Portal venous gas: an unusual finding in child abuse.
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Abdominal Injuries↗
Biomedical subjects
J W Wu
Publications and source records attributed to J W Wu.
Imaging findings of chronic pulmonary embolism.
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Adult↗
Effect of the factor V Leiden mutation on the clinical expression of severe hemophilia A.
To determine whether the factor V Leiden mutation is associated with decreased bleeding in individuals with severe hemophilia A, factor concentrate utilization, maximum annual number of bleeding episodes, and the prevalence of hemophilic arthropathy between carriers and non-carriers of the factor V Leiden mutation were compared. Heterozygosity for the factor V Leiden mutation was found in 6 of 137 subjects (4.4%). Carriers of the factor V Leiden mutation utilized less factor concentrate (geometric mean: 310 vs. 1185 units/kg/year) and had fewer bleeding episodes than non-carriers (proportion with 10 or fewer bleeding episodes in their worst year: 50 vs. 11%). However, the factor V Leiden mutation was not associated with the absence of arthropathy. The intron 22 inversion mutation of the factor VIII gene was tested for in a subgroup of 80 subjects, but it was not found to be a significant variable for any of the bleeding endpoints. The results of this small study are consistent with the hypothesis that the factor V Leiden mutation imparts a protective effect; however, a larger confirmatory study in which the factor VIII molecular defects can be controlled for is needed. Furthermore, most severe hemophiliacs who used fewer than 200 units/kg/year of factor concentrate or who had experienced 10 or fewer bleeding episodes per year did not carry the factor V Leiden mutation, suggesting that the proportion of severe hemophiliacs whose mild clinical course can be attributed to the factor V Leiden mutation is small.
Adolescent↗
Recombinant factor VIIa is effective for bleeding and surgery in patients with Glanzmann thrombasthenia.
Recombinant activated factor VII (rFVIIa) was found to be effective and safe in treating 24 bleeding episodes and to prevent bleeding during one bilateral herniorrhaphy in four children with Glanzmann thrombasthenia. One of the patients had alloantibodies to platelet membrane glycoprotein (GP) IIb/IIIa and was refractory to platelet transfusion. rFVIIa was administered at 89 to 116 microg/kg per injection every 2 hours, in association with antifibrinolytic drugs. Bleeding stopped in all cases, but platelet transfusion was required in one. Two bleeding episodes recurred 36 and 63 hours after discontinuation of rFVIIa, but were successfully treated with additional doses. No adverse effects of rFVIIa were observed. Although the number of patients is small, our study suggests that rFVIIa may be an alternative to platelet transfusions in patients with a severe congenital thrombocytopathy.
Blood Loss, Surgical↗
New evidence for the denaturant binding model.
Denaturation with guanidine hydrochloride (GdnHCl) or urea is one of the primary ways of measuring the conformational stability of proteins and comparing the stability of mutant proteins. Despite the widespread use of these two denaturants to provide quantitative data for the free energies of unfolding, the mode of action of these agents is not well understood. We are not even certain whether the action of these agents on proteins is direct and can be regarded as ligand binding, or indirect and involves a change in the properties of solvent (water) in the presence of GdnHCl and urea. In this paper, an extensive kinetic study of the inhibition of ribonuclease A and papain by urea has been performed. The results suggest that the effect of urea on activities of these enzymes can be well described by the denaturant binding model. The binding constants of urea determined by the present method are nearly identical to that determined from a variety of different studies on model compounds and proteins.
Hydrogen-Ion Concentration↗
Variant bronchial anatomy: CT appearance and classification.
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Bronchi↗
The inactivation kinetics of papain by guanidine hydrochloride: a re-analysis.
The kinetic theory of the substrate reaction during modification of enzyme activity has been applied to study the inactivation kinetics of enzymes by denaturant. However, an important problem related to the determination of the inactivation rate constants has not been considered in a previous publication (Xiao, et al., Biochim. Biophys. Acta, 1164 (1993) 54-60). In most denaturation experiments, the high concentrations of denaturants may greatly affect the kinetic behavior of the system to preclude the use of the kinetic parameters determined in the absence of denaturant. In the present study, the kinetic equation of substrate reaction in presence of denaturant has been derived. A re-examination of the effect of high concentrations of guanidine hydrochloride on the inactivation of papain, taking into consideration the effect of high concentrations of guanidine hydrochloride on the Michaelis constant, showed that, for papain, the substrate gives no protection on its inactivation. It is the purpose of the present communication to stress the importance of observing the effect of the denaturant on the kinetic parameters for kinetic analysis of enzyme inactivation by denaturants generally.
Guanidine↗
Activation mechanism and modification kinetics of Chinese hamster dihydrofolate reductase by p-chloromercuribenzoate.
Substrate effects on the activation kinetics of Chinese hamster dihydrofolate reductase by p-chloromercuribenzoate (pCMB) have been studied. On the basis of the kinetic equation of substrate reaction in the presence of pCMB, all modification kinetic constants for the free enzyme and enzyme-substrate binary and ternary complexes have been determined. The results of the present study indicate that the modification of Chinese hamster dihydrofolate reductase by pCMB shows single-phase kinetics, and that changes in the enzyme activity and tertiary structure proceed simultaneously during the modification process. Both substrates, NADPH and 7,8-dihydrofolate, protect dihydrofolate reductase against modification by pCMB. In the presence of a saturating concentration of NADPH, the value of kcat for 7,8-dihydrofolate in the enzyme-catalysed reaction increased four-fold on modification of Cys-6, accompanied by a two-fold increase in Km for the modified enzyme. The utilization of the binding energy of a group to increase kcat rather than reduce Km implies that the full binding energy of the group is not realized in the formation of the enzyme-substrate complex, but is used to stabilize the enzyme-transition-state complex.
Animals↗
Three-state kinetic analysis of Chinese hamster dihydrofolate reductase unfolding by guanidine hydrochloride.
The unfolding behavior of dihydrofolate reductase from Chinese hamster in solutions of guanidine hydrochloride (GdnHCl) was studied. The GdnHCl-induced unfolding of the dihydrofolate reductase monitored by intrinsic fluorescence shows a biphasic transition, while the change in the enzyme activity is a single exponential process. The rate constant of inactivation is consistent with that of the fast conformational change. Therefore, the kinetic intermediate of protein unfolding should be a partially folded and inactive form. On the basis of the kinetic equation of substrate reaction in the presence of GdnHCl, all microscopic kinetic constants for the free enzyme and enzyme-substrate complexes have been determined. Both substrates, NADPH and 7,8-dihydrofolate, protect dihydrofolate reductase against inactivation.
Animals↗
Inactivation kinetics of the reduced spinach chloroplast fructose-1,6-bisphosphatase by subtilisin.
The course of inactivation of the reduced spinach chloroplast fructose-1,6-bisphosphatase by digestion with subtilisin has been followed by the progress curve method [Tsou, C. L. (1988) Adv. Enzymol. 61, 381-436] and found to follow first-order kinetics. On the basis of the hydrolysis of the substrate, fructose 1,6-bisphosphate, at different concentrations during proteolysis by subtilisin, the first-order inactivation rate constants for the free enzyme and the enzyme-substrate complex can both be determined. The ratio between the inactivation rate constants for the free enzyme and the enzyme-substrate complex indicates strong protection against subtilisin proteolysis by the substrate. It is proposed that the above ratio can be used as a quantitative measure of substrate protection for enzyme inactivation generally. As it has been found that the site of proteolysis is located in a loop region near the N-terminus and well away from the active site, the substrate protection indicates a conformation change of the enzyme away from the substrate binding site.
Binding Sites↗
Inactivation kinetics of dihydrofolate reductase from Chinese hamster during urea denaturation.
The kinetic theory of substrate reaction during modification of enzyme activity has been applied to the study of inactivation kinetics of Chinese hamster dihydrofolate reductase by urea [Tsou (1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61, 381-436]. On the basis of the kinetic equation of substrate reaction in the presence of urea, all microscopic kinetic constants for the free enzyme and enzyme-substrate binary and ternary complexes have been determined. The results of the present study indicate that the denaturation of dihydrofolate reductase by urea follows single-phase kinetics, and changes in enzyme activity and tertiary structure proceed simultaneously in the unfolding process. Both substrates, NADPH and 7,8-dihydrofolate, protect dihydrofolate reductase against inactivation, and enzyme-substrate complexes lose their activity less rapidly than the free enzyme.
Animals↗
Localization of proteins to the apico-lateral junctions of Drosophila epithelia.
We have examined the distribution of proteins in the apico-lateral cell junctions in Drosophila imaginal discs. The subcellular distribution of these proteins in normal and mutant proliferating cells was analyzed with marker antibodies and confocal microscopy. Antibodies to phosphotyrosine (PY), Armadillo (Arm) and Drosophila E-cadherin (DE-cad) as well as FITC phalloidin marking filamentous actin, labeled the site of the adherens junction, whereas antibodies to Discs large (DIg), Fasciclin III (FasIII) and Coracle (Cor) labeled the more basal septate junction. The junctional proteins labeled by these antibodies underwent specific changes in distribution during the cell cycle. We have previously shown that a loss-of-function dlg mutation, which causes neoplastic imaginal disc overgrowth, leads to loss of the septate junctions and the formation of what appear to be ectopic adherens junctions [Woods et al., 1996]. We therefore extended this study to examine the effects of mutations in other genes that also cause imaginal disc overgrowth. Based on staining with PY and DIg antibodies, the apico-lateral junctional complexes appeared normal in tissue from the hyperplastic overgrowth mutants fat, dco, gd and wts. However, imaginal disc tissue from the neoplastic overgrowth mutants dlg and lgl showed abnormal distribution of the junctional markers including a complete loss of apico-basal polarity in loss-of-function dlg mutations. These results support the idea that some of the proteins of apico-lateral junctions are required both for apico-basal cell polarity and for the signalling mechanisms controlling cell proliferation, whereas others are required more specifically in cell-cell signalling.
Animals↗
The hepatitis C virus NS4A protein: interactions with the NS4B and NS5A proteins.
Hepatitis C virus encodes a large polyprotein precursor that is proteolytically processed into at least 10 distinct products, in the order NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B -COOH. A serine proteinase encoded in the N-terminal 181 residues of the NS3 nonstructural protein is responsible for cleavage at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the nonstructural region. NS4A, a 54-residue nonstructural protein which forms a stable complex with the NS3 proteinase, is required as a cofactor for cleavage at the 3/4A and 4B/5A sites and enhances processing at the 4A/4B and 5A/5B sites. Recently reported crystal structures demonstrated that NS4A forms an integral part of the NS3 serine proteinase. In this report, we present evidence that NS4A forms a nonionic-detergent-stable complex with the NS4B5A polyprotein substrate, which may explain the requirement of NS4A for the 4B/5A cleavage. Isoleucine-29 of NS4A, which has been previously shown to be essential for its proteinase cofactor activity and formation of the NS3 complex, was found to be important for the interaction between NS4A and the NS4B5A substrate. In addition, two more hydrophobic residues in the NS4A central region (valine-23 and isoleucine-25) were also shown to be essential for the cofactor activity and for the interaction with either the NS3 proteinase or the NS4B5A polyprotein substrate. Finally, the possible mechanisms by which these viral proteins interact with each other are discussed.
Animals↗
Diagnostic value of serum gamma-glutamyl transferase isoenzyme for hepatocellular carcinoma: a 10-year study.
Serum gamma-glutamyl transferase (GGT) was separated into nine to 11 isoenzyme bands (designated as GGT I-XI) by vertical slab electrophoresis on polyacrylamide gradient gel. The diagnostic value of GGT isoenzyme II (GGT II) for hepatocellular carcinoma (HCC) was studied, and the results were as follows: 1) GGT II was positive in 90% of 90 cases of HCC, and negative in most patients with acute and chronic viral hepatitis, extrahepatic tumors, in pregnant women, and in healthy controls; 2) the positive rate of GGT II assay was higher than that of alkaline phosphatase isoenzyme I (ALP I), alpha-fetoprotein (AFP), and alpha 1-antitrypsin (AAT) in 101 cases of HCC. In cases in which the AFP was greater than 50 ng/ml or less than 50 ng/ml, the positive rates of GGT II were 70.8% and 75-100%, respectively; 3) of 14 cases of small-size HCC, the positive rate of GGT II was 78.6%, which was higher than that of AFP (50%), AAT (28.6%), and ALP I (0%); 4) of 62 cases that were false-positive for GGT II assay, 24.2% developed into HCC during a follow-up of 2.1-20 months. In subjects with persistent and recurrent positivity of GGT II, 86.7% and 22.2%, respectively, developed HCC. No patient with temporal positivity of GGT II developed HCC. The results show that GGT II can be applied as an additional marker for HCC, and is valuable not only for the diagnosis of clinical HCC, but for the detection of small or subclinical HCC. Periodic follow-up with assay of GGT II in patients at high risk for HCC may predict the development of hepatoma.
Biomarkers, Tumor↗
Fracture of the patella treated by open reduction and external compressive skeletal fixation.
Open reduction combined with external compressive skeletal fixation was used to treat twenty-seven patients who had a separated fracture of the patella. Fixation was obtained by the use of two compressive clamps applied to stainless-steel pins that were inserted just proximal and distal to the proximal and distal poles of the patella. Range-of-motion exercises for the knee were begun at two weeks and the pins were removed at three to four weeks. All of the fractures healed. Twenty-four patients regained a range of motion that was equal to that of the opposite knee. There was no evidence of chronic osteomyelitis. Osteoarthritis was noted in one patient who refused excision of a portion of the patella.
Adult↗
Quantitation of haptens by homogeneous immunoprecipitation. 2. Centrifugal analysis for tobramycin, phenobarbital, and theophylline in serum.
Our homogeneous immunoprecipitation inhibition assay (Clin. Chem. 28:659-661, 1982) is applied here to tobramycin, phenobarbital, and theophylline. Only 10-50 microL of test sample is needed. No sample treatment, dilution, or extraction is required. A test serum sample is simultaneously mixed with a drug conjugate and its specific antiserum in a centrifugal analyzer. The subsequent reaction and the measurement are completed in 3 min. Within-run and between-run CVs for clinically relevant concentrations were well below 10%. Results for patients' samples correlated well with those by enzyme immunoassay.
Anti-Bacterial Agents↗
Quantitation of haptens by homogeneous immunoprecipitation. 1. Automated analysis of gentamicin in serum.
A new homogeneous immunoprecipitation inhibition assay has been developed to quantitate concentrations of hapten in human serum or plasma without the use of radioactive isotopes, enzymes, fluorescent markers, or laser nephelometers. This immunoprecipitation is based on spectrophotometric measurement of the inhibition by free hapten of the precipitating reaction between antihapten antibody and polyhaptenic antigen. The immunoprecipitation analysis of the antibiotic gentamicin in human serum is reported here. A serum test sample is mixed with gentamicin-human serum albumin polyhaptenic conjugate and rabbit antiserum to gentamicin on a centrifugal analyzer, and the subsequent reaction monitored for 3 min. No sample dilution or pretreatment is required. The within-run and between-run coefficients of variation are well below 10%. The results on patients' test samples correlate well with those obtained by commercially available radioimmunoassay and enzyme immunoassay kits.
Antigen-Antibody Complex↗