Limited role for CXC chemokines in the pathogenesis of alpha-naphthylisothiocyanate-induced liver injury

Alpha-naphthylisothiocyanate (ANIT) is a hepatotoxin that causes severe neutrophilic inflammation around portal tracts and bile ducts. The chemotactic signals that provoke this inflammatory response are unknown. In this study, we addressed the possibility that ANIT upregulates CXC chemokines in the liver and that these compounds mediate hepatic inflammation and tissue injury after ANIT treatment. Mice treated with a single dose of ANIT (50 mg/kg) exhibited rapid hepatic induction of the CXC chemokine macrophage inflammatory protein-2 (MIP-2). MIP-2 derived primarily from hepatocytes, with no apparent contribution by biliary cells. In ANIT-treated mice, the induction of MIP-2 coincided with an influx of neutrophils to portal zones; this hepatic neutrophil recruitment was suppressed by 50% in mice that lack the receptor for MIP-2 (CXCR2(-/-)). Interestingly, despite their markedly reduced degree of hepatic inflammation, CXCR2(-/-) mice displayed just as much hepatocellular injury and cholestasis after ANIT treatment as wild-type mice. Moreover, after long-term exposure, ANIT CXCR2(-/-) mice developed liver fibrosis that was indistinguishable from that in wild-type mice. In summary, our data show that CXC chemokines are responsible for some of the hepatic inflammation that occurs in response to ANIT but that these compounds are not essential to the pathogenesis of either acute or chronic ANIT hepatotoxicity.     

Von Hippel-Lindau disease

Germline mutations in the VHL tumour suppressor gene may cause a variety of phenotypes including von Hippel-Lindau (VHL) disease, familial phaeochromocytoma and inherited polycythaemia. VHL disease is a multisystem familial cancer syndrome and is the commonest cause of familial renal cell carcinoma (RCC). VHL disease provides a paradigm for illustrating how studies of a rare familial cancer syndrome can produce advances in clinical medicin and important insights into basic biological processes. Thus the identification of the VHL gene has improved the diagnosis and clinical management of VHL disease and provided insights into the pathogenesis of sporadic clear cell RCC. Functional investigations of the VHL gene product have provided novel information on how cells sense oxygen and the role of hypoxia-response pathways in human tumourigenesis. Such information offers prospects of novel therapeutic interventions for VHL disease and common cancers including RCC.     

Homozygosity for a missense mutation in the 67 kDa isoform of glutamate decarboxylase in a family with autosomal recessive spastic cerebral palsy: parallels with Stiff-Person Syndrome and other movement disorders

Background  

Cerebral palsy (CP) is an heterogeneous group of neurological disorders of movement and/or posture, with an estimated incidence of 1 in 1000 live births. Non-progressive forms of symmetrical, spastic CP have been identified, which show a Mendelian autosomal recessive pattern of inheritance. We recently described the mapping of a recessive spastic CP locus to a 5 cM chromosomal region located at 2q24-31.1, in rare consanguineous families.     

Solution measurement of DNA curvature in papillomavirus E2 binding sites

‘Indirect readout’ refers to the proposal that proteins can recognize the intrinsic three-dimensional shape or flexibility of a DNA binding sequence apart from direct protein contact with DNA base pairs. The differing affinities of human papillomavirus (HPV) E2 proteins for different E2 binding sites have been proposed to reflect indirect readout. DNA bending has been observed in X-ray structures of E2 protein–DNA complexes. X-ray structures of three different E2 DNA binding sites revealed differences in intrinsic curvature. DNA sites with intrinsic curvature in the direction of protein-induced bending were bound more tightly by E2 proteins, supporting the indirect readout model. We now report solution measurements of intrinsic DNA curvature for three E2 binding sites using a sensitive electrophoretic phasing assay. Measured E2 site curvature agrees well the predictions of a dinucleotide model and supports an indirect readout hypothesis for DNA recognition by HPV E2.     

Identification of the gene for beta-fructofuranosidase of Bifidobacterium lactis DSM10140(T) and characterization of the enzyme expressed in Escherichia coli

Bifidobacterium lactis is a moderately oxygen-tolerant, saccharolytic bacterium often used in combination with fructooligosaccharides (FOS) as a probiotic supplement in diverse dairy products. This is the first report describing the gene structure and enzymatic properties of a beta-fructofuranosidase [EC 3.2.1.26] from Bifidobacteria. BfrA was identified in Bifidobacterium lactis DSM 10140(T) and heterologously expressed in Escherichia coli. The G+C content was identical with the G+C content as determined for the total genomic DNA (61.9 mol %). The gene codes for a 532-aa residue polypeptide of 59.4 kDa. Surprisingly, the deduced aa sequence revealed only minor similarity to other fructofuranosidases (18% to E. coli cscA). The enzyme was purified to homogeneity after incorporation of a C-terminal 6 x HIS affinity tag. It hydrolased sucrose, 1-kestose, Raftilose, Actilight, inulin, and raffinose (100%, 91%, 84%, 80%, 37%, 4%). Fructose moieties were released in an exo-type fashion. Substrates with alpha-glycosidic linkages or residues other than fructose were not attacked. The kinetic parameters K(m) and V(max) for sucrose hydrolysis were 10.3 m M and 0.031 microM/min (pH 7.6; 37 degrees C). The activity was abolished by Zn(2+) (1 m M) and significantly inhibited by Fe(2+) and Ni(2+) (10 m M). The enzyme showed its maximal activity at 40 degrees C.     

Subcellular localization of Pseudomonas pyocyanin cytotoxicity in human lung epithelial cells

The Pseudomonas aeruginosa secretory product pyocyanin damages lung epithelium, likely due to redox cycling of pyocyanin and resultant superoxide and H(2)O(2) generation. Subcellular site(s) of pyocyanin redox cycling and toxicity have not been well studied. Therefore, pyocyanin's effects on subcellular parameters in the A549 human type II alveolar epithelial cell line were examined. Confocal and electron microscopy studies suggested mitochondrial redox cycling of pyocyanin and extracellular H(2)O(2) release, respectively. Pyocyanin decreased mitochondrial and cytoplasmic aconitase activity, ATP levels, cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, and mitochondrial membrane potential. These effects were transient at low pyocyanin concentrations and were linked to apparent cell-mediated metabolism of pyocyanin. Overexpression of MnSOD, but not CuZnSOD or catalase, protected cellular aconitase, but not ATP, from pyocyanin-mediated depletion. This suggests that loss of aconitase activity is not responsible for ATP depletion. How pyocyanin leads to ATP depletion, the mechanism of cellular metabolism of pyocyanin, and the impact of mitochondrial pyocyanin redox cycling on other cellular events are important areas for future study.     

ST7 is a novel low-density lipoprotein receptor-related protein (LRP) with a cytoplasmic tail that interacts with proteins related to signal transduction pathways

In 1997, McCormick and co-workers identified a novel putative tumor suppressor gene, designated ST7, encoding a unique protein with transmembrane receptor characteristics [Qing et al. (1999) Oncogene 18, 335-342]. Using degenerate primers corresponding to the highly conserved region of the ligand-binding domains of members of the low-density lipoprotein receptor (LDLR) superfamily, Ishii et al. [Genomics (1998) 51, 132-135] discovered a low-density lipoprotein receptor-related protein (LRP) that closely resembles ST7. Later, another LRP closely resembling ST7 and LRP3 was found (murine LRP9) [Sugiyama et al. (2000) Biochemistry 39, 15817-15825]. These results strongly suggested that ST7 was also a novel member of the low-density lipoprotein receptor superfamily. Proteins of this superfamily have been shown to function in endocytosis and/or signal transduction. To evaluate the relationship of ST7 to the LDLR superfamily proteins and to determine whether ST7 may function in endocytosis and/or signal transduction, we used proteomic tools to analyze the functional motifs present in the protein. Our results indicate that ST7 is a member of a subfamily of the LDLR superfamily and that its cytoplasmic domain contains several motifs implicated in endocytosis and signal transduction. Use of the yeast two-hybrid system to identify proteins that associate with ST7's cytoplasmic domain revealed that this domain interacts with three proteins involved in signal transduction and/or endocytosis, viz., receptor for activated protein C kinase 1 (RACK1), muscle integrin binding protein (MIBP), and SMAD anchor for receptor activation (SARA), suggesting that ST7, like other proteins in the LDLR superfamily, functions in these two pathways. Clearly, ST7 is an LRP, and therefore, it should now be referred to as LRP12.     

Mutagenesis in eukaryotes dependent on DNA polymerase zeta and Rev1p

DNA polymerase zeta (Pol zeta) and Rev1p carry out translesion replication in budding yeast, Saccharomyces cerevisiae, and are jointly responsible for almost all base pair substitution and frameshift mutations induced by DNA damage in this organism. In addition, Pol zeta is responsible for the majority of spontaneous mutations in yeast and has been proposed as the enzyme responsible for somatic hypermutability. Pol zeta, a non-processive enzyme that lacks a 3' to 5' exonuclease proofreading activity, is composed of Rev3p, the catalytic subunit, and a second subunit encoded by REV7. In keeping with its role, extension by Pol zeta is relatively tolerant of abnormal DNA structure at the primer terminus and is much more capable of extension from terminal mismatches than yeast DNA polymerase alpha (Pol alpha). Rev1p is a bifunctional enzyme that possesses a deoxycytidyl transferase activity that incorporates deoxycytidyl opposite abasic sites in the template and a second, at present poorly defined, activity that is required for the bypass of a variety of lesions as well as abasic sites. Human homologues of the yeast REV1 and REV3 have been identified and, based on the phenotype of cells producing antisense RNA to one or other of these genes, their products appear also to be employed in translation replication and spontaneous mutagenesis. We suggest that Pol zeta is best regarded as a replication enzyme, albeit one that is used only intermittently, that promotes extension at forks the progress of which is blocked for any reason, whether the presence of an unedited terminal mismatch or unrepaired DNA lesion.     

Mutation analysis in Rett syndrome

Rett syndrome is an X-linked dominant neurodevelopmental disorder caused by mutations in the MECP2 gene. Mutations have been demonstrated in more than 80% of females with typical features of Rett syndrome. We identified mutations in the MECP2 gene and documented the clinical manifestations in 65 Rett syndrome patients to characterize the genotype-phenotype spectrum. Bidirectional sequencing of the entire MECP2 coding region was performed. We diagnosed 65 patients with MECP2 mutations. Of these, 15 mutations had been reported previously and 13 are novel. Two patients have multiple deletions within the MECP2 gene. Eight common mutations were found in 43 of 65 patients (66.15%). The majority of patients with identified mutations have the classic Rett phenotype, and several had atypical phenotypes. MECP2 analysis identified mutations in almost all cases of typical Rett syndrome, as well as in some with atypical phenotypes. Eleven (20.4%) of the 54 patients with defined mutations and in whom phenotypic data were obtained did not develop acquired microcephaly. Hence, microcephaly at birth or absence of acquired microcephaly does not obviate the need for MECP2 analysis. We have initiated cascade testing starting with PCR analysis for common mutations followed by sequencing, when necessary. Analysis of common mutations before sequencing the entire gene is anticipated to be the most efficacious strategy to identify Rett syndrome gene mutations.     

Molecular and functional characterization of genes encoding horse MHC class I antigens

Sequence and functional analyses were undertaken on two cDNAs and a genomic clone encoding horse major histocompatibility complex (MHC) class I molecules. All of the clones were isolated from a single horse that is homozygous for all known horse MHC class I and class II antigens. The two cDNAs (clones 8-9 and 1-29) were isolated from a lymphocyte library and encode polymorphic MHC antigens from two loci. The genomic cosmid clone, isolated from a sperm library, contains the 8-9 gene. All three genes were expressed in mouse L-cells and were recognized by alloantisera and, for the cDNAs, by alloreactive cytotoxic T lymphocytes. A total of 3815 bp of the genomic clone were sequenced, extending from 429 bp upstream (5') of the leader peptide through the 3' untranslated region. Promoter region motifs and an intron-exon structure characteristic of MHC class I genes of other species were found. A subclone containing 407 bp of the promoter region was inserted into a chloramphenicol acetyl transferase reporter plasmid, tested in transient transfection assays, and found to have promoter activity in heterologous cells. This genomic clone will enable detailed studies of MHC class I gene regulation in horse trophoblasts, and in horse retroviral infections.