Interaction of electron acceptors with the excited triplet state of Zn cytochrome c

The decay rate of the excited triplet state of Zn cytochrome c was enhanced by electron acceptors including methyl viologen and ferric complexes of cyanide, oxalate, EDTA and cytochrome c at room temperature. Ferrous compounds were several orders of magnitude less effective than the respective ferric form in quenching the phosphorescence. In the presence of ferricytochrome c and ferricyanide the semilogarithmic plots of the decay curve showed an anomalous decay profile in which the rate of interaction appeared to accelerate after excitation. One explanation is that the quenching process was accelerated by a conformational change of the polypeptide chain around the excited triplet state porphyrin. Another explanation is that quenching occurs via an intermediate.     

Ultrasonic studies of lipid bilayer. Phase transition in synthetic phosphatidylcholine liposomes

The ultrasonic velocity at 3 MHz and the density in the nonsonicated and sonicated liposomes of dipalmitoylphosphatidylcholine have been measured in the temperature range from 0 degrees C to 55 degrees C. The results indicate that nonsonicated multilamellar vesicles undergo a weak first order transition which is analogous to the nematic-isotropic transition of liquid crystals. A sharp change in the ultrasonic velocity associated with the first order transition disappears when the multilamellar vesicles are sonicated. The bulk modulus of the lipid bilayer calculated from the ultrasonic velocity and the density of sonicated liposomes has a value of 3.0 X 10(10) dyne/cm2 at 20 degrees C, reaches a minimum value of 2.1 X 10(10) dyne/cm2 at its transition temperature and increases slightly to 2.2 X 10(10) dyne/cm2 at 50 degrees C.     

Phospholipid transmethylation in the membrane of human neutrophils and lymphocytes

Phospholipid transmethylation in the microsomal fraction of stimulated and unstimulated human leukocytes was measured in a recently developed assay system. Microsomal fraction was prepared from neutrophils, unseparated lymphocytes, T lymphocytes, and non-T lymphocytes by sonication and subsequent ultracentrifugation. Two hundred micrograms of microsomal protein was reacted with S-adenosyl-L-[methyl-3H]methionine. In unstimulated cells, incorporation of methyl-3H into phospholipid was 0.60 +/- 0.06 pmol min-1 mg protein in neutrophil membrane, 0.84 +/- 0.075 in unseparated lymphocytes, 1.23 +/- 0.17 in T lymphocytes, and 0.71 +/- 0.085 in non-T lymphocytes (mean +/- SE). Stimulation of neutrophils with opsonized zymosan or concanavalin A (Con A), and of lymphocytes with Con A, phytohemagglutinin, or pokeweed mitogen increased 15 to 30%. The resulting methylated phospholipids were identified and quantitated by two-dimensional thin-layer chromatography. The inhibitor 5'-S-isobutyl-5'-deoxyadenosine (SIBA) inhibited transmethylation 47-55%. This assay system appears to measure specifically the activity of methyltransferases which mediate the transmethylation of membrane phospholipid; the assay should find important applications in the study of membrane lipid metabolism in human health and disease.     

Epidermal protein metabolism directed toward keratinization by hydrocortisone in the chick embryonic skin growing in a chemically defined medium.

The metabolism of the epidermal structural and nonstructural proteins was studied in hydrocortisone-induced in vitro keratinization of 13-day chick embryonic skin growing in a chemically defined medium. The protein metabolism of the epidermis was examined by determining the amounts of radioactivity incorporated into the fractions of reduced, S-carboxymethylated epidermal proteins (SCMEp) which were separated by polyacrylamide gel electrophoresis. A group of high molecular weight, glycine-rich derivatives of the epidermal fibrous protein called SCMEpA were found to be actively synthesized in the hydrocortisone-treated epidermis alone, while a group of undefined protein derivatives called SCMEpX was shown to be synthesized exclusively in the nontreated epidermis. Chase-culture of the prelabeled explants revealed that hydrocortisone accelerated the degradation of general proteins including SCMEpX while SCMEpA remained metabolically stable throughout the culture. Actinomycin D did not significantly affect the hydrocortisone-induced synthesis of SCMEpA but greatly inhibited that of SCMEpX of the nontreated epidermis, suggesting the induction by the steroid of relatively stable mRNA for SCMEpA. From these findings, it is concluded that hydrocortisone directed the cultured epidermis toward keratinization through acceleration of the synthesis of epidermal structural proteins and degradation of other proteins.     

Proliferative kinetics of human lymphocytes in culture measured by autoradiography and sister chromatid differential staining

A simple combination of autoradiography, to determine when a cell synthesized DNA, and sister chromatid differential staining, to determine how many times a cell has divided, was used to follow up the proliferating fate of human lymphocytes in culture. Cells were incubated continuously with 5-bromodeoxyuridine (BrdU) and pulse-labelled with 0.1 muCi/ml [3H]thymidine at various times after stimulation with phytohemagglutinin (PHA). The cells were then harvested at 4 h intervals up to 72 h, and the percentage of labelled mitoses was determined separately in first, second, or third division cells. The data showed that the cycling cells, whether they began cycling at earlier or later times after stimulation, had about the same generation times of 12--14 h. This confirms that the heterogeneity of cell generations seen in short-term lymphocyte cultures is in large part due to the difference in the times when cells began cell cycling in response to PHA.     

Chemical analysis of the female sex pheromone in Palpita nigropunctalis (Lepidoptera: Crambidae)

The lilac pyralid, Palpita nigropunctalis Bremer (Lepidoptera: Crambidae), is a common pest of Oleaceae plants. A crude extract of the female sex pheromone glands was examined by gas chromatography-electroantennogram detection (GC-EAD) and GC coupled to a mass spectrometer (GC/MS). The GC-EAD analysis revealed three EAG-active components (I–III) in a ratio of 1:0.2:0.01 (I: II: III). GC/MS analysis successfully recorded the mass spectra of I and II. For I, ions at m/z 238 (M⁺) and 220 ([M-18]⁺) indicated the structure of a monoenyl aldehyde with a 16-carbon chain. For II, M⁺ was not detected, but ions at m/z 222 ([M-60]⁺) and 61 ([AcOH+1]⁺) suggested that II was a monoenyl acetate with a 16-carbon chain. Further GC/MS analysis of the extract treated with dimethyl disulfide revealed that the double bonds in both I and II are located at the same position of 11th-carbon. In addition, the pheromone extract was examined by GC/Fourier transform-infrared spectrophotometer (GC/FT-IR). An IR spectrum of I showed characteristic absorption at 1716 and 966?cm^?1, indicating a formyl group and E configuration of the double bond, respectively. In the case of II, absorption at 1745 and 968?cm^?1 indicated an ester carbonyl and E configuration, respectively. Taken together and by comparison with authentic standards, I and II were confirmed as (E)-11-hexadecenal and (E)-11-hexadecenyl acetate, respectively; while III was speculated as (E)-11-hexadecen-1-ol. The synthetic I, II and III all coincided well with those of the natural components in chemical data, and elicited strong electroantennographic activity in male P. nigropunctalis.     

Adenylate kinase bound to the envelope membranes of spinach chloroplasts.

The activity of adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) in both the forward (2ADP → ATP + AMP) and backward (ATP + AMP → 2ADP) reactions was found to be associated with the envelope membranes which were isolated from spinach chloroplasts. Sonication and repeated washing in a medium of high ionic strength were unable to release the enzymes from the envelope membranes. Adenylate kinase bound to the envelope is stable in the cold and inactivated by heat and acid treatments. The enzyme requires magnesium ion as an activator. The pH-activity profile of the forward reaction catalyzed by membrane-bound adenylate kinase gave a maximal activity at pH 8.5. The apparent Michaelis constant, K_m, value for ADP in the forward reaction was estimated to be 1.3 ± 0.2 × 10^?4m. A Lineweaver-Burk plot of the forward reaction gave a straight line when the reciprocal of the reaction rate was plotted versus the reciprocal, and not the square of the reciprocal, of the concentration of substrate ADP. This favors the view that the adenylate kinase bound to the chloroplast envelope has a single or equivalent binding site of Mg-ADP^?. The probable involvement of adenylate kinase bound to the chloroplast envelope in controlling the energy pool and adenylate translocation in chloroplasts is suggested.     

Kinetics of repair of O6-methylguanine in DNA by O6-methylguanine-DNA methyltransferase in, vitro and in, vivo

The demethylation of O⁶-methylguanine in double stranded DNA catalyzed by rat liver O⁶-methylguanine-DNA transmethylase was found to proceed much more rapidly when the DNA substrate was methylated to a high extent. When the content of O⁶-methylguanine in DNA was equal to 1 in 2000 guanines, the reaction was 90% complete within 2 min, but when the content was 1 in 500,000 it required 27 min at 37°C. These results suggest that the repair protein either moves along the DNA substrate or else has little selectivity for binding specifically to the sites containing O⁶-methylguanine rather than to the normal DNA. The repair of O⁶-methylguanine in rat liver $\text{in}\text{,}\text{vivo}$ occurred at rates comparable to those seen $\text{in}\text{,}\text{vitro}$ with the substrates alkylated to low extents and was virtually complete within 3 hours. These results provide strong evidence that this protein is the factor responsible for O⁶-methylguanine removal $\text{in}\text{,}\text{vivo}$ and explain the wide variation in time courses reported in the literature since substrates methylated to greatly different extents have been used for such experiments.     

Ultrastructural changes of the teleostean hatching gland cell during natural and electrically induced precocious secretion.

Ultrastructural changes of the hatching gland during electrically induced precocious secretion were compared with those during natural secretion in the medaka, Oryzias latipes. The gland cells are covered by a layer of epithelial cells, which adjoin one another just on the apical center of each gland cell. When the natural as well as the precocious secretion occurred, each gland cell was swollen upward and rounded, and separation of the epithelial joints occurred, giving rise to an exposure of the apical portion of the gland cells. There were marked differences between these two kinds of secretion process in the behavior of the secretory granules prior to secretion and in the mode of discharge of the secretory substances. The changes which occurred during both types of secretion and which, therefore, seemed to be essential to the secretory processes of this gland cell were the swelling up of the gland cells in the initiation of secretion and the reduction of the electron density of the zymogen granules. These secretion-associated ultrastructural changes are discussed in view of the difference in the maturation of the gland cells.     

Organ-specific difference in the sugar chains of gamma-glutamyltranspeptidase

Sugar chains of gamma-glutamyltranspeptidase purified from neonatal mouse liver and adult mouse kidney were quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis. A comparative study of the structures of the oligosaccharides has revealed that the GlcNAc beta 1 leads to 4Man beta 1 leads to group is found in the sugar chains of kidney enzyme but not in those of liver enzyme. This is considered as an organ-specific difference common to mammals because the same phenomenon was found in bovine and rat enzymes.     

Effect of glucose on protein phosphorylation in rat pancreatic islets

Isolated rat pancreatic islets, incubated in the presence of extracellular ³²Pi to steady state ³²P incorporation into cellular phosphopeptides, were exposed to glucose for 10 min. Glucose (16.7 mM) significantly stimulated the phosphorylation of six phosphoproteins with molecular weights of 15,000, 35,000, 49,000, 64,000, 93,000 and 138,000. Mannoheptulose (16.7 mM) markedly inhibited glucose-stimulated phosphorylation of these six phosphoproteins. This protein phosphorylation might be important in mediating glucose-stimulated insulin release.     

The crystal structure of uncomplexed-hydrated cyclooctaamylose

Cyclooctaamylose crystallizes from aqueous solution with space-group symmetry P2₁ and lattice parameters: $\text{a}\text{= 20.253(8),}\text{b}\text{= 10.494(5),}\text{c}\text{= 16.892(6)}$ A and β = 105.32(1)^o, Z = 2; the apparent formular per asymmetric unit is C₄₈H₈₀O₄₀·17H₂O. The macrocycle is in an open conformation but displays significant deviations from ideal eight fold molecular symmetry. Of the 19 water molecules thus far located, four of which have occupancy factors of one half, 12 may be characterized as being in the torus of the cycloamylose.     

Ultrasonic measurements of two-component lipid bilayer suspensions

Two-component lipid bilayers of dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine were studied by measuring, ultrasonic velocity and absorption at 3 MHz. The phase diagram of the two-component lipid bilayers is discussed based upon the transition anomalies of the ultrasonic velocity as well as absorption, and it is suggested that this binary system has two critical points. The bulk modulus of lipid bilayers was determined from the ultrasonic velocity to be (2.2–3.0) × 10¹⁰$\text{dyne}\text{cm}{}^2$, whereas the bulk viscosity calculated from the absorption was 10–20 P except for the transition regions.     

A Polydeoxythymidylic acid-specific deoxyribonuclease from rat ascites hepatoma cells.

A deoxyribonuclease has been purified 950-fold from rat ascites hepatoma cells and has been separated from another deoxyribonuclease that appears to have DNase III type activity. The enzyme preferentially degrades single stranded poly(dT), requires Mg²⁺ for maximum activity and has a pH optimum at 8.5 in Tris-HCl buffer. Poly(dA), poly(dC), poly(rA), and poly(rU) are not effective substrates. The hydrolysis of poly(dT) is strongly inhibited when poly(dA) or poly(rA) is annealed with poly(dT). Poly(dT) is degraded ultimately into 5′-deoxythymidylic acid via the formation of oligodeoxythymidylate intermediates.     

Tyrosine hydroxylase and tryptophan hydroxylase activities and its cofactor biopterin level in brain regions of the rolling mouse: The influence of thyrotropin releasing hormone

Biopterin, the cofactor for tyrosine hydroxylase and tryptophan hydroxylase, was decreased in caudate nucleus, hypothalamus and cerebellum of the rolling mouse. Though there were not significant differences of tyrosine hydroxylase and tryptophan hydroxylase activities between the rolling and normal control mouse in the hypothalamus, the rolling showed significant increase of biopterin concentration and tyrosine hydroxylase activity after administration of thyrotropin releasing hormone (TRH). These results suggest that ataxic gait of the rolling mouse may be partly due to some abnormalities of catecholaminergic neurons, especially noradrenergic neurons, and that TRH may improve the abnormalities of catecholaminergic neurons. The changes of biopterin concentration by TRH administration indicate that biopterin may be a regulatory factor in catecholamine biosynthesis.     

Resistance to tolerance induction in B cells of autoimmune mice—Abnormal expansion of low affinity IgG-producing B-cell population

Several strains of mice are known to develop spontaneous autoimmune diseases like lupus erythematosus and they show various immunological abnormalities as well. Despite different genetic backgrounds, they manifest various immunological abnormalities in common, e.g., polyclonal B-cell activation (PBA) and resistance to tolerance induction. To elucidate mechanisms of the development of autoimmunity, tolerance inducibility was examined in autoimmune and normal mice using trinitrophenylated carboxymethyl cellulose (TNP-CMC) as tolerogen which is known to induce TNP-specific B-cell tolerance without the participation of T cells. NZB and MRL/Mp-lpr/lpr mice were used as autoimmune mice and C57BL/6, BALB/c, and MRL/Mp-+/+ mice as nonautoimmune mice. When TNP-CMC-injected mice were challenged with T-independent antigens, all of the mice tested were shown to be tolerant. In contrast, when TNP-CMC-injected mice were challenged with T-dependent antigen and secondary IgG responses were assessed, autoimmune mice showed rather hyperreactivity, while nonautoimmune mice showed hyporesponsiveness. Cyclophosphamide improved this defective tolerance inducibility. By the solid-phase radioimmunoassay it was revealed that average affinity of serum anti-TNP antibodies produced in TNP-CMC-injected mice was low. Such low affinity antibodies were produced in large amount in autoimmune mice. Hence, it was suggested that B-cell clones destined to produce low affinity IgG antibodies were responsible for the resistance to tolerance induction and such clones were expanding in autoimmune mice.     

Polysaccharides sulfated at the time of gastrulation in embryos of the sea urchin Clypeaster japonicus

Based on the fact that the development of sea urchin embryos is arrested at the blastula stage in sulfate-free sea water (SFSW), we attempted in the present study to elucidate the nature of sulfated polysaccharides (PSs) which appear at the time of gastrulation in embryos of the sea urchin Clypeaster japonicus. Electrophoretic analysis of PSs prepared from embryos at different developmental stages revealed that three kinds of PSs (3A, 3B, 3C) appear de novo at the gastrula stage, and that these PSs are not found in embryos at the hatching blastula stage, nor are they found in permanent blastula reared in SFSW. These, three PSs were mostly of extracellular matrix origin. Among them, 3C was identified as dermatan sulfate on the basis of its electrophoretic mobility and sensitivity to enzymatic digestion. 3A and 3B remained to be identified. Further, a plausible precursor of 3C, which was sulfated under normal conditions, was detected as 6D in the embryos reared in SFSW. Autoradiographic analysis using [35S]sulfate revealed that these three PSs, accounted for more than 90% of [35S]sulfate incorporated into the acid PS fraction during gastrulation.     

Initial development of conduction pattern of spontaneous action potential in early embryonic precontractile chick heart

The conduction of spontaneous action potentials in the 7-10 somite embryonic developing chick hearts was monitored optically using a potential-sensitive merocyanine-rhodanine dye. Spontaneous optical action signals from 5 to 12 different regions of the primitive heart were recorded simultaneously. Short delays were observed among firing times of the absorption signals which were nearly synchronized among the different regions. From these delays, we estimated the conduction velocity of the spontaneous excitatory waves. Usually, in the 7-somite to the beginning of the 9-somite stage, (i) excitatory waves conducted radially over one side of the prebeating heart, at a uniform rate; (ii) the "radially" spreading electrical wave slowed considerably within the primordial fusion line at the midline of the heart; and (iii) this delay disappeared in the later period of the 9-somite stage to the 10-somite stage. These observations suggest that electrical coupling among the cells within the primordial fusion line is poor during the 7 to 9-somite stage, and that the coupling is strengthened by the late 9th or 10th somite stage.     

Solubilization of the enzyme catalyzing CDP-diglyceride-independent incorporation of myo-inositol into phosphatidyl inositol and its comparison to CDP-diglyceride:inositol transferase.

A solubilized preparation with activity for catalyzing the incorporation of free myo-inositol into phosphatidyl inositol was obtained from a rat liver microsomal fraction. The incorporation took place both in the presence and in the absence of cytidine diphosphodiglyceride (CDP-DG). The pH optimum of the incorporation in the absence of CDP-DG was 7.4–7.5, while that of the incorporation in its presence was 8.5–8.6. The incorporation in the absence of CDP-DG was activated by Mn²⁺ but not by Mg²⁺, while that in the presence of CDP-DG was activated by either Mn²⁺ or Mg²⁺. These results indicated that the incorporation in the absence of CDP-DG and the incorporation in its presence were catalyzed by different enzymes. Before Solubilization, the CDP-DG-independent enzyme was bound to endoplasmic reticulum. The CDP-DG-dependent enzyme also was bound mainly to endoplasmic reticulum and, to a minor extent, to plasma membrane. The CDP-DG-independent enzyme was more easily solubilized by sodium cholate than the CDP-DG-dependent enzyme. There were also differences between these two enzyme activities of the solubilized preparation with respect to their sensitivity to various detergents and their dependence on exogenous lipids. The CDP-DG-independent incorporation was inhibited by CDP-DG, by some nucleotides, and by phosphatidyl serine, while the CDP-DG-dependent incorporation was not inhibited by these substances. Both activities were both inhibited by thiol-reactive compounds.