The cell wall and plasma membrane of Halobacterium halobium (R1) were examined with electron microscopy of thin sections and “freeze replicas. Deep-etched replicas from freshly frozen-fixed specimens showed filamentous structures, arising in the cell wall to anchor at the plasmic fracture face and in the cytoplasm to reach the ectoplasmic fracture face, and rarely penetrating the red membrane. The red membrane contained cylindrical or tubular particles in complementary freeze replicas. In the region of the purple membrane, in contrast, similar filaments or particles did not occur with comparable sharpness. The deep-etched filaments and freeze-fracture particles possibly represent a part of the transmembranous system connecting the biostatic cytoplasm with the ectoplasm or cell wall which is under continuous exposure to the extreme environment of brine.

Binding sites showing the characteristics of receptors for angiotensin II have been solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) from bovine adrenal membranes. Binding of ¹²⁵I-angiotensin II to the solubilized receptor was of high affinity and saturable. Angiotensin II analogs inhibited the binding competitively, whereas unrelated peptides did not. Binding activity of the receptor was completely abolished by treatment with the disulfide-reducing agent β-mercaptoethanol. In CHAPS, the receptor formed a complex with ¹²⁵I-angiotensin II, which is sufficiently stable to permit physicochemical characterization. The CHAPS-solubilized receptor has a Stokes radius of 7.5 nm when measured by gel filtration on Ultrogel AcA 34. On isoelectric focusing, ¹²⁵I-angiotensin-receptor complexes gave a major (pI=6.1) and a minor (pI=4.3) peaks. Angiotensin II-bound and free receptors exhibited marked differences in their stability against acid and β-mercaptoethanol.

5-Hydroxytryptamine (serotonin), 5-hydroxytryptophan, tryptophan, and 5-hydroxyindoleacetic acid were determined together with the enzymic activity of tryptophan hydroxylase and aromatic L-amino acid decarboxylase in a comparative way with two lines of transplantable mouse mastocytoma, P-815 and FMA3. These cells had been maintained more than 10 years in our laboratories. 5-Hydroxytryptamine and the other related compounds were determined simultaneously by high performance liquid chromatography (HPLC) with fluorescence detection. The HPLC was also used in the assay of tryptophan hydroxylase and aromatic L-amino acid decarboxylase. For the comparison of tryptophan hydroxylase between the two cell lines, the activity was measured both with and without an in vitro activation by anaerobic preincubation with dithiothreitol. Both lines of the cell contained a large amount of serotonin (1.27 and 0.24 nmol/10⁶ cells in FMA3 and P-815, respectively). The tryptophan hydroxylase activity of FMA3 cells was about one third of that of P-815 when the activity was measured after the activation with dithiothreitol. The activity measured without the activation was comparable in these two cells (0.64 and 0.95 nmol/min/mg protein in FMA3 and P-815, respectively). The biosynthesis of serotonin in P-815 cells was almost completely blocked at the decarboxylation step; the decarboxylase activity of P-815 cells was only 1/400 of FMA3 cells (0.0043 vs.1.53 nmol/min/mg protein).

Immunoreactive collagenase was localized -in isolated mouse bone cells by indirect immunofluorescent and immunoperoxidase antibody techniques using goat antimouse bone collagenase antibody. Immunoreactive collagenase was localized primarily in cells with osteoblastic characteristics (osteoblast-like cells) based on biochemical parameters; i.e., cAMP production and alkaline and acid phosphatase activity changes in response to parathyroid hormone. There was no collagenase immunofluorescence in cell populations rich in osteoclastic cells. Both parathyroid hormone and heparin appeared to stimulate the synthesis and/or release of immunoreactive collagenase by isolated osteoblast-like cells. These findings were consistent with the results of our biochemical studies (subsequent paper) using the same isolated bone cell preparations. Collagenase immunofluorescence of isolated bone cells was observed initially as fine granular fluorescence packed at perinuclear regions. This fine granular fluorescence then appeared to diffuse through the cytoplasm and to be secreted extracellularly.

Latent collagenase was isolated by heparin-Sepharose affinity chromatography from the culture medium of isolated mouse bone cells. Collagenase was synthesized by osteoblast-like cells rather than by osteoclastic cells. Collagenase synthesis by osteoblast-like cells was significantly stimulated by the addition of parathyroid hormone and/or heparin to the culture medium at concentrations that induce bone resorption in bone organ culture systems. The results were consistent with immunocytochemical findings reported in the preceding paper. The cellular origin of the isolated collagenase was confirmed by demonstrating the characteristic 3/4 and 1/4 fragments of collagen α-chains, as well as inhibition of the enzyme by anti-mouse bone collagenase antibody.

Rats with or without lesions of the suprachiasmatic nucleus (SCN) were kept under a 12-h light (0800-2000) and 12-h dark (2000-0800) cycle, and oral and intravenous glucose tolerance tests were performed in the light period (at 1000) and dark period (at 2200). In control rats, glucose tolerance was higher in the dark period than in the light period in both tests, though increase in plasma insulin was significantly higher in the dark period than in the light period only in the intravenous test. Lesions involving the bilateral SCN markedly increased glucose tolerance and insulin secretion after oral and intravenous glucose loads in both the light and the dark period, and there was no difference between the results of either test in the light and the dark period. These findings suggest that in rats, glucose tolerance shows a daily rhythm and that the SCN may be involved in generation of this rhythm through photoperiodic control of insulin secretion.

Effect of the suprachiasmatic nucleus (SCN) on photoperiod-dependent hyperglycemia due to 2-deoxy-D-glucose (2DG) was investigated using SCN-injured rats. Lesions involving the SCN caused not only complete suppression of photoperioddependent hyperglycemia due to 2DG, but also hyperinsulinemia without hyperphagia, irrespective of the lighting conditions. We propose that besides acting as a circadian clock, the SCN is involved in the central glucoreceptor mechanism and also in the regulation of insulin secretion.

The concentration of glucagon-like materials, i.e., glucagon immunoreactivity (GI) and glucagon-like immunoreactivity (GLI), in the canine brain was measured by radioimmunoassay before and after injection of insulin or concomitant infusion of glucose and insulin. The amount of GI in the thalamus-hypothalamus was increased in insulin-induced hypoglycemic dogs (hypoglycemia and hyperinsulinemia), and decreased in glucose-insulin infused dogs (hyperglycemia and hyperinsulinemia). These results indicated that brain GI responds inversely to plasma glucose levels in the presence of hyperinsulinemia. Our previous observation suggested that the brain GI was increased in both hyperglycemia and hypoglycemia when insulin was absent. It is deduced that the brain GI increases in either hypoglycemia or hypoinsulinemia which may induce intracellular glycopenia.

Type C retrovirus-like particles were found in some pancreatic B-cells in the nonobese diabetic (NOD) mice which manifest overt diabetes associated with severe insulitis. In six animals examined, ranging in age from 2 days to 19 weeks, the virus-like particles were localized in the cisternae of rough endoplasmic reticulum, where the outer surface partly lacked ribosomes. The virus-like particles were contained in 1-10 % of all the B-cells in the NOD mice, and they occurred in every mouse of the NOD strain. Each particle, membrane-limited and cylindrical (or spherical) in shape, had a hollow and cylindrical core 50-85 nm in outer diameter and 15-50 nm in inner diameter. The vertical infection of this virus-like particles from mother to child could be implicated with the pathogenesis of insulitis and insulitis-associated diabetes in these diabetic mice.

The characterization of mononuclear cells in human peripheral blood stained by anti-myelin-associated glycoprotein (MAG) antiserum was examined in a two-color immunofluorescence study and by rosetting with K-562 cells, the target cells of human natural killer cells. Human mononuclear cells consisting of more than 95% of MAG positive cells were also stained with Leu 7, a monoclonal antibody against human natural killer cells, but only 50 to 90 % of Leu 7 positive cells were stained with anti-MAG antiserum. The cells that expressed T cell antigen or monocyte antigen were also stained with anti-MAG antiserum. The percentage of MAG positive cells in OKMI positive cells (monocytes) was larger than in OKT3 positive cells (T lymphocytes). The percentage of MAG positive cells in B cells was less than 5% Human mononuclear cells conjugated to K-562 cells were stained by Leu 7 and also by anti-MAG antiserum. These results indicate that MAG positive cells are closely related to human natural killer cells.

In the rat parotid gland, stimulation of its β-adrenergic receptors causes alterations in the phosphorylation state of three endogenous proteins (17, 19, and 30 kDa). The β-adrenergic receptors involved were characterized. Rat parotid slices were prelabeled with [³²P]orthophosphate and incubated with several concentrations of l-norepinephrine, l-epinephrine, and l-isoproterenol. The three catecholamines increased the ³²P content of the 19 and 30 kDa proteins and decreased that of the 17 kDa protein with the potency order of l-isoproterenol > l-norepinephrine ≥ l-epinephrine. Practolol was more potent in blocking the l-isoproterenol-induced alterations in the ³²P content than butoxamine. These results indicate that β₁-adrenergic receptors are involved in regulation of the phosphorylation state of the three parotid gland proteins, supporting the view that at least one of the three proteins is involved in parotid amylase secretion that is also regulated through β₁-adrenergic receptors.

Adrenal chromaffin cells release acetylcholinesterase (AChE) in parallel with catecholamines upon stimulation with nicotine, high K⁺, and veratridine. The origin of this released AChE was investigated. Cultured chromaflin cells were treated with permeant and non-permeant AChE inhibitors to selectively inactivate intracellular and external AChE, and then the modified cells were stimulated with　veratridine. Our results suggest that AChE is released partly from intracellular compartment(s) and partly from the external surface of the plasma membrane.