Hydrogen production from glucose by anaerobes

Various anaerobes were cultivated in media containing glucose. When 100 mL of thioglycollate medium containing 2.0% (w/v) glucose was used, Clostridium butyricum ATCC 859, NBRC 3315, and NBRC 13949 evolved 227-243 mL of biogas containing about 180 mL of hydrogen in 1 day. Although some strains had some resistance against oxygen, C. butyricum ATCC 859 and 860 did not have it. C. butyricum NBRC 3315 and Enterobacter aerogenes NBRC 13534 produced hydrogen in the presence of glucose or pyruvic acid, and E. aerogenes NBRC 13534 produced hydrogen by not only glucose and pyruvic acid but also dextrin, sucrose, maltose, galactose, fructose, mannose, and mannitol. When a medium containing 0.5% (w/v) yeast extract and 2.0% (w/v) glucose was used, E. aerogenes NBRC 13534 evolved more biogas and hydrogen than C. butyricum NBRC 3315 in the absence of reducing agent.     

Hsk1-Dfp1/Him1, the Cdc7-Dbf4 kinase in Schizosaccharomyces pombe, associates with Swi1, a component of the replication fork protection complex

The protein kinase Hsk1 is essential for DNA replication in Schizosaccharomyces pombe. It associates with Dfp1/Him1 to form an active complex equivalent to the Cdc7-Dbf4 protein kinase in Saccharomyces cerevisiae. Swi1 and Swi3 are subunits of the replication fork protection complex in S. pombe that is homologous to the Tof1-Csm3 complex in S. cerevisiae. The fork protection complex helps to preserve the integrity of stalled replication forks and is important for activation of the checkpoint protein kinase Cds1 in response to fork arrest. Here we describe physical and genetic interactions involving Swi1 and Hsk1-Dfp1/Him1. Dfp1/Him1 was identified in a yeast two-hybrid screen with Swi1. Hsk1 and Dfp1/Him1 both co-immunoprecipitate with Swi1. Swi1 is required for growth of a temperature-sensitive hsk1 (hsk1ts) mutant at its semi-permissive temperature. Hsk1ts cells accumulate Rad22 (Rad52 homologue) DNA repair foci at the permissive temperature, as previously observed in swi1 cells, indicating that abnormal single-stranded DNA regions form near the replication fork in hsk1ts cells. hsk1ts cells were also unable to properly delay S-phase progression in the presence of a DNA alkylating agent and were partially defective in mating type switching. These data suggest that Hsk1-Dfp1/Him1 and Swi1-Swi3 complexes have interrelated roles in stabilization of arrested replication forks.     

An axonemal dynein particularly important for flagellar movement at high viscosity. Implications from a new Chlamydomonas mutant deficient in the dynein heavy chain gene DHC9

Ciliary and flagellar axonemes contain multiple inner arm dyneins of which the functional difference is largely unknown. In this study, a Chlamydomonas mutant, ida9, lacking inner arm dynein c was isolated and shown to carry a mutation in the DHC9 dynein heavy chain gene. The cDNA sequence of DHC9 was determined, and its information was used to show that >80% of it is lost in the mutant. Electron microscopy and image analysis showed that the ida9 axoneme lacked electron density near the base of the S2 radial spoke, indicating that dynein c localizes to this site. The mutant ida9 swam only slightly slower than the wild type in normal media. However, swimming velocity was greatly reduced when medium viscosity was modestly increased. Thus, dynein c in wild type axonemes must produce a significant force when flagella are beating in viscous media. Because motility analyses in vitro have shown that dynein c is the fastest among all the inner arm dyneins, we can regard this dynein as a fast yet powerful motor.     

The mouse ortholog of EFHC1 implicated in juvenile myoclonic epilepsy is an axonemal protein widely conserved among organisms with motile cilia and flagella

The gene product of EFHC1 recently implicated in juvenile myoclonic epilepsy (JME) was found to be a homolog of Chlamydomonas axonemal protein Rib72, whose homologs are present in a wide variety of organisms that have motile cilia and flagella. Western blot analyses and immunofluorescence localization of the mouse ortholog mRib72-1/Efhc1 indicated that it is indeed abundantly present in sperm flagella and tracheal cilia but only in a small amount in the brain. It is not present in immotile primary cilia. These observations raise the possibility that malfunction of motile cilia is involved in the development of JME.     

Pnlip encoding pancreatic lipase is possible candidate for obesity QTL in the OLETF rat

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat exhibits polygenic obesity, and one of quantitative trait loci (QTLs) responsible for a susceptibility to obesity in the OLETF, Nidd6/of, has been mapped to the approximately 10-cM genomic region between D1Rat166 and D1Rat90 on chromosome 1 in (OLETF x normal) F2 intercross. In this study, we have attempted to identify the causal gene for the Nidd6/of QTL. A Nidd6/of congenic strain, constructed by introgressing the OLETF allele on the mapped Nidd6/of region in the normal F344 rat strain, confirmed the existence of the Nidd6/of as obesity QTL. The Nidd6/of region was refined to a approximately 2.3-cM genomic region between D1Rat225 and D1Rat90, using informative recombinants selected from (Nidd6/of congenic x F344) F1 x Nidd6/of congenic backcross progenies. Among 46 genes located within the approximately 2.3-cM region, pancreatic lipase gene, Pnlip, was regarded as the most prominent and physiologically relevant positional candidate for the Nidd6/of QTL. We found that Pnlip possesses an OLETF allele-specific increase of mRNA levels in the pancreas, and that the OLETF allele is longer in variable number of tandem repeat (VNTR) within the 5'-flanking region than normal alleles. We further showed that the Nidd6/of QTL completely cosegregates with Pnlip VNTR in the informative recombinants from (Nidd6/of congenic x F344) F1 x Nidd6/of congenic backcross progenies. These results suggest that Pnlip is possible candidate for the Nidd6/of QTL.     

Sequential immune escape and shifting of T cell responses in a long-term survivor of melanoma

Immune-mediated control of tumors may occur, in part, through lysis of malignant cells by CD8(+) T cells that recognize specific Ag-HLA class I complexes. However, tumor cell populations may escape T cell responses by immune editing, by preventing formation of those Ag-HLA complexes. It remains unclear whether the human immune system can respond to immune editing and recognize newly arising escape variants. We report an example of shifting immune responses to escape variants in a patient with sequential metastases of melanoma and long-term survival after surgery alone. Tumor cells in the first metastasis escaped immune recognition via selective loss of an HLA haplotype (HLA-A11, -B44, and -Cw17), but maintained expression of HLA-A2. In the second metastasis, immune escape from an immunodominant MART-1-specific T cell response was mediated by HLA class I down-regulation, resulting in a failure to present this epitope, but persistent presentation of a tyrosinase-derived epitope. Consequent to this modification in tumor Ag presentation, the dominant CTL response shifted principally toward a tyrosinase-targeted response, even though tyrosinase-specific CTL had been undetectable during the initial metastatic event. Thus, in response to immune editing of tumor cells, a patient's spontaneous T cell response adapted, gaining the ability to recognize and to lyse "edited" tumor targets. The observation of both immune editing and immune adaptation in a patient with long-term survival after surgery alone demonstrates an example of immune system reactivity to counteract the escape mechanism(s) developed by tumor cells, which may contribute to the clinical outcome of malignant disease.     

Kinetics and mechanism of a reaction catalyzed by PST-01 protease from Pseudomonas aeruginosa PST-01

The initial rates of carboxybenzoyl-alanyl-l-leucyl-amide (Z-L-Ala-L-Leu-NH(2)) synthesis from carboxybenzoyl-L-alanine (Z-L-Ala) and L-leucineamide (L-Leu-NH(2)) and Z-L-Ala-L-Leu-NH(2) hydrolysis in a homogeneous dimethyl sulfoxide-aqueous buffer solution [1:1 (v/v)] system catalyzed by PST-01 protease from Pseudomonas aeruginosa were measured under a wide range of Z-L-Ala, L-Leu-NH(2) and Z-L-Ala-L-Leu-NH(2) concentrations. The initial rates of the synthetic reaction, in which Z-L-Ala-L-Leu-NH(2) was produced from Z-L-Ala and L-Leu-NH(2), were inhibited by the substrates. Furthermore, the initial rates of the synthetic reaction were not inhibited by the product Z-L-Ala-L-Leu-NH(2), and those of the hydrolytic reaction were inhibited by Z-L-Ala and L-Leu-NH(2). All the initial rate data of the synthetic and hydrolytic reactions were well correlated with the rate equation derived based on the proposed reaction scheme.     

Slow ADP-dependent acceleration of microtubule translocation produced by an axonemal dynein

Dynein has four nucleotide binding sites, of which the functional significance is unknown except for the single catalytic site. To obtain clues to the function of non-catalytic nucleotide binding, we examined the effect of ADP on the in vitro motility of Chlamydomonas inner-arm dynein species 'a'. Upon continuous perfusion with ATP and ADP, microtubules glided on a dynein-coated glass surface with a velocity that gradually increased over a few minutes. The velocity increased faster at higher ADP concentrations. These results suggest that this dynein is activated by nucleotide binding to regulatory site(s) through an extremely slow process.     

Discrete PIH proteins function in the cytoplasmic preassembly of different subsets of axonemal dyneins

Axonemal dyneins are preassembled in the cytoplasm before being transported into cilia and flagella. Recently, PF13/KTU, a conserved protein containing a PIH (protein interacting with HSP90) domain, was identified as a protein responsible for dynein preassembly in humans and Chlamydomonas reinhardtii. This protein is involved in the preassembly of outer arm dynein and some inner arm dyneins, possibly as a cofactor of molecular chaperones. However, it is not known which factors function in the preassembly of other inner arm dyneins. Here, we analyzed a novel C. reinhardtii mutant, ida10, and found that another conserved PIH family protein, MOT48, is responsible for the formation of another subset of inner arm dyneins. A variety of organisms with motile cilia and flagella typically have three to four PIH proteins, including potential homologues of MOT48 and PF13/KTU, whereas organisms without them have no, or only one, such protein. These findings raise the possibility that multiple PIH proteins are commonly involved in the preassembly of different subsets of axonemal dyneins.     

Phylogenetic analysis of manganese-oxidizing fungi isolated from manganese-rich aquatic environments in Hokkaido, Japan

Three strains of Mn-oxidizing fungi were isolated from manganese-rich aquatic environments: sediment in a stream (Komanoyu) in Mori-machi and inflow to an artificial wetland in Kaminokuni-cho, Hokkaido, Japan. The characteristics of each strain were then established. Genetic analysis based on the ribosomal RNA (rRNA) gene was performed to clarify their classification. The sequences of the 18S rRNA and internal transcribed spacer (ITS1)-5.8S rRNA-ITS2 genes showed that all three strains are Ascomycetes. Based on its morphology, it seems probable that the KY-1 strain from Mori-machi belongs to the genus Phoma or Ampelomyces. The phylogenetic analysis indicates that this strain belongs to Phoma rather than Ampelomyces. Morphological identification of WL-1 and WL-2 strains from Kaminokuni-cho was impossible because of the lack of a sexual stage and specific organs. Phylogenetic analysis of the sequence in the ITS1-5.8S rRNA-ITS2 gene suggests that the WL-1 strain corresponds to Paraconyothyrium sporulosum and that WL-2 also belongs to the genus Paraconiothyrium. Because the ability to oxidize Mn has not been evaluated for most species of Phoma or Paraconiothyrium (Coniothyrium), further study is needed to confirm the status of these three strains.