A novel hydrogenase has recently been found in methanogenic archaea. It catalyzes the reversible dehydrogenation of methylenetetrahydromethanopterin (CH2=H4MPT) to methenyltetrahydromethanopterin (CHEH4MPT') and H2 and was therefore named H2-forming methylenetetrahydromethanopterin dehydrogenase. The hydrogenasc, which is composed of only one polypeptide with an apparent molecular mass of 43 kDa, does not mediate the reduction of viologen dyes with either Hz or CH2=H4MPT. We report here that the purified enzyme from Methanobacterium thernioautotrophicum exhibits the following other unique properties : (a) the colorless protein with a specific activity or 2000 Uimg (V,,,,,) did not contain iron-sulfur clusters, nickel, or flavins; (b) the activity was not inhibited by carbon monoxide, acetylene. nitrite, cyanide, or azide; (cj the enzyme did not catalyze an isotopic exchange between 'H, and 'H+ ; (d) the enzyme catalyzed the reduction of CHFH4MPT+ with 'H, generating [n2ethylenc-'HH]CH,=H4MPT; and (e) the primary structure contained at most four conserved cysteines as revealed by a comparison of the DNA-deduced amino acid sequence of [he proteins from M . theumuautotrophicum and Methanopyrus kundleri. None of the four cysteines were closely spaced as would be indicative for a (NiFe) hydrogenase or a ferredoxintype iron-sulfur protein.Properties of the H,-forming methylenetetrahydromethanopterin dehydrogenase from Methmobacterium i v d f e i are also described indicating that thc enzyme from this methanogenic archaeon is very similar to the enzyme from M . thermoautotrophicum with respect both to molecular and catalytic properties.

An integration vector for use in Methanococcus voltae was constructed, based on the Escherichia coli vector pUC18. It carries the structural gene for puromycin transacetylase from Streptomyces alboniger, which is flanked by expression signals of M. voltae structural genes and hisA gene sequences of this bacterium. Transformed M. voltae cells are puromycin resistant. Several types of integration of the vector into the chromosome were found. Only one case was due to nonhomologous recombination. The integrated sequences were stable under selective pressure but were slowly lost in some cases in the absence of the selective drug. The vector could be excised from M. voltae chromosomal DNA, recircularized and transformed back into E. coli.

The extracellular matrix molecule tenascin-C (TNC) is a major component of the cancer-specific matrix, and high TNC expression is linked to poor prognosis in several cancers. To provide a comprehensive understanding of TNC's functions in cancer, we established an immune-competent transgenic mouse model of pancreatic β-cell carcinogenesis with varying levels of TNC expression and compared stochastic neuroendocrine tumor formation in abundance or absence of TNC. We show that TNC promotes tumor cell survival, the angiogenic switch, more and leaky vessels, carcinoma progression, and lung micrometastasis. TNC downregulates Dickkopf-1 (DKK1) promoter activity through the blocking of actin stress fiber formation, activates Wnt signaling, and induces Wnt target genes in tumor and endothelial cells. Our results implicate DKK1 downregulation as an important mechanism underlying TNC-enhanced tumor progression through the provision of a proangiogenic tumor microenvironment.

The Neuromutagenesis Facility at the Jackson Laboratory generated a mouse model of retinal vasculopathy, nmf223, which is characterized clinically by vitreal fibroplasia and vessel tortuosity. nmf223 homozygotes also have reduced electroretinogram responses, which are coupled histologically with a thinning of the inner nuclear layer. The nmf223 locus was mapped to chromosome 17, and a missense mutation was identified in Lama1 that leads to the substitution of cysteine for a tyrosine at amino acid 265 of laminin ␣1, a basement membrane protein.Despite normal localization of laminin ␣1 and other components of the inner limiting membrane, a reduced integrity of this structure was suggested by ectopic cells and blood vessels within the vitreous. Immunohistochemical characterization of nmf223 homozygous retinas demonstrated the abnormal migration of retinal astrocytes into the vitreous along with the persistence of hyaloid vasculature. The Y265C mutation significantly reduced laminin N-terminal domain (LN) interactions in a bacterial twohybrid system. Therefore, this mutation could affect interactions between laminin ␣1 and other laminin chains. To expand upon these findings, a Lama1 null mutant, Lama1 tm1.1Olf , was generated that exhibits a similar but more severe retinal phenotype than that seen in nmf223 homozygotes. The increased severity of the Lama1 null mutant phenotype is probably due to the complete loss of the inner limiting membrane in these mice. This first report of viable Lama1 mouse mutants emphasizes the importance of this gene in retinal development. The data presented herein suggest that hypomorphic mutations in human LAMA1 could lead to retinal disease.