The presence of partially degraded collagen fragments in the resorbing granulation tissue in rats.

Insoluble collagen of granulation tissue produced by carrageenin injection was solubilized by pepsin treatment and purified. The pepsin-solubilized insoluble collagen contained partially degraded collagen fragments and the amounts of these small fragments of collagen were much greater in the resorbing granulation tissues than in the growing tissues, suggesting that these small fragment were formed in the course of resorption of granulation tissue, including collagen breakdown.     

Effects of taurine and γ-aminobutyric acid on akinesia and analgesia induced by D-Ala2-Met-enkephalinamide in rats

Effects of taurine or γ-aminobutyric acid (GABA) on akinesia and analgesia induced by D-Ala²-Met-enkephalinamide were investigated in rats. Administration of taurine (dose range: 2.375×10^?2 M–9.5×10^?2 M/10 μl) into the left lateral ventricle 10 min prior to the injection of D-Ala²-Met-enkephalinamide (50 μg/10 μl) produced a dose-dependent reduction in the duration of akinesia and to some extent of analgesia, as estimated at 30 min and 60 min following the enkephalinamide injection; at the first estimation-time (10 min), taurine did not alter the duration of akinesia or that of analgesia. The median effective dose (ED₅₀) for akinesia determined at 60 min after D-Ala²-Met-enkephalinamide was 5 times greater and that for analgesia assessed at the same time was 1.7 times greater in taurine-treated rats than the respective doses in control animals. Administration of GABA under similar experimental conditions produced a dose-dependent reduction in the duration of analgesia from the initial estimation time (10 min) following the injection of D-Ala²-Met-enkephalinamide. The ED₅₀ for analgesia determined at 30 min after D-Ala²-Met-enkephalinamide was 3 times greater in GABA-treated rats than in control animals. Unlike the effects of taurine, GABA did not alter the duration of akinesia. Neither the duration of akinesia nor that of analgesia was modified by taurine or GABA alone in rats tested 9 min after the injection of each amino acid. These findings suggest that taurine may promote a recovery from both akinesia and analgesia, while GABA decreases only the analgesia induced by D-Ala²-Met-enkephalinamide.     

Studies on the function of proplastids in the metabolism of in vitro-cultured tobacco cells III. Source of reducing power for amino acid synthesis from nitrite

Evidence to show the presence of glucose-6-phosphate dehydrogenase,6-phospho-gluconate dehydrogenase, and NADP-dependent malicenzyme in proplastids of in vitro-cultured tobacco cells wasobtained. Amino acid synthesis from nitrite and 2-oxoglutaratein the proplastids was stimulated by addition of 20 mM glucose-6-phosphate.6-Phosphogluconate, malate, and isocitrate did not affect thesynthesis. Nitrite reduction and glutamate synthesis in theproplastids are assumed to be supplied with NADPH₂ as the sourceof reducing power through the reactions catalyzed by glucose-6-phosphatedehyrdogenase and 6-phosphoglyconate dehydrogenase. (Received March 22, 1977; )     

Production of anti-self-H-2 antibodies by C3D2F1 mice hyperimmune to L cell/L1210 hybrids and L1210 leukemia cells

During the course of immunization of (C3H × DBA/2)F₁ mice (genotype H-2^k/b) with L cell (H-2^k/k)/L1210 leukemia cell (H-2^d/d) hybrids and L1210 leukemia cells, some of them produced a good titer of anti-self-H-2 (H-2^d) antibodies. Antigens recognized by this anti-self-H-2 antiserum were shown to be controlled by the H-2K-IA-IB-IJ-IE subregions of the H-2^d but not H-2^k nor H-2^b haplotypes of parental as well as F₁ origins and to have a tissue distribution identical to that of class 1 H-2 (H-2K/D) antigens.     

The ret oncogene can induce melanogenesis and melanocyte development in W/W mice

We recently reported the establishment of transgenic mouse lines carrying the mouse metallothionein/ret fusion gene in which severe melanosis and melanocytic tumors developed. In the present study, we demonstrate that a significant number of pigmented hairs developed in W^v/W^v mice crossed to one of the transgenic mouse lines. The pigmented hair of W^v/W^v mice carrying the ret oncogene did not lose color during aging and reappeared after shaving, indicating that the melanocytes in the hair follicle function. The melanocytic tumors also developed in these mice, although the incidence was lower than that in the wild transgenic mice. Furthermore, the neural tube culture of mouse embryos indicated that neural crest cells of the transgenic mice gave rise to a cell population that autonomously produced melanin even in the absence of melanocyte stimulating hormone. These results strongly suggested that the introduced ret oncogene could compensate for the defect of c-kit in W^v mice during both embryogenesis and postnatal life and induce a high level of melanin synthesis in the process of melanocyte development.     

Possible involvement of protein kinase C and calcium in GSH efflux from Hep G2 cells.

We investigated the effects of protein kinase C modulations and calcium mobilization on GSH efflux in Hep G2 cells. GSH efflux from Hep G2 cells was increased by a phorbol ester. Staurosporine, an inhibitor of protein kinase C, diminished phorbol ester-stimulated GSH efflux from the cells. GSH efflux was negatively correlated with extracellular calcium concentrations. Verapamil enhanced GSH efflux, whereas ATP decreased GSH efflux. The latter effect was diminished in the absence of extracellular calcium. Protein kinase C and calcium mobilization may be crucial factors in GSH efflux from human hepatocytes.     

Spontaneous and agonist-induced calcium oscillations in pituitary gonadotrophs

Basal and receptor-regulated changes in cytoplasmic calcium concentration ([Ca2+]i) were monitored by fluorescence analysis in individual rat pituitary gonadotrophs loaded with the calcium-sensitive dye indo-1. Most gonadotrophs exhibited low amplitude spontaneous oscillations in basal [Ca2+]i that were interspersed by quiescent periods and abolished by removal of extracellular Ca2+ or addition of calcium channel blockers. Such random fluctuations in [Ca2+]i, which reflect the operation of a plasma membrane oscillator, were not coupled to basal gonadotropin secretion. The physiological agonist GnRH induced high amplitude [Ca2+]i oscillations; when a threshold [Ca2+]i level was reached, a cytoplasmic oscillator began to generate extremely regular Ca2+ transients. The time required to reach the threshold [Ca2+]i level was inversely correlated with agonist dose; the frequency, but not the amplitude, of agonist-induced Ca2+ spiking increased with agonist concentration. The duration of the latent period decreased and the frequency of Ca2+ spiking increased with the increase in ambient temperature. At high GnRH concentrations, the calcium transients merged into biphasic responses similar to those observed in cell suspensions at all GnRH concentrations. The presence of spontaneous fluctuations in basal [Ca2+]i did not significantly change the patterns of agonist-induced [Ca2+]i responses. Also, removal of extracellular Ca2+ did not interfere with the frequency or amplitude of Ca2+ spikes, but caused the loss of the plateau phase. Blockade of intracellular Ca(2+)-ATPase pumps by thapsigargin was usually accompanied by a subthreshold increase in [Ca2+]i. In such cells the agonist-induced oscillatory pattern was transformed into the biphasic response. In about 10% of the cells, however, high thapsigargin concentrations induced coarse [Ca2+]i oscillations; subsequent stimulation of such cells with GnRH was ineffective. The cytoplasmic oscillatory and biphasic responses may represent a mechanism for differential activation of Ca(2+)-dependent enzymes and their dependent cellular processes, including hormone secretion. The membrane oscillator is probably responsible for refilling of agonist-sensitive pools during and after agonist stimulation.     

Purification and characterization of hydroxypyruvate reductase from a serine-producing methylotroph, Hyphomicrobium methylovorum GM2

Hydroxypyruvate reductase of a serine-producing methylotroph, Hyphomicrobium methylovorum GM2, was purified to complete homogeneity, crystallized and characterized, the first time for an enzyme from a methylotroph. The enzyme was found to be a dimer composed of identical subunits (38 kDa), the molecular mass of the enzyme being about 70 kDa. The enzyme was stable against heating at 25 degrees C for 10 min at pH values between 5 and 9. Optimal activity was observed at pH 6.8 and around 45 degrees C. The enzyme catalyzed the reduction of hydroxypyruvate with the oxidation of only NADH. Other than hydroxypyruvate, only glyoxylate served as a substrate. The Km values were found to be 0.175 mM for hydroxypyruvate and 10.8 mM for glyoxylate. Taking advantage of the high substrate specificity of this enzyme, a means of enzymatic determination of hydroxypyruvate was established.