The influence of magnesium on calcium-activated, vascular smooth muscle actomyosin ATPase activity

Histamine activation of adenylyl cyclase activity in sonicated enriched rat gastric parietal cells showed a time, temperature, and concentration dependence upon guanine diphosphoimide (Gpp(NH)p). Enzyme activation was first order with Gpp(NH)p alone or Gpp(NH)p plus histamine. The K_a for Gpp(NH)p was ~2 μm and was not influenced by histamine. GTP and GDP were inactive alone or with histamine and were competitive with Gpp(NH)p, showing apparent K_i's of near 0.4 and 0.3 μm, respectively. In the presence of Gpp(NH)p, parietal cell adenylyl cyclase was activated by histamine with an EC₅₀ of 24 μm, the most potent in a series of histamine analogs, further substantiating an H₂-receptor classification for this response. H₂-Receptor antagonists were competitive inhibitors with submicromolar K_i's. Preincubation of parietal cells with histamine and Gpp(NH)p resulted in adenylyl cyclase activity up to 15 times the basal level. The activated state was retained after washing the cells free of histamine and Gpp(NH)p and was not reversed by the subsequent addition of either histamine, cimetidine, or GTP. The other gastric acid secretagogues, pentagastrin and carbamylcholine, were without effect upon histamine activation or the activated state of adenylyl cyclase. These results describe a level of control of histamine-sensitive adenylyl cyclase that requires consideration in the activation of the parietal cell H₂-receptor system by histamine to modulate acid secretion.     

Collagen in developing chick muscle in vivo and in vitro

Because of the known role of collagen in chick skeletal muscle differentiation the collagen synthesized by embryonic chick muscle was studied. The major collagen synthesized by this muscle was found to be type I collagen. In addition, the effectiveness of types I, II, III and IV collagens in promoting myoblast fusion in vitro was compared. These collagens were found to be equally effective as in vitro substrates.     

Purification of lipoamide dehydrogenase from Ascaris muscle mitochondria and its relationship to NADH:NAD+ transhydrogenase activity

Lipoamide dehydrogenase (NADH:lipoamide oxidoreductase EC 1.6.4.3) has been isolated from Ascaris suum muscle mitochondria. This activity has been purified to apparent homogeneity from both the pyruvate dehydrogenase complex and from 150,000g mitochondrial supernatants which were devoid of pyruvate dehydrogenase complex activity. The enzymes from both sources exhibited similar kinetic, catalytic, and regulatory properties and appear to be identical as judged by polyacrylamide gel electrophoresis. The native enzyme acts as a dimer, containing 2 mol of FAD, and has a subunit molecular weight of 54,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel chromatography. The enzyme also possesses substantial NADH:NAD⁺ transhydrogenase activity. Heat denaturation and differential solubilization experiments imply that the transhydrogenase activity previously reported is, in fact, associated with the lipoamide dehydrogenase moiety of the Ascaris pyruvate dehydrogenase complex. Whether or not this activity functions physiologically in hydride ion translocation, as previously suggested, remains to be demonstrated.     

Appearance of contractile activity in muscular dysgenesis (mdgmdg) mouse myotubes during coculture with normal spinal cord cells

Muscular dysgenesis (mdg) in the mouse is an autosomal recessive mutation expressed in the homozygous mutant as lack of skeletal muscle contraction. To test the ability of normal neurons to form neuromuscular contacts with, and/or possibly induce contractions in $\text{mdg}\text{mdg}$ muscle, dispersed cell cultures of normal and dysgenic muscle from newborn mice were cocultured with normal embryonic rat, mouse, and chick dissociated spinal cord cells. Contraction was induced in $\text{mdg}\text{mdg}$ muscle 1 to 10 days (depending upon the species of the neuronal source) following establishment of the cocultures. Control experiments indicated that the dispersed spinal cord preparations were free of myoblasts capable of fusing with $\text{mdg}\text{mdg}$ muscle. The establishment of neuromuscular contacts in the rat neuron cocultures was monitored by cytochemical staining of acetylcholinesterase (AChE), autoradiography of ¹²⁵I-α-bungarotoxin-bound acetylcholine receptors (AChR), and electrophysiological study of muscle membrane activity. Patches of high AChE activity were similar in size and distribution to high-density clusters of AChR on both control and $\text{mdg}\text{mdg}$ myotubes cocultured with rat neurons. The resting membrane potentials of normal myotubes and those of $\text{mdg}\text{mdg}$ myotubes in the presence of neurons were similar (? ?52 mV). The mepp frequency and the mepp amplitude distribution were the same for both control and mutant cocultured muscle. Thus, normal rat spinal cord neurons were capable of forming normal, functional neuromuscular junctions with $\text{mdg}\text{mdg}$ myotubes, and contractions were induced under coculture conditions, in otherwise noncontracting mutant muscle.     

Carbonic anhydrase activity in developing sea urchin embryos

A 13-fold increase in carbonic anhydrase specific activity was found during the first 24 h in developing embryos of the sea urchin, Strongylocentrotus purpuratus. Carbonic anhydrase activity was sensitive to inhibition by 10⁻⁴ M acetazolamide. Roles for carbonic anhydrase activity in intracellular pH regulation and spicule formation are discussed.     

Coexistence of fast-type and slow-type C-proteins in neonatal chicken breast muscle

Two different C-protein variants which selectively react with either monoclonal anti-fast C-protein antibody (MF-1) or monoclonal anti-slow C-protein antibody (ALD-66) were separated from neonatal chicken pectoralis muscle by hydroxylapatite column chromatography. Myofibrils isolated from the neonatal chicken muscle reacted with both monoclonal antibodies as examined by an indirect immunofluorescence method. These observations strongly indicate that both fast-type and slow-type C-proteins are expressed in the neonatal chicken skeletal muscle. Both of them are intermingled and assembled in the same myofibrils.     

Genotype modulates testosterone-dependent activity and reactivity in male mice

Adult castration significantly reduced the homecage locomotor activity of both inbred C57BL/6J and DBA/2J and outbred Rockland-Swiss (R-S) male mice. Castrated C57BL animals exhibited greater reductions in this behavior than did the other genotypes. Locomotor activity in a novel environment (reactivity) was also reduced by castration but only for inbred males. In both test situations, postcastration reductions in ambulation were prevented by implants of testosterone (T)-containing Silastic capsules. Thus, testicular hormones promote activity and reactivity in the male mouse in a genotype-dependent fashion.     

Timing, localization, and control of wheat germ agglutinin synthesis in developing wheat embryos

The lectin, wheat germ agglutinin (WGA), is synthesized de novo by developing wheat (Triticum aestivum, L.) embryos but is not synthesized or localized in developing endosperm as shown by radioimmunoassay. Young embryos removed from the grain and cultured on a defined medium germinate precociously and concomitantly cease WGA synthesis. In vitro precocious germination of young embryos is reversibly inhibited by low levels (1–100 μM) of the plant growth substance abscisic acid (ABA). Embryos inhibited from germinating by this growth regulator not only continue synthesizing WGA, but do so at an accelerated rate when compared with embryos left associated with the grain.     

Oxytocin: anti-insulin-like effects in isolated fat cells

Oxytocin has both insulin-like and insulin antagonistic actions in fat cells in vitro. The anti-insulin-like effects of oxytocin in dispersed rat fat cells have been studied. The magnitude of the anti-insulin-like activity varies with the metabolic pathway of glucose utilization; oxidation [14CO2 production], 32%; glycogen synthesis (D-[U-14C] glucose incorporation into glycogen), 77%. In addition, direct inhibition of the activation of fat cell glycogen synthase has been shown. These inhibitory effects depend upon an intact disulfide ring, since the ability of N-ethylmaleimide-reacted oxytocin to inhibit insulin-stimulated processes was reduced by more than 90% when compared to the intact molecule.     

Small circular DNA complexes in eucaryotic cells

A small number of eucaryotic cells (100 to 1000 cells) were pressed by mica sheet; then the extruded contents were adsorbed on mica and processed for electron microscopy. In the absence of divalent cation, small polydisperse circular DNA molecules bound to proteins or membrane material were preferentially adsorbed. The small circular DNA complexes have been found in every eucaryotic cell, primary lymphoid tissue cells of bursa and thymus, primary cell lines of retina and liver, and established cultured cell lines of embryonal teratocarcinoma, F9 and PCC3, HeLa and 3T6. Size distribution of these DNA complexes varies, depending on the cell source. The circles less than 1 μm in contour length predominate in cultured cell lines and the larger ones in primary cell lines and cells in situ. Polydisperse covalently closed circular DNAs were recovered from thymus lymphocytes by the conventional dye-CsCl buoyant density method. Their size distribution was similar to that of the small circular DNA complexes detected by the mica-press-adsorption method. They are present in several tens to hundreds of copies per cell representing, at a maximum, 0.02% of the total cellular DNA. The possibility that small circular DNA complexes may result from gene rearrangement as well as from replicon “misfiring” (A. Varshavsky, 1981, Proc. Nat. Acad. Sci. USA 78, 3673–3677) are discussed.     

Spectrophotometric determination of myoglobin in cardiac and skeletal muscle: separation from hemoglobin by subunit-exchange chromatography.

Myoglobin is extracted from muscle and separated from blood hemoglobin by subunit-exchange chromatography on a column of Sepharose 4B to which hemoglobin α-β subunits are linked covalently. Hemoglobin is retained on the column. Myoglobin in the effluent is determined spectrophotometrically as ferrous myoglobin or as carbon monoxide ferrous myoglobin. The method is applicable to cardiac, smooth, or skeletal muscle from mammals, reptiles, birds, and teleost fish, but failed with the one amphibian and the one shark tested.     

Selectivity in metabolic cooperation between cultured mammalian cells

Selective communication between cultured mammalian cells was detected as selectivity in metabolic cooperation. Whilst the majority of the cell types examined (human skin fibroblast, PC13, G3, Don, PyY) showed metabolic cooperation at almost all (>95%) of their homotypic cell-to-cell contacts, they did not necessarily show cooperation at such a high proportion of their heterotypic contacts. Less than 10% of G3/human fibroblast contacts, and usually less than 30% of G3/PC13 contacts were observed to be positive for metabolic cooperation. L cells differed from these other cell types in that they formed permeable junctions at a greater proportion of their heterotypic cell-to-cell contacts (contacts between L and PyY cells) than their homotypic contacts. We question why it was that the contacts between any two poorly-compatible cell types were positive for metabolic cooperation in only a small proportion of cases. We could find no indication that this phenomenon was attributable to heterogeneity within the cell stocks. Time course studies upon PC13 and G3 cells showed that the proportion of cooperation-positive contacts was not constant but that it continued to increase over many hours of co-culture. In comparing the homotypic and heterotypic interactions of these cell types, selectivity manifested as a difference in the rate of appearance of permeable junctions. We discuss possible explanations for these findings.     

Extrachromosomal circular DNAs from murine hemopoietic tissue cells

Extrachromosomal circular DNA complexes from cells of murine hemopoietic organs, bone marrow, thymus, spleen, and lymph nodes were examined by mica-press-adsorption method (H. Yamagishi, T. Kunisada, and T. Tsuda, 1982, Plasmid 8, 299-306). They showed wide size distribution, from 0.3 to 10 micron. The large-size DNAs of more than 1 micron (3.1 kb) in contour length were more abundant in bone marrow and thymus than they were in spleen and lymph nodes. The appearance of the large size DNAs was examined on splenocytes of athymic nude mice during ontogeny. The large-size DNAs first became detectable after 2 weeks of age and the amount increased thereafter until 9 weeks of age. It appears that large-size circular DNAs appear during differentiation from the hemopoietic stem cells into several descendent cells. Possible immunological implications for the appearance of extrachromosomal circular DNAs are discussed.     

Effect of vitamin D deficiency on skeletal development during early growth in the rat

To define the role of vitamin D in early development, female weanling rats were grown to maturity on a vitamin D-deficient diet and mated with normal males. At Day 20 of pregnancy the weight and total body calcium of fetuses were determined. At various times after parturition, pups were sacrificed. Plasma samples were analyzed for calcium and phorphorus, and femurs were characterized as to volume, dry weight, ash weight, and total calcium. The results indicate that vitamin D deficiency with its accompanying hypocalcemia does not impair placental transfer of calcium nor weight gain of the fetus. Vitamin D deficiency does appear to increase calcium accumulation in the fetus. After parturition vitamin D is functional in maintaining a normocalcemia as early as 3 days postpartum and its importance increases with age of the neonate. Bone mineralization is clearly disrupted by Day 14 as judged by calcium content per unit bone volume and the severity of the defect increases with age. Both vitamin D and normal concentrations of calcium and phosphorus appear to be essential for proper skeletal development during early growth postpartum.     

Purification and properties of a lectin from the seeds of Croton tiglium with hemolytic activity toward rabbit red cells

The pH dependence of the chemical shift of the inorganic phosphate (P_i) inside mitochondria observable by ³¹P nmr has been examined and used for the measurement of the internal pH. The pH gradient and the P_i concentration gradient were in the simple relation expected for the neutral exchange process of H₂PO₄^? and OH^?. This P_i distribution across the mitochondrial membrane was not influenced by the cross-membrane electrical potential. Both the P_i, distribution and the pH titration curve of the internal P_i indicate that the activity of the internal P_i can be well represented by the concentration of P_i measured by ³¹P nmr peak intensity. The present results give a sound base for applying ³¹P nmr to study bioenergetics and cell metabolism.     

Immunocytochemical localization of a calcium-activated protease in skeletal muscle cells

The 80 000-D subunit of a calcium-activated protease from skeletal muscle was purified to homogeneity using preparative sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and was used to elicit antibody production in rabbits. Antiserum was purified using affinity chromatography to yield a monospecific antibody fraction (anti-80K) directed against the 80 000-D subunit. Localization studies showed that the 80 000-D subunit is present in or near the sarcolemma of cultured myoblasts and sectioned muscle tissue, in discrete areas of the cytoplasm of myoblasts, and in the Z disks of the myofibril. The location of the calcium-activated protease in the cell suggests that the enzyme may be involved in myofibril degradation and in membrane alterations in developing and mature muscle cells.     

Effect of zinc deficiency on autologous rosette-forming cells

Ability to form autologous rosettes (A-rosettes) is characteristic of a certain population of immature T cells, present in the thymus of various species including man. In mice, few A-rosettes are normally found in peripheral blood and spleen but their number increases markedly in spleen following thymectomy. In our studies, A-rosette formation could be demonstrated to be significantly enhanced in the spleen of C57B1/Ks mice after the animals had been maintained 3, 6, and 8 weeks on a zinc-deficient diet (Zn^?) and to increase progressively with duration of feeding the Zn^? diet. These changes were quickly reversed by feeding a normal diet containing zinc but could not be eliminated by complete adrenalectomy, a finding that ruled out their dependence on pituitary-adrenal as is function attributable to stress. Pair-fed controls and mice fed a zinc-supplemented diet ad libitum showed few A-rosettes, as expected.     

Gangliosides mediate association of tetanus toxin with neural cells in culture

The effects of human serum albumin (HSA) on the rate of dithionite reduction of iron(III)deuteroporphyrin (iron(III)Dp) have been investigated in order to further characterize the porphyrin binding site and the changes manifested in this site under various conditions. These studies were performed under pseudo-first-order conditions, and in the presence of carbon monoxide as a "trapping agent" for the reduced iron(II)porphyrin. The rate of reduction of the free iron(III)Dp in phosphate buffer at pH 7.4 follows second-order kinetics with a rate constant (4.2 X 10(9) M-1 s-1) suggestive of a diffusion-controlled process. A six-orders of magnitude decrease in the rate of reduction was observed with iron(III)Dp was complexed with HSA. This result is consistent with HSA-bound porphyrin being less accessible to the aqueous environment. Additional studies demonstrated that both pH and anions induce various alterations in the complex that are reflected in the rate of reduction of iron(III)porphyrin.     

Properties of rat 6-N-trimethyl-l-lysine hydroxylases: Similarities among the kidney,liver, heart,and skeletal muscle activities

Hydroxylation of 6-N-trimethyl-l-lysine(lys(Me₃)) to 3-hydroxy-6-N-trimethyl-l-lysine(3-HO-lys(Me₃)) by several rat tissues has been examined and compared. The kidney enzyme, which previously was shown to require molecular oxygen and α-ketoglutarate as cosubstrates, ferrous iron and ascorbate as cofactors, and to be stimulated by catalase, has a broad pH optimum ranging between 6.5 to 7.5 at 37 °C. As determined with crude tissue extracts from kidney, liver, heart, and skeletal muscle, similar apparent K_m values were obtained for substrate, cosubstrates, and cofactors. In view of similar kinetic parameters among the several lys(Me₃) hydroxylases examined in rat tissues, and the fact that the level of skeletal muscle lys(Me₃) hydroxylase activity is comparable to that of heart, liver, and kidney, because of its large total mass, skeletal muscle may contribute significantly to the biosynthesis of l-carnitine from lys(Me₃). The most effective inhibitors found, competitive with lys(Me₃), were 2-N-acetyl-6-N-trimethyl-l-lysine, 6-N-monomethyl-l-lysine, and 6-N-dimethyl-l-lysine. l-2-Amino-6-N-trimethylammonium-4-hexynoate, d-2-amino-6-N-trimethylammonium-4-hexynoate, and dl2-amino-6-N-trimethylammonium-cis-4-hexenoate, also inhibited hydroxylase activity but by a yet undetermined mechanism. Oxalacetate, succinate, and citrate inhibited the hydroxylation reaction by competing with α-ketoglutarate. The binding of ferrous iron to the enzyme was competitively inhibited by ions of “soft metals” (e.g., Cd²⁺, Zn²⁺) but not by those of “hard metals” (e.g., Ca²⁺, Mg²⁺). Preincubation of the crude kidney enzyme for 15 min at 37 °C with mercuriphenylsulfonate, N-ethylmaleimide, iodoacetate, or iodoacetamide resulted in considerable inhibition of 3-HO-lys(Me₃) formation. The degree of inhibition by N-ethylmaleimide could be reduced by including Zn (II) during preincubation of the enzyme. The effects of “soft” metals and sulfhydryl reagents on the enzyme suggest that sulfhydryl groups are required for ferrous iron binding in the active site.     

A rapid assay for epoxide hydratase activity with benzo(a)pyrene 4,5-(K-region-)oxide as substrate

A rapid radiometric assay for epoxide hydratase activity has been developed using the highly mutagenic [³H]benzo(a)pyrene 4,5-(K-region-)oxide as substrate. By addition of dimethylsulfoxide after the incubation, conditions were found where the unreacted substrate could be separated from the product benzo(a)pyrene-4,5-dihydrodiol(trans) simply by extraction into petroleum ether. The product is then extracted into ethyl acetate and, radioactivity is measured by scintillation spectrometry. This assay allows a rapid measurement of epoxide hydratase activity with an epoxide derived from a carcinogenic polycyclic hydrocarbon as substrate and is at the same time sensitive enough for accurate determination of epoxide hydratase activity in preparations with extremely low enzyme levels such as rat skin homogenate (8–14 pmol of product/mg of protein/min).