Alterations in cardiac function and subcellular membrane activities after hypervitaminosis D3

The present study was designed to induce massive accumulation of calcium in the myocardium and to evaluate the effect of calcium overload on myocardial contractile function and biochemical activity of cardiac subcellular membranes. Rats were treated with an oral administration of 500,000 units/kg of vitamin D₃ for 3 consecutive days, and their hearts were sampled on the 5th day for biochemical analysis. On the 4th and 5th days, heart rate, mean aortic pressure, left ventricular systolic pressure and left ventricular dP/dt were significantly lowered in vitamin D₃-treated rats, demonstrating the existence of appreciable myocardial contractile dysfunction. Marked increases in the myocardial calcium (67-fold increase) and mitochondrial calcium contents (24-fold increase) were observed by hypervitaminosis D3. Mitochondrial oxidative phosphorylation and ATPase activity were significantly reduced by this treatment. A decline in sarcolemmal Na⁺, K⁺-ATPase activity was also observed, while relatively minor or insignificant changes in calcium uptake and ATPase activities of sarcoplasmic reticulum were detectable. Electron microscopic examination revealed calcium deposits in the mitochondria after vitamin D₃ treatment. The results suggest that hypervitaminosis D₃ produces massive accumulation of calcium in the myocardium, particularly in the cardiac mitochondrial membrane, which may induce an impairment in the mitochondrial function and eventually may lead to a failure in the cardiac contractile function.     

A monoclonal antibody that recognizes sialyl-Lea oligosaccharide, but is distinct from NS 19-9 as to epitope recognition

A murine monoclonal antibody, designated as MSW 113, was generated using a human colonic cancer cell line, SW 1116, as the immunogen. MSW 113 was shown to be directed mainly to mucin-type oligosaccharide with sialyl-Lea antigens. The reactivity of MSW 113 to sialyl-Lea was stronger than that of NS 19-9, which is believed to be raised against the same determinant group. MSW 113 binds to sialyl-Lea-ol, LS-tetrasaccharide a, and disialyllacto-N-tetraose with higher affinities, compared to NS 19-9. These two antibodies could clearly be distinguished in that MSW 113 bound to sialic acid but not to fucose, whereas NS 19-9 bound to fucose but not to sialic acid. Thus, MSW 113 is directed more toward sialic acid-containing terminal structures while NS 19-9 is directed toward fucose-containing internal structures. MSW 113 was found to be useful for detecting antigens in the bloodstream of patients, especially those with pancreas cancer. Even NS 19-9 negative patient sera were positive for MSW 113.     

Nucleotide sequences of Saccharomycopsis fibuligera genes for extracellular beta-glucosidases as expressed in Saccharomyces cerevisiae.

We isolated two genes for extracellular beta-glucosidase, BGL1 and BGL2, from the genomic library of the yeast Saccharomycopsis fibuligera. Gene products (BGLI and BGLII) were purified from the culture fluids of Saccharomyces cerevisiae transformed with BGL1 and BGL2, respectively. Molecular weights of BGLI and BGLII were estimated to be 220,000 and 200,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The two beta-glucosidases showed the same enzymatic characteristics, such as thermo-denaturation kinetics and dependencies on pH and temperature, but quite different substrate specificities: BGLI hydrolyzed cellobiose efficiently, but BGLII did not. This result is consistent with the observation that the S. cerevisiae transformant carrying BGL1 fermented cellobiose to ethanol but the transformant carrying BGL2 did not. Southern blot analysis revealed that the two beta-glucosidase genes were derived from Saccharomycopsis fibuligera and that the nucleotide sequences of the two genes are closely related. The complete nucleotide sequences of the two genes were determined. BGL1 and BGL2 encode 876- and 880-amino-acid proteins which were shown to be highly similar to each other. The putative precursors begin with hydrophobic segments that presumably act as signal sequences for secretion. Amino acid analysis of the purified proteins confirmed that BGL1 and BGL2 encode BGLI and BGLII, respectively.     

Hemostatic effect of a heat-treated factor VIII concentrate (Haemate P) in von Willebrand's disease

A heat-treated factor VIII (F VIII) concentrate (Haemate P) has been administered to patients with various types of von Willebrand's disease (vWD). The 4 activities of F VIII/vWF as well as change in the multimeric structure of vWF were then studied. In 4 patients with type I vWF who were given a Ristocetin cofactor (Rcof) dose of 42-78 U/kg, there was a clear reduction of the bleeding time and an increase of F VIII: C, F VIII: Ag, Rcof and vWF: Ag for several hours. The recovery of Rcof. after 1 h was 50-75%. Although the multimeric composition of vWF in these patients was similar to that of normal plasma, the density of each multimer band was very low. After infusion, however, the density of all multimer bands increased for several hours, to decrease again after 24 h. In 4 patients with type II A vWD who received a dose of Rcof of 55-76 U/kg, the 4 activities of F VIII/vWF increased similarly as was the case in type I. All patients had only 3-4 smaller multimer bands. New larger and intermediate multimers appeared for several hours after infusion of the preparation. Two patients with type III vWD who received doses of Rcof of 52 and 65 U/kg showed also a similar increase in the 4 activities of F VIII/vWF after infusion. All the multimers lacking in these patients appeared for several hours after infusion.     

Purification of a phospholipase C from rat liver cytosol that acts on phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate

A soluble phospholipase C from rat liver was purified to homogeneity using phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate. After ammonium sulfate fractionation, the purification involved chromatography on phosphocellulose, DEAE-Sepharose CL-6B, hydroxylapatite, Reactive Blue 2 dye-linked agarose, and Mono S cation exchanger. Under the conditions of the assay, the pure enzyme had a specific activity of 407 mumol/mg protein/min. It migrated as a single band with a molecular mass of 87 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The water-soluble product formed during the hydrolysis of PIP2 by the purified enzyme was inositol 1,4,5-trisphosphate. The enzyme shows one-half of maximum velocity at 2 microM Ca2+ with PIP2 as substrate. Between 0 and 100 microM Ca2+, the enzyme shows approximately the same activity with phosphatidylinositol 4-phosphate (PIP) as it does with PIP2, and very low activity with phosphatidylinositol. The enzyme is activated by low concentrations of basic proteins; for example, with PIP2 as substrate, 1 microgram/ml histone activates the enzyme 3.6-fold. The enzyme shows an almost absolute requirement for monovalent salts which can be met by different alkali metal halides. A second, minor peak of PIP2-hydrolyzing phospholipase C activity was resolved during chromatography of the enzyme on hydroxylapatite. The substrate specificity suggests that PIP and PIP2 are normal substrates of this enzyme. Under physiological conditions of activation, the enzyme may therefore generate inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate in amounts determined by the ratio of PIP and PIP2 present in the cellular membranes.     

Modification of lactate dehydrogenase by pyridoxal phosphate and adenosine polyphosphopyridoxal

Pyridoxal phosphate reacts with not only the lysyl residue(s) essential for enzymatic activity but also other reactive lysyl residues in rabbit muscle lactate dehydrogenase (EC 1.1.1.27). To raise the specificity of pyridoxal phosphate, adenosine diphospho-, triphospho-, and tetraphosphopyridoxals have been newly synthesized and used for modification of the enzyme. Incubation of the enzyme for 30 min with the diphospho, triphospho, and tetraphospho compounds all at 1 mM followed by reduction by sodium borohydride resulted in the loss of enzymatic activity by 64, 51, and 34%, respectively. NADH almost completely protected the enzyme from inactivation, whereas pyruvate showed no protection. Binding of the reagents to the enzyme subunit in an equimolar amount corresponds to the complete inactivation. The adenosine diphosphopyridoxal modified enzymes with different residual activities were chromatographed on a Blue Toyopearl affinity column. The results showed the presence of at least four enzyme species besides the intact enzyme that are significantly different from one another in the amount of the reagent bound, the affinity for NADH, and the specific activity. The decrease in the affinity of the enzyme for NADH and the loss of enzymatic activity paralleled in the modification by adenosine diphosphopyridoxal, whereas, in the modification by pyridoxal phosphate, the decrease in the affinity for NADH preceded the inactivation. It is concluded that modification by adenosine polyphosphopyridoxal compounds are specific for the active site lysyl residue(s) in lactate dehydrogenase.     

The effect of quercetin, a mutagenicity-enhancing agent, on the metabolism of 2-acetylaminofluorene with mammalian metabolic activation systems

The effect of quercetin as the comutagen on 2-acetylaminofluorene (AAF) was investigated. AAF was metabolized with mammalian metabolic systems (S9 mix) in the presence or absence of quercetin in vitro, and its metabolites were determined by high-performance liquid chromatography. In the presence of quercetin, the total metabolic rate of AAF decreased compared with that in the absence of quercetin, whereas the formation of N-hydroxy-AAF (N-OH-AAF) and 2-aminofluorene (AF) increased. Since the main metabolic pathway of AAF is aryl-hydroxylation, it is suggested that the decrease of total metabolic rate of AAF is due to the inhibition of aryl-hydroxylation by quercetin. From these results, it seems probable that the comutagenic effect of quercetin on AAF is due to the inhibition of aryl-hydroxylation (the detoxifying pathway) and the promotion of N-hydroxylation and deacetylation (the activating pathway) in the AAF metabolism with S9 mix.     

Relationship between mutagenic potency in Salmonella typhimurium strains and the chemical structure of nitro biphenyls

Most of the positional isomers of mono-, di-, tri- and tetranitrobiphenyls were synthesized and assayed for their mutagenicity in Salmonella typhimurium strains TA98, TA98NR and TA98/1,8DNP6 in the absence of S9 mix. In mono- and dinitrobiphenyls, the structure requirements favoring mutagenic activity are the presence of a nitro group at the 4-position and its absence at the 2-position. TA98 and TA98/1,8DNP6 were reverted by 2-position-free 4-nitro analogues, but TA98NR was not reverted. The results suggest that direct-acting mutagenicity involves the reduction of the nitro group by bacterial nitroreductase but does not involve specific esterification enzymes. Some of the tri- and tetranitrobiphenyls e.g. 3,4,3'-, 3,4,4'-, 3,4,3',4'- and 3,4,2',4'-derivatives reverted not only TA98 and TA98/1,8DNP6 but also TA98NR. Those derivatives commonly have 2 nitro groups at an adjoining position (3,4-dinitro group), whereas 2,4,2',4'-tetranitrobiphenyl, which has strong potency not only in TA98 and TA98/1,8DNP6 but also in TA98NR, possesses 2 nitro groups at the 2-position of each benzene ring.     

Evaluation of tri-combinant vaccine for feline herpesvirus, calicivirus and panleukopenia virus infections in Japanese native cats

Tri-combinant vaccine consisting of attenuated feline herpesvirus (FHV) and feline calicivirus (FCV) and inactivated feline panleukopenia virus (FPLV), were evaluated for safety and efficacy, using Japanese native cats and the viral strains isolated in Japan. Thirty-eight 9- to 12-week-old kittens were inoculated intramuscularly and subcutaneously with the vaccine. Consequently, no adverse reaction was found, and protective efficacy was confirmed by challenge tests with the virulent strains of each virus. Serum-neutralizing antibodies against FCV and FPLV were maintained for at least one year after vaccination, whereas antibody against FHV disappeared in two cases at 24 weeks after vaccination. Application of this vaccine seemed effective for control of feline viral disease in cats for experimental use.     

Cloning of the Escherichia coli gene for the stringent starvation protein

Summary In order to clone the Escherichia coli gene for the stringent starvation protein (SSP), we determined its N-terminal sequence as well as the sequence of two peptide fragments obtained by cyanogen bromide cleavage of the protein. We then chemically synthesized four sets of oligodeoxyribonucleotide mixtures that represented possible codon combinations for parts of these amino acid sequences. The synthetic oligonucleotides were labelled with ³²P at their 5-termini and used as hybridization probes to detect DNA fragments containing the complementary sequences. Genomic Southern hybridization of E. coli chromosomal DNA gave up to ten DNA fragments hybridizing with each probe but only a few hybridized with two or more of the probes. The latter fragments were coloned in pBR322. By determining partial base sequences with a rapid method and examining proteins encoded by the DNA fragments, we were able to show that we had isolated a clone containing the complete SSP structural gene.Abbreviations SSP stringent starvation protein - PTH phenylthiohydantoin