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1.
Enzymatic modifications of human plasma fibronectin in relation to opsonizing activity 总被引:1,自引:0,他引:1
Plasma fibronectin is one of the largest plasma proteins (Mr approximately 440 000), comprising two approximately equal polypeptide chains which are held together by a disulfide linkage near the C-terminal end of the molecule. The binding of gelatinized latex beads to liver slices as well as the internalization of these particles by macrophages, in the presence of heparin, is greatly enhanced by fibronectin. The question as to whether the entire covalent structure of fibronectin was necessary for opsonizing activity was approached by limited proteolytic degradations of the molecule. Patterns of controlled digestion with trypsin, cathepsin D, Staphylococcus aureus protease, and plasmin all indicate that the minimal unit necessary for retention of opsonic activity is some large (Mr 200 000 and 190 000) single-chain entity. Treatment with plasmin proved to be the most reliable procedure for generating the active split product which could be readily separated from the inactive, disulfide-containing C-terminal fragment. Incorporation of dansylcadaverine into plasma fibronectin (3.5 mol/mol of protein) by fibronoligase (coagulation factor XIIIa) did not affect the opsonic activity of the protein. 相似文献
2.
Okadaic acid (OA), a protein phosphatase inhibitor, was found to induce hyperphosphorylation and reorganization of vimentin intermediate filaments in 9L rat brain tumor cells. The process was dose dependent. Vimentin phosphorylation was initially enhanced by 400 nM OA in 30 min and reached maximal level (about 26-fold) when cells were treated with 400 nM OA for 90 min. Upon removal of OA, dephosphorylation of the hyperphosphorylated vimentin was observed and the levels of phosphorylation returned to that of the controls after the cells recovered under normal growing conditions for 11 h. The phosphorylation and dephosphorylation of vimentin induced by OA concomitantly resulted in reversible reorganization of vimentin filaments and alteration of cell morphology. Cells rounded up as they were entering mitosis in the presence of OA and returned to normal appearance after 11 h of recovery. Immuno-staining with anti-vimentin antibody revealed that vimentin filaments were disassembled and clustered around the nucleus when the cells were treated with OA but subsequently returned to the filamentous states when OA was removed. Two-dimensional electrophoresis analysis further revealed that hyperphosphorylation of vimentin generated at least seven isoforms having different isoelectric points. Furthermore, the enhanced vimentin phosphorylation was accompanied by changes in the detergent-solubility of the protein. In untreated cells, the detergent-soluble and -insoluble vimentins were of equal amounts but the solubility could be increased when vimentins were hyperphosphorylated in the presence of OA. Taken together, the results indicated that OA could be involved in reversible hyperphosphorylation and reorganization of vimentin intermediate filaments, which may play an important role in the structure-function regulation of cytoskeleton in the cell. 相似文献
3.
Siew Choo Lim Matthew W. Bowler Ting Feng Lai Haiwei Song 《Nucleic acids research》2012,40(21):11009-11022
Mutations in immunoglobulin µ-binding protein 2 (Ighmbp2) cause distal spinal muscular atrophy type 1 (DSMA1), an autosomal recessive disease that is clinically characterized by distal limb weakness and respiratory distress. However, despite extensive studies, the mechanism of disease-causing mutations remains elusive. Here we report the crystal structures of the Ighmbp2 helicase core with and without bound RNA. The structures show that the overall fold of Ighmbp2 is very similar to that of Upf1, a key helicase involved in nonsense-mediated mRNA decay. Similar to Upf1, domains 1B and 1C of Ighmbp2 undergo large conformational changes in response to RNA binding, rotating 30° and 10°, respectively. The RNA binding and ATPase activities of Ighmbp2 are further enhanced by the R3H domain, located just downstream of the helicase core. Mapping of the pathogenic mutations of DSMA1 onto the helicase core structure provides a molecular basis for understanding the disease-causing consequences of Ighmbp2 mutations. 相似文献
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Breast microcysts are considered to be a normal findings in the adult female breast without any increased risk of developing carcinomatous change. Breast cysts fluid contains steroid but not studies have been reported on the ability of breast microcysts to metabolise steroid hormones. It was, therefore, the aim of this study to identify the metabolites formed on incubation of radiolabelled testosterone with microcysts. In all instances dihydrotestosterone and androstenedione were formed. Oestrogens were not identified. Tis study, therefore, provides evidence for th presence of 5-alpha-reductase and 17-oxidoreductase enzyme systems in breast microcysts. 相似文献
7.
Investigation into the association of insurance status with the outcomes of patients undergoing neurosurgical intervention has been limited: this is the first nationwide study to analyze the impact of primary payer on the outcomes of patients with aneurysmal subarachnoid hemorrhage who underwent endovascular coiling or microsurgical clipping. The Nationwide Inpatient Sample (2001–2010) was utilized to identify patients; those with both an ICD-9 diagnosis codes for subarachnoid hemorrhage and a procedure code for aneurysm repair (either via an endovascular or surgical approach) were included. Hierarchical multivariate regression analyses were utilized to evaluate the impact of primary payer on in-hospital mortality, hospital discharge disposition, and length of hospital stay with hospital as the random effects variable. Models were adjusted for patient age, sex, race, comorbidities, socioeconomic status, hospital region, location (urban versus rural), and teaching status, procedural volume, year of admission, and the proportion of patients who underwent ventriculostomy. Subsequent models were also adjusted for time to aneurysm repair and time to ventriculostomy; subgroup analyses evaluated for those who underwent endovascular and surgical procedures separately. 15,557 hospitalizations were included. In the initial model, the adjusted odds of in-hospital mortality were higher for Medicare (OR 1.23, p<0.001), Medicaid (OR 1.23, p<0.001), and uninsured patients (OR 1.49, p<0.001) compared to those with private insurance. After also adjusting for timing of intervention, Medicaid and uninsured patients had a reduced odds of non-routine discharge (OR 0.75, p<0.001 and OR 0.42, p<0.001) despite longer hospital stays (by 8.35 days, p<0.001 and 2.45 days, p = 0.005). Variations in outcomes by primary payer–including in-hospital post-procedural mortality–were more pronounced for patients of all insurance types who underwent microsurgical clipping. The observed differences by primary payer are likely multifactorial, attributable to varied socioeconomic factors and the complexities of the American healthcare delivery system. 相似文献
8.
Silvia Penuela Alexander W Lohman Wesley Lai Laszlo Gyenis David W Litchfield Brant E Isakson Dale W Laird 《Channels (Austin, Tex.)》2014,8(2):124-130
The pannexin family of channel-forming proteins is composed of 3 distinct but related members called Panx1, Panx2, and Panx3. Pannexins have been implicated in many physiological processes as well as pathological conditions, primarily through their function as ATP release channels. However, it is currently unclear if all pannexins are subject to similar or different post-translational modifications as most studies have focused primarily on Panx1. Using in vitro biochemical assays performed on ectopically expressed pannexins in HEK-293T cells, we confirmed that all 3 pannexins are N-glycosylated to different degrees, but they are not modified by sialylation or O-linked glycosylation in a manner that changes their apparent molecular weight. Using cell-free caspase assays, we also discovered that similar to Panx1, the C-terminus of Panx2 is a substrate for caspase cleavage. Panx3, on the other hand, is not subject to caspase digestion but an in vitro biotin switch assay revealed that it was S-nitrosylated by nitric oxide donors. Taken together, our findings uncover novel and diverse pannexin post-translational modifications suggesting that they may be differentially regulated for distinct or overlapping cellular and physiological functions. 相似文献
9.
An isothermal titration calorimetric (ITC) method was developed to measure the kinetic parameters of ribonuclease A catalytic hydrolysis of cytidine 2',3'-cyclic monophosphate. Employing the inhibition of product as a probe, the K(m), K(i), k(c), and DeltaH(m) can be determined by two simple calorimetric measurements. First, the substrate was titrated into the cell containing high concentration of enzyme. The molar reaction heat was calculated from the titration peak area divided by substrate moles per titration, and the initial catalytic reaction rate in the presence of various concentrations of product can be calculated from the peak height and the molar reaction heat. From Michaelis-Menten function in the presence of inhibitors, the relationship between K(m) and K(i) can be obtained. Then, the dissociation constant, which is equal to K(i), was measured by a regular ITC experiment. Thus, K(m) and k(c) can be calculated. The method developed here can be applied in other enzyme catalytic systems with inhibitive products. 相似文献
10.
Sequence and translation of the murine coronavirus 5''-end genomic RNA reveals the N-terminal structure of the putative RNA polymerase. 总被引:20,自引:15,他引:5
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A 28-kilodalton protein has been suggested to be the amino-terminal protein cleavage product of the putative coronavirus RNA polymerase (gene A) (M.R. Denison and S. Perlman, Virology 157:565-568, 1987). To elucidate the structure and mechanism of synthesis of this protein, the nucleotide sequence of the 5' 2.0 kilobases of the coronavirus mouse hepatitis virus strain JHM genome was determined. This sequence contains a single, long open reading frame and predicts a highly basic amino-terminal region. Cell-free translation of RNAs transcribed in vitro from DNAs containing gene A sequences in pT7 vectors yielded proteins initiated from the 5'-most optimal initiation codon at position 215 from the 5' end of the genome. The sequence preceding this initiation codon predicts the presence of a stable hairpin loop structure. The presence of an RNA secondary structure at the 5' end of the RNA genome is supported by the observation that gene A sequences were more efficiently translated in vitro when upstream noncoding sequences were removed. By comparing the translation products of virion genomic RNA and in vitro transcribed RNAs, we established that our clones encompassing the 5'-end mouse hepatitis virus genomic RNA encode the 28-kilodalton N-terminal cleavage product of the gene A protein. Possible cleavage sites for this protein are proposed. 相似文献