Prenatal development of rat thyroid C-cells was immunocytochemically investigated in culture. Immunoreactivities for calcitonin (CT) and CT gene-related peptide (CGRP) were used as the markers of C-cell differentiation. The thyroid glands at various gestational ages were enzymatically dispersed and cultured in Dulbecco’s modified Eagle medium supplemented with 5% fetal calf serum and 4% rat embryo extract. In the dispersed thyroid cells of day 14 embryos, CT- and CGRP-immunoreactive cells appeared after 2 days of incubation in 4 cases out of 7 independent experiments. CT- and CGRP-immunoreactive cells appeared without exception in the dispersed thyroid cells of day 15 or later stages of embryos. We have succeeded in establishing the culture condition under which C-cell precursors taken from the embryos as early as day 14 initiate the synthesis of CT and CGRP. These findings suggest that one of the key events participating in C-cell differentiation takes place at day 14 in the rat embryo.
New spider toxins, nephilatoxins, were purified from the venom of Joro spider, Nephila clavata, and characterized. Nephilatoxins are structurally similar to each other and possess a 6-hydroxy-indole-3-acetyl or indole-3-acetyl moiety linked with N-termini of the polycationic part composed of basic amino acids and polyamines. The toxins induce histamine release from rat peritoneal mast cells. Some of the toxins suppress the excitatory postsynaptic potential in the lobster walking leg. In addition, the toxins show insecticidal action against wriggler of mosquitos when they were added in the water.
The effects of recently developed nonpeptide cholecystokinin (CCK) antagonists L-364, 718 and CR 1392 on pancreatic exocrine secretion were studied in both isolated pancreatic acini and the isolated perfused pancreata of rats. In the isolated acini, both L-364, 718 and CR 1392 caused a concentration-dependent rightward shift of the dose-response curve for amylase secretion stimulated by CCK octapeptide (CCK-8) without altering the maximal increase. Both antagonists significantly inhibited amylase release in response to CCK-8 and caerulein but had no effect on amylase release stimulated by other receptor secretagogues or by agents bypassing receptors. L-364, 718 and CR 1392 added 20 min after the CCK-8 stimulation rapidly abolished pancreatic exocrine secretions in the isolated perfused pancreas. The suppressive effects of these antagonists on pancreatic exocrine secretion persisted even after the terminations of their infusion. These results indicate that both L-364, 718 and CR 1392 are extremely patent, competitive, specific and longacting antagonists of CCK in rat pancreas. These compounds are thus suitable for investigations on the physiological and pharmacological actions of CCK.
In fruit fly, Drosophila melanogaster, three eye proteins are known to be phosphorylated by light stimulation (8, 9). Among them, 49 K protein is most abundant. Matsumoto et al. (8, 9) suggested their possible involvement in the visual procasses. In this paper, we describe its cellular localization revealed by immunohistological methods. The protein is localized in all the visual receptor cells including retinular cells (R1-R8) of the adult compound eye, adult ocelli and larval photoreceptor organ. It also exists in their axons. The antigen is shown to be enriched in microvillous rhabdomere and multivesicular cells suggests that it plays an important role in the processes following photoreception and transduction.
The interaction of a protein-bound polysaccharide (PSK) isolated from Basidiomycetes with myosin subfragment-1 (S-1) was examined by measuring the changes in ATPase activity, sedimentation assay, affinity chromatography, limited digestion and chemical cross-linking. At low ionic strength, S-1 bound to PSK immobilized on Sepharose 4B and the bound S-1 was released at 150-200 mM KCl. PSK inhibited remarkably the Mg2+-ATPase activity of acto-S-1, while it hardly affected the activity of S-1 itself. The amount of S-1 bound to actin was reduced by addition of PSK in the presence of ATP, but not in its absence. Limited tryptic digestion of S-1 heavy chain produced mainly three fragments of 27, 50 and 20 kDa. The cleavage of an intermediate 77 kDa fragment (27-50 kDa) was delayed when PSK was present. The chemical cross-linking experiments using 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC) indicated that about 2 mol of PSK could bind per mol of S-1 heavy chain at the maximum. When tryptic S-1 and PSK were mixed and then incubated with EDC, PSK was predominantly cross-linked with the 70 kDa fragment (50-20 kDa) rather than with the 77 kDa fragment or with further cleaved fragments (50 and 20 kDa). Moreover, the 27 kDa fragment hardly bound to PSK. Taken together, the binding regions of PSK in the S-1 molecule appear to be present in the surrounding of N-terminal of the 50 kDa fragment and in the environment of the 50-20 kDa hinge region.
We previously demonstrated the increased activity of fatty acid elongation in the microsomal fraction of adrenoleukodystrophy (ALD) fibroblasts. In the present study, detailed pathways for the synthesis of hexacosanoic acid (C26:0) were delineated using deuterium labeling and mass fragmentography. Replacement rate or fractional turnover rate of C26:0 in ALD fibroblasts increased by 67% of the control, while the rate of replacement from C16:0 to C24:0 decreased in ALD. This may indicate an activation of fatty acid synthesis specific for C26:0. When two elongation activities using endogenously formed precursors and exogenous or preexisting fatty acids were separately measured in ALD, elongation products starting from exogenous fatty acids increased in C16:0, C18:0, C20:0, C22:0 and C24:0, but the corresponding fraction in C26:0 decreased as compared to the control. These results suggest that the synthesis of C26:0 may take place in a compartment different from those for the synthesis of fatty acids whose chains are shorter than C26:0, and that the activity of C26:0 synthesis is enhanced in vivo in ALD.
Effects of α-adrenoceptor antagonists and α,β-methylene ATP (mATP) on electrical responses elicited by perivascular nerve stimulation were studied in smooth muscle cells of the rat tail artery, and the results were compared with those of exogenously applied noradrenaline (NA) or ATP. Nerve stimulation elicited an excitatory junction potential (e.j.p.) and slow depolarization. The latter component and NA-induced depolarization were blocked by α-adrenoceptor antagonists; prazosin and phentolamine were more potent than yohimbine. The membrane resistance was increased during the slow depolarization and the NA-induced depolarization. mATP depolarized the membrane, decreased the membrane resistance, reduced the time constant of the falling phase of the e.j.p., inhibited the amplitude of the e.j.p. and the ATP-induced depolarization to a similar extent, and enhanced amplitude of the slow depolarization and the NA-induced depolarization. The results provided evidence that in this artery, both α1- and α2-adrenoceptors are involved in the NA-induced depolarization. Inhibition by mATP of the e.j.p. cannot be concluded solely due to desensitization of the postjunctional ATP-receptor.
Transmembrane electrical responses produced by noradrenaline (NA) were recorded from intrapulmonary artery and vein muscles of the rat. In the artery, NA (above 1 nM) depolarized the membrane and, in high concentrations (above 1 μM), generated slow oscillatory potentials. The excitatory action of NA was antagonized by prazosin but not by yohimbine. The amplitude of electrotonic potentials was increased during the NA-induced depolarization. Separate stimulation of α1,- and β-adrenoceptors by NA potentiated and inhibited the high-K induced contraction, respectively. In the vein, NA produced a biphasic response, i.e., an initial transient (1-2 min) hyperpolarization followed by a slowly developing depolarization which required 15-20 min to reach the steady potential. The initial hyperpolarization was blocked by propranolol and the following depolarization by prazosin but not by yohimbine. Amplitude of electrotonic potential was decreased or increased during the NA-induced hyperpolarization and depolarization, respectively. The results provide evidence that in the intrapulmonary artery and vein of rats, stimulation of the α1-adrenoceptor depolarizes the membrane with associated increase in membrane resistance. Stimulation of β-adrenoceptors in the artery inhibited the muscle contraction, with no alteration of the membrane potential. The β-adrenoceptormediated hyperpolarization in the vein was accompanied by a decrease in membrane resistance.
The distribution and morphology of the cells containing S-100 protein and/or nonneuronal enolase (NNE) were investigated in rat, mouse, hamster, guinea pig, rabbit, chinchilla, pika, mole, suncus, pig, cow, goat, dog, cat, monkey and human pituitary glands using the peroxidase-antiperoxidase and the avidin-biotin-peroxidase complex methods. The immunoreactivity for S-100 protein was detected in the glands of the rat, cat, cow, pig, goat, moneky and human, and recognized in folliculo-stellate cells in the rat, monkey and human pituitary glands. On the other hand, this immunoreactivity was not detected in any anterior pituitary cells including the folliculo-stellate cells in the mouse, guinea pig, hamster, rabbit, pika, chinchilla, suncus, mole and dog. The antiserum against NNE stained some types of pituitary cells in the rat, mouse, guinea pig, pika, cat, goat and monkey, but not in other animals examined. The cells immunostained for S-100 protein were distinct from the cells immunostained for NNE in the cat, goat and monkey. immunoreactivities for S-100 protein and NNE were detected in the same folliculo-stellate cells only in rats. It was concluded that S-100 protein and NNE were not always specific markers of a particular kind ofpituitary cells in mammals, with the exception of the rat whose folliculo-stellate cells contained both proteins.
3Y1tsF121 cells, a temperature-sensitive mutant of rat 3Y1 fibroblasts, are arrested in the G1 phase of the cell cycle when the cells proliferating at permissive temperature are placed at restrictive temperature. The cells arrested in the G1 phase are induced to enter the S phase when simply placed at permissive temperature or when added with a large dose of serum into the culture medium at restrictive temperature. This G1 phase-arrest overcoming activity in serum was not dialyzable and was lost after exposure of serum to 90°C for 30 min. Among the growth factors examined, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF) were found to replace the serum for the G1 arrest overcoming activity. In any of the three factors, the maximal efficiency of the overcome of the G1-phase arrest (in terms of percentage of cells which entered the S phase) was lower than the maximal efficiency in serum. When PDGF and EGF were added in combination, the efficiency increased to the same level as the maximal efficiency seen in serum. In this case, however, the efficiency was lower than the sum seen with single applications of PDGF and EGF. Administration of EGF during the first half of the incubation period (0-24 h), followed by that of PDGF during the second half (24-48 h), or vice versa, resulted in the entry into S phase with an efficiency much the same as that seen with the simultaneous administration of PDGF and EGF during the entire incubation period. These results suggest that 1) PDGF and EGF either independently or cooperatively act on the events controlling the entry into S phase and involving the functional lesion in 3Y1tsF121 mutant. 2) PDGF and EGF have both common and unique actions in the events involving the lesion in 3Y1stF121, and complement each other in their unique actions. 3) PDGF and EGF need not be simultaneously administered for their cooperative action, and the ordered sequential stimulation of cells is not required. It was also found that, between EGF and PDGF, and between FGF and EGF, there was no detectable cooperative action. Dexamethasone perfectly suppressed the overcome of the G1 arrest mediated by serum or byEGF. The expression of the G1-arrest overcoming activity of each of PDFG, EGF required the simultaneous presence of a small quantity of serum which alone did not have the overcoming activity, suggesting that a serum component is required for the successful action of each of the three growth factor. This growth-factor helping activity in serum was not dialyzable and was resistant to heat (100℃).
The effects of autophosphorylation of brain Ca2+/calmodulin-dependent protein kinase II were investigated using several substrates. The autophosphorylation of the enzyme increased the Ca2+/calmodulin-independent activity with any of the substrates used. When myelin basic protein or calcineurin was used as substrate, the total kinase activity was prominently increased by autophosphorylation, suggesting that the enzyme is activated by autophosphorylation. Since this effect was not observed with chicken gizzard myosin light chain as substrate, the activation of the enzyme by autophosphorylation was dependent on substrate used. The addition of calmoduilin at higher concentrations inhibited the total activity of the autophosphorylated enzyme. The results suggest that the substrate specificity of the enzyme changes by autophosphorylation and that thereby the selectivity of the enzyme for substrates increases.
The ultrastructures of follicular or folliculo-stellate cells in fetal and adult bovine pituitaries were studied by electron microscopy. The structure of an anterior lobe was further investigated immunohistochemically with the antiserum for bovine type IV collagen. Follicular cells are divided into three categories on the basis of fine structural properties: One group consists of a cluster of agranular and spherical cells in which the ductule-like pseudolumina are equipped with microvilli; the seond group consists of spherical cells with a few small secretory granules (50-150 nm in diameter), which encircles pseudolumina into which all the cells project microvilli; and the third group consists of agranular stellate cells protruding their thin cytoplasmic processes between glandular cells. These stellate cells make a closed pseudolumen, and bear numerous microvilli which interdigitate with the microvilli from other cells along the apical region of the opposing cells. These three types of follicular cells contact each other with either desmosomes or gap junctions. The former two types of the follicular cells sometimes mix together making clusters with ductule-like pseudolumina, and they are commonly observed in the fetal pituitary. The second type of follicular cell is rare in adult pituitaries. The third type of folliculo-stellate cell is infrequently observed in fetal pituitaries, and frequently in adult glands. The culsters of follicular cells, granulated glandular cells and the encircling basement membranes immunostained with type IV collagen antiserum form lobule-like units in anterior lobes. The size of the units in fetal pituitary glands varies depending on the number of granulated glandular cells which are distributed within the cluster of follicular cells and the basement membranes. In adult anterior lobes, however, units are uniformly large and are composed of a cluster of folliculostellate cells, heavily granulated glandular cells, and basement membranes. These observations suggest that during development some follicular cells in a cluster surrounded with basement membrane start to make minute secretory granules in their cytoplasms, and then leave the cluster of follicular cells, and squeeze toward basement membranes. Thus the large lobule-like units are formed by increasing the number of granulated cells during development. It is also suggested that some of follicular cells keep their agranularity after the maturation of the animal and become stellate in shape and form the folliculo-stellate cells of adult pituitary gland.
Slow moving protease (SMP), a non-pepsinogen type acid protease present in human gastric muscosa, was purified to apparent homogeneity by an improved purification procedure including chromatography on DEAE-cellulose, concanavalin A-Sepharose and Sephadex G-150. Upon DEAE-cellulose chromatography, SMP was separated into three fractions, designated SMP-I, SMP-II, and SMP-III. SMP-I was the major component and the other two appeared to be autodigestion products of SMP-I. Investigation on the specificity of action of SMP on the B chain of oxidized insulin revealed that SMP resembles cathepsin E and pepsin A in substrate specificity rather than cathepsin D and pepsin C.
Developmental changes in the activity of glutathione S-transferase (GST) and its immunocytochemical localization were studied in the cerebella of homozygous (j/j) and heterozygous (j/+) Gunn rats. The activity of jaundiced j/j rats began to increase after 8 days of life and reached twice as high as that of non-jaundiced j/+ rats at 15-20 days, chronologically coincident with the development of bilirubin cerebellopathy. When j/j rats were prevented from cerebellopathy by photoirradiation to the newborns from 4 to 7 days, the increase did not occur in spite of yet prolonged jaundice. DE-52 chromatography of the cerebellar 105,000 ｇ supernatant from either j/+ or j/j rats revealed four main activity peaks, and the relative amount of each peak did not differ significantly between the two groups of rats. Immunocytochemical studies with anti-GST antibody showed staining of astoroglial and ependymal cells in the cerebella of both groups of rats. Staining was more extensive in j/j rats than in j/+ rats. The present results suggest that the increase in the cerebellar GST activity in jaundiced Gunn rats is ascribed to hypertrophy or proliferation of astroglial cells, a consepuence of bilirulin neurotoxicity.
Mitochondria from the muscle of a patient with mitochondrial myopathy contained paracrystalline inclusion bodies. To characterize the mitochondrial fraction containing the crystalline materials, the muscle homogenate was subjected to a Ficoll density gradient centrifugation. The mitochondrial fraction was identified by immunoblotting with specific antisera against the F1-ATPase β-subunit and lipoamide acetyltransferase of the pyruvate dehydrogenase complex. The mitochondria from the patient had a density higher than in mitochondria from controls. This method is useful for isolating abnormal mitochondria from muscle specimens of about 0.1 ｇ.