The effect of platelet activating factor on ovulation.

The mechanism of ovulation has been compared to an inflammatory reaction. Platelet activating factor (PAF) is an important mediator of inflammation as it may induce the production of prostaglandins and lysosomal enzyme. We evaluated the potential role of PAF in PMSG-HCG induced ovulation using CV3988, a specific PAF receptor antagonist in a superovulated ICR mice (9-12 weeks old). CV3988 blocked the ovulation in a dose dependent manner, and the significant reduced ovulatory efficiency was observed at more than 500 micrograms dose (p less than 0.001). The ovulatory efficiency reduced by CV3988 was reversed by PAF in a dose dependent manner. In vitro fertilization (IVF) rate of follicular oocytes with treatment of CV3988 was not different from that of ovulated ova without treatment. These results suggest that PAF may be involved in the ovulation process but the presence of PAF may not be essential for the fertilization of the ova as IVF.     

Further characterization of the in vitro hydrolysis of [Leu]- and [Met]enkephalin in rat plasma: HPLC-ECD measurement of substrate and metabolite concentrations.

Hydrolysis of [Leu]- and [Met]enkephalin was determined in whole rat plasma in vitro by using HPLC-ECD to measure Tyr, Tyr-Gly and Tyr-Gly-Gly formation. Although [Leu]- and [Met]enkephalin did not differ in Tyr or Tyr-Gly accumulation, the amount of Tyr-Gly-Gly resulting from [Met]enkephalin hydrolysis was greater than that resulting from [Leu]enkephalin hydrolysis, and [Met]enkephalin's half-life in plasma was slightly shorter than that of [Leu]enkephalin. By comparing metabolite formation in the presence and absence of peptidase inhibitors with high selectivity for their respective enzymes, these studies demonstrated that aminopeptidase M and angiotensin converting enzyme are the major peptidases that hydrolyze enkephalins in rat plasma.     

Inhibitory Ca(2+)-regulation of myosin light chain kinase in the lower eukaryote, Physarum polycephalum: role of a Ca(2+)-dependent inhibitory factor.

ATP-dependent interactions between myosin and actin in the lower eukaryote, Physarum polycephalum, are inhibited by micromolar levels of Ca2+. This inhibition is mediated by the binding of Ca2+ to myosin, the phosphorylation of which is required if Ca2+ is to inhibit the activities of myosin (Kohama, K., Trends Pharmacol. Sci. 11, 433-435 (1990)). As the first step to examine whether Ca2+ also regulates phosphorylation in the actomyosin system, we purified myosin light chain kinase (MLCK) of 55 kDa almost to homogeneity. The MLCK activity was high whether or not Ca2+ was present. However, a Ca(2+)-dependent inhibitory factor (CIF) purified from Physarum (Okagaki et al., Biochem. Biophys. Res. Commun. 176, 564-570 (1991)) was shown to reduce the MLCK activity in a Ca(2+)-dependent manner. Using crude preparations, not only MLCK but also myosin heavy chain kinase and actin kinase were shown to be inhibited by Ca2+ half-maximally at micromolar levels. Since CIF is the only Ca(2+)-binding protein in the preparations, we propose that this inhibitory Ca(2+)-regulation of the kinases for actomyosin is mediated by CIF.     

Cloning and sequence analyses of cDNAs encoding vasotocin and isotocin precursors of chum salmon,Oncorhynchus keta: evolutionary relationships of neurohypophysial hormone precursors

Summary The nucleotide sequences of cloned cDNAs were used to determine the primary structures of the precursors of vasotocin (sVT) and isotocin (sIT) from the hypothalamus of the chum salmon,Oncorhynchus keta. Two different cDNAs were obtained for each of sVT and sIT precursors (sVT-I and sVT-II; sIT-I and sIT-II). Both sVT and sIT precursors were found to contain a signal peptide and hormone that is connected to a neurophysin by a Gly-Lys-Arg sequence. Northern and Southern blot analyses showed that the sVT and sIT genes are expressed by the same chum salmon hypothalamus, but not by the liver and kidney. Microheterogeneity was found in the nucleotide and amino acid sequences of sVT precursors between our results and the previously reported data (Heierhorst et al. 1990). The conspicuous difference is the occurrence of a stop codon in the middle of sVT-II cDNA. The carboxyl termini of both sVT and sIT neurophysins are about 30 amino acids longer than neurophysins of toad and mammalian neurohypophysial hormone precursors. Although these extended regions do not contain a glycosylation site, they show striking similarity with the glycopeptide moiety (copeptin) of toad vasotocin and mammalian vasopressin precursors. The central portion of the neurophysins shows highest homology among corresponding regions of sVT and sIT precursors. Moreover, calculation of nucleotide substitution rates suggests that a recent gene conversion may have occurred which encompasses the exon that encodes the central segment of the sVT and sIT precursors. A possible pathway for the evolution of precursor molecules of neurohypophysial hormones is discussed.Abbreviations AVP vasopressin - C carboxyl - h human - IT isotocin - MT mesotocin - N amino - OXT oxytocin - S chum salmon - SDS sodium dodecyl sulfate - t toad - VT vasotocin     

Killer cells induced by stimulation with allogeneic tumor cells and subsequent culture with recombinant interleukin-2

Summary Peripheral blood lymphocytes were cultured for 5 days with allogeneic tumor cells (allogeneic mixed lymphocyte/tumor cell culture), and subsequently cultured with recombinant interleukin-2 for 12 days. These cultured cells were found to be cytotoxic to autologous tumor cells. Results of two-color analysis using monoclonal antibodies to cell markers showed that more than 80% of their cultured cells were CD3⁺ cells, and CD4⁺ cells showed a higher distribution than CD8⁺ cells. However, CD8⁺ cells had a much higher killing activity with autologous tumor than did CD4⁺ cells, when estimated by an elimination study using monoclonal antibodies to T cell phenotypes and complement. The cold-target inhibition test showed that the cytotoxicity of these cells for autologous tumor cells was inhibited by unlabeled autologous tumor cells but not by unlabeled stimulator cells. Furthermore, about 40% of the cytotoxicity was suppressed by blocking of HLA class I antigen with a monoclonal antibody on autologous tumor cells. Thus, cytotoxic activity of lymphocytes to autologous tumor restricted by target cell HLA class I antigen is possibly induced by allogeneic tumor-stimulation.     

Sensitive sandwich enzyme immunoassay of human growth hormone (hGH) in serum and urine using monoclonal antibody-coated polystyrene balls

A sensitive sandwich enzyme immunoassay for human growth hormone (hGH) using monoclonal antibody is described. A monoclonal anti-hGH IgG-coated polystyrene ball was incubated with hGH and subsequently with affinity-purified rabbit anti-hGH Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorimetry using 3-(4-hydroxyphenyl) propionic acid as a substrate. The detection limits of hGH in serum and urine were 1.5 ng/l using 20 microliters of serum and 0.2 ng/l using 0.15 ml of urine, respectively. The specificity and assay precision were satisfactory. hGH levels in serum and urine determined by the present sandwich enzyme immunoassay using monoclonal anti-hGH IgG-coated polystyrene balls were well correlated to those determined by the previous sandwich enzyme immunoassay using rabbit anti-hGH IgG-coated polystyrene balls. Levels of hGH in urine collected as first morning voids from healthy subjects aged 19-28 yr were 6.4 +/- 3.2 (SD) ng/g creatinine. However, the present assay gave lower hGH levels than the previous assay. This was at least partly explained by the fact that hGH in urine was less efficiently bound to monoclonal anti-hGH IgG-polystyrene balls than standard hGH, while the binding of hGH in urine and standard hGH to rabbit anti-hGH IgG-coated polystyrene balls was equally efficient. In addition, gel filtration showed that 22K hGH, a major component, in urine was less efficiently bound to monoclonal anti-hGH IgG-coated polystyrene balls than standard 22K hGH. The nature of hGH in serum and urine remains to be investigated.     

The subunit structure of a major glutathione S-transferase form, MT, in rat testis. Evidence for a heterodimer consisting of subunits with different isoelectric points

A major glutathione S-transferase form (pI 5.7) in rat testis (MT) purified by S-hexyl-glutathione affinity chromatography, followed by chromatofocusing, showed two polypeptide of pI 6.7 (Yn1) and 6.0 (Yn2), having apparently the same molecular mass of 26 kDa on two-dimensional gel electrophoresis. Rechromatofocusing of the MT preparation after 4 M guanidine hydrochloride treatment revealed two additional protein peaks (pI 6.2 and 5.4). These were identified as the two homodimers consisting of the subunits of MT, Yn1Yn1 and Yn2Yn2, respectively. Furthermore, MT could be reconstituted from Yn1Yn1 and Yn2Yn2. These results indicate that MT is a heterodimer, Yn1Yn2, consisting of subunits with very similar molecular masses but different isoelectric points. The Yn1Yn1 form had glutathione S-transferase activities towards 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene. However, the Yn2Yn2 form had no activity towards any of the substrates examined. N-terminal amino acid sequences of subunits Yn1 and Yn2 revealed differences at two positions in the first 20 residues; the amino acid compositions of these subunits were also similar but not identical, indicating that these two subunits are different in the primary structure. Subunits Yn1 and Yn2 are immunologically related to each other and also to subunits 3 (Yb1) and 4 (Yb2) but they are not identical. These four subunits also showed a high degree of similarity in N-terminal amino acid sequences. Subunits Yn1 and Yn2 seem to belong to the rat GST 3-4 family or class mu. Subunits Yn1 and 4 can make a heterodimer, which is detectable not only in rat testis, but also in the heart, kidney and lung. The Yn1Yn1 form was not detected in the testis, but is present in rat brain [Tsuchida et al. (1987) Eur. J. Biochem. 170, 159-164]. The Yn2Yn2 form seemed to differ from GST 5-5 and may be a new form of rat glutathione S-transferase.     

Localization of the regulatory subunit of cAMP-dependent protein kinase in Saccharomyces cerevisiae

The subcellular distribution of the regulatory subunit of cAMP-dependent protein kinase in Saccharomyces cerevisiae cells was determined by subcellular fractionation and indirect immunofluorescence microscopy using the bcy1 mutant deficient in the regulatory subunit as control. The regulatory subunit of cAMP-dependent protein kinase showing cAMP-binding activity was identified as a single protein of 50 kDa by photoaffinity labeling and immunoblotting. The regulatory subunit was concentrated in a nuclear fraction in addition to a cytoplasmic fraction. By comparison of the regulatory subunit distribution with the DNA localization, the area detected by the indirect immunofluorescence was identified as the nucleus.     

Movement of water in conjunction with plant movement visualized by NMR imaging

The water distribution in the pulvinus of Mimosa can be visualized by an NMR imaging technique. After stimulation of a Mimosa plant, water in the lower half of the main pulvinus disappeared, the water previously contained in this area seeming to be transferred to the upper half of the main pulvinus. Movement of the water in conjunction with Mimosa movement was visualized sequentially by a non-invasive NMR imaging procedure.