Identification of a novel endochitinase from a marine bacterium Vibrio proteolyticus strain No. 442

Chitin binding proteins prepared from Vibrio proteolyticus were purified and the N-terminal amino-acid sequence of a protein from a 110-kDa band on SDS-PAGE was found to be 85-90% identical to the 22nd-41st residues of the N-termini of chitinase A precursor proteins from other vibrios. We cloned the corresponding gene, which encodes a putative protein of 850 amino acids containing a 26-residue signal sequence. The chitinase precursor from V. proteolyticus was 78-80% identical to those from Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio carchariae. However, the proteolytic cleavage site for C-terminal processing between R597 and K598 in the chitinase precursor of other vibrios was not observed in the amino acid sequence of V. proteolyticus, which instead had the sequence R600 and A601. Subsequently, full-length and truncated chitinases were generated in Escherichia coli. The specific activity of full-length chitinase expressed in E. coli was 17- and 20-folds higher for colloidal and alpha-chitins (insoluble substrate), respectively, than that of the C-terminal truncated enzyme. However, both recombinants showed similar hydrolysis patterns of hexa-N-acetyl-chitohexaose (soluble substrate), producing di-N-acetyl-chitobiose as major product on TLC analysis. We showed that the C-terminus of the V. proteolyticus chitinase A was important for expression of high specific activity against insoluble chitins.     

Effect of linear alkylbenzene sulfonate on Cu2+ removal by Spirulina platensis strain (FACHB‐834)

The removal efficiency of Cu²⁺ by Spirulina platensis (strain FACHB‐834), in viable and heat‐inactivated forms, was investigated in the presence and absence of linear alkylbenzene sulfonate (LAS). When the initial Cu²⁺ concentration was in the range of 0.5–1.5 mg · L^?1, a slight increase in growth rate of FACHB‐834 was observed. In contrast, when Cu²⁺ or LAS concentrations were at or higher than 2.0 or 6.0 mg · L^?1, respectively, the growth of FACHB‐834 was inhibited and displayed yellowing and fragmentation of filaments. The presence of LAS improved Cu²⁺ removal by ~20%, and accelerated attainment of Cu²⁺ retention equilibrium. For the 2‐ mg · L^?1 Cu²⁺ treatments, retention equilibrium occurred within 2 d and showed maximum Cu²⁺ removal of 1.83 mg · L^?1. In the presence of LAS, the ratio of extracellular bound Cu²⁺ to intracellular Cu²⁺ taken up by the cells was lower (1.05–2.26) than corresponding ratios (2.46–7.85) in the absence of LAS. The percentages of extracellular bound Cu²⁺ to total Cu²⁺ removal (both bound and taken up by cells) in the presence of LAS ranged from 51.2% to 69.3%, which was lower than their corresponding percentages (71.1%–88.7%) in the absence of LAS. LAS promoted biologically active transport of the extracellular bound form of Cu²⁺ into the cell. In contrast, the addition of LAS did not increase the maximum removal efficiency of Cu²⁺ (61.4% ± 5.6%) by heat‐inactivated cells compared to that of living cells (59.6% ± 6.0%). These results provide a theoretical foundation for designing bioremediation strategies using FACHB‐834 for use in surface waters contaminated by both heavy metals and LAS.     

Unexpectedly complex gradation of coral population structure in the Nansei Islands,Japan

To establish effective locations and sizes of potential protected areas for reef ecosystems, detailed information about source and sink relationships between populations is critical, especially in archipelagic regions. Therefore, we assessed population structure and genetic diversity of Acropora tenuis, one of the dominant stony coral species in the Pacific, using 13 microsatellite markers to investigate 298 colonies from 15 locations across the Nansei Islands in southwestern Japan. Genetic diversity was not significant among sampling locations, even in possibly peripheral locations. In addition, our results showed that there are at least two populations of A. tenuis in the study area. The level of genetic differentiation between these populations was relatively low, but significant between many pairs of sampling locations. Directions of gene flow, which were estimated using a coalescence‐based approach, suggest that gene flow not only occurs from south to north, but also from north to south in various locations. Consequently, the Yaeyama Islands and the Amami Islands are potential northern and southern sources of corals. On the other hand, the Miyako Islands and west central Okinawa Island are potential sink populations. The Kerama Islands and the vicinity of Taketomi Island are potential contact points of genetic subdivision of coral populations in the Nansei Islands. We found that genetic population structure of A. tenuis in the Nansei Islands is more complex than previously thought. These cryptic populations are very important for preserving genetic diversity and should be maintained.     

Comparison of lung cancer cell lines representing four histopathological subtypes with gene expression profiling using quantitative real-time PCR

Background  

Lung cancers are the most common type of human malignancy and are intractable. Lung cancers are generally classified into four histopathological subtypes: adenocarcinoma (AD), squamous cell carcinoma (SQ), large cell carcinoma (LC), and small cell carcinoma (SC). Molecular biological characterization of these subtypes has been performed mainly using DNA microarrays. In this study, we compared the gene expression profiles of these four subtypes using twelve human lung cancer cell lines and the more reliable quantitative real-time PCR (qPCR).     

Molecular basis for the inhibition of β-hydroxyacyl-ACP dehydratase HadAB complex from Mycobacterium tuberculosis by flavonoid inhibitors

Dehydration is one of the key steps in the biosynthesis of mycolic acids and is vital to the growth of Mycobacterium tuberculosis (Mtb). Consequently, stalling dehydration cures tuberculosis (TB). Clinically used anti-TB drugs like thiacetazone (TAC) and isoxyl (ISO) as well as flavonoids inhibit the enzyme activity of the β-hydroxyacyl-ACP dehydratase HadAB complex. How this inhibition is exerted, has remained an enigma for years. Here, we describe the first crystal structures of the MtbHadAB complex bound with flavonoid inhibitor butein, 2’,4,4’-trihydroxychalcone or fisetin. Despite sharing no sequence identity from Blast, HadA and HadB adopt a very similar hotdog fold. HadA forms a tight dimer with HadB in which the proteins are sitting side-by-side, but are oriented anti-parallel. While HadB contributes the catalytically critical His-Asp dyad, HadA binds the fatty acid substrate in a long channel. The atypical double hotdog fold with a single active site formed by MtbHadAB gives rise to a long, narrow cavity that vertically traverses the fatty acid binding channel. At the base of this cavity lies Cys61, which upon mutation to Ser confers drug-resistance in TB patients. We show that inhibitors bind in this cavity and protrude into the substrate binding channel. Thus, inhibitors of MtbHadAB exert their effect by occluding substrate from the active site. The unveiling of this mechanism of inhibition paves the way for accelerating development of next generation of anti-TB drugs.     

Mrakia terrae sp. nov. and Mrakia soli sp. nov., Two Novel Basidiomycetous Yeast Species Isolated from Soil in Korea

Three strains, YP416^T, YP421^T, and Y422, were isolated from soil samples in Pocheon City, Gyeonggi province, South Korea. The strains belong to two novel yeast species in the genus Mrakia. Molecular phylogenetic analysis showed that the strain YP416^T was closely related to Mrakia niccombsii. Still, it differed by 9 nucleotide substitutions with no gap (1.51%) in the D1/D2 domain of the LSU rRNA gene and 14 nucleotide substitutions with 7 gaps (2.36%) in the ITS region. The strain YP421^T differed from the type strain of the most closely related species, Mrakia aquatica, by 5 nucleotide substitutions with no gap (0.81%) in the D1/D2 domain of the LSU rRNA gene and 9 nucleotide substitutions with one gap (1.43%) in the ITS region. The names Mrakia terrae sp. nov. and Mrakia soli sp. nov. are proposed, with type strains YP416^T (KCTC 27886^T) and YP421^T (KCTC 27890^T), respectively. MycoBank numbers of the strains YP416^T and YP421^T are MB 836844 and MB 836847, respectively.     

A Study on Spin-Labelled Oligonucleotide Synthesis and its Electron Spin Resonance Behavior in Solution

An oligonucleotide spin-labelled with 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl (4-amino-TEMPO) at the internucleotide bond (d-Tp(L)TpTpTpT) prepared by oxidation of the pentanucleotide containing the H-phosphonate diester (d-Tp(H)TpTpTpT) in the presence of 4-amino-TEMPO, was separated and identified by high-performance, reverse-phase liquid chromatography combined with detection by electron spin resonance spectroscopy. This spin-labelled oligonucleotide produced a triplet with the slightly broadened M₁ = — I ESR component, while a triplet with almost equal intensities was obtained from the spin-label. The M₁ = —1 component from the labelled oligonucleotide was further broadened in the presence of poly(A) which forms a complementary double strand with this molecule.