Spin Trapping Isotopically-Labelled Nitric Oxide Produced from [15n]L-Arginine and [17ojdioxygen by Activated Macrophages Using a Water Soluble Fe++-Dithiocarbamate Spin Trap

The unique capabilities of EPR spin trapping of nitric oxide based on a ferrous-dithiocarbamate spin trap have been demonstrated in a study verifying the source of the nitrogen and oxygen atoms in nitric oxide produced from activated macrophages. Spin trapping experiments were performed during nitric oxide generation from activated mouse peritoneal macrophages using the ferrous complex of N-methyl D-glucam-ine dithiocarbamate as a spin trap. When ¹⁵N-substituted arginine was given to the activated macrophages in the presence of the spin trap, a characteristic EPR spectrum of the nitric oxide spin adduct was obtained, which indicates the presence of the ^l5N atom in the nitric oxide molecule. The hyperfine splitting (hfs) constant of the ^l5N nucleus was 17.6 gauss. When ^l7O-containing dioxygen (55%) was supplied to the medium, an EPR spectrum consistent with the “O-substituted nitric oxide spin adduct was observed in the composite spectrum. The hfs of “O was estimated to be 2.5 gauss. The ^l4NO spin adduct observed after prolonged incubation in the medium which contains [^l5N]L-arginine as the only extracellular source of arginine demonstrates that arginine is recycled through its metabolite in activated macrophages.     

Inhibition of the Bacterial Cell Wall Synthesis in vitro by Enduracidin,a New Polypeptide Antibiotic

Adenosine 5′-phosphosulfate (APS) kinase from a thermophilic bacterium, Bacillus st ear other mophilus, was purified to apparent homogeneity. The apparent molecular weight was 50 kDa, consisting of two 26-kDa subunits. The enzyme was very thermostable and lacked cysteine and methionine residues. Enzyme activity was more stimulated with Mn^{2 +} , Zn^{2 +}, or Co^{2 +} than with Mg^{2 +} and the K_m for ATP and APS were 220 µM and 42 µM, respectively.     

Isolation and characterization of a cDNA encoding mitochondrial chaperonin 10 from Arabidopsis thaliana by functional complementation of an Escherichia coli groES mutant

Chaperonin (Cpn) is one of the molecular chaperones. Cpn10 is a co-factor of Cpn60, which regulates Cpn60-mediated protein folding. It is known that Cpn10 is located in mitochondria and chloroplasts in plant cells. The Escherichia coli homologue of Cpn10 is called GroES. A cDNA for the Cpn10 homologue was isolated from Arabidopsis thaliana by functional complementation of the E. coli groES mutant. The cDNA was 647 bp long and encoded a polypeptide of 98 amino acids. The deduced amino acid sequence showed approximately 50% identity to mammalian mitochondrial Cpn10s and 30% identity to GroES. A Northern blot analysis revealed that the mRNA for the Cpn10 homologue was expressed uniformly in various organs and was markedly induced by heat-shock treatment. The Cpn10 homologue was constitutively expressed in transgenic tobaccos. Immunogold and immunoblot analyses following the subcellular fractionation of leaves from transgenic tobaccos revealed that the Cpn10 homologue was localized in mitochondria and accumulated at a high level in transgenic tobaccos.     

Genetic analysis of human lymphocyte proteins by two-dimensional gel electrophoresis: 4. Genetic polymorphism of cytosol 100k polypeptide

Summary We describe a genetic polymorphism of a human cellular polypeptide with mol. wt. 100,000, detected in peripheral blood lymphocytes by high resolution two-dimensional electrophoresis. Three different electrophoretic types (1-1, 2-1, and 2-2) of the polypeptide have been identified. Family and population studies indicate that the three phenotypes of the polypeptide are determined by two common alleles at a single autosomal locus. The polypeptide occurs in the cytosol and is one of the abundant polypeptides of B-lymphoblastoid cells, T-lymphoblastoid cells, fibroblasts, and HeLa cells. The data indicate that the cytosol polypeptide with mol.wt. 100,000 shows a genetic polymorphism determined by aew autosomal locus. It is proposed that the polypeptide and its locus be temporarily designated cytosol 100k polypeptide (C100k polypeptide) and C100P, respectively. In a Japanese population, the gene frequencies of C100P ¹ and C100P ² were 0.907 and 0.093, respectively.     

Interleukin 6 Receptor Is an Independent Prognostic Factor and a Potential Therapeutic Target of Ovarian Cancer

Ovarian cancer remains the most lethal gynecologic cancer and new targeted molecular therapies against this miserable disease continue to be challenging. In this study, we analyzed the expressional patterns of Interleukin-6 (IL-6) and its receptor (IL-6R) expression in ovarian cancer tissues, evaluated the impact of these expressions on clinical outcomes of patients, and found that a high-level of IL-6R expression but not IL-6 expression in cancer cells is an independent prognostic factor. In in vitro analyses using ovarian cell lines, while six (RMUG-S, RMG-1, OVISE, A2780, SKOV3ip1 and OVCAR-3) of seven overexpressed IL-6R compared with a primary normal ovarian surface epithelium, only two (RMG-1, OVISE) of seven cell lines overexpressed IL-6, suggesting that IL-6/IL-6R signaling exerts in a paracrine manner in certain types of ovarian cancer cells. Ovarian cancer ascites were collected from patients, and we found that primary CD11b⁺CD14⁺ cells, which were predominantly M2-polarized macrophages, are the major source of IL-6 production in an ovarian cancer microenvironment. When CD11b⁺CD14⁺ cells were co-cultured with cancer cells, both the invasion and the proliferation of cancer cells were robustly promoted and these promotions were almost completely inhibited by pretreatment with anti-IL-6R antibody (tocilizumab). The data presented herein suggest a rationale for anti-IL-6/IL-6R therapy to suppress the peritoneal spread of ovarian cancer, and represent evidence of the therapeutic potential of anti-IL-6R therapy for ovarian cancer treatment.     

Suppression of Hydroxycinnamate Network Formation in Cell Walls of Rice Shoots Grown under Microgravity Conditions in Space

Network structures created by hydroxycinnamate cross-links within the cell wall architecture of gramineous plants make the cell wall resistant to the gravitational force of the earth. In this study, the effects of microgravity on the formation of cell wall-bound hydroxycinnamates were examined using etiolated rice shoots simultaneously grown under artificial 1 g and microgravity conditions in the Cell Biology Experiment Facility on the International Space Station. Measurement of the mechanical properties of cell walls showed that shoot cell walls became stiff during the growth period and that microgravity suppressed this stiffening. Amounts of cell wall polysaccharides, cell wall-bound phenolic acids, and lignin in rice shoots increased as the shoot grew. Microgravity did not influence changes in the amounts of cell wall polysaccharides or phenolic acid monomers such as ferulic acid (FA) and p-coumaric acid, but it suppressed increases in diferulic acid (DFA) isomers and lignin. Activities of the enzymes phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) in shoots also increased as the shoot grew. PAL activity in microgravity-grown shoots was almost comparable to that in artificial 1 g-grown shoots, while CW-PRX activity increased less in microgravity-grown shoots than in artificial 1 g-grown shoots. Furthermore, the increases in expression levels of some class III peroxidase genes were reduced under microgravity conditions. These results suggest that a microgravity environment modifies the expression levels of certain class III peroxidase genes in rice shoots, that the resultant reduction of CW-PRX activity may be involved in suppressing DFA formation and lignin polymerization, and that this suppression may cause a decrease in cross-linkages within the cell wall architecture. The reduction in intra-network structures may contribute to keeping the cell wall loose under microgravity conditions.     

Relationship between body condition of American alligators and water depth in the Everglades, Florida

Feeding opportunities of American alligators (Alligator mississippiensis) in freshwater wetlands in south Florida are closely linked to hydrologic conditions. In the Everglades, seasonally and annually fluctuating surface water levels affect populations of aquatic organisms that alligators consume. Since prey becomes more concentrated when water depth decreases, we hypothesized an inverse relationship between body condition and water depth in the Everglades. On average, condition of adult alligators in the dry season was significantly higher than in the wet season, but this was not the case for juveniles/subadults. The correlation between body condition and measured water depth at capture locations was weak; however, there was a significant negative correlation between the condition and predicted water depth prior to capture for all animals except for spring juveniles/subadults which had a weak positive condition–water depth relationship. Overall, a relatively strong inverse correlation occurred at 10–49 days prior to the capture day, suggesting that current body condition of alligators may depend on feeding opportunities during that period. Fitted regression of body condition on water depth (mean depth of 10 days when condition-water depth correlation was greatest) resulted in a significantly negative slope, except for spring adult females and spring juveniles/subadults for which slopes were not significantly different from zero. Our results imply that water management practices may be critical for alligators in the Everglades since water depth can affect animal condition in a relatively short period of time.     

African American-preponderant single nucleotide polymorphisms (SNPs) and risk of breast cancer

Background: African American women more often present with more aggressive types of breast cancer than Caucasian women, but little is known whether genetic polymorphisms specific to or disproportionate in African Americans are associated with their risk of breast cancer. Methods: A population-based case-control study was conducted including 194 cases identified through the Metropolitan Detroit Cancer Surveillance System and 189 controls recruited through random digit dialing to examine polymorphisms in genes involved in estrogen metabolism and action. Results: The African American-specific CYP1A1 5639C allele was associated with an increased risk of breast cancer (odds ratio (OR) = 2.34, 95% confidence interval (CI) 1.23–4.44) and this association with the CYP1A1 5639 locus was dependent on another polymorphism in the CYP3A4 gene (P = 0.043 for the interaction). In addition, African American-predominant CYP1B1 432 Val allele was significantly more often found in the cases than in the controls overall and the HSD17B1 312 Gly allele was specifically associated with premenopausal breast cancer risk (OR = 3.00, 95%CI 1.29–6.99). Conclusion: These observations need to be confirmed in larger studies due to the limited statistical power of the study based on a small number of cases.