NADH dehydrogenase [EC 1.6.99.3] in membranes of Bacillus caldotenax was solubilized with sodium N-lauroylsarcosinate and purified 50-fold from membranes to 75-80% homogeneity, as judged by SDS-polyacrylamide gel electrophoresis. The enzyme was considered to be located on the electron transport chain and to be an FAD-containing protein. The molecular weight of the subunit was estimated to be 44,000. The enzyme (or the enzyme bound to the B. caldotenax membrane lipids) follows a ping-pong mechanism. The enzyme can oxidize NADH, but not NADPH, with 2,6-dichlorophenol indophenol, ferricyanide, menadione, and cytochrome c as electron acceptors. Membrane lipids or Triton X-100 stimulated the enzyme activity, except that with menadione. Lipids decreased the apparent affinity of electron acceptors and NADH to the enzyme, and increased the maximum velocity, except when menadione was used as the electron acceptor. Lipids partially protected the enzyme from thermal inactivation. The enzyme exhibited a continuous Arrhenius plot, while the lipids- or membrane-bound enzyme exhibited a discontinuous plot.

Bacillus↗

PubMed1980

Change in chemical composition of membrane of Bacillus caldotenax after shifting the growth temperature.

Membranes from Bacillus caldotenax contain neutral lipids and phospholipids such as phosphatidyl-ethanolamine, phosphatidyl glycerol and cardiolipin. Each of the lipids has almost the same fatty acid composition. When the growth temperature decreases, not only the fatty acid composition but also the lipid composition changes such that the membrane fluidity increases, and the composition of membrane-bound proteins also changes. On shifting the growth temperature from 65 degrees to 45 degress C, the bacterium grows immediately with a doubling time at 45 degrees C, but the compositions of proteins and lipids in membranes gradually change and reach the compositions typical of cells growing at 45 degrees C one doubling time after the temperature shift, respectively. It is concluded that the change in chemical composition of membrane of the bacterium on the temperature shift from 65 degrees to 45 degrees C is not prerequisite for growth at 45 degrees C.

Bacillus↗

PubMedDate not supplied

Effects of arachidonic acid metabolites and interleukin-1 on platelet activating factor production by hepatic sinusoidal endothelial cells from mice.

The effects of interleukin-1 (IL-1) and arachidonic acid metabolites, prostaglandin (PG) E1, leukotriene (LT) B4 and LTC4, on the amount of platelet activating factor (PAF) produced and released by mouse hepatic sinusoidal endothelial cells were investigated. When hepatic sinusoidal endothelial cells were incubated with 1 microgram/mL of calcium ionophore (CaI) A23187, PAF production increased until 20 min after the start of incubation, but when the cells were incubated with PGE1 and CaI A23187, PAF production was significantly suppressed. On the other hand, when hepatic sinusoidal endothelial cells were incubated with IL-1 beta, LTB4 or LTC4 alone, PAF production significantly increased compared with that of the cells incubated without these substances. However, when the cells were incubated with PGE1 alone or with IL-1 beta, LTB4 or LTC4 and CaI A23187, these effects were not observed. These results indicated that PAF produced by hepatic sinusoidal endothelial cells is affected by IL-1 and arachidonic acid metabolites, suggesting that immune and inflammatory reactions in these cells may be regulated by chemical mediators produced by the cells themselves.

Alprostadil↗

PubMedDate not supplied

Establishment and characteristics of human hepatocellular carcinoma cells with metastasis to lymph nodes.

BACKGROUND/AIMS: Although the mechanism of cancer metastasis has been gradually elucidated, less is known concerning the characteristics of human hepatocellular carcinomas (HCCs) with metastatic potential. We examined the expression of molecules that mediate cell-cell or cell-substrate interaction, nm23-H1 expression, and ultrastructural features of several human HCC cell lines. METHODOLOGY: Expression of E-cadherin, integrin (alpha3beta1), intracellular adhesion molecule-1 (ICAM-1), and nm23-H1 protein was analyzed by immunocytochemistry or Western blotting, and ultrastructural features were further studied by electron microscopy in 4 human HCC cell lines, PLC/PRF/5, HuH-7, OCUH-16, and Nuk-1 which were originally established from metastatic cells in lymph nodes at our institute. RESULTS: Neither E-cadherin, integrin, nor ICAM-1 was immunocytochemically detected in any of the 4 cell lines. Expression of nm23-H1 protein was weakly detected in OCUH-16, Nuk-1, and Huh-7 cells by Western blotting, but was clearly detected in PLC/PRF/5 cells by Western blotting. Ultrastructurally, metastatic Nuk-1 cells exhibited the intracytoplasmic canaliculus-like structures found in fibrolamellar carcinoma and the intracytoplasmic glandular lumina found in bile-duct carcinoma, while the other 3 cell lines did not. In addition, Nuk-1 cells expressed neither cytokeratin 8 nor cytokeratin 19. CONCLUSIONS: Nuk-1 cells, which are human HCC cells with metastasis to lymph nodes, alone exhibited intracytoplasmic canaliculus-like structures and glandular lumina, as well as a marked reduction of nm23-H1 protein, but did not express E-cadherin, integrin, or ICAM-1. Formation of both intracytoplasmic canaliculus-like structures and intracytoplasmic glandular lumina is one of the characteristics that may be involved in metastasis of HCC cells to lymph nodes, as is reduction of nm23-H1 protein.

Biomarkers, Tumor↗

PubMedDate not supplied

Angiotensinogen gene polymorphism of threonine/methionine at position 235-potential problems of the modified restriction endonuclease (Tth111-I) digestion method.

There are four methods for detecting the angiotensinogen gene Agt T235/M235; 1) allele-specific oligonucleotide hybridization (PCR-ASO); 2) mutagenically separated PCR (MS-PCR) using three primers to amplify all possible alleles in on PCR reaction; 3) restriction endonuclease Tth111-I; 4) SfaN-I digestion method using mismatched primer for the PCR (modified PCR-RFLP). Two of these four methods have been used in Japanese studies. The reported allelic frequencies of Agt T235/M235 in normal controls in the Japanese population are around 0.75-0.84/0.25-0.16 (as a whole 0.80/0.20) by PCR-ASO and 0.70-0.65/0.30-0.35 (as a whole 0.67/0.33) by the modified PCR-RFLP (Tth111-I). The present study tested how these methods contribute to the differences in Agt T235/M235. By PCR-ASO, the genotypes could be clearly determined. However, it is hard to complete every digestive reaction under the experimental conditions described for modified PCR-RFLP (Tth111-I). Thus, for studying Agt T235/M235, PCR-ASO or some method other than PCR-RFLP (Tth111-I) can be recommended. Our findings suggest that the allele frequency of Agt T235/M235 in the normal Japanese population is closer to 0.75-0.84/0.25-0.16. Although a strong association was reported between the Agt T235 allele and essential hypertension or myocardial infarction, using the modified PCR-RFLP (Tth111-I), two of three studies using PCR-ASO found no or only a weak association. The relationship between the Agt T235 allele and essential hypertension or myocardial infarction in the Japanese population needs to be assessed.

Angiotensinogen↗