Characterization of a HMT2-like enzyme for sulfide oxidation from Pseudomonas putida

The open reading frame pp0053, which has a high homology with the sequence of mitochondrial sulfide dehydrogenase (HMT2) conferring cadmium tolerance in fission yeast, was amplified from Pseudomonas putida KT2440 and expressed in Escherichia coli JM109(DE3). The isolated and purified PP0053-His showed absorption spectra typical of a flavin adenine dinucleotide (FAD)--binding protein. The PP0053-His catalyzed a transfer of sulfide-sulfur to the thiophilic acceptor, cyanide, which decreased the Km value of the enzyme for sulfide oxidation and elevated the sulfide-dependent quinone reduction. Reaction of the enzyme with cyanide elicited a dose-dependent formation of a charge transfer band, and the FAD-cyanide adduct was supposed to work for a sulfur transfer. The pp0053 deletion from P. putida KT2440 led to activity declines of the intracellular catalase and ubiquinone-H2 oxidase. The sulfide-quinone oxidoreductase activity in P. putida KT2440 was attributable to the presence of pp0053, and the activity showed a close relevance to enzymatic activities related to sulfur assimilation.     

Early candidacy for differentiation into heterocysts in the filamentous cyanobacterium Anabaena sp. PCC 7120

The filamentous cyanobacterium Anabaena sp. PCC 7120 fixes dinitrogen facultatively. Upon depletion of combined nitrogen, about 10% of vegetative cells within the filaments differentiate terminally into nitrogen-fixing cells. The heterocyst has been studied as a model system of prokaryotic cell differentiation, with major focus on signal transduction and pattern formation. The fate of heterocyst differentiation is determined at about the eighth hour of induction (point of no return), well before conspicuous morphological or metabolic changes occur. However, little is known about how the initial heterocysts are selected after the induction by nitrogen deprivation. To address this question, we followed the fate of every cells on agar plates after nitrogen deprivation with an interval of 4 h. About 10% of heterocysts were formed without prior division after the start of nitrogen deprivation. The intensity of fluorescence of GFP in the transformants of hetR-gfp increased markedly in the future heterocysts at the fourth hour with respect to other cells. We also noted that the growing filaments consisted of clusters of four consecutive cells that we call quartets. About 75% of initial heterocysts originated from either of the two outer cells of quartets at the start of nitrogen deprivation. These results suggest that the future heterocysts are loosely selected at early times after the start of nitrogen deprivation, before the commitment. Such early candidacy could be explained by different properties of the outer and inner cells of a quartet, but the molecular nature of candidacy remains to be uncovered.     

Interaction of Cytotoxic Bicyclic Peptides, Theonellamides A and F, with Glutamate Dehydrogenase and 17β-Hydroxysteroid Dehydrogenase IV

Theonellamide A, a bicyclic peptide isolated from a Theonella sponge, was fixed on hydrazide-containing gel beads and screened for its binding proteins from rabbit liver tissues. Analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis revealed that two major proteins of 80 kDa and 55 kDa interacted with theonellamide A. The interaction between theonellamide A and two proteins was confirmed by competition experiments in which these two proteins failed to bind to theonellamide A–conjugated gel beads in the presence of theonellamide A or F. Amino-terminal amino acid sequence analysis of peptide fragments derived from the binding proteins by lysylendopeptidase digestion demonstrated that the 80-kDa and 55-kDa proteins were 17β-hydroxysteroid dehydrogenase IV and glutamate dehydrogenase, respectively. In an in vitro assay system, amination of α-ketoglutarate by glutamate dehydrogenase was activated with theonellamide F, although this effect was weaker than that with adenosine diphosphate, a well-known activator. Received October 15, 1999; accepted January 4, 2000.     

Identification and characterization of d-arabinose reductase and d-arabinose transporters from Pichia stipitis

d-xylose and l-arabinose are the major constituents of plant lignocelluloses, and the related fungal metabolic pathways have been extensively examined. Although Pichia stipitis CBS 6054 grows using d-arabinose as the sole carbon source, the hypothetical pathway has not yet been clarified at the molecular level. We herein purified NAD(P)H-dependent d-arabinose reductase from cells grown on d-arabinose, and found that the enzyme was identical to the known d-xylose reductase (XR). The enzyme activity of XR with d-arabinose was previously reported to be only 1% that with d-xylose. The k_cat/K_m value with d-arabinose (1.27 min^?1 mM^?1), which was determined using the recombinant enzyme, was 13.6- and 10.5-fold lower than those with l-arabinose and d-xylose, respectively. Among the 34 putative sugar transporters from P. stipitis, only seven genes exhibited uptake ability not only for d-arabinose, but also for d-glucose and other pentose sugars including d-xylose and l-arabinose in Saccharomyces cerevisiae.     

The levels of mature glycosylated nicastrin are regulated and correlate with gamma-secretase processing of amyloid beta-precursor protein

Nicastrin, a type-I transmembrane glycoprotein, is a necessary component of the high molecular weight presenilin (PS) complexes that mediate intramembranous cleavage of beta-amyloid precursor protein (betaAPP) and Notch. Nicastrin undergoes trafficking-dependent glycosylation maturation, and PS1 interacts preferentially with these maturely glycosylated forms of nicastrin. We investigated the effects of differing levels of the immature and mature endoglycosidase-H-resistant forms of nicastrin on Abeta40- and Abeta42-peptide secretion in several cell lines stably expressing a mutant nicastrin (D336A/Y337A) that increases Abeta secretion. There was no correlation between Abeta secretion and the level of over-expression of the immature forms of nicastrin. The total level of mature nicastrin remained constant, but mutant nicastrin replaced endogenous mature nicastrin in varying degrees. Differences in the levels of mature mutant nicastrin positively correlated with Abeta secretion, but did not influence either betaAPP trafficking or processing by alpha- and beta-secretases. Proper trafficking and terminal maturation of nicastrin is therefore a necessary event for the regulated intramembranous proteolysis of betaAPP.     

Genome-wide identification of chromatin-enriched RNA reveals that unspliced dentin matrix protein-1 mRNA regulates cell proliferation in squamous cell carcinoma

Chromatin-enriched noncoding RNAs (ncRNAs) have emerged as key molecules in epigenetic processes by interacting with chromatin-associated proteins. Recently, protein-coding mRNA genes have been reported to be chromatin-tethered, similar with ncRNA. However, very little is known about whether chromatin-enriched mRNA is involved in the chromatin modification process. Here, we comprehensively examined chromatin-enriched RNA in squamous cell carcinoma (SQCC) cells by RNA subcellular localization analysis, which was a combination of RNA fractionation and RNA-seq. We identified 11 mRNAs as highly chromatin-enriched RNAs. Among these, we focused on the dentin matrix protein-1 (DMP-1) gene because its expression in SQCC cells has not been reported. Furthermore, we clarified that DMP-1 mRNA was retained in chromatin in its unspliced form in SQCC in vitro and in vivo. As the inhibition of the unspliced DMP-1 mRNA (unspDMP-1) expression resulted in decreased cellular proliferation in SQCC cells, we performed ChIP-qPCR to identify cell cycle-related genes whose expression was epigenetically modified by unspDMP-1, and found that the CDKN1B promoter became active in SQCC cells by inhibiting unspDMP-1 expression. This result was further validated by the increased CDKN1B gene expression in the cells treated with siRNA for unspDMP-1 and by restoration of the decreased cellular proliferation rate by simultaneously inhibiting CDKN1B expression in SQCC cells. Further, to examine whether unspDMP-1 was able to associate with the CDKN1B promoter region, SQCC cells stably expressing PP7-mCherry fusion protein were transiently transfected with the unspDMP-1 fused to 24 repeats of the PP7 RNA stem loop (unspDMP-1-24xPP7) and we found that unspDMP-1-24xPP7 was efficiently precipitated with the antibody against mCherry and was significantly enriched in the CDKN1B promoter region. Thus, unspDMP-1 is a novel chromatin-enriched RNA that epigenetically regulates cellular proliferation of SQCC.     

Development of a novel tocopheryl ester for suppression of lipid accumulation without cytotoxicity by optimization of dicarboxylic ester moiety

Tocopheryl succinate (Tsuc) is a succinic acid ester of the well-known antioxidant α-tocopherol (T). Tsuc exhibits various biological activities, including tumor growth suppression via activation of cell signaling and prevention of lipid accumulation in mouse adipocyte 3T3-L1 cells. The latter findings suggest that Tsuc may be a drug candidate for the treatment of obesity. However, Tsuc was found to induce apoptosis of normal cells (in addition to cancer cells), demonstrating the need to reduce the cytotoxicity of Tsuc without losing the suppression effect on lipid accumulation. Based on our previous findings, we focused on the ester structure of Tsuc for controlling cytotoxicity. Herein, we examined the cytotoxicity and lipid accumulation suppression effect of various T ester derivatives. We found that the terminal carboxylic group is necessary for suppression of lipid accumulation. We synthesized tocopheryl glutarate (Tglu) and tocopheryl adipate (Tadi) by elongation of carbon atoms 1 and 2 of the dicarboxylic moiety, respectively. Tglu and Tadi did not show any cytotoxicity, and both esters suppressed lipid accumulation, although their suppression activities were weaker than that of Tsuc. Tadi showed a more potent lipid accumulation inhibitory effect than Tglu. Although Tadi inhibited lipogenesis and promoted lipolysis, lipolysis was induced at lower concentrations than inhibition of lipogenesis, suggesting that Tadi mainly affects lipolysis. Taken together, we succeeded in the reduction of cytotoxicity, without loss of the suppression effect on lipid accumulation, by elongation of the dicarboxylic moiety of Tsuc. Tadi may be a promising candidate as an anti-obesity drug.     

Two new taxa of Achnanthidium and Encyonema (Bacillariophyceae) from the Yom River,Thailand, with special reference to the areolae occlusions implying ontogenetic relationship

We discovered two new taxa of Achnanthidium and Encyonema occurring with high abundance in the upstream of the Yom River, which is one of four major rivers in Northern Thailand. Achnanthidium pseudoconspicuum var. yomensis var. nov. closely resembles Achnanthidium pseudoconspicuum var. pseudoconspicuum in scanning electron microscopy (SEM), but has clearly different valve morphometry under the light microscopy (LM). Achnanthidium pseudoconspicuum var. yomensis is also similar to A. japonicum in valve morphology under LM, but differs to this species based on SEM observation. Encyonema yuwadeeanum sp. nov. shows similarities to Encyonema leei and Encyonema subkukenanum, but differs in valve shape and/or polar fissure shape. We discuss the possible valve ontogenetic relationship between Achnanthidium and Cymbellales based on the detailed areolae structures of these two new taxa.     

Quantification of Cellular NEMO Content and Its Impact on NF-κB Activation by Genotoxic Stress

NF-κB essential modulator, NEMO, plays a key role in canonical NF-κB signaling induced by a variety of stimuli, including cytokines and genotoxic agents. To dissect the different biochemical and functional roles of NEMO in NF-κB signaling, various mutant forms of NEMO have been previously analyzed. However, transient or stable overexpression of wild-type NEMO can significantly inhibit NF-κB activation, thereby confounding the analysis of NEMO mutant phenotypes. What levels of NEMO overexpression lead to such an artifact and what levels are tolerated with no significant impact on NEMO function in NF-κB activation are currently unknown. Here we purified full-length recombinant human NEMO protein and used it as a standard to quantify the average number of NEMO molecules per cell in a 1.3E2 NEMO-deficient murine pre-B cell clone stably reconstituted with full-length human NEMO (C5). We determined that the C5 cell clone has an average of 4 x 10⁵ molecules of NEMO per cell. Stable reconstitution of 1.3E2 cells with different numbers of NEMO molecules per cell has demonstrated that a 10-fold range of NEMO expression (0.6–6x10⁵ molecules per cell) yields statistically equivalent NF-κB activation in response to the DNA damaging agent etoposide. Using the C5 cell line, we also quantified the number of NEMO molecules per cell in several commonly employed human cell lines. These results establish baseline numbers of endogenous NEMO per cell and highlight surprisingly normal functionality of NEMO in the DNA damage pathway over a wide range of expression levels that can provide a guideline for future NEMO reconstitution studies.     

A GC polymorphism associated with serum 25-hydroxyvitamin D level is a risk factor for hip fracture in Japanese patients with rheumatoid arthritis: 10-year follow-up of the Institute of Rheumatology,Rheumatoid Arthritis cohort study

Introduction

Vitamin D deficiency has been reported to be common in patients with rheumatoid arthritis (RA) who have a higher prevalence of osteoporosis and hip fracture than healthy individuals. Genetic variants affecting serum 25-hydroxyvitamin D (25(OH)D) concentration, an indicator of vitamin D status, were recently identified by genome-wide association studies of Caucasian populations. The purpose of this study was to validate the association and to test whether the serum 25(OH)D-linked genetic variants were associated with the occurrence of hip fracture in Japanese RA patients.

Methods

DNA samples of 1,957 Japanese RA patients were obtained from the Institute of Rheumatology, Rheumatoid Arthritis (IORRA) cohort DNA collection. First, five single nucleotide polymorphisms (SNPs) that were reported to be associated with serum 25(OH)D concentration by genome-wide association studies were genotyped. The SNPs that showed a significant association with serum 25(OH)D level in the cross-sectional study were used in the longitudinal analysis of hip fracture risk. The genetic risk for hip fracture was determined by a multivariate Cox proportional hazards model in 1,957 patients with a maximum follow-up of 10 years (median, 8 years).

Results

Multivariate linear regression analyses showed that rs2282679 in GC (the gene encoding group-specific component (vitamin D binding protein)) locus was significantly associated with lower serum 25(OH)D concentration (P = 8.1 × 10^-5). A Cox proportional hazards model indicated that rs2282679 in GC was significantly associated with the occurrence of hip fracture in a recessive model (hazard ratio (95% confidence interval) = 2.52 (1.05-6.05), P = 0.039).

Conclusions

A two-staged analysis demonstrated that rs2282679 in GC was associated with serum 25(OH)D concentration and could be a risk factor for hip fracture in Japanese RA patients.