from a common ancestral gene, that the divergence was closely associated with the evolutionary divergence of V. hollisae from other species of genus Vibrio, and that strain-to-strain variation of the Vh-tdh gene exists in V. hollisae.     

Purification and characterization of a heat-stable enterotoxin of Vibrio mimicus

A heat-stable enterotoxin produced by Vibrio mimicus (VM-ST) was studied. VM-ST was purified from a culture supernatant of V. mimicus strain AQ-0915 by ammonium sulfate fractionation, hydroxyapatite treatment, ethanol extraction, column chromatography on both SP-Sephadex C-50 and DEAE-Sephadex A-25, and HPLC, and the recovery rate was about 15%. Purified VM-ST was heat-stable. VM-ST activity was cross-neutralized by anti-STh antiserum. The amino acid composition of the purified VM-ST was determined 17 amino acid residues in the following sequence: Ile-Asp-Cys-Cys-Glu-Ile-Cys-Cys-Asn-Pro-Ala-Cys-Phe-Gly-Cys-Leu-Asn. This composition and sequence were identical to those of V. cholerae non-O1-ST. These results clearly demonstrate the production of a characteristic VM-ST by V. mimicus.     

The effect of inosine on the post ischemic heart as bio-energy recovering factor in 31P-MRS

Perfused guinea-pig hearts, which were analyzed by 31P-MRS, were subjected to 30 and 60 minute ischemia and reperfused using two perfusates, one containing 200 microM inosine, and the other without inosine. After 4 hour reperfusion with inosine, ATP levels increased to 95.5% of preischemic value (30 minute ischemia) and 76.2% (60 minute ischemia). However, after 4 hour reperfusion without inosine, ATP levels increased only to 72.2% (30 minute ischemia) and to 48.2% (60 minute ischemia). In 60 minute ischemic hearts reperfused with inosine, left ventricular maximal positive dp/dt (LV dp/dt) was improved significantly to 82.4% after 6 hour reperfusion in contrast to hearts reperfused without inosine (43.1%). Administration of inosine was very useful for increasing myocardial gross energy product and improving cardiac performance.     

Purification and characterization of ATP:AMP phosphotransferase from Mycobacterium marinum

ATP:AMP phosphotransferase (EC 2.7.4.3) (adenylate kinase) has been purified 1746-fold from Mycobacterium marinum (ATCC 927) by successive column chromatography on DEAE-cellulose (DE-53), Reactive Blue agarose, Sephadex G-75, hydroxyapatite and, finally, DEAE-Sephadex A-50. The final enzyme preparation had a specific activity of 576 mumol/min per mg protein with an overall yield of 51%. The preparation was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was estimated to have an Mr of 29500 and an isoelectric point of 6.7, properties which generally resemble those of the mitochondrial enzyme. Indeed, the two enzymes failed to separate when subjected to polyacrylamide gel electrophoresis under denaturing conditions. The extinction coefficient (at 276 nm) was calculated to be 3.114 X 10(4) M-1 X cm-1 and E1%1cm = 10.556. Adenylate kinase was present at a concentration of 0.06 mg/g (wet weight) bacteria. Enzyme was stable for months in 60% glycerol in the freezer; at 4 degrees C, less than 5% of the activity was lost over a 7 day period.     

Concentrations and origin of oxytocin in breast milk

Samples of human milk obtained from lactating women in the early postpartum period were assayed for oxytocin concentrations by specific RIA, following extraction procedures with Florisil. Mean oxytocin concentrations in human milk at postpartum day 1 to 5 were 4.5 +/- 1.1, 4.7 +/- 1.1, 4.0 +/- 1.3, 3.2 +/- 0.4, 3.3 +/- 0.6 microunits/ml (+/- SE), respectively. Oxytocin levels in milk were significantly increased by nursing (3.1 +/- 0.6, 5.3 +/- 1.0 microunits/ml, respectively). 3H-oxytocin in human milk was stable even after incubation at 37 degrees C for 2 hours. The dilution curve for milk was parallel to the curve for the standard oxytocin. The chromatographic fraction of immunoreactive oxytocin was identical to that of 3H-oxytocin. 3H-oxytocin was administered to lactating rats. Radioactivity in the neonatal gastric contents and plasma were 12.8% and 4.4% of the counts in the maternal plasma. It was made clear that oxytocin is stable in milk and that oxytocin in maternal blood can be transferred to mik and then to neonates.     

Population Genetics of an Expanding Family of Mobile Genetic Elements

A model of an expanding family of dispersed repetitive DNA was studied. Based on the previous result of the model of duplicative transposition, an approximate solution to give allelism and identify coefficients as functions of time was obtained, and theoretical predictions were verified by Monte Carlo experiments. The results show that, even if the copy number per genome increases very rapidly, allelism and identity coefficients may take a long time to reach equilibrium. The changes of allelism and allelic identity are similar to that of homozygosity at an ordinary single locus, whereas that of nonallelic identity can be much slower, particularly when the copy number per genome is large. Thus, many existing families of highly repetitive sequences may represent nonequilibrium states for nonallelic identity. The present model may be extended to include other evolutionary forces such as gene conversion or the recurrent insertion from normal gene copies.     

Protein kinase C activity in platelets from spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY)

Calcium-activated phospholipid dependent protein kinase (protein kinase C) activity in platelets was measured in 4, 12, and 20-week-old SHR and WKY. At age 4-weeks, there was no significant difference in protein kinase C activity and systolic blood pressure between SHR and WKY. In 12 and 20-week-old SHR, both protein kinase C activity and systolic blood pressure were significantly higher than in the age-matched WKY. These results suggest that protein kinase C may be involved in the control of blood pressure in SHR and WKY.     

Isolation, and catalytic and immunochemical properties of cathepsin D-like acid proteinase from rat erythrocytes

An erythrocyte membrane-associated cathepsin D-like acid proteinase, termed "EMAP," was purified to homogeneity from freshly collected rat blood in a yield of 60-65%. The molecular weight of the enzyme was determined to be 80,000-82,000 by Sephadex G-100 gel filtration. The enzyme was inhibited strongly by pepstatin and partially by HgCl2, Pb(NO3)2, and iodoacetic acid. The preferred substrate for the enzyme was hemoglobin. The enzyme also hydrolyzed serum albumin and casein, but to lesser extents, with an optimum pH of 3.5-4.0. However, it could not hydrolyze leucyl-2-naphthylamide, benzyloxycarbonyl-Phe-Arg-4-methyl-7-coumarylamide or other synthetic substrates at pH values ranging from 3.5 to 9.5. The enzyme was very similar to human EMAP in a number of enzymatic properties, whereas it differed from rat cathepsin D in several respects, such as pH stability, molecular weight, isoelectric point, and chromatographic properties. Immunologically, the enzyme cross-reacted with the rabbit antibody prepared against human EMAP. The patterns of immunoelectrophoresis, immunoblotting, and immunoprecipitation of the enzyme were remarkably similar, if not identical, to those of human EMAP. In contrast, rat EMAP showed no reaction with the rabbit antibody raised to rat spleen cathepsin D. These results indicate that EMAP is a unique cathepsin D-like acid proteinase different from ordinary cathepsin D.     

Conformational changes of papain induced on interaction with thiol proteinase inhibitors from newborn rat epidermis

The conformational changes of the papain molecular on interaction with two thiol proteinase inhibitors (TPI(1) and TPI(2] from newborn rat epidermis were studied by measuring circular dichroism (CD), the difference absorption spectrum, and the fluorescence spectrum due to tryptophan residues in papain. The far-ultraviolet CD band of papain between 210 and 230 nm was distinctly reduced on interaction with both inhibitors. Also, the near-ultraviolet CD spectrum of TPI(1)-bound papain changed between 285 and 320 nm as well as that of the TPI(2)-bound enzyme. The difference absorption spectrum for TPI(1)-bound papain exhibited two distinct peaks at 276.5 and 282 nm, indicating perturbation of aromatic amino acid residues. The fluorescence intensity of papain was significantly decreased on interaction with both inhibitors, which showed pH-dependency on an ionizable group, with pK values of 8.5 and 7.9 for TPI(1) and TPI(2), respectively. The complex formation of papain with both inhibitors caused a reduction of the susceptibility of a tryptophan residue, probably tryptophan-177, to chemical modification with N-bromosuccinimide. These results suggest that the active site involving histidine-159 in the papain molecule was much influenced by the alteration of the microenvironment of tryptophan-177 as a part of the interaction site for these two thiol proteinase inhibitors.     

Different interactions used by Cro repressor in specific and nonspecific DNA binding

The mode of interaction of Cro repressor with specific and nonspecific sites on DNA was explored by chemical modification and protection of lysine and tyrosine residues. Cro has 8 lysines. In the presence of DNA, lysines 32 and 56 are fully protected and lysines 21, 62, and 63 are partially protected from alkylation. However, the terminal amino group and lysines 8, 18, and 39 are not protected. Location of the protected and unprotected lysines on the three-dimensional Cro structure defines a DNA-binding region. The results provide direct experimental support for a mode of interaction between Cro and DNA, in which Cro buries its 2-fold related alpha-helices in consecutive DNA major grooves (Anderson, W. F., Ohlendorf, D. H., Takeda, Y., and Matthews, B. W. (1981) Nature 290, 754-758; Ohlendorf, D. H., Anderson, W. F., Fisher, R. G., Takeda, Y., and Matthews, B. W. (1982) Nature 298, 718-723). In the model, the carboxyl-terminal part of Cro was tentatively presumed to interact with the DNA minor groove. Protection of lysines 62 and 63 confirms the involvement of the carboxyl terminus in DNA binding. Although nonspecific and specific DNA protect the same lysine residues, there are differences in the nature of the interaction of Cro with nonspecific and specific DNA. Cro-nonspecific DNA interaction is salt-sensitive, suggesting that the interaction is predominantly electrostatic. On the other hand, Cro-specific DNA interaction is salt-resistant, suggesting that the interaction may include nonelectrostatic components (hydrogen bonds and hydrophobic interactions) as well. Protection experiments of tyrosine residues (against iodination) suggest that the conformation of Cro repressor changes in two stages: first, when Cro binds at nonspecific sites, and, second, when Cro binds to specific sites on DNA.