Methyl viologen hydrogenase II, a new member of the hydrogenase family from Methanobacterium thermoautotrophicum delta H.

Two methyl viologen hydrogenase (MVH) enzymes from Methanobacterium thermoautotrophicum delta H have been separated (resolution, Rs at 1.0) on a Mono Q column after chromatography on DEAE-Sephacel and Superose 6 Prep Grade. The newly discovered MVH (MVH II) was eluted at 0.5 M NaCl with a linear gradient of 0.45 to 0.65 M NaCl (100 ml). The previously described MVH (MVH I) eluted in a NaCl gradient at 0.56 M. The specific activities of MVH I and MVH II were 184.8 and 61.3 U/mg of protein, respectively, when enzyme activity was compared at pH 7.5, the optimal pH for MVH II. Gel electrophoresis in nondenaturing systems indicated that MVH I and MVH II had a similar molecular mass of 145 kDa. Denatured MVH II showed four protein bands (alpha, 50 kDa; beta, 44 kDa; gamma, 36 kDa; delta, 15 kDa), similar to MVH I. The N-terminal amino acid sequences of the alpha, gamma, and delta subunits of MVH II were identical with the sequences of the equivalent subunits of MVH I. However, the N-terminal amino acid sequence of the beta subunit of MVH II was totally different from the sequence of the beta subunit of MVH I. Both MVH I and MVH II had the same optimal temperature of 60 degrees C for maximum activity. The pH optima of MVH I and MVH II were 9.0 and 7.5, respectively. Most of the divalent metal ions tested significantly inhibited MVH I activity, but MVH II activity was only partially inhibited by some divalent cations. Both hydrogenases were shown to be stable for over 8 days at --20 degrees C under anaerobic conditions. When exposed to air, 90% of MVH I activity was lost within 2 min; however, MVH II lost only 50% of its activity in 3 h.     

Dynamic heat and moisture transfer through multiple clothing layers

1. 1. The purposes of this study are to find out the arrangement effects on the vapor pressure gradient across the cotton–nylon double layer and to elucidate changes in the vapor pressure gradient when an additional third layer covers the double layer.

2. 2. Model tests for single, double and triple layer system and wear test for triple layer clothing were conducted.

3. 3. It was found that up to the second layer, dryness of innermost microclimate could be maintained when cotton faced the skin (C/N).

4. 4. However, when more permeable and hydrophobic third layer (UWF) covers the double layer, the microclimate of C/N is no longer drier than N/C.

5. 5. When nylon is exposed to the skin, a larger drop in vapor pressure across the first two layers occurred for both model and wear test.

6. 6. The innermost microclimate was not necessarily kept dry when the outermost layer dissipated more moisture due to the inefficient distribution of moisture.

Metabolic engineering of Mannheimia succiniciproducens for malic acid production using dimethylsulfoxide as an electron acceptor

Microbial production of various TCA intermediates and related chemicals through the reductive TCA cycle has been of great interest. However, rumen bacteria that naturally possess strong reductive TCA cycle have been rarely studied to produce these chemicals, except for succinic acid, due to their dependence on fumarate reduction to transport electrons for ATP synthesis. In this study, malic acid (MA), a dicarboxylic acid of industrial importance, was selected as a target chemical for mass production using Mannheimia succiniciproducens, a rumen bacterium possessing a strong reductive branch of the TCA cycle. The metabolic pathway was reconstructed by eliminating fumarase to prevent MA conversion to fumarate. The respiration system of M. succiniciproducens was reconstructed by introducing the Actinobacillus succinogenes dimethylsulfoxide (DMSO) reductase to improve cell growth using DMSO as an electron acceptor. Also, the cell membrane was engineered by employing Pseudomonas aeruginosa cis-trans isomerase to enhance MA tolerance. High inoculum fed-batch fermentation of the final engineered strain produced 61 g/L of MA with an overall productivity of 2.27 g/L/h, which is the highest MA productivity reported to date. The systems metabolic engineering strategies reported in this study will be useful for developing anaerobic bioprocesses for the production of various industrially important chemicals.     

Novel (E)-3-(3-Oxo-4-substituted-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxypropenamides as Histone Deacetylase Inhibitors: Design,Synthesis and Bioevaluation

Herein, we report the design, synthesis and evaluation of novel (E)-3-(3-oxo-4-substituted-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxypropenamides ( 4 a – i , 7 a – g ) targeting histone deacetylases. Three human cancer cell lines were used to test the cytotoxicity of the synthesized compounds (SW620, colon; PC-3, prostate; NCI−H23, lung cancer); inhibitory activity towards HDAC; anticancer activity; as well as their impact on the cell cycle and apoptosis. As a result, compounds 4 a – i bearing the alkyl substituents seemed to be less potent than the benzyl-containing compounds 7 a – g in all biological assays. Compounds 7 e – f were found to be the most active HDAC inhibitors with IC₅₀ of 1.498±0.020 μM and 1.794±0.159 μM, respectively. In terms of cytotoxicity and anticancer assay, 7 e and 7 f also showed good activity with IC₅₀ values in the micromolar range. In addition, the cell cycle and apoptosis of SW620 were affected by compound 7 f in almost a similar manner to that of reference compound SAHA. Docking assays were carried out for analysis the binding mode and selectivity of this compound toward 8 HDAC isoforms. Overall, our data confirmed that the inhibition of HDAC plays a pivotal role in their anticancer activity.     

In vitro oxygen consumption and motility of Cryptobia salmositica, Cryptobia bullocki, and Cryptobia catostomi (Sarcomastigophora: Kinetoplastida).

Cryptobia salmositica (pathogenic and vaccine strains), Cryptobia bullocki (pathogenic), and Cryptobia catostomi (nonpathogenic) have similar oxygen consumption rates (0.17 +/- 0.01 nm O2/10(6) parasites). Incubation with sodium azide (5 microliters of a 1-M solution to 1 ml of parasite suspension, i.e., a 5-mM final concentration) reduced the oxygen consumption by approximately 4.5-fold. Motility of the parasites was also greatly reduced in sodium azide. The oxygen consumption and motility of the parasites returned to preazide treatment levels when the azide was removed even after 24 hr of incubation in sodium azide. The activities of hexokinase, pyruvate kinase, and cytochrome C oxidase were not detected in the 3 species of Cryptobia.     

Vector specificity of Trypanosoma catostomi and its infectivity to freshwater fishes.

Trypanosoma catostomi was found in 36.2% of 558 white suckers (Catostomus commersoni) from Ontario, Canada. The abundance of Actinobdella inequiannulata was 35% (68 leeches/197 suckers examined for leeches). The susceptibility of 3 species of leeches (Hemiclepsis marginata, Desserobdella phalera, and A. inequiannulata) and 7 species of fishes (C. commersoni, Amia calva, Anguilla rostrata, Ictalurus nebulosus, Oncorhynchus mykiss, Perca flavescens, and Esox lucius) to infection with T. catostomi was examined. Metatrypanosomes were found in the crop and proboscis sheath of 13 of 21 A. inequiannulata and in the crop of 10 of 12 H. marginata and 1 of 21 D. phalera. Only flagellates from A. inequiannulata were infective to C. commersoni. Cultured T. catostomi infected C. commersoni and A. calva but not any other fish species. Laboratory-reared C. commersoni were more susceptible than wild-caught specimens. Cultured Trypanosoma phaleri did not infect its natural host, A. calva. Host specificity should be established experimentally before a specific diagnosis is made. Cultures may be useful in simulating factors that affect development in the vector.     

Construction and characterization of two rice bacterial artificial chromosome libraries from the parents of a permanent recombinant inbred mapping population

Rice is a leading grain crop and the staple food for over half of the world population. Rice is also an ideal species for genetic and biological studies of cereal crops and other monocotyledonous plants because of its small genome and well developed genetic system. To facilitate rice genome analysis leading to physical mapping, the identification of molecular markers closely linked to economic traits, and map-based cloning, we have constructed two rice bacterial artificial chromosome (BAC) libraries from the parents of a permanent mapping population (Lemont and Teqing) consisting of 400 F9 recombinant inbred lines (RILs). Lemont (japonica) and Teqing (indica) represent the two major genomes of cultivated rice, both are leading commercial varieties and widely used germplasm in rice breeding programs. The Lemont library contains 7296 clones with an average insert size of 150 kb, which represents 2.6 rice haploid genome equivalents. The Teqing library contains 14208 clones with an average insert size of 130 kb, which represents 4.4. rice haploid genome equivalents. Three single-copy DNA probes were used to screen the libraries and at least two overlapping BAC clones were isolated with each probe from each library, ranging from 45 to 260 kb in insert size. Hybridization of BAC clones with chloroplast DNA probes and fluorescent in situ hybridization using BAC DNA as probes demonstrated that both libraries contain very few clones of chloroplast DNA origin and are likely free of chimeric clones. These data indicate that both BAC libraries should be suitable for map-based cloning of rice genes and physical mapping of the rice genome.     

Internal pH crisis, lysine decarboxylase and the acid tolerance response of Salmonella typhimurium

Salmonella typhimurium possesses an adaptive response to acid that increases survival during exposure to extremely low pH values. The acid tolerance response (ATR) includes both log-phase and stationary-phase systems. The log-phase ATR appears to require two components for maximum acid tolerance, namely an inducible pH homeostasis system, and a series of acid-shock proteins. We have discovered one of what appears to be a series of inducible exigency pH homeostasis systems that contribute to acid tolerance in extreme acid environments. The low pH-inducible lysine decarboxylase was shown to contribute significantly to pH homeostasis in environments as low as pH 3.0. Under the conditions tested, both lysine decarboxylase and σ^s-dependent acid-shock proteins were required for acid tolerance but only lysine decarboxylase contributed to pH homeostasis. The cadBA operon encoding lysine decarboxylase and a lysine/cadaverine antiporter were cloned from S. typhimurium and were found to be 79% homologous to the cadBA operon from Escherichia coli . The results suggest that S. typhimurium has a variety of means of fulfilling the pH homeostasis requirement of the ATR in the form of inducible amino acid decarboxylases.     

Gene identification in a complex chromosomal continuum by local genomic cross-referencing

Most higher plants have complex genomes containing large quantities of repetitive DNA interspersed with low-copy-number sequences. Many of these repetitive DNAs are mobile and have homology to RNAs in various cell types. This can make it difficult to identify the genes in a long chromosomal continuum. It was decided to use genic sequence conservation and grass genome co-linearity as tools for gene identification. A bacterial artificial chromosome (BAC) clone containing sorghum genomic DNA was selected using a maize Adh1 probe. The 165 kb sorghum BAC was tested for hybridization to a set of clones representing the contiguous 280 kb of DNA flanking maize Adh1. None of the repetitive maize DNAs hybridized, but most of the low-copy-number sequences did. A low-copy-number sequence that did cross-hybridize was found to be a gene, while one that did not was found to be a low-copy-number retrotransposon that was named Reina. Regions of cross-hybridization were co-linear between the two genomes, but closer together in the smaller sorghum genome. These results indicate that local genomic cross-referencing by hybridization of orthologous clones can be an efficient and rapid technique for gene identification and studies of genome organization.