A Study on 5′-Nucleotides in D2O Solution by Proton Magnetic Resonance Spectroscopy

Nucleotides, 5′-AMP, 5′-GMP, 5′-UMP, 5′-CMP and 5′-TMP, in D₂O solution have been investigated by proton magnetic resonance spectroscopy. The concentration and the pD dependences of the proton chemical shifts of the nucleotides have been examined in detail. These results indicate that intermolecular association of vertical stacking of the base rings and intramolecular association between base protons and ionized phosphate group occur in solution. The effects of the temperature and lithium ion on 5′-AMP and 5′-UMP have been also investigated. The increase of temperature causes to reduce the intramolecular association for 5′-UMP and the both intra- and intermolecular association for 5′-AMP. Lithium ion reduces the intramolecular association for both 5′-AMP and 5′-UMP, and at the same time promotes the intermolecular one for the former. This can be interpreted by the ion-pair formation of lithium ion with the ionized phosphate group.     

Establishment of hepatic and neural differentiation platforms of Wilson’s disease specific induced pluripotent stem cells

The combination of disease-specific human induced pluripotent stem cells (iPSC) and directed cell differentiation offers an ideal platform for modeling and studying many inherited human diseases. Wilson’s disease (WD) is a monogenic disorder of toxic copper accumulation caused by pathologic mutations of the ATP7B gene. WD affects multiple organs with primary manifestations in the liver and central nervous system (CNS). In order to better investigate the cellular pathogenesis of WD and to develop novel therapies against various WD syndromes, we sought to establish a comprehensive platform to differentiate WD patient iPSC into both hepatic and neural lineages. Here we report the generation of patient iPSC bearing a Caucasian population hotspot mutation of ATP7B. Combining with directed cell differentiation strategies, we successfully differentiated WD iPSC into hepatocyte-like cells, neural stem cells and neurons. Gene expression analysis and cDNA sequencing confirmed the expression of the mutant ATP7B gene in all differentiated cells. Hence we established a platform for studying both hepatic and neural abnormalities of WD, which may provide a new tool for tissue-specific disease modeling and drug screening in the future.     

Effect of cigarette smoke on the mutagenic activation of environmental carcinogens by rodent liver.

In order to assess the effect of cigarette smoke (CS) on metabolic enzymes, male hamsters and rats were exposed for two weeks to smoke produced in a Hamburg type II smoking machine. The livers were then used for Ames liquid incubation and western immunoblot assays. Mutagenic activities of seven heterocyclic amines (HCAs) in Salmonella typhimurium TA98 in the presence of rat or hamster liver S9 were elevated up to 3.7 times above controls (including sham smoke control). Enhancement of mutagenic activities of PhIP and aflatoxin B(1) was observed only in CS-exposed hamster, whereas no significant alteration of mutagenicity was observed with 2-aminofluorene, benzo[a]pyrene, and 3'-hydroxymethyl-N, N-dimethyl-4-aminoazobenzene in strain TA98 or with six N-nitrosodialkylamines in strain TA100. 7,8-Benzoflavone and/or furafylline considerably inhibited the mutagenic activation of IQ and Trp-P-1 in the presence of liver S9 from untreated hamsters and sham smoke- or CS-exposed hamsters and rats, indicating the predominant involvement of hamster cytochrome P450 (CYP) 1A enzymes in the metabolic activation of HCAs. In addition, the data suggest that CS-exposure may selectively induce hepatic CYP1A1/1A2 isoforms. Western immunoblot analyses of liver microsomes using anti-rat CYP antibodies revealed that CS-exposure increased the levels of hamster CYP1A2 (3.9-fold) and rat CYP1A2 (3.0-fold) and CYP1A1, without significant change in the levels of CYP2E1 and CYP2B and 3A isoforms in each species. The presently observed selective induction of HCA activation and CYP isozymes due to CS supports the idea that CS may contribute to enhancing effects on initiation by carcinogens which are metabolically activated by hepatic CYP1A1/1A2. In conjunction with results observed for smokers, the present findings indicate that the hamster is a good animal for studies with CS, and that cigarette smoking in combination with intake of heating protein-rich foods as a life style may markedly contribute to the human carcinogenesis by HCAs.     

Gliclazide protects pancreatic beta-cells from damage by hydrogen peroxide

Oxidative stress is induced under diabetic conditions and possibly causes various forms of tissue damage in patients with diabetes. Recently, it has become aware that susceptibility of pancreatic beta-cells to oxidative stress contributes to the progressive deterioration of beta-cell function in type 2 diabetes. A hypoglycemic sulfonylurea, gliclazide, is known to be a general free radical scavenger and its beneficial effects on diabetic complications have been documented. In the present study, we investigated whether gliclazide could protect pancreatic beta-cells from oxidative damage. One hundred and fifty microM hydrogen peroxide reduced viability of mouse MIN6 beta-cells to 29.3%. Addition of 2 microM gliclazide protected MIN6 cells from the cell death induced by H(2)O(2) to 55.9%. Glibenclamide, another widely used sulfonylurea, had no significant effects even at 10 microM. Nuclear chromatin staining analysis revealed that the preserved viability by gliclazide was due to inhibition of apoptosis. Hydrogen peroxide-induced expression of an anti-oxidative gene heme oxygenase-1 and stress genes A20 and p21(CIP1/WAF1), whose induction was suppressed by gliclazide. These results suggest that gliclazide reduces oxidative stress of beta-cells by H(2)O(2) probably due to its radical scavenging activity. Gliclazide may be effective in preventing beta-cells from the toxic action of reactive oxygen species in diabetes.     

Reproduction,grazing, and development of the large subarctic calanoid <Emphasis Type="Italic">Eucalanus bungii</Emphasis>: is the spring diatom bloom the key to controlling their recruitment?

The response of large calanoid, Eucalanus bungii, to environmental fluctuation, particularly in relation to the spring diatom bloom in the Oyashio region, western subarctic Pacific Ocean, was examined by investigating egg production, grazing, development and starvation tolerance. Mean in situ egg production rate increased with ambient chlorophyll-a concentration, ranging from 0 to 47 eggs female⁻¹ d⁻¹, while no diurnal synchronous spawning behavior was observed. Under the spring bloom condition, E. bungii showed prey preference for less mobile and larger-sized prey (≥30 μm ESD) and bloom-forming diatom Thalassiosira spp. accounted for >80% of ingested carbon. In the laboratory, E. bungii was successfully reared from newly hatched nauplii to adult with the diatom, Thalassiosira nordenskioldi, as a food resource. Nauplii newly hatched from eggs reached the adult stage in ca. 150 days (5°C) with a sigmoidal developmental pattern and no sexual difference in development pattern. Starvation experiments indicated that the starved copepodids (C1–C4) became more vulnerable to high temperature with the progression of developmental stage, suggesting that the post-bloom condition with low food availability and increased temperature is harsh for their copepodids. The results of this study in conjunction with previous findings suggest that E. bungii is well adapted to utilize large-sized phytoplankton, such as a bloom-forming diatoms and, therefore, their recruitment processes, including egg production, development and mortality would be strongly affected by the duration and intensity of the spring bloom.     

The large linear plasmid pSLA2-L of Streptomyces rochei has an unusually condensed gene organization for secondary metabolism

The complete nucleotide sequence of the large linear plasmid pSLA2-L in Streptomyces rochei strain 7434AN4 has been determined. pSLA2-L was found to be 210 614 bp long with a GC content of 72.8% and carries 143 open reading frames. It is especially noteworthy that three-quarters of the pSLA2-L DNA is occupied by secondary metabolism-related genes, namely two type I polyketide synthase (PKS) gene clusters for lankacidin and lankamycin, a mithramycin synthase-like type II PKS gene cluster, a carotenoid biosynthetic gene cluster and many regulatory genes. In particular, the lankacidin PKS is unique, because it may be a mixture of modular- and iterative-type PKSs and carries a fusion protein of non-ribosomal peptide synthetase and PKS. It is also interesting that all the homologues of the afsA, arpA, adpA and strR genes in the A-factor regulatory cascade in Streptomyces griseus were found on pSLA2-L, and disruption of the afsA homologue caused non-production of both lankacidin and lankamycin. These results, together with the finding of three possible replication origins at 50-63 kb from the right end, suggest that the present form of pSLA2-L might have been generated by a series of insertions of the biosynthetic gene clusters into the left side of the original plasmid.     

Structures and Syntheses of Two Phenolic Amides from Piper nigrum L.

Two phenolic amides were isolated from the fruits of white pepper (Piper nigrum L.) and identified to be N-trans-feruloyl tyramine (2a) and N-5-(4-hydroxyphenyl)-2E, 4E-pentadienoyl piperidine (6a) on the basis of chemical and specrtal evidence. Both compounds were synthesized.     

Peroxynitrite augments fibroblast-mediated tissue remodeling via myofibroblast differentiation

Irreversible airflow limitation in asthma is associated with airway remodeling in which the differentiation of fibroblasts to myofibroblasts plays a pivotal role. In asthmatic airways, excessive production of reactive nitrogen species (RNS) has been observed. The aim of this study is to evaluate whether peroxynitrite, one of the RNS, can affect the differentiation of fibroblasts to myofibroblasts. Human fetal lung fibroblasts were treated with various concentrations of authentic peroxynitrite or a peroxynitrite donor 3-morpholinosydnonimine hydrochloride (SIN-1), and the expressions of alpha-smooth muscle actin (alpha-SMA) and desmin, markers of myofibroblast differentiation, were evaluated. The releases of transforming growth factor-beta(1) (TGF-beta(1)) and ECM proteins including fibronectin and collagen I were assessed. To clarify the mechanism in this differentiation, the effect of anti-TGF-beta antibody or NF-kappaB inhibitors on the alpha-SMA expression and ECM production was assessed. Peroxynitrite and SIN-1 significantly augmented the alpha-SMA expression compared with control in a concentration-dependent manner (P < 0.01 and P < 0.05, respectively). Peroxynitrite significantly increased desmin and TGF-beta(1) production (P < 0.01). Peroxynitrite enhanced the translocation of NF-kappaB into the nucleus confirmed by immunocytostaining and immunoblotting. Peroxynitrite-augmented alpha-SMA expression was blocked by NF-kappaB inhibitors, MG132 and caffeic acid phenethyl ester (CAPE), and anti-TGF-beta antibody. CAPE completely inhibited the peroxynitrite-augmented TGF-beta(1) release. The production of fibronectin and collagen I was significantly increased by peroxynitrite (P < 0.01) and inhibited by anti-TGF-beta antibody. These results suggest that RNS can affect the differentiation to myofibroblasts and excessive ECM production via a NF-kappaB-TGF-beta(1)-dependent pathway.