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961.
962.
The molecular weights of the native polysaccharides of Erwinia chrysanthemi strains range from 1.8 to 7.1 x 10(6) and their hydrodynamic properties are those of polydisperse, polyanionic biopolymers with pseudoplastic, non-thixotropic flow characteristics in aqueous solutions. The effect on the hydrodynamic properties of the polysaccharides by adding carboxyl groups to increase the charge density is studied, with particular reference to their molecular weight (MW), viscosity and conformation. In general, it is found that periodate oxidation of the extracellular polysaccharides of E. chrysanthemi strains, Ech9Sm6 and Ech6S+, introduces little change in the hydrodynamic properties of the resulting polyaldehydes. However, bromine oxidation at neutral pH of the polyaldehydes results in polycarboxylate biopolymers that show significant reduction in MW and viscosity, but they are still characteristic polyanions. 相似文献
963.
Isolated Trichinella spiralis nurse cells transport a significantly greater amount of glucose/mg of protein than the normal skeletal muscle cell line (L6). V(max) and K(m) estimations revealed that nurse cells have a much higher saturation point than L6 cells for glucose. The effects of numerous physiological conditions (Na(+) concentration, pH, and temperature) on nurse cell glucose uptake were investigated. It was determined that sodium concentration had no effect on glucose uptake. Low (<6.5) and high (>7.3) pH and low (5 degrees C) temperatures significantly effected glucose uptake. The two hormones, insulin and epinephrine, appeared to have little, if any, influence on the rate of glucose uptake by nurse cells. Glucose uptake was inhibited in the presence of 6-carbon carbohydrates. The H(+)/glucose symport inhibitors, dicyclohexylcarbodiimide (DCCD) and Carbonyl cyanide 4-trifluoromethoxyphenlhydrazone (FCCP), and the facilitated diffusion inhibitor phloretin also inhibited glucose uptake. Oubain, a Na(+)/glucose symport inhibitor, did not inhibit glucose uptake. These data, in conjunction with Western blot analyses, revealed that the transport of glucose occurs via H(+)/glucose symport and facilitated diffusion, perhaps through the glucose transport proteins GLUT 1 and/or 4. It was also demonstrated that nurse cells are capable of synthesising glycogen. It appears that glycogen is in a constant state of flux and physiological conditions, such as glucose concentration, significantly influence the synthesis of this macromolecule. We conclude that these results are consistent with the hypothesis that nurse cells, at least maintained in vitro, are metabolically highly active but show significant divergence from normal muscle cells in several fundamental aspects of sugar metabolism. 相似文献
964.
Montgomery HJ Bartlett R Perdicakis B Jervis E Squier TC Guillemette JG 《Biochemistry》2003,42(25):7759-7768
Several calmodulin (CaM) mutants were engineered in an effort to identify the functional implications of the oxidation of individual methionines in CaM on the activity of the constitutive isoforms of nitric oxide synthase (NOS). Site-directed mutagenesis was used to substitute the majority of methionines with leucines. Substitution of all nine methionine residues in CaM with leucines had minimal effects on the binding affinity or maximal enzyme activation for either the neuronal (nNOS) or endothelial (eNOS) isoform. Selective substitution permitted determination of the functional consequences of the site-specific oxidation of Met(144) and Met(145) on the regulation of electron transfer within nNOS and eNOS. Site-specific oxidation of Met(144) and Met(145) resulted in changes in the CaM concentration necessary for half-maximal activation of nNOS and eNOS, suggesting that these side chains are involved in stabilizing the productive association between CaM and NOS. However, the site-specific oxidation of Met(144) and Met(145) had essentially no effect on the maximal extent of eNOS activation in the presence of saturating concentrations of CaM. In contrast, the site-specific oxidation of Met(144) (but not Met(145)) resulted in a reduction in the level of nNOS activation that was associated with decreased rates of electron transfer within the reductase domain. Thus, nNOS and eNOS exhibit different functional sensitivities to conditions of oxidative stress that are expected to oxidize CaM. This may underlie some aspects of the observed differences in the sensitivities of proteins in vasculature and neuronal tissues to nitration that are linked to NOS activation and the associated generation of peroxynitrite. 相似文献
965.
Stewart JM Medow MS Montgomery LD 《American journal of physiology. Heart and circulatory physiology》2003,285(6):H2749-H2756
Postural tachycardia syndrome (POTS) is defined by orthostatic intolerance associated with abnormal upright tachycardia. Some patients have defective peripheral vasoconstriction and increased calf blood flow. Others have increased peripheral arterial resistance and decreased blood flow. In 14 POTS patients (13-19 yr) evenly subdivided among low-flow POTS (LFP) and high-flow POTS (HFP) we tested the hypothesis that myogenic, venoarteriolar, and reactive hyperemic responses are abnormal. We used venous occlusion plethysmography to measure calf venous pressure and blood flow in the supine position and when the calf was lowered by 40 cm to evoke myogenic and venoarteriolar responses and during venous hypertension by 40-mmHg occlusion to evoke the venoarteriolar response. We measured calf reactive hyperemia with plethysmography and cutaneous laser-Doppler flowmetry. Baseline blood flow in LFP was reduced compared with HFP and control subjects (0.8 +/- 0.2 vs. 4.4 +/- 0.5 and 2.7 +/- 0.4 ml.min-1.100 ml-1) but increased during leg lowering (1.2 +/- 0.5 ml.min-1. 100 ml-1) while decreasing in the others. Baseline peripheral arterial resistance was increased in LFP and decreased in HFP compared with control subjects (39 +/- 13 vs. 15 +/- 3 and 22 +/- 5 mmHg.ml-1. 100 ml. min) but decreased to 29 +/- 13 mmHg.ml-1.100 ml. min in LFP during venous hypertension. Resistance increased in the other groups. Maximum calf hyperemic flow and cutaneous flow were similar in all subjects. The duration of hyperemic blood flow was curtailed in LFP compared with either control or HFP subjects (plethysmographic time constant = 20 +/- 2 vs. 29 +/- 4 and 28 +/- 4 s; cutaneous time constant = 60 +/- 25 vs. 149 +/- 53 s in controls). Local blood flow regulation in low-flow POTS is impaired. 相似文献
966.
Effect of microbial heat shock proteins on airway inflammation and hyperresponsiveness 总被引:5,自引:0,他引:5
Rha YH Taube C Haczku A Joetham A Takeda K Duez C Siegel M Aydintug MK Born WK Dakhama A Gelfand EW 《Journal of immunology (Baltimore, Md. : 1950)》2002,169(9):5300-5307
Microbial heat shock proteins (hsp) have been associated with the generation and induction of Th1-type immune responses. We tested the effects of treatment with five different microbial hsp (Mycobacterium leprae, Streptococcus pneumoniae, Helicobacter pylori, bacillus Calmette-Guérin, and Mycobacterium tuberculosis) in a murine model of allergic airway inflammation and airway hyperresponsiveness (AHR). Mice were sensitized to OVA by i.p. injection and then challenged by OVA inhalation. Hsp were administered to each group by i.p. injection before sensitization and challenge. Sensitized and challenged mice developed increased serum levels of OVA-specific IgE with significant airway eosinophilia and heightened responsiveness to methacholine when compared with nonsensitized animals. Administration of M. leprae hsp prevented both development of AHR as well as bronchoalveolar lavage fluid eosinophilia in a dose-dependent manner. Treatment with M. leprae hsp also resulted in suppression of IL-4 and IL-5 production in bronchoalveolar lavage fluid, while IL-10 and IFN-gamma production were increased. Furthermore, M. leprae hsp treatment significantly suppressed OVA-specific IgE production and goblet cell hyperplasia/mucin hyperproduction. In contrast, treatment with the other hsp failed to prevent changes in airway responsiveness, lung eosinophilia, or cytokine production. Depletion of gamma/delta T lymphocytes before sensitization and challenge abolished the effect of M. leprae hsp treatment on AHR. These results indicate selective and distinctive properties among the hsp, and that M. leprae hsp may have a potential therapeutic role in the treatment of allergic airway inflammation and altered airway function. 相似文献
967.
David CA Middleton T Montgomery D Lim HB Kati W Molla A Xuei X Warrior U Kofron JL Burns DJ 《Journal of biomolecular screening》2002,7(3):259-266
A novel high-throughput strand transfer assay has been developed, using Microarray Compound Screening (microARCS) technology, to identify inhibitors of human immunodeficiency virus (HIV) integrase. This technology utilizes agarose matrices to introduce a majority of the reagents throughout the assay. Integration of biotinylated donor DNA with fluorescein isothiocyanate (FITC)-labeled target DNA occurs on a SAM membrane in the presence of integrase. An anti-FITC antibody conjugated to alkaline phosphatase (AP) was used to do an enzyme-linked immunosorbent assay with the SAM. An agarose gel containing AttoPhos, a substrate of AP, was used for detection of the integrase reactions on the SAM. For detection, the AttoPhos gel was separated from the SAM after incubation and then the gel was imaged using an Eagle Eye II closed-circuit device camera system. Potential integrase inhibitors appear as dark spots on the gel image. A library of approximately 250,000 compounds was screened using this HIV integrase strand transfer assay in microARCS format. Compounds from different structural classes were identified in this assay as novel integrase inhibitors. 相似文献
968.
Hewett D Samuelsson L Polding J Enlund F Smart D Cantone K See CG Chadha S Inerot A Enerback C Montgomery D Christodolou C Robinson P Matthews P Plumpton M Wahlstrom J Swanbeck G Martinsson T Roses A Riley J Purvis I 《Genomics》2002,79(3):305-314
Psoriasis is a chronic inflammatory disease of the skin with both genetic and environmental risk factors. Here we describe the creation of a single-nucleotide polymorphism (SNP) map spanning 900-1200 kb of chromosome 3q21, which had been previously recognized as containing a psoriasis susceptibility locus, PSORS5. We genotyped 644 individuals, from 195 Swedish psoriatic families, for 19 polymorphisms. Linkage disequilibrium (LD) between marker and disease was assessed using the transmission/disequilibrium test (TDT). In the TDT analysis, alleles of three of these SNPs showed significant association with disease (P<0.05). A 160-kb interval encompassing these three SNPs was sequenced, and a coding sequence consisting of 13 exons was identified. The predicted protein shares 30-40% homology with the family of cation/chloride cotransporters. A five-marker haplotype spanning the 3' half of this gene is associated with psoriasis to a P value of 3.8<10(-5). We have called this gene SLC12A8, coding for a member of the solute carrier family 12 proteins. It belongs to a class of genes that were previously unrecognized as playing a role in psoriasis pathogenesis. 相似文献
969.
Willem M Miosge N Halfter W Smyth N Jannetti I Burghart E Timpl R Mayer U 《Development (Cambridge, England)》2002,129(11):2711-2722
Basement membrane assembly is of crucial importance in the development and function of tissues and during embryogenesis. Nidogen 1 was thought to be central in the assembly processes, connecting the networks formed by collagen type IV and laminins, however, targeted inactivation of nidogen 1 resulted in no obvious phenotype. We have now selectively deleted the sequence coding for the 56 amino acid nidogen-binding site, gamma1III4, within the Lamc1 gene by gene targeting. Here, we show that mice homozygous for the deletion die immediately after birth, showing renal agenesis and impaired lung development. These developmental defects were attributed to locally restricted ruptures in the basement membrane of the elongating Wolffian duct and of alveolar sacculi. These data demonstrate that an interaction between two basement membrane proteins is required for early kidney morphogenesis in vivo. 相似文献
970.
Fine-tuning function: correlation of hinge domain interactions with functional distinctions between LacI and PurR
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Swint-Kruse L Larson C Pettitt BM Matthews KS 《Protein science : a publication of the Protein Society》2002,11(4):778-794
LacI and PurR are highly homologous proteins. Their functional units are homodimers, with an N-terminal DNA binding domain that comprises the helix-turn-helix (HTH), N-linker, and hinge regions from both monomers. Hinge structural changes are known to occur upon DNA dissociation but are difficult to monitor experimentally. The initial steps of hinge unfolding were therefore examined using molecular dynamics simulations, utilizing a truncated, chimeric protein comprising the LacI HTH/N-linker and PurR hinge. A terminal Gly-Cys-Gly was added to allow "dimerization" through disulfide bond formation. Simulations indicate that differences in LacI and PurR hinge primary sequence affect the quaternary structure of the hinge x hinge' interface. However, these alternate hinge orientations would be sterically restricted by the core domain. These results prompted detailed comparison of recently available DNA-bound structures for LacI and truncated LacI(1-62) with the PurR structure. Examination revealed that different N-linker and hinge contacts to the core domain of the partner monomer (which binds effector molecule) affect the juxtapositions of the HTH, N-linker, and hinge regions in the DNA binding domain. In addition, the two full-length repressors exhibit significant differences in the interactions between the core and the C-linker connection to the DNA binding domain. Both linkers and the hinge have been implicated in the allosteric response of these repressors. Intriguingly, one functional difference between these two proteins is that they exhibit opposite allosteric response to effector. Simulations and observed structural distinctions are correlated with mutational analysis and sequence information from the LacI/GalR family to formulate a mechanism for fine-tuning individual repressor function. 相似文献