Biomass production and energy source of thermophiles in a Japanese alkaline geothermal pool

Microbial biomass production has been measured to investigate the contribution of planktonic bacteria to fluxations in dissolved organic matter in marine and freshwater environments, but little is known about biomass production of thermophiles inhabiting geothermal and hydrothermal regions. The biomass production of thermophiles inhabiting an 85°C geothermal pool was measured by in situ cultivation using diffusion chambers. The thermophiles' growth rates ranged from 0.43 to 0.82 day^?1, similar to those of planktonic bacteria in marine and freshwater habitats. Biomass production was estimated based on cellular carbon content measured directly from the thermophiles inhabiting the geothermal pool, which ranged from 5.0 to 6.1 μg C l^?1 h^?1. This production was 2–75 times higher than that of planktonic bacteria in other habitats, because the cellular carbon content of the thermophiles was much higher. Quantitative PCR and phylogenetic analysis targeting 16S rRNA genes revealed that thermophilic H₂‐oxidizing bacteria closely related to Calderobacterium and Geothermobacterium were dominant in the geothermal pool. Chemical analysis showed the presence of H₂ in gases bubbling from the bottom of the geothermal pool. These results strongly suggested that H₂ plays an important role as a primary energy source of thermophiles in the geothermal pool.     

Withaferin A Targets Intermediate Filaments Glial Fibrillary Acidic Protein and Vimentin in a Model of Retinal Gliosis

Gliosis is a biological process that occurs during injury repair in the central nervous system and is characterized by the overexpression of the intermediate filaments (IFs) glial fibrillary acidic protein (GFAP) and vimentin. A common thread in many retinal diseases is reactive Müller cell gliosis, an untreatable condition that leads to tissue scarring and even blindness. Here, we demonstrate that the vimentin-targeting small molecule withaferin A (WFA) is a novel chemical probe of GFAP. Using molecular modeling studies that build on the x-ray crystal structure of tetrameric vimentin rod 2B domain we reveal that the WFA binding site is conserved in the corresponding domain of tetrameric GFAP. Consequently, we demonstrate that WFA covalently binds soluble recombinant tetrameric human GFAP at cysteine 294. In cultured primary astrocytes, WFA binds to and down-regulates soluble vimentin and GFAP expression to cause cell cycle G₀/G₁ arrest. Exploiting a chemical injury model that overexpresses vimentin and GFAP in retinal Müller glia, we demonstrate that systemic delivery of WFA down-regulates soluble vimentin and GFAP expression in mouse retinas. This pharmacological knockdown of soluble IFs results in the impairment of GFAP filament assembly and inhibition of cell proliferative response in Müller glia. We further show that a more severe GFAP filament assembly deficit manifests in vimentin-deficient mice, which is partly rescued by WFA. These findings illustrate WFA as a chemical probe of type III IFs and illuminate this class of withanolide as a potential treatment for diverse gliosis-dependent central nervous system traumatic injury conditions and diseases, and for orphan IF-dependent pathologies.     

Genome Sequence of the Cellulosome-Producing Mesophilic Organism Clostridium cellulovorans 743B

Clostridium cellulovorans 743B was isolated from a wood chip pile and is an anaerobic and mesophilic spore-forming bacterium. This organism degrades native substrates in soft biomass such as corn fiber and rice straw efficiently by producing an extracellular enzyme complex called the cellulosome. Here we report the genome sequence of C. cellulovorans 743B.The biotechnological potential of polysaccharolytic enzymes has resulted in the isolation and characterization of a large number of anaerobic, Gram-positive, spore-forming bacteria, the majority of which have been allocated to the genus Clostridium. Among clostridia, the cellulosomes produced by Clostridium species are particularly designed for efficient degradation of plant cell wall polysaccharides. The component parts of the multicomponent complex are integrated by virtue of a unique family of integrating modules, the cohesins and the dockerins, whose distribution and specificity dictate the overall cellulosome architecture (3). The cellulosome system in Clostridium cellulovorans 743B (ATCC 35296) has been studied extensively for the last 20 years and has resulted in providing basic information about mesophilic cellulosomes. This organism was isolated from a wood chip pile and is an anaerobic spore-forming bacterium whose optimal growth temperature is 37°C (9). It has the ability to utilize cellulose, xylan, pectin, cellobiose, glucose, fructose, galactose, and mannose as carbon sources for growth. Its fermentation products include H₂, CO₂, acetate, butyrate, formate, lactate, and ethanol. When it is grown in the presence of cellulose, electron micrographs have shown that large protuberances are present on its cell surface (4), while small or no protuberances are evident when cells are grown in the presence of glucose or cellobiose (5).We sequenced a total length of 101,749,598 bp and analyzed 381,514 reads by Genome Sequencer FLX 454./Roche sequencing (8) (GS-FLX version) to highly oversample the genome (20× coverage) and generated 123,892 paired-end sequence tags, to enable the assembly of all tags using the GS De Novo Assembler version 1.1.03.24 (Roche Diagnostics) and the Genome Analyzer II and sequencing kit 36-Cycle Run (Illumina). Finally, we assembled 30 scaffolds (sets of 601 ordered and oriented contigs; total length of 5,123,527 bp) to generate approximately 5.1 Mbp of nearly contiguous Clostridium botulinum E3 strain Alaska E43 (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_010723","term_id":"188587536","term_text":"NC_010723"}}NC_010723) complete genome sequence. We analyzed a number of predicted genes included in the C. cellulovorans genome using CRITICA (version 1.05b) (2) and Glimmer 2 (version 2.10) (6) to find regions in proteins with known functions. We annotated and classified according to Gene Ontology (GO) (1). In silico Molecular Cloning Genomic Edition ver. 3.0.26 software (In Silico Biology, Co., Ltd., Japan) was used for individual genomic analysis.The C. cellulovorans 743B (ATCC 35296) genome consists of 5,123,527 bp. A total of 4,220 polypeptide-encoding open reading frames (ORFs) were identified using CRITICA, while 4,297 ORFs were identified using Glimmer 2. The number of ORFs identical between CRITICA and Glimmer 2 was 2,773. Sixty-three tRNAs and 33 anticodons were also identified using tRNAscan-SE (7). In comparison of the genome sizes among cellulosomal clostridia such as Clostridium cellulolyticum H10 (4.07 Mbp) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"CP001348","term_id":"219997787","term_text":"CP001348"}}CP001348) and Clostridium thermocellum ATCC 27405 (3.84 Mbp) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"CP000568","term_id":"125712750","term_text":"CP000568"}}CP000568), the C. cellulovorans genome was over 1 Mbp larger than the other genomes. Moreover, the number of predicted genes (4,220 by CRITICA) in the C. cellulovorans genome was the largest among them. On the other hand, the G+C content in C. cellulovorans was 31.1%, similar to that (30.9%) in Clostridium acetobutylicum ATCC 824 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AE001437","term_id":"25168256","term_text":"AE001437"}}AE001437), while the G+C contents in C. cellulolyticum and C. thermocellum were 37.7% and 39.0%, respectively.A protein BLAST search against the database of clusters of orthologous groups (COGs) of proteins indicated that 4,171 genes were annotated by 4,220 predicted coding sequences using CRITICA while 4,098 genes were identified by 4,297 predicted coding sequences using Glimmer 2. On the other hand, a protein BLAST search against the NCBI nr database indicated that 4,184 genes were annotated by 4,220 predicted coding sequences using CRITICA while 4,071 genes were identified by 4,297 predicted coding sequences using Glimmer 2. Interestingly, 57 cellulosomal genes were found in the C. cellulovorans genome and coded for not only carbohydrate-active enzymes but also lipases, peptidases, and proteinase inhibitors. Moreover, two novel genes encoding a scaffolding protein were found in the genome. Thus, by examining genome sequences from multiple Clostridium species, comparative genomics offers new insight into genome evolution and the way in which natural selection molds functional DNA sequence evolution. Our analysis, coupled with the genome sequence data, will provide a road map for constructing enhanced cellulosome-producing Clostridium strains for industrial applications such as biofuel production.     

Identification of a two-component VirR/VirS regulon in Clostridium perfringens

Clostridium perfringens, a Gram-positive anaerobic pathogen, is a causative agent of human gas gangrene that leads to severe rapid tissue destruction and can cause death within hours unless treated immediately. Production of several toxins is known to be controlled by the two-component VirR/VirS system involving a regulatory RNA (VR-RNA) in C. perfringens. To elucidate the precise regulatory network governed by VirR/VirS and VR-RNA, a series of microarray screening using VirR/VirS and VR-RNA-deficient mutants was performed. Finally, by qRT-PCR analysis, 147 genes (30 single genes and 21 putative operons) were confirmed to be under the control of the VirR/VirS-VR-RNA regulatory cascade. Several virulence-related genes for alpha-toxin, kappa-toxin, hyaluronidases, sialidase, and capsular polysaccharide synthesis were found. Furthermore, some genes for catalytic enzymes, various genes for transporters, and many genes for energy metabolism were also found to be controlled by the cascade. Our data indicate that the VirR/VirS-VR-RNA system is a global gene regulator that might control multiple cellular functions to survive and multiply in the host, which would turn out to be a lethal flesh-eating infection.     

Complexity of the genomic diversity in enterohemorrhagic Escherichia coli O157 revealed by the combinational use of the O157 Sakai OligoDNA microarray and the Whole Genome PCR scanning.

Escherichia coli O157, an etiological agent of hemorrhagic colitis and hemolytic uremic syndrome, is one of the leading worldwide public health threats. Genome sequencing of two O157 strains have revealed that the chromosome is comprised of a 4.1 Mb backbone shared by K-12 and a total of 1.4 Mb O157-specific sequences. Most of the large O157-specific sequences are prophages and prophage-like elements, which have carried many virulence genes into the O157 genome. This suggests that bacteriophages have played the key roles in the emergence of O157. The Whole Genome PCR Scanning (WGPScanning) analysis of O157 strains, on the other hand, revealed a high level of genomic diversity in O157. Variation of prophages has also been suggested as a major factor generating such diversity. In this study, we analyzed the gene content of O157 strains, by an oligoDNA microarray, using the same set of strains as examined by the WGPScanning method. Although most of the strains were typical O157 : H7, they differed remarkably in gene composition, particularly in those on prophages, and we identified more than 400 'variably absent or present' genes which included virulence-related genes. This confirms the role of prophages in generating the genomic diversity, and raises a possibility that some level of variation in potential virulence is present among O157 strains. Fine comparison of the two datasets obtained by microarray and WGPScanning provided much further details on the O157 genome diversity than illustrated by each method alone, indicating the usefulness of this combinational approach in the genomic comparison of closely related strains.     

Stellate cell apoptosis by a soluble mediator from immortalized human hepatocytes

Activated hepatic stellate cells (HSCs) are the major source of extracellular matrix in fibrosis and cirrhosis. In this study, we have investigated the role of hepatitis C virus (HCV) core protein induced immortalized human hepatocytes (IHH) on HSC growth. Preferential growth of IHH and apoptosis of activated human hepatic stellate cells (LX2) were observed upon coculture of these two cell types in a dual chamber or in the presence of conditioned medium (CM) from IHH. CM did not display a growth inhibitory role on other hepatic (Huh-7, HepG2, Hep3B and THLE) and non-hepatic (HeLa, MCF-7, and BHK) epithelial cells, indicating that the soluble mediator from IHH does not have a generalized effect on cell lines examined in our study. Further studies suggested that CM from IHH increased the expression of TRAIL receptors on LX2 cell surface, and induced apoptosis by a caspase dependent mechanism. Peptide mass fingerprinting of the purified soluble mediator from CM suggested that gelsolin fragments may play a role in apoptosis of LX2 cells. Taken together, our results suggested that a soluble mediator secreted from immortalized human hepatocytes plays an important role in hepatic stellate cell growth regulation.     

Mathematical modelling and experiments for the proliferation and differentiation of Drosophila intestinal stem cells I

We study the proliferation and differentiation of stem cells in the Drosophila posterior midgut epithelium, which mainly consists of intestinal stem cells (ISCs); semi-differentiated cells, i.e. enteroblasts (EBs); and two types of fully differentiated cells, i.e. enteroendocrine cells (EEs) and enterocytes (ECs). The cellular system of ISCs is controlled by Wnt and Notch signalling pathways. In this article, we experimentally show that EBs are not capable of efficiently differentiating into ECs in the absence of Wnt signalling. On the basis of the experimental results and known facts, we propose a scheme and a simple ordinary differential equation (ODE) model for the proliferation and differentiation of ISCs. This is a first step towards understanding the universal mechanism for the maintenance of the cellular system of tissue stem cells controlled by signalling pathways.     

Screening for possible miRNA–mRNA associations in a colon cancer cell line

MicroRNAs (miRNAs) are small non-coding RNAs mediating the regulation of gene expression in various biological contexts, including carcinogenesis. Here, we screened putative associations between 34, 45, and 103 miRNAs and 164, 391, and 81 mRNAs via Argonaute1 (Ago1) or Ago2 immunoprecipitation (IP) experiments in a colon cancer cell line. We used a combination of RIP Seq analysis. RNAs that were co-immunoprecipitated with Ago1 or Ago2 were used for massively parallel small RNA and mRNA sequencing. The detected miRNAs and mRNAs were further associated with one another based on in silico target predictions. Analysis of the putative associations indicated that, although Ago1 and Ago2 shared a similar repertory of miRNAs, the mRNAs possibly regulated by those miRNAs seemed different. The mRNAs detected with Ago1 IP were indicated to be frequently associated with genes having constitutive cellular functions, regulated by a smaller number of miRNAs, and appeared to receive more stringent translational regulation. In contrast, putative miRNA-mRNA associations detected with Ago2 IP appeared to be related to signal transduction genes, which had a larger number of possible miRNA binding sites. We then conducted a similar analysis using the colon cancer cells cultured under hypoxia and identified potential hypoxia-induced miRNA-mRNA associations, which included several well-characterized cancer-related genes as novel putative miRNA targets.     

Development of Sink Capacity of the "Storage Root" in a Radish Cultivar with a High Ratio of "Storage Root" to Shoot

The mechanisms that control sink capacity are poorly understood.in radish, a major sink is the "storage root", which beginsto thicken early in development, mainly as a result of thickeningof the hypocotyl. We investigated changes in the accumulationof dry matter, sink activity (increase in dry weight of thehypocotyl per unit of dry weight present per unit of time),carbohydrate content, levels of metabolites, activities of enzymesrelated to the breakdown of sucrose, and the profile of solubleproteins, as well as changes in anatomy, using hypocotyls ofa cultivar with a high ratio of "storage root" to shoot. Wefound that sink activity was strongly related to the level andactivity of sucrose synthase but not to the activity of invertase.We also found a significant correlation between sucrose contentand the level and activity of sucrose synthase. Our resultssuggest that sucrose synthase, but not invertase, might be criticalfor the development of the sink activity of the radish hypocotyland that the level of sucrose might regulate the expressionof sucrose synthase. A discussion of sink capacity is presentedthat includes consideration of structural changes in the hypocotyl. (Received December 14, 1998; Accepted January 27, 1999)     

TNF-alpha and insulin, alone and synergistically, induce plasminogen activator inhibitor-1 expression in adipocytes

Obesity is associated with hyperinsulinemia and elevatedconcentrations of tumor necrosis factor- (TNF-) inadipose tissue. TNF- has been implicated as an inducer of thesynthesis of plasminogen activator inhibitor-1 (PAI-1), the primaryphysiological inhibitor of fibrinolysis, mediated by plasminogenactivators in cultured adipocytes. To identify mechanism(s) throughwhich TNF- induces PAI-1, 3T3-L1 preadipocytes were differentiatedinto adipocytes and exposed to TNF- for 24 h. TNF- selectivelyincreased the synthesis of PAI-1 without increasing activity ofplasminogen activators. Both superoxide (generated by xanthine oxidaseplus hypoxanthine) and hydrogen peroxide were potent inducers of PAI-1, and hydroxyl radical scavengers completely abolished the TNF- induction of PAI-1. Exposure of adipocytes to TNF- or insulin aloneover 5 days increased PAI-1 production. These agonists exert synergistic effects. Results obtained suggest that TNF- stimulates PAI-1 production by adipocytes, an effect potentiated by insulin, andthat adipocyte generation of reactive oxygen centered radicals mediatesthe induction of PAI-1 production by TNF-. Because induction ofPAI-1 by TNF- is potentiated synergistically by insulin, both agonists appear likely to contribute to the impairment of fibrinolytic system capacity typical in obese, hyperinsulinemic patients.