Light and electron microscopic observation on the appearance of immunoreactive LHRH in perinatal rat hypothalamus

Summary The appearance and localization of LHRH were studied in the developing hypothalamus of perinatal rats using the unlabelled antibody method. By light microscopy, immunoreactive LHRH was first detected as brown dots on day 18.5 of gestation in the OVLT and on day 19.5 in the median eminence, respectively. When the median eminence was examined by the preembedding immunohistochemistry technique for electron microscopy, the occurrence of immunoreactive LHRH fibers could be demonstrated on day 18.5. These fibers were thin and very occasionally encountered near the surface of the lateral regions of the median eminence. The axoplasm contained a few immunopositive secretory granules and also extragranular immunoreactive products. With development, a gradual increase was noted both in number and size of nerve fibers with a concomitant accumulation of secretory granules within the axoplasm.A possible physiological significance of LHRH is discussed in relation to the onset of hypothalamo-hypophysial system in fetal life.     

Classification of the responses of a giant neuron from the snail Acatina fulica Férussac, caused by inhibitory sugstances depending on chlorine ions]

GABA, three of its derivatives (l-GABOB, d-GABOB and delta-amino valeric acid), acetycholine (Ach), dopamine (DA) and l-Phe-Tyr all inhibit an identifiable giant neurone (the TAN, tonically autoactive neurone) of Achatina fulica. These effects were examined by microdrop application in two different conditions: in physiological solution and in the absence of chloride ions. The results show that the relatively transient (rapid) inhibitions caused by GABA, by its derivatives and by Ach are dependent on chloride ions; the relatively maintained (long-lasting) inhibitions, caused by DA and l-Phe-Tyr, are independent of chloride ions.     

Myosin and actin from Escherichia coli K12 C600.

Myosin-like protein and actin-like protein from E. coli formed filaments very similar in structure to those of myosin and actin from skeletal muscle. At 0.2 M KCl, a large number of "thick filaments" of uniform size (about 0.6-0.7 micron long and about 20 nm wide) was present. These thick filaments aggregated as the KCl concentration decreased to less than 0.2 M. Filaments of actin-like protein were decorated with muscle heavy meromyosin, showing "arrowheads". The arrowhead structure disappeared in the presence of ATP. A mixture of E. coli myosin-like protein and rabbit skeletal actin exhibited a gelation phenomenon on the additon of ATP. The phenomenon was reversible and showed ATP specificity. However, the gelation phenomenon was not observed with the mixture of E. coli actin-like protein and E. coli myosin-like protein. These results provide compelling evidence that the E. coli myosin-like protein and actin-like protein we isolated are essentially identical to myosin and actin, respectively.     

Calcium sensitivity of contractile proteins from chicken gizzard muscle.

Actin, myosin, and "native" tropomyosin (NTM) were separately isolated from chicken gizzard muscle and rabbit skeletal muscle. With various combinations of the isolated contractile proteins, Mg-ATPase activity and superprecipitation activity were measured. It was thus found that gizzard myosin and gizzard NTM behaved differently from skeletal myosin and skeletal NTM, whereas gizzard actin functioned in the same wasy as skeletal actin. It was also found that gizzard myosin preparations were often Ca-sensitive, that is, that the two activities of gizzard myosin plus actin without NTM were activated by low concentrations of Ca2+. The Mg-ATPase activity of a Ca-insensitive preparation of gizzard myosin was not activated by actin even in the presence of Ca2+. When Ca-sensitive gizzard myosin was incubated with ATP (and Mg2+) in the presence of Ca2+, a light-chain component of gizzard myosin was phosphorylated. The light-chain phosphorylation also occurred when Ca-insensitive myosin was incubated with gizzard NTM and ATP (plus Mg2+) in the presence of Ca2+. In either case, the light-chain phosphorylation required Ca2+. Phosphorylated gizzard myosin in combination with actin was able to exhibit superprecipitation, and Mg-ATPase of the phosphorylated gizzard myosin was activated by actin; the actin activation and superprecipitation were found to occur even in the absence of Ca2+ and NTM or tropomyosin. The phosphorylated light-chain component was found to be dephosphorylated by a partially purified preparation of gizzard myosin light-chain phosphatase. Gizzard myosin thus dephosphorylated behaved exactly like untreated Ca-insensitive gizzard myosin; in combination with actin, it did not superprecipitate either in the presence of Ca2+ or in its absence, but did superprecipitated in the presence of NTM and Ca2+. Ca-activated hydrolysis of ATP catalyzed by gizzard myosin B proceeded at a reduced rate after removal of Ca2+ (by adding EGTA), whereas that catalyzed by a combination of actin, gizzard myosin, and gizzard NTM proceeded at the same rate even after removal of Ca2+. However, addition of a partially purified preparation of gizzard myosin light-chain phosphatase was found to make the recombined system behave like myosin B. Based on these findings, it appears that myosin light-chain kinase and myosin light-chain phosphatase can function as regulatory proteins for contraction and relaxation, respectively, of gizzard muscle.     

Isolation and characterization of adult rat hearts cells.

Adult rat heart muscle cells were isolated after simultaneous perfusion of multiple (two to eight) hearts with buffered salt solutions containing collagenase and hyaluronidase. Yields (35 to 50% of ventricular weight with approximately 70% viability) are quantitatively suitable for metabolic studies. Viability has been determined by the ability of intact cells to exclude trypan blue and the inability of intact cells to oxidize exogenous succinate. Micrographs show that the fine structure of the isolated cells is well ordered. Cell concentrations of glycogen, glucose 6-phosphate, citrate, and various enzymes were similar to those of intact heart. ATP and creatine phosphate concentrations were lower than in whole hearts. Adenosine 3′,5′-monophosphate concentrations were somewhat elevated. Deoxyribonucleic acid was lower than in whole tissue. The isolated cells retain certain metabolic control mechanisms. The uncoupler of oxidative phosphorylation, 2,4-dinitrophenol, increased oxygen consumption severalfold, whereas exogenous ADP had no effect on respiration. Under anaerobic conditions the rates of glucose utilization and lactate production were faster than in the presence of oxygen, indicating retention of the Pasteur effect. The addition of glucose and insulin caused a decrease in oxygen uptake or the Crabtree effect. Exogenously added pyruvate decreased glycolytic flux and produced a pronounced increase in intracellular citrate and glucose 6-phosphate. Isoproterenol stimulated adenylate cyclase activity of the isolated cells at the same concentrations effective with intact heart preparations. Isoproterenol and glucagon caused the activation of phosphorylase. The cells deteriorated as a function of incubation time, as indicated by a decrease in ATP content and a loss of lactate dehydrogenase into the medium. Cell deterioration was greatly accelerated by Ca²⁺ at concentrations greater than 10^?5m.     

Microbial Resolution of alpha-Hydroxy Acids by Enantiospecifically Dehydrogenating Bacteria from Soil

Thirty-three bacterial strains were isolated from soil, utilizing optically asymmetric degradation of dl-2-hydroxy-4-methylpentanoic acid (dl-HMPA) as the screening probe. Those strains were distributed in the following group and genera: Coryneform and Bacillus, Pseudomonas, and Streptomyces. Among them, the most potent strains, Bacillus freudenreichii NRS-137KH20B and Brevibacterium albidum NRS-130KH20B, could perform the resolution of more than 30 g of dl-HMPA per liter within 4 to 5 days of fermentation. Optically pure l- and d-HMPA enantiomers were obtained in more than 80% theoretical yield, whereas the transformed enantiomer was almost quantitatively recovered as 2-oxo-4-methyl-pentanoic acid in the culture broth. The enantiospecific dehydrogenation responsible for this resolution reaction had a rather wide substrate specificity on straight or branched aliphatic C(4) to C(16) 2-hydroxy acids, exhibiting the optima at chain lengths of either C(7) or C(5), although the enantiospecificity was not changed by chain length. The process was thus successfully extended to the preparation of optically pure C(5) to C(9) 2-hydroxy acids.     

X-ray diffraction data for (1→3)-α-d-glucan triacetate

The crystal structures of (1→3)-α-d-glucan triacetates were studied by X-ray diffraction measurements on fibre diagrams. The oriented films annealed in water at high temperature were of higher crystallinity and occurred as two crystalline polymorphs (GTA I and GTA II) depending on the samples and also the annealing temperature. All reflections in GTA I were indexed with a pseudo-orthorhombic unit cell with a = 1·753, b = 3·018 and c(fibre axis) = 1·205 nm. From the fibre repeat data coupled with the density data and the presence of only the (003) reflection on the meridian, an extended three-fold helical structure was proposed. Although some reflections in GTA II split from the layer lines, the basic unit cell was a monoclinic system with a = 1·685, b = 3·878, c (fibre axis) = 1·210 nm and γ = 112·2°. A similar three-fold structure to GTA I was proposed from the almost identical fibre repeat and the conformational analysis on (1→3)-α-d-glucan. It was concluded that, on acetylation, the d-glucan structure changed from the fully extended two-fold helix to the extended three-fold accompanied by some extent of chain shrinking.     

Molecular weight determination of phospholamban oligomer in the presence of sodium dodecyl sulfate: application of low-angle laser light scattering photometry

The number of polypeptides constituting the oligomeric structure of canine phospholamban (a putative regulator of Ca(2+)-ATPase of cardiac sarcoplasmic reticulum) stable even in the presence of sodium dodecyl sulfate was estimated through determination of the molecular weight of the oligomer. Owing to the small molecular size, the low UV-absorptivity and the limited availability, the molecular weight determination required very sophisticated application of the following technique, used as the only recourse: low-angle laser light scattering measurement combined with high-performance gel chromatography. The molecular weight of phospholamban oligomer was found to be 30,400 and the number of subunits was concluded to be five after correction for the dependence of the apparent molecular weights on the protein concentration.