Prostaglandin-E2-induced activation of adenosine 3'-5' cyclic monophosphate-dependent protein kinases of a murine macrophage-like cell line (P388D1)

Changes in the activities of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinases in response to prostaglandin (PG)E2-induced elevation of intracellular cAMP level were investigated with a murine macrophage-like cell line, P388D1. Photoaffinity labeling with 8-azido-[32P]cAMP showed that untreated P388D1 cells possess two types of cAMP-binding proteins of m.w. 49,000 and 52,000, respectively, in the cytosol fraction in a ration of 1:8. They must represent regulatory subunits (RI and RII, respectively) of cAMP-dependent protein kinases, because affinity chromatography on a column of omega-aminohexyl-agarose of the cytosol fraction clearly separated two fractions that exhibited the enzymatic activities and cAMP-binding activities. Photoaffinity labeling of these fractions with 8-azido-[32P]cAMP confirmed the separation of two types of isoenzymes, because each cAMP-dependent protein kinase active fraction was associated with only one type of regulatory subunit. The exposure of P388D1 cells to exogenously added PGE2 (1 microM) caused about 7.5-fold increase in the intracellular cAMP level within 30 sec. The cAMP level then sharply dropped to about 100 pmol/10(7) cells, remained at this level for about 20 min, and then gradually increased to 200 pmol/10(7) (about fivefold over the control). The enzyme assay of the cytosol demonstrated that the activation of cAMP-dependent protein kinases closely follows the kinetics of the intracellular cAMP level. The activation of the enzyme was specific for PGE2 and was not triggered by 1 microM PGF2 alpha or PGD2 which have been shown to be unable to activate adenylate cyclase of P388D1 cells. The PGE2-induced increase in the intracellular cAMP level appeared to activate preferentially the type I isoenzyme, inasmuch as the enzymatic activity of this type separated by the affinity chromatography of the cytosol of PGE2-exposed cells was lower in the presence than in the absence of cAMP, whereas the type II enzyme activity remained responsive to exogenously added cAMP.     

Circular dichroism of cephalopod rhodopsin and its intermediates in the bleaching and photoregeneration process.

In the bleaching process of cephalopod rhodopsin, a new intermediate was found in the conversion process from lumirhodopsin to metarhodopsin. This intermediate of octopus has an absorption peak at about 475 nm and has been named as M475. The circular dichroism value of M475 is too small to be evaluated. On the other hand, lumirhodopsin shows a negative CD at 470 nm, a positive CD at 350 nm and a large positive CD band with three peaks at 280, 287 and 295 nm. Such a large CD band in the ultraviolet region is not observed in rhodopsin, M475 and metarhodopsin. This CD seems to be mainly due to tryptophan and tyrosine residues restricted in free rotation in the protein moiety of lumirhodopsin. The intermediate in the photoregeneration process of cephalopod rhodopsin, P380, has a positive CD band at the main peak, 380 nm, and also a large positive CD band in the ultraviolet region like lumirhodopsin.     

Membranes of mammary gland : XV. 5-Thio- -glucose decreases lactose content and inhibits secretory vesicle maturation in lactating rat mammary gland

Short-term administration of the glucose analog 5-thio- -glucose to primiparous lactating rats reduced mammary tissue lactose concentrations to half of control levels. Treatment with colchicine alone caused slight reductions in mammary tissue lactose content. These treatments did not alter the morphology or degree of development of rough endoplasmic reticulum or Golgi apparatus, but did cause alterations in secretory vesicles. In mammary tissue from untreated lactating animals, large, swollen secretory vesicles were abundant in apical regions of epithelial cells. After thioglucose administration secretory vesicles in the apical cytoplasm were smaller and were more densely packed with contents. While administration of colchicine alone caused accumulation of large numbers of nearly fully swollen vesicles, treatment with both colchicine and thioglucose induced accumulation of smaller, less fully developed secretory vesicles which contained morphologically recognizable casein micelles. Mammary tissue from late gestation rats was low in lactose; vesicles in this tissue resembled secretory vesicles in tissue from rats treated with thioglucose in that they were small and densely packed. These observations suggest that lactose, an osmoregulator in mammary gland, is transferred from Golgi apparatus to the apical cell surface within secretory vesicles. Lactose appears to be important for secretory vesicle maturation in mammary epithelial cells.     

Anti-hypertensive activity of genetically modified soybean seeds accumulating novokinin

Novokinin (Arg-Pro-Leu-Lys-Pro-Trp), which has been designed based on the structure of ovokinin (2-7), significantly reduces the systolic blood pressure at a dose of 100 microg/kg after oral administration in spontaneously hypertensive rats (SHRs). In this study, we generated a transgenic soybean which accumulates novokinin. A vector encoding a modified beta-conglycinin alpha' subunit (4novokinin-alpha') in which four novokinin sequences have been incorporated by site-directed mutagenesis was introduced into somatic embryos by whisker-mediated gene transformation to produce a transgenic soybean. The 4novokinin-alpha' occupied 0.5% of total soluble protein and 5% of the beta-conglycinin alpha' subunit in the transgenic soybean seeds. Protein extracted from the transgenic soybean reduced systolic blood pressure after single oral administration in SHRs at a dose of 0.15 g/kg. Defatted flour from the transgenic soybean also reduced the systolic blood pressure at a dose of 0.25 g/kg. Thus, the 4novokinin-alpha' produced in soybean exhibited an anti-hypertensive activity in SHRs after oral administration.     

The carboxy-terminal region of mammalian HSP90 is required for its dimerization and function in vivo.

The majority of mouse HSP90 exists as alpha-alpha and beta-beta homodimers. Truncation of the 15-kDa carboxy-terminal region of mouse HSP90 by digestion with the Ca(2+)-dependent protease m-calpain caused dissociation of the dimer. When expressed in a reticulocyte lysate, the full-length human HSP90 alpha formed a dimeric form. A plasmid harboring human HSP90 alpha cDNA was constructed so that the carboxy-terminal 49 amino acid residues were removed when translated in vitro. This carboxy-terminally truncated human HSP90 alpha was found to exist as a monomer. In contrast, loss of the 118 amino acid residues from the amino terminus of human HSP90 alpha did not affect its in vitro dimerization. Introduction of an expression plasmid harboring the full-length human HSP90 alpha complements the lethality caused by the double mutations of two HSP90-related genes, hsp82 and hsc82, in a haploid strain of Saccharomyces cerevisiae. The carboxy-terminally truncated human HSP90 alpha neither formed dimers in yeast cells nor rescued the lethal double mutant.     

Endogenous gibberellins in clover broomrape,a parasitic plant,and its host,clover: Dependency of the parasite on the host for gibberellin production

Endogenous gibberellins were analyzed from a parasitic plant, clover broomrape (Orobanche minor Smith), and its host, clover (Trifolium repens L.). Members of both the early-13- and the early-non-hydroxylation pathways were identified from both the parasite and the host (GA₁₂, GA₂₄, GA₉ GA₄, GA₄₄, GA₁₉, GA₂₀, and GA₁ from clover broomrape; GA₉, GA₄, GA₄₄, GA₁₉, GA₂₀, and GA₁ from clover). Quantitative analyses showed that GA₄₄ was present at high levels in both host and parasite. The similarity in the gibberellins suggests the possibility that the major gibberellins in clover broomrape are transported from clover. However gibberellins such as GA₅₈, GA₃₈, and notably GA₄₇ which was identified from a plant for the first time were detected only from clover broomrape, suggesting that the parasite may have the ability to produce at least those gibberellins     

Drug Resistance of Enteric Bacteria XIII. Distribution of R Factors in Escherichia coli Strains Isolated from Livestock

Escherichia coli strains isolated from 151 swine and 108 fowl, which were kept at the Animal Health Center, Maebashi, Japan, were surveyed for drug resistance and distribution of R factors. All of the swine and 38% of the fowl excreted E. coli strains resistant to tetracycline, chloramphenicol, streptomycin, and sulfanilamide, or certain combinations thereof. Among 278 resistant cultures isolated from swine, 13% were found to be resistant to one antibiotic, whereas 87% were resistant to more than one antibiotic. Among these resistant strains, 40% carried R factors which were transferable by the usual conjugal process. The resistance patterns of these R factors included 36% which were singly resistant and 64% which were multiply resistant. Among 54 resistant cultures isolated from fowl, 24% were singly resistant and 76% were multiply resistant. Of the resistant strains from fowl, 22% carried R factors. The resistance patterns of R factors included 50% of the singly resistant type and 50% which were multiply resistant. In spite of feeding with dairy products containing only tetracycline, a high incidence of multiple resistance was observed in the E. coli strains and the R factors isolated from these animals.     

Length-dependent activation and auto-oscillation in skeletal myofibrils at partial activation by Ca2+

The length-dependent activation of skeletal myofibrils was examined at the single-sarcomere level with phase-contrast microscopy at sarcomere length (SL) >2.2 μm. At the maximal activation by Ca²⁺ (pCa 4.5) the active force linearly decreased with increasing SL, while at partial activation by Ca²⁺ (pCa 6.1-6.5) the larger active force was generated at longer SL. Throughout these experiments, the distribution of SL was kept homogeneous upon activation. In addition, we found that the spontaneous oscillation of force and SL frequently occurs in the SL range 2.2-2.6 μm at pCa 6.1-6.2. Either changes in [Ca²⁺] or osmotic compression of the myofilament lattice induced by the addition of dextran T-500, affected both the length dependence of activation and the occurrence of auto-oscillation. These results suggest that the force-generating properties of sarcomeres in striated muscle are determined not only by [Ca²⁺], but also by the lattice spacing as a function of SL.     

Molecular mechanisms for Kv1.3 potassium channel current inhibition by CD3/CD28 stimulation in Jurkat T cells

In T lymphocyte, activation of Kv1.3 channel, the major voltage-dependent K⁺ channel, is an essential step for cell proliferation in immune responses. Here, effects of anti-CD3 and anti-CD28 antibodies on Kv1.3 current were examined in three types of human T lymphocyte derived cell lines, Jurkat E6-1, p56lck-kinase deficient mutant JCaM.1, and CD45-phosphatase deficient mutant J45.01. Kv1.3 current was partly reduced by CD3 stimulation and more strongly by addition of anti-CD28 antibody in E6-1. In JCaM.1, Kv1.3 current responses to anti-CD28/CD3 antibodies were similar to those in E6-1. In J45.01, CD3 stimulation partly inhibited Kv1.3 current, but the additive reduction by CD28 stimulation was not significant. The inhibition of tyrosine phosphatase in E6-1 abolished the additional inhibition by anti-CD28 antibody in a similar manner as in J45.01. In conclusion, the stimulation of CD28 in addition to CD3 strongly inhibits Kv1.3 current and this additive inhibition is mediated by CD45 activation.