Impact of acute ascites on the action of the canine abdominal muscles.

Although ascites causes abdominal expansion, its effects on abdominal muscle function are uncertain. In the present study, progressively increasing ascites was induced in supine anesthetized dogs, and the changes in abdominal (DeltaPab) and airway opening (DeltaPao) pressure obtained during stimulation of the internal oblique and transversus abdominis muscles were measured; the changes in internal oblique muscle length were also measured. As ascites increased from 0 to 100 ml/kg body wt, Pab and muscle length during relaxation increased. DeltaPab also showed a threefold increase (P < 0.001). However, DeltaPao decreased (P < 0.001). When ascites increased further to 200 ml/kg, resting muscle length continued to increase and muscle shortening during stimulation became very small so that active muscle length was 155% of the resting muscle length in the control condition. Concomitantly, DeltaPab returned to the control value, and DeltaPao continued to decrease. Similar results were obtained with the animals in the head-up posture, although the decrease in DeltaPao appeared only when ascites was greater than 125 ml/kg. It is concluded that 1) ascites adversely affects the expiratory action of the abdominal muscles on the lung; 2) this effect results primarily from the increase in diaphragm elastance; and 3) when ascites is severe, the abdomen cross-sectional area is also increased and the abdominal muscles are excessively lengthened so that their active pressure-generating ability itself is reduced.     

Superoxide Dismutase Is a Virulence Factor Produced by the Coral Bleaching Pathogen <Emphasis Type="Italic">Vibrio shiloi</Emphasis>

Coral bleaching is a disease that threatens coral reefs throughout the world. The disease is correlated with higher-than-normal seawater temperatures. Data have been reported showing that bleaching of the coral Oculina patagonica during the summer in the Mediterranean Sea is the result of an infection with Vibrio shiloi. The summer temperatures induce the expression of virulence factors in the pathogen. We report here that V. shiloi produces an extracellular superoxide dismutase (SOD) at 30°C, but not at 16°C. An SOD⁻ mutant was avirulent. The mutant adhered to corals, penetrated into coral cells, multiplied intracellularly for a short time, and then died. These data support the hypothesis that SOD protects the intracellular V. shiloi from oxidative stress caused by the high concentration of oxygen produced by intracellular zooxanthellae photosynthesis. Received: 3 July 2002 / Accepted: 27 July 2002     

Tryptophan synthase: the workings of a channeling nanomachine

Substrate channeling between enzymes has an important role in cellular metabolism by compartmentalizing cytoplasmic synthetic processes. The bacterial tryptophan synthases are multienzyme nanomachines that catalyze the last two steps in L-tryptophan biosynthesis. The common metabolite indole is transferred from one enzyme to the other in each alphabeta-dimeric unit of the alpha2beta2 complex via an interconnecting 25-A-long tunnel. Recent solution studies of the Salmonella typhimurium alpha2beta2 complex coupled with X-ray crystal-structure determinations of complexes with substrates, intermediates and substrate analogs have driven important breakthroughs concerning the identification of the linkages between the bi-enzyme complex structure, catalysis at the alpha- and beta-active sites, and the allosteric regulation of substrate channeling.     

SNP-specific extraction of haplotype-resolved targeted genomic regions

The availability of genotyping platforms for comprehensive genetic analysis of complex traits has resulted in a plethora of studies reporting the association of specific single-nucleotide polymorphisms (SNPs) with common diseases or drug responses. However, detailed genetic analysis of these associated regions that would correlate particular polymorphisms to phenotypes has lagged. This is primarily due to the lack of technologies that provide additional sequence information about genomic regions surrounding specific SNPs, preferably in haploid form. Enrichment methods for resequencing should have the specificity to provide DNA linked to SNPs of interest with sufficient quality to be used in a cost-effective and high-throughput manner. We describe a simple, automated method of targeting specific sequences of genomic DNA that can directly be used in downstream applications. The method isolates haploid chromosomal regions flanking targeted SNPs by hybridizing and enzymatically elongating oligonucleotides with biotinylated nucleotides based on their selective binding to unique sequence elements that differentiate one allele from any other differing sequence. The targeted genomic region is captured by streptavidin-coated magnetic particles and analyzed by standard genotyping, sequencing or microarray analysis. We applied this technology to determine contiguous molecular haplotypes across a ~150 kb genomic region of the major histocompatibility complex.     

Crystal structure of human XLF/Cernunnos reveals unexpected differences from XRCC4 with implications for NHEJ

The recently characterised 299-residue human XLF/Cernunnos protein plays a crucial role in DNA repair by non-homologous end joining (NHEJ) and interacts with the XRCC4-DNA Ligase IV complex. Here, we report the crystal structure of the XLF (1-233) homodimer at 2.3 A resolution, confirming the predicted structural similarity to XRCC4. The XLF coiled-coil, however, is shorter than that of XRCC4 and undergoes an unexpected reverse in direction giving rise to a short distorted four helical bundle and a C-terminal helical structure wedged between the coiled-coil and head domain. The existence of a dimer as the major species is confirmed by size-exclusion chromatography, analytical ultracentrifugation, small-angle X-ray scattering and other biophysical methods. We show that the XLF structure is not easily compatible with a proposed XRCC4:XLF heterodimer. However, we demonstrate interactions between dimers of XLF and XRCC4 by surface plasmon resonance and analyse these in terms of surface properties, amino-acid conservation and mutations in immunodeficient patients. Our data are most consistent with head-to-head interactions in a 2:2:1 XRCC4:XLF:Ligase IV complex.     

Cholest-4-En-3-One-Δ1-Dehydrogenase, a Flavoprotein Catalyzing the Second Step in Anoxic Cholesterol Metabolism

The anoxic metabolism of cholesterol was studied in the denitrifying bacterium Sterolibacterium denitrificans, which was grown with cholesterol and nitrate. Cholest-4-en-3-one was identified before as the product of cholesterol dehydrogenase/isomerase, the first enzyme of the pathway. The postulated second enzyme, cholest-4-en-3-one-Δ¹-dehydrogenase, was partially purified, and its N-terminal amino acid sequence and tryptic peptide sequences were determined. Based on this information, the corresponding gene was amplified and cloned and the His-tagged recombinant protein was overproduced, purified, and characterized. The recombinant enzyme catalyzes the expected Δ¹-desaturation (cholest-4-en-3-one to cholesta-1,4-dien-3-one) under anoxic conditions. It contains approximately one molecule of FAD per 62-kDa subunit and forms high molecular aggregates in the absence of detergents. The enzyme accepts various artificial electron acceptors, including dichlorophenol indophenol and methylene blue. It oxidizes not only cholest-4-en-3-one, but also progesterone (with highest catalytic efficiency, androst-4-en-3,17-dione, testosterone, 19-nortestosterone, and cholest-5-en-3-one. Two steroids, corticosterone and estrone, act as competitive inhibitors. The dehydrogenase resembles 3-ketosteroid-Δ¹-dehydrogenases from other organisms (highest amino acid sequence identity with that from Pseudoalteromonas haloplanktis), with some interesting differences. Due to its catalytic properties, the enzyme may be useful in steroid transformations.     

The tight junction proteins claudin-1, -6, and -9 are entry cofactors for hepatitis C virus

Hepatitis C virus (HCV) is a major cause of liver disease in humans. The CD81 tetraspanin is necessary but not sufficient for HCV penetration into hepatocytes, and it was recently reported that the tight junction protein claudin-1 is a critical HCV entry cofactor. Here, we confirm the role of claudin-1 in HCV entry. In addition, we show that claudin-6 and claudin-9 expressed in CD81(+) cells also enable the entry of HCV pseudoparticles derived from six of the major genotypes. Whereas claudin-1, -6, and -9 function equally well as entry cofactors in endothelial cells, claudin-1 is more efficient in hepatoma cells. This suggests that additional cellular factors modulate the ability of claudins to function as HCV entry cofactors. Our work has generated novel and essential means to investigate the mechanism of HCV penetration into hepatocytes and the role of the claudin protein family in HCV dissemination, replication, and pathogenesis.     

Engineering and use of (32)P-labeled human recombinant interleukin-11 for receptor binding studies.

Human interleukin-11 (hIL-11) is a pleiotropic cytokine that is involved in numerous biological activities such as hematopoiesis, osteoclastogenesis, neurogenesis and female fertility. IL-11 is obviously a key reagent to study the IL-11 receptors. However, conventional radio-iodination techniques lead to a loss of IL-11 bioactivity. Here, we report the construction and the production of a new recombinant human IL-11 (FP Delta IL-11). In this molecule, a specific phosphorylation site (RRASVA) has been introduced at the N-terminus of rhIL-11. It can be specifically phosphorylated by bovine heart protein kinase and accordingly, easily radiolabeled with (32)P. A high radiological specific activity (250,000 c.p.m x ng(-1) of protein) was obtained with the retention of full biological activity of the protein. The binding of (32)P-labeled FP Delta IL-11 to Ba/F3 cells stably transfected with plasmids encoding human IL-11 receptors alpha and beta chains (IL-11R alpha and gp130) was specific and saturable with a high affinity as determined from Scatchard plot analysis. Availability of this new ligand should prompt further studies on IL-11R structure, expression and regulation.     

Obesity modulates the expression of haptoglobin in the white adipose tissue via TNFalpha.

Increase in adipose mass results in obesity and modulation of several factors in white adipose tissue (WAT). Two important examples are tumor necrosis factor alpha (TNFalpha) and leptin, both of which are upregulated in adipose tissue in obesity. In order to isolate genes differentially expressed in the WAT of genetically obese db/db mice compared to their lean littermates, we performed RNA fingerprinting and identified haptoglobin (Hp), which is significantly upregulated in the obese animals. Hp is a glycoprotein induced by a number of cytokines, LPS (Lipopolysaccharide), and more generally by inflammation. A significant upregulation of WAT Hp expression was also evident in several experimental obese models including the yellow agouti (/) A(y), ob/ob and goldthioglucose-treated mice (10-, 8-, and 7-fold, respectively). To identify the potential signals for an increase in Hp expression in obesity, we examined leptin and TNFalpha in vivo. Wild type animals treated with recombinant leptin did not show any alteration in WAT Hp expression compared to controls that were food restricted to the level of intake of the treated animals. On the other hand, Hp expression was induced in mice transgenically expressing TNFalpha in adipose tissue. Finally, a significant downregulation of WAT Hp mRNA was observed in ob/ob mice deficient in TNFalpha function, when compared to the ob/ob controls. These results demonstrate that haptoglobin expression in WAT is increased in obesity in rodents and TNFalpha is an important signal for this regulation.