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71.
Summary Extended and enhanced production of swainsonine was achieved from fed-batch fermentations of the fungus Metarhizium anisopliae. A complex medium based on oatmeal extract was intermittently fed with D-glucose and/or lysine. Swainsonine titres were improved eleven-fold and the duration of production extended was from 240 to 550 hours compared with a batch culture under the same conditions. 相似文献
72.
Dihydroxypropylation of amino groups of proteins: use of glyceraldehyde as a reversible agent for reductive alkylation 总被引:1,自引:0,他引:1
The mode of derivatization of amino groups of proteins by glyceraldehyde, an aldotriose, depends on the presence or absence of reducing agent. In the presence of sodium cyanoborohydride, the Schiff base adducts of the aldehyde with the amino groups are reduced, and dihydroxypropylation of amino groups takes place (reductive mode). The reductively glycated lysine residue, N epsilon-(2,3-dihydroxypropyl)lysine, is a substituted alpha-amino alcohol. This alpha-amino alcoholic function of the derivatized lysine should be susceptible to periodate oxidation, and this oxidation is anticipated to result in the regeneration of the lysine residue. This aspect has been now investigated. Indeed, on mild periodate oxidation (15 mM periodate, 15 min at room temperature) of dihydroxypropylated ribonuclease A, nearly 95% of its N epsilon-(2,3-dihydroxypropyl)lysine residues were regenerated to lysine residues. The removal of the dihydroxypropyl groups by periodate oxidation could be accomplished within a wide pH range with little variation in the recovery of lysines. The possible usefulness of this reversible chemical modification procedure in the primary structural studies of proteins was investigated with a tryptic peptide of dihydroxypropylated streptococcal M5 protein, namely, DHP-T4. This 12-residue tryptic peptide contains one internal N epsilon-(dihydroxypropyl)lysine. The dihydroxypropylated peptide released most of its dihydroxypropyl groups on mild periodate oxidation. Redigestion of the periodate-treated peptide with trypsin generated the two expected peptides, demonstrating the generation of a trypsin-susceptible site. Reductive dihydroxypropylation of amino groups of RNase A resulted in the loss of its enzyme activity, the extent of inactivation increasing with the concentration of the glyceraldehyde used.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
73.
D G Streefkerk B N Manjula C P Glaudemans 《Journal of immunology (Baltimore, Md. : 1950)》1979,122(2):537-541
Five murine myeloma immunoglobulins have been studied for their binding with fructose-containing homopolysaccharides. The results indicate that the anti-(2 leads to 1)-D-fructofuranans are capable of binding (2 leads to 6)-D-fructofuranans with reduced affinity. An anti-(2 leads to 6)-D-fructofuranan (U10) was incapable of binding a (2 leads to 1)-D-fructofuranan. By using molecular models of fructofuranans of both linkage types, and a hypothetical molecular model of the combining region of E109, an anti-(2 leads to 1)-D-fructofuranan, a rationalization of these date can be proposed. 相似文献
74.
Saravanan Manjula Magudeeswaran Sivanandam 《Journal of biomolecular structure & dynamics》2019,37(9):2440-2456
The NS5B RdRp polymerase is a prominent enzyme for the replication of Hepatitis C virus (HCV). During the HCV replication, the template RNA binding takes place in the “fingers” sub-domain of NS5B. The “fingers” domain is a new emerging allosteric site for the HCV drug development. The inhibitors of the “fingers” sub-domain adopt a new antiviral mechanism called RNA intervention. The details of essential amino acid residues, binding mode of the ligand, and the active site intermolecular interactions of RNA intervention reflect that this mechanism is ambiguous in the experimental study. To elucidate these details, we performed molecular docking analysis of the fingers domain inhibitor quercetagetin (QGN) with NS5B polymerase. The detailed analysis of QGN-NS5B intermolecular interactions was carried out and found that QGN interacts with the binding pocket amino acid residues Ala97, Ala140, Ile160, Phe162, Gly283, Gly557, and Asp559; and also forms π?π stacking interaction with Phe162 and hydrogen bonding interaction with Gly283. These are found to be the essential interactions for the RNA intervention mechanism. Among the strong hydrogen bonding interactions, the QGN?Ala140 is a newly identified important hydrogen bonding interaction by the present work and this interaction was not resolved by the previously reported crystal structure. Since D559G mutation at the fingers domain was reported for reducing the inhibition percentage of QGN to sevenfold, we carried out molecular dynamics (MD) simulation for wild and D559G mutated complexes to study the stability of protein conformation and intermolecular interactions. At the end of 50?ns MD simulation, the π?π stacking interaction of Phe162 with QGN found in the wild-type complex is altered into T-shaped π stacking interaction, which reduces the inhibition strength. The origin of the D559G resistance mutation was studied using combined MD simulation, binding free energy calculations and principal component analysis. The results were compared with the wild-type complex. The mutation D559G reduces the binding affinity of the QGN molecule to the fingers domain. The free energy decomposition analysis of each residue of wild-type and mutated complexes revealed that the loss of non-polar energy contribution is the origin of the resistance.
Communicated by Ramaswamy H. Sarma 相似文献
75.
Truncated human LMP-1 triggers differentiation of C2C12 cells to an osteoblastic phenotype in vitro 总被引:4,自引:0,他引:4
Fei Q Boden SD Sangadala S Viggeswarapu M Liu Y Titus L 《Acta biochimica et biophysica Sinica》2007,39(9):693-700
LIM mineralization protein-1 (LMP-1) is a novel intracellular osteoinductive protein that has been shown to induce bone formation both in vitro and in viva. LMP-1 contains an N-terminal PDZ domain and three C-terminal LIM domains. In this study, we investigated whether a truncated form of human LMP-1 (hLMP-1 [t]), lacking the three C-terminal LIM domains, triggers the differentiation of pluripotent myoblastic C2C12 cells to the osteoblast lineage. C2C12 cells were transiently transduced with AdS-hLMP-1 (t)-green fluorescent protein or viral vector control. The expression of hLMP-1 (t) RNA and the truncated protein were examined. The results showed that hLMP-1 (t) blocked myotube formation in C2C12 cultures and significantly enhanced the alkaline phosphatase (ALP) activity. In addition, the expressions of ALP, osteocalcin, and bone morphogenetic protein (BMP)-2 and BMP-7 genes were also increased. The induction of these key osteogenic markers suggests that hLMP- 1 (t) can trigger the pluripotent myoblastic C2C12 cells to differentiate into osteoblastic lineage, thus extending our previous observation that LMP-1 and LMP-1 (t) enhances the osteoblastic phenotype in cultures of cells already committed to the osteoblastic lineage. Therefore, C2C12 cells are an appropriate model system for the examination of LMP-1 induction of the osteoblastic phenotype and the study of mechanisms of LMP-1 action. 相似文献
76.
Deng J Wang X Qian F Vogel S Xiao L Ranjan R Park H Karpurapu M Ye RD Park GY Christman JW 《Journal of immunology (Baltimore, Md. : 1950)》2012,188(11):5734-5740
Reactive oxygen species (ROS) generated by NADPH oxidase are generally known to be proinflammatory, and it seems to be counterintuitive that ROS play a critical role in regulating the resolution of the inflammatory response. However, we observed that deficiency of the p47(phox) component of NADPH oxidase in macrophages was associated with a paradoxical accentuation of inflammation in a whole animal model of noninfectious sepsis induced by endotoxin. We have confirmed this observation by interrogating four separate in vivo models that use complementary methodology including the use of p47(phox-/-) mice, p47(phox-/-) bone marrow chimera mice, adoptive transfer of macrophages from p47(phox-/-) mice, and an isolated perfused lung edema model that all point to a relationship between excessive acute inflammation and p47(phox) deficiency in macrophages. Mechanistic data indicate that ROS deficiency in both cells and mice results in decreased production of IL-10 in response to treatment with LPS, at least in part, through attenuation of the Akt-GSK3-β signal pathway and that it can be reversed by the administration of rIL-10. Our data support the innovative concept that generation of ROS is essential for counterregulation of acute lung inflammation. 相似文献
77.
78.
Sangeeta R. Kashyap Dima L. Diab Allison R. Baker Lisa Yerian Harpreet Bajaj Courtney Gray‐McGuire Philip R. Schauer Manjula Gupta Ariel E. Feldstein Stanley L. Hazen Catherine M. Stein 《Obesity (Silver Spring, Md.)》2009,17(9):1696-1701
Although nonalcoholic fatty liver disease (NAFLD) is frequent in obesity, the metabolic determinants of advanced liver disease remain unclear. Adipokines reflect inflammation and insulin resistance associated with obesity and may identify advanced NAFLD. At the time of obesity surgery, 142 consecutive patients underwent liver biopsy and had their preoperative demographic and clinical data obtained. Liver histology was scored by the NAFLD activity score, and patients subdivided into four groups. Concentrations of retinol‐binding protein 4 (RBP4), adiponectin, tumor necrosis factor‐α (TNF‐α), and leptin were determined ~1 week prior to surgery and results were related to liver histology. The prevalence of no NAFLD was 30%, simple steatosis 23%, borderline nonalcoholic steatohepatitis (NASH) 28%, and definitive NASH 18%. Type 2 diabetes mellitus (T2DM) and metabolic syndrome (MS) prevalence were 39 and 75%, respectively, and did not differ across the four histological groups (P = NS). Triglyceride (TG) and alanine transaminase (ALT) levels, strongly associated with advanced stages of NAFLD and NASH (P = 0.04). TG levels >150 mg/dl, increased the likelihood of NASH 3.4‐fold, whereas high‐density lipoprotein (HDL) levels predicted no NAFLD (P < 0.01). Concentrations of TNF‐α, leptin, and RBP4 did not differ among histological groups and thus did not identify NASH; however, there was a trend for adiponectin to be lower in NASH vs. no NAFLD (P = 0.061). In summary, both TG and ALT levels assist in identification of NASH in an obesity surgery cohort. These findings underscore the importance of fatty acid delivery mechanisms to NASH development in severely obese individuals. 相似文献
79.
80.
Proteins from thermophilic microorganisms are stabilized by various mechanisms to preserve their native folded states at higher
temperatures. A thermostable glucose-6-phosphate dehydrogenase (tG6PDH) from the hyperthermophilic bacterium Aquifex aeolicus was expressed as a recombinant protein in Escherichia coli. The A. aeolicus G6PDH is a homodimer exhibiting remarkable thermostability (t1/2=24 hr at 90°C). Based on homology modeling and upon comparison of its structure with human G6PDH, it was predicted that cysteine
184 of one subunit could form a disulfide bond with cysteine 352 of the other subunit resulting in reinforced intersubunit
interactions that hold the dimer together. Site-directed mutagenesis was performed on tG6PDH to convert C184 and C352 to serines.
The tG6PDH double mutant exhibited a dramatic decrease in the half-life from 24 hr to 3 hr at 90°C. The same decrease in half-life
was also found when either C184 or C352 was mutated to serine. The result indicates that C184 and C352 may play a crucial
role in strengthening the dimer interface through disulfide bond formation, thereby contributing to the thermal stability
of the enzyme. 相似文献