Catecholamine-containing pancreatic islet cells of the domestic fowl

Summary In an attempt to identify pancreatic islet cells emitting formaldehyde-induced fluorescence (FIF), the pancreatic islets of the domestic fowl were studied by combined fluorescence, ultrastructural, silver-impregnation and immunohistochemical methods in the same section or in consecutive semi-thin and ultra-thin sections. The results indicate that islet cells emitting intense FIF exhibit a strongly argyrophil reaction with the Grimelius' silver method and also immunohistochemical reaction with anti-glucagon serum, but not with anti-5-HT serum. Therefore, the fowl islet A cell, a peptide hormone-producing cell, stores simultaneously catecholamine as biogenic amine. The islet B and D cells did not display any FIF, any argyrophil reaction with the Grimelius' silver method, or any immunoreactivity with anti-glucagon or anti-5-HT sera. The fluorescent but non-argyrophil cells dispersed in the exocrine acinus may well be PP cells.     

The Lipid Phase of Thylakoid and Cytoplasmic Membranes from the Blue-Green Algae (Cyanobacteria), Anacystis nidulans and Anabaena variabilis

The lipid phases of the thylakoid and cytoplasmic membranesfrom the blue-green alga, Anacystis nidulans, were studied bya spin-probe method using 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl.The thylakoid and cytoplasmic membranes of this alga were bothin the liquid crystalline state at growth temperature, and inthe phase separation state at about 0?C. The thylakoid membranesentered the phase separation state at a temperature higher thanthe cytoplasmic membranes. The lipid phase of the thylakoidmembranes from Anabaena variabilis was studied in a similarway, and these membranes were found also to undergo the phasetransition. The temperature for the onset of the phase separationand the fluidity of the membrane lipids of both algae dependedon the growth temperature of the culture. (Received April 9, 1984; Accepted June 1, 1984)     

Purification and partial characterization of cytochrome b5 from Tetrahymena pyriformis

With the use of detergents and successive column chromatographies, Tetrahymena b-type cytochrome was purified from microsomes to a specific content of 36.0 nmol per mg of protein. The purified form showed a single band on SDS-polyacrylamide gel with molecular weight of 22,000. The spectral properties of the reduced b-type cytochrome, the α-peak of which is situated at 560 nm and asymmetric with a shoulder at 556 nm, was different from that of rat liver microsomal cytochrome b₅. However, it was reducible by NADH in the presence of NADH-cytochrome b₅ reductase purified from rat liver microsomes.The results indicated that the microsomal b-type cytochrome should be designated as cytochrome b₅ of a ciliated protozoan, Tetrahymena pyriformis.     

Identification of cultured and uncultured picocyanobacteria from a mesotrophic freshwater lake based on the partial sequences of 16S rDNA

Eight cultured strains (OK01, OK02, OK03, OK05, OK07, OK08, OK09, and OK10) of picocyanobacteria were isolated from Lake Okutama. Five cyanobacterial DNA fragments (DGGE bands; B4, B5, B6, B7, and B8) were obtained from the lake water samples by denaturing gradient gel electrophoresis (DGGE) after polymerase chain reaction (PCR) amplification of 16S ribosomal genes. To classify the picocyanobacterial strains and the DGGE bands, a partial sequence of 16S rDNA was used. Among seven strains, OK01, OK07, and OK09 were identified as the genus Synechococcus and OK02 and OK05 as the genus Phormidium. OK03 was identified as the genus Oscillatoria and was closely related to B4 (100% homology). B5, B6, B7, and B8 were related to the genus Synechococcus. These results revealed that the picocyanobacteria in the lake are phylogenetically diverse. PCR-DGGE analysis is a useful tool to determine picocyanobacterial community structure in freshwater environments. Received: February 25, 2001 / Accepted: July 27, 2001     

Thrombin activates p38 mitogen-activated protein kinase in vascular smooth muscle cells

Thrombin is a potent mitogen for vascular smooth muscle cells. However, the signaling pathways by which thrombin mediates its mitogenic response are not fully understood. The ERK (extracellular signal-regulated protein kinase) and JNK (c-Jun N-terminal kinase) members of the mitogen-activated protein kinase (MAPK) family are reported to be activated by thrombin. We have investigated the response to thrombin of another member of the MAPK family, p38 MAPK, which has been suggested to be activated by both stress and inflammatory stimuli in vascular smooth muscle cells. We found that thrombin induced time- and dose-dependent activation of p38 MAPK. Maximal stimulation of p38 MAPK was observed after a 10-min incubation with 1 unit ml(-1) thrombin. GF109203X, a protein kinase C inhibitor, and prolonged treatment with phorbol 12-myristate 13-acetate partially inhibited p38 MAPK activation. A tyrosine kinase inhibitor, genistein, also inhibited p38 MAPK activation in a dose-dependent manner. p38 MAPK activation was inhibited by overexpression of betaARK1ct (beta-adrenergic receptor kinase I C-terminal peptide). p38 MAPK activation was also inhibited by expression of dominant-negative Ras, not by dominant-negative Rac. We next examined the effect of a p38 MAPK inhibitor, SB203580, on thrombin-induced proliferation. SB203580 inhibited thrombin-induced DNA synthesis in a dose-dependent manner. These results suggest that thrombin activates p38 MAPK in a manner dependent on Gbetagamma, protein kinase C, a tyrosine kinase, and Ras, that p38 MAPK has a role in thrombin-induced mitogenic response in the cells.     

Heavy meromyosin and subfragment-1 from squid mantle myosin, and Ca-sensitivity of their Mg-ATPases

Heavy meromyosin (HMM) and subfragment-1 (S1) were obtained from squid mantle myosin by tryptic digestion and chymotryptic digestion, respectively. Squid HMM(T) and S1(CT) preparations contained stoichiometric amounts of the two types of light chain subunit; regulatory light chain, LC-2, and essential light chain, LC-1. No difference was detected in the chymotryptic digestibilities of squid mantle myosin in Ca-medium and in EDTA-medium. This is in contrast to the digestibility of scallop adductor myosin. The Mg-ATPase activity of HMM(T) alone and that of acto-HMM(T) were both sensitive to calcium ions. In contrast, the activity of S1(CT) alone and that of acto-S1(CT) were both insensitive to calcium ions. The affinity of HMM(T) for actin was not affected by calcium ions, but the amount of HMM(T) bound to actin was increased by calcium ions from 20% to 60% of the total amount of HMM(T). On the other hand, the actin affinity of S1(CT) and the amount of S1(CT) bound to actin were both unaffected by calcium ions. The role of calcium ions in the regulation of contraction in molluscan muscles is discussed.     

Somatic and germ cell chimerism in chimeric mice between strains of high litter size and regular litter size

CFO is an inbred strain of mice showing a high litter size. The high fertility of CFO is due mainly to a very low embryonic death rate during uterine development. C57BL and 129 strains are characterized by regular litter size. Crosses were made and CFO----C57BL and CFO----129 chimeras were produced. In CFO----C57BL mice, coat color of the C57BL predominated over that of CFO; internal chimerism except for that of the gonads was observed to be in the same proportion as the two genotypes, but the genotypic component of the gonads was almost entirely CFO. Germ cells undergoing gametogenesis in the CFO----C57BL mice were almost all derived from the CFO genotype, and the chimeric females showed high fertility just as did the CFO females. In the CFO----129 mice no obvious skewing toward one genotype was observed in the coat color, but the germ cells undergoing oogenesis in the two types of chimeric females were recognizable as nearly all of the 129 genotype but with the females showing the same high fertility as do the CFO females. This fact suggests that the genotypes of germ cells in the ovary or in developing embryos do not influence fertility, but rather that the litter size is controlled mainly by the uterine environment.     

Proteome approach for identification of schistosomiasis japonica vaccine candidate antigen

Experimental vaccination with radiation-attenuated cercariae (RAC) confers possible practical levels of resistance to challenge infection by humoral and by cellular mechanism. Here, we aimed to identify possible vaccine antigens by using specific IgG antibody from RAC vaccinated miniature pig. Two milligrams of soluble egg antigen (SEA) or schistosomal worm antigen preparation (SWAP) was fractionated using two dimensional liquid chromatography (proteome PF 2D) consisted of high performance chromatofocusing (HPCF) and high resolution reversed phase chromatography (HPRP). Of the 42 HPCF fractions of SEA or SWAP, 26 (61.9%) or 15 (35.7%) showed positive dot blot reaction with RAC vaccinated serum respectively. The dot blot positive fractions were applied to the second HPRP column. One hundred and seven out of 26 x 96 of SEA fractions and 18 out of 15 x 96 SWAP fractions reacted with RAC vaccinated serum. From the positive fractions we chose 17 of SEA and 10 of SWAP that had no reactivity with normal cercariae infected (NCI) sera and had single peak of 214 nm; and automated N-terminal amino acid sequence based on in situ Edman Reaction was conducted. Four sequences were obtained and applied to the homology search in NCBI database. A total of eight candidate genes were listed up and their cDNA clones from schistosomula stage were obtained. Two of the recombinant proteins (AAW27472.1 and AXX25883.1) showed strong reactivity with the RAC vaccinated serum but marginal with NCI serum. This protocol using proteome PF 2D could be applicable in identifying immunoreactive proteins from crude extract for the development of vaccines or for diagnostics.