Enhancement of Nitrogen Dioxide-Induced Lipid Peroxidation and Dna Strand Breaking by Cysteine and Glutathione

Nitrogen dioxide less than 100 ppm in air induced lipid peroxidation of liposome composed of l-palmitoyl-2-arachidonylphosphatidylcholine as assessed by thiobarbituric acid reactivity. The nitrogen dioxide-induced lipid peroxidation was enhanced by cysteine, glutathione and bovine serum albumin. While the activity of nitrogen dioxide in air to induce single strand breaks of supercoiled plasmid DNA was low, the breaking was remarkably enhanced by cysteine, glutathione and bovine serum albumin. ESR spin trapping using 5,5-dimethyl-1-pyrroline N-oxide showed that certain strong oxidant(s) were generated by interaction of nitrogen dioxide and cysteine. The spin trapping using 3,5-dibromo-4-nitrosobenzene-sulfonate suggested that sulfur-containing radicals were generated by interaction of nitrogen dioxide and cysteine or glutathione. Hence, certain sulfur-containing radicals generated by the interaction which could effectively induce lipid peroxidation and DNA strand breaks.     

Biosynthesis of various types of collagen by human hepatoma cells in vitro

A cloned human hepatoma cell line (HH2-1) produced and formed collagen fibers in vitro. The relative rate of collagen synthesis by the cells was increased with an enhancement of the cell density. An analysis of the components of the collagen using sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the cells synthesized interstitial collagen, types I and III, and other collagenous proteins. Thus, human hepatoma cells may play an important role in the formation of stromal collagen in the tumor.     

Digestibility of the hydrogenated derivative of an isomaltooligosaccharide mixture by rats.

The digestibility of the hydrogenated derivative of an isomaltooligosaccharide mixture (IMO-H) was investigated. In an in vitro experiment, the digestibility of IMO-H was examined by models of the digestive system. IMO-H was resistant to two types of alpha-amylase and to artificial gastric juice. Enzymes in the rat small intestinal mucosa hydrolyzed tri-, tetra- and higher saccharide alcohols to disaccharide alcohol, removing successive glucose units from the non-reducing ends of the chains. The hydrolysis ratio for IMO-H was intermediate between the values for maltose and maltitol. In an in vivo study, growing rats were fed on an experimental diet containing IMO-H, maltitol, or hydrogenated palatinose in the range from 5% to 20%. The growth parameters of the rats fed on the test sugar show that the availability of IMO-H was about 1.2 to 1.25 times that of maltitol or hydrogenated palatinose.     

Beta-sheet folding of fragment (16-36) of bovine pancreatic trypsin inhibitor as predicted by Monte Carlo simulated annealing.

A tertiary structure prediction is described using Monte Carlo simulated annealing for the peptide fragment corresponding to residues 16-36 of bovine pancreatic trypsin inhibitor (BPTI). The simulation starts with randomly chosen initial conformations and is performed without imposing experimental constraints using energy functions given for generic interatomic interactions. Out of 20 simulation trials, seven conformations show a sheet-like structure--two strands connected by a turn--although this sheet-like structure is not as rigid as that observed in native BPTI. It is also shown that these conformations are mostly looped and exhibit a native-like right-handed twist. Unlike the case with the C-peptide of RNase A, no conspicuous alpha-helical structure is found in any of the final conformations obtained in the simulation. However, the lowest-energy conformation does not resemble exactly the native structure. This indicates that the rigid beta-sheet conformation of native BPTI merely corresponds to a local minimum of the energy function if the fragment with residues 16-36 is isolated from the native protein. A statistical analysis of all 20 final conformations suggests that the tendency for the peptide segments to form extended beta-strands is strong for those with residues 18-24, and moderate for those with residues 30-35. The segment of residues 25-29 does not tend to form any definite structure. In native BPTI, the former segments are involved in the beta-sheet and the latter in the turn. A folding scenario is also speculated from this analysis.     

Characterization of transverse tubule membrane proteins: tentative identification of the Mg-ATPase

Vesiculated fragments of chicken skeletal muscle transverse tubule (TT) membranes were analyzed for their content of loosely associated and integral membrane proteins. Of particular interest was the identification of the magnesium-stimulated ATPase (Mg-ATPase), which is characteristically located in native isolated TT vesicles of chicken skeletal muscle [R. A. Sabbadini and V. R. Okamoto (1983) Arch. Biochem. Biophys. 223, 107-119]. A number of the proteins found in vesicular TT preparations were found to be extractable by a mild Triton-X100 treatment and were identified as aldolase, enolase, creatine kinase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and pyruvate kinase. Approximately 60% of TT-associated protein was extracted with Triton, resulting in a twofold enrichment of the Mg-ATPase. Concommitantly, one core integral membrane protein possessing a Mr of 102,000 was enriched, suggesting that it is responsible for the Mg-ATPase activity present in chicken skeletal muscle TT membranes.     

MiR-376c Down-Regulation Accelerates EGF-Dependent Migration by Targeting GRB2 in the HuCCT1 Human Intrahepatic Cholangiocarcinoma Cell Line

MicroRNA miR-376c was expressed in normal intrahepatic biliary epithelial cells (HIBEpiC), but was significantly suppressed in the HuCCT1 intrahepatic cholangiocarcinoma (ICC) cell line. The biological significance of the down-regulation of miR-376c in HuCCT1 cells is unknown. We hypothesized that miR-376c could function as a tumor suppressor in these cells. To test this hypothesis, we sought the targets of miR-376c, and characterized the effect of its down-regulation on HuCCT1 cells. We performed proteomic analysis of miR-376c-overexpressing HuCCT1 cells to identify candidate targets of miR-376c, and validated these targets by 3′-UTR reporter assay. Transwell migration assays were performed to study the migratory response of HuCCT1 cells to miR-376c overexpression. Furthermore, microarrays were used to identify the signaling that were potentially involved in the miR-376c-modulated migration of HuCCT1. Finally, we assessed epigenetic changes within the potential promoter region of the miR-376c gene in these cells. Proteomic analysis and subsequent validation assays showed that growth factor receptor-bound protein 2 (GRB2) was a direct target of miR-376c. The transwell migration assay revealed that miR-376c significantly reduced epidermal growth factor (EGF)-dependent cell migration in HuCCT1 cells. DNA microarray and subsequent pathway analysis showed that interleukin 1 beta and matrix metallopeptidase 9 were possible participants in EGF-dependent migration of HuCCT1 cells. Bisulfite sequencing showed higher methylation levels of CpG sites upstream of the miR-376c gene in HuCCT1 relative to HIBEpiC cells. Combined treatment with the DNA-demethylating agent 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor trichostatin A significantly upregulated the expression of miR-376c in HuCCT1 cells. We revealed that epigenetic repression of miR-376c accelerated EGF-dependent cell migration through its target GRB2 in HuCCT1 cells. These findings suggest that miR-376c functions as a tumor suppressor. Since metastasis is the major cause of death in ICC, microRNA manipulation could lead to the development of novel anti-cancer therapy strategies for ICC.     

Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe

Integration of an external gene into a fission yeast chromosome is useful to investigate the effect of the gene product. An easy way to knock-in a gene construct is use of an integration plasmid, which can be targeted and inserted to a chromosome through homologous recombination. Despite the advantage of integration, construction of integration plasmids is energy- and time-consuming, because there is no systematic library of integration plasmids with various promoters, fluorescent protein tags, terminators and selection markers; therefore, researchers are often forced to make appropriate ones through multiple rounds of cloning procedures. Here, we establish materials and methods to easily construct integration plasmids. We introduce a convenient cloning system based on Golden Gate DNA shuffling, which enables the connection of multiple DNA fragments at once: any kind of promoters and terminators, the gene of interest, in combination with any fluorescent protein tag genes and any selection markers. Each of those DNA fragments, called a ‘module’, can be tandemly ligated in the order we desire in a single reaction, which yields a circular plasmid in a one-step manner. The resulting plasmids can be integrated through standard methods for transformation. Thus, these materials and methods help easy construction of knock-in strains, and this will further increase the value of fission yeast as a model organism.     

PISTIL DEVELOPMENT AND PARIETAL PLACENTATION IN THE PSEUDOMONOMEROUS OVARY OF ZELKOVA SERRATA (ULMACEAE)

Development of female flowers in Zelkova serrata was observed using epi-illuminated microscopy and scanning electron microscopy, with particular attention given to placentation. After the inception of staminodial primordia, the floral apex becomes flat, and the first and subsequently the second carpel primordia appear at opposite comers of the pistil primordium. Inside each carpel primordium a fossette forms. Through differential growth this depression becomes clear and the carpel wall encircles one side of the future placental region. The placental region is detectable even in early stages, but clear signs of ovule inception appear late when the placental region is elevated onto one side of the ovary wall by intercalary growth. Although the relative size of the two carpels varies among flowers, the placental position always appears to be the border between the two carpels and the floral apex. This suggests that the placentation of Zelkova is parietal. The ovule position in tricarpellate ovaries also suggests an evolutionary derivation from ovaries with parietal placentation. Parietal placentation appears to be the original condition in Urticales.     

Roles of neonatal and prepubertal testicular androgens on androgen-induced proliferative response of seminal vesicle cells in adult mice

Male mice were castrated at 0, 10, 20, 30, 40 and 60 days of age; daily injections of testosterone propionate (TP, 4 micrograms/g b. wt) were started from day 90. On various days after starting the TP injections, the incorporation of 5-[125I]iodo-2'-deoxyuridine into the whole seminal vesicles was determined as an index for proliferation. The seminal vesicle cells in mice castrated on days 0 and 20 were characterized by low weight (0.5-1 mg) before TP injection, long duration of androgen-induced proliferation (greater than 20 days) with a low peak, and involvement of both epithelial and fibromuscular cells (neonatal castration type). The seminal vesicle cells in mice castrated on days 60 and 40 were characterized by relatively high weight (5-10 mg) before TP injection, short duration of androgen-induced proliferation (10 days) with a high peak, and involvement of only the epithelial cells (adult castration type). In mice castrated on days 0 and 20, the neonatal castration type of androgen-induced proliferation was completely changed to the adult castration type when TP pretreatment (2 micrograms/g b. wt per 12 h) had been given from day 20 to day 40. However, the TP pretreatment given from day 90 to day 110 instead of days 20-40 had no such effect in 140-day old mice castrated on day 0. The present findings suggest that testicular androgens secreted from day 20 to day 40 play an indispensable role in the induction of irreversible proliferative response of the mouse seminal vesicle. The activity of the prepubertal androgens may not be completely compensated by androgen activity at adulthood.     

Aldosterone biosynthesis and cytochrome P-45011 beta: evidence for two different forms of the enzyme in rats

Whereas cytochrome P-45011 beta has been recently shown to catalyze the two-step conversion of corticosterone to aldosterone in the bovine and porcine adrenal cortex, the identity of the enzyme involved in the two final steps of aldosterone biosynthesis in the rat adrenal cortex is as yet unknown. Mitochondria from capsular adrenals of sodium-deficient, potassium-replete rats converted corticosterone to 18-hydroxycorticosterone and aldosterone at markedly higher rates than mitochondria from capsular adrenals of sodium-replete, potassium-deficient rats. However, the same preparations exhibited no difference in the 11 beta-hydroxylase activity, i.e. the conversion of deoxycorticosterone to corticosterone. Only mitochondria of zona glomerulosa from rats with stimulated aldosterone biosynthesis contained a 49K protein which showed a strong cross-reactivity with a monoclonal antibody raised against purified bovine cytochrome P-45011 beta. By contrast, a crossreactive protein with a molecular weight of 51K was found in mitochondria of zona fasciculata and in mitochondria of zona glomerulosa from rats with a suppressed aldosterone biosynthesis. These findings indicate the existence of two different forms of cytochrome P-45011 beta in the rat adrenal cortex, with only one of them, i.e. the 49K form, being capable of catalyzing the two final steps of aldosterone biosynthesis in situ.