ABSTRACT
We have previously cloned cDNAs encoding the N-terminally extended class III human ubiquitin-conjugating enzymes (E2s), UBE2E2 and UBE2E3, the biological functions of which are not known. In this study, we performed yeast two-hybrid screening for protein(s) interacting with UBE2E2, and two RING-finger proteins, ARA54 and RNF8, were identified. Both ARA54, a ligand-dependent androgen receptor coactivator, and RNF8 interacted with class III E2s (UBE2E2, UbcH6, and UBE2E3), but not with other E2s (UbcH5, UbcH7, UbcH10, hCdc34, and hBendless) in the yeast two-hybrid assay. The use of various deletion mutants of UBE2E2 and RING-finger proteins and two RING point mutants, ARA54 C(220)S and RNF8 C(403)S, in which the RING structure is disrupted, showed that the UBC domain of UBE2E2 and the RING domain of these RING-finger proteins were involved in this association. Wild-type ARA54 and RNF8, expressed in insect Sf9 cells, catalyzed E2-dependent autoubiquitination in vitro, whereas the point mutated proteins showed markedly reduced activity. Ubiquitination of wild-type ARA54 and RNF8, expressed in COS-7 cells, was also observed, and a proteasome inhibitor, MG132, prevented the degradation of these wild-type proteins, but was much less effective in protecting the RING mutants. Transfection of COS-7 cells with a green fluorescent protein chimera showed that RNF8 was localized in the nucleus, and ARA54 in both the cytoplasm and nucleus. Our results suggest that ARA54 and RNF8 possibly act as Ub-ligases (E3) in the ubiquitination of certain nuclear protein(s).
Subject(s)
Carrier Proteins/metabolism , Intracellular Signaling Peptides and Proteins , Ligases/chemistry , Ligases/metabolism , Amino Acid Sequence , Animals , COS Cells , Carrier Proteins/chemistry , Cysteine Endopeptidases/metabolism , DNA, Complementary/genetics , Humans , In Vitro Techniques , Ligases/genetics , Molecular Sequence Data , Multienzyme Complexes/metabolism , Proteasome Endopeptidase Complex , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Deletion , Sequence Homology, Amino Acid , Two-Hybrid System Techniques , Ubiquitin-Conjugating Enzymes , Zinc FingersABSTRACT
We identified three mutations in four Japanese patients with central type 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency. One missense mutation was a C-to-T transition, resulting in the substitution of Pro by Ser at codon 87 (P87S) in exon 5. Another missense mutation was a G-to-A transition, resulting in the substitution of Asp by Asn at codon 96 (D96N) in exon 5. A splicing mutation was found by skipping of exon 4 on PTPS mRNA analysis, and a G-to-A transition at the third base of codon 81 (E81E) and at the terminal base in exon 4 were detected on genomic PTPS DNA analysis. The E81E mutation affected the splice donor site of exon 4 and caused the splicing error. In COS cell expression analysis, the P87S and D96N mutant constructs revealed, respectively, 52% and 10% of wild-type activity. Patients with P87S/P87S (52%/52% in-vitro PTPS activity) exhibited 0.11 and 0 microU/g hemoglobin [Hb] in erythrocyte PTPS activity (wild-type control: 11-29 microU/gHb) erythrocyte PTPS activity, and the patient with P87S/D96N mutations (52%/10%) had 0.97 microU/gHb in PTPS erythrocyte activity. The PTPS erythrocyte activity did not coincide with the in-vitro PTPS activity based on patient genotype.
Subject(s)
Amino Acid Metabolism, Inborn Errors/genetics , Asian People/genetics , Mutation, Missense , Phosphorus-Oxygen Lyases/deficiency , Phosphorus-Oxygen Lyases/genetics , DNA, Complementary/genetics , Gestational Age , Humans , Infant , Infant, Newborn , Japan , Male , Phenylalanine/blood , Sequence Analysis, DNAABSTRACT
We report two cases of failure of fluoroquinolone treatment of urinary tract infections with Klebsiella pneumoniae strains harboring quinolone resistance-associated alterations in GyrA and ParC and in vivo selection of posttreatment isolates with enhanced fluoroquinolone resistance. Active efflux leading to decreased accumulation of a drug enhanced fluoroquinolone resistance in one posttreatment isolate, and an additional mutation in parC resulting in an additional amino acid change in ParC was associated with increased resistance in the other.
Subject(s)
Anti-Infective Agents, Urinary/therapeutic use , Anti-Infective Agents/therapeutic use , Klebsiella pneumoniae/drug effects , Selection, Genetic , Urinary Tract Infections/drug therapy , Aged , Fluoroquinolones , Humans , Klebsiella pneumoniae/genetics , Male , Microbial Sensitivity Tests , Species SpecificityABSTRACT
Sixty-three patients with unresectable hepatocellular carcinoma (HCC) were treated with cisplatin-phosphatidyl-choline-Lipiodol (CPL) suspension. Partial response (PR) and minor response (MR) were obtained in 3 of 14 cases (21.4%) by one shot therapy, and in 13 of 43 cases (30.2%) by TAE therapy. AFP decreased in 11 of 15 patients (73.3%) by one shot therapy, and in 32 of 33 patients (97%) by TAE therapy. PIVKA II also decreased. The one-year survival rate was 74% in TAE therapy, and 52% in one shot therapy. The two-year survival rate was 53% in TAE therapy, and 28% in one shot therapy. Nausea, vomiting and fever were noted in most cases as adverse effects, but they were slight. The concentration of free-CDDP in the peripheral venous blood was lower and continued longer than that of CDDP on the market. These results suggest that CPL was useful as an anticancer agent for arterial chemotherapy or TAE therapy for unresectable HCC.
Subject(s)
Carcinoma, Hepatocellular/drug therapy , Cisplatin/administration & dosage , Iodized Oil/administration & dosage , Liver Neoplasms/drug therapy , Phosphatidylcholines/administration & dosage , Carcinoma, Hepatocellular/mortality , Drug Administration Schedule , Female , Humans , Infusion Pumps, Implantable , Infusions, Intra-Arterial , Injections, Intra-Arterial , Liver Neoplasms/mortality , Male , Middle Aged , Survival RateABSTRACT
The effect of selenium ions on the catabolism of prostaglandin E2 by 15-hydroxy prostaglandin dehydrogenase and prostaglandin delta 13 reductase in rabbit kidney cortex was examined. Selenium ions had no effect on the 15-hydroxy prostaglandin dehydrogenase activity. On the other hand, selenium ions inhibited the prostaglandin delta 13 reductase activity at concentrations ranging from 0.05 to 0.5 mM. The effect was concentration-dependent. The inhibition by selenium ions was non-competitive with regard to NADH and 15-keto PGE2, respectively. These results suggest that selenium ions have the potential to increase 15-keto PG levels in the kidney by inhibiting the prostaglandin delta 13 reductase activity and that such action of selenium ions may participate in renal damage of selenium poisoning.
Subject(s)
Dinoprostone/metabolism , Kidney Cortex/metabolism , Selenium/pharmacology , Animals , Hydroxyprostaglandin Dehydrogenases/metabolism , Kidney Cortex/enzymology , Male , RabbitsABSTRACT
The serum zinc concentration was examined in 61 patients with acute myocardial infarction who were admitted within twenty-four hours after the onset. Forty-two of 61 patients were admitted within twelve hours. The zinc level fell sharply as early as within three hours after the onset. The minimum values were attained two or three days after infarction and then rose to normal values within five to ten days. There was a mild degree of change and early recovery in 9 patients who underwent major abdominal surgery. The pathologic factors that could influence these two conditions are discussed. In those patients whose serum zinc concentrations were remarkably lowered, clinically estimated severity was far graver, and prognosis seemed to be much worse. It should be asked whether zinc supplementation would be of therapeutic benefit to patients with acute infarction.
Subject(s)
Myocardial Infarction/blood , Zinc/blood , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Prognosis , Time FactorsABSTRACT
Twenty-seven patients with unresectable hepatocellular carcinoma (HCC) were treated with Cisplatin-Phosphatidyl-choline-Lipiodol (CPL) suspension. PR was obtained in two of ten cases (20%) by one shot therapy. AFP decreased in 9 of 10 patients by one shot therapy with a 62.1% rate of decrease. In all of 13 patients by TAE, AFP decreased and the rate of decrease was 64.8%. The concentration of CDDP in the peripheral venous blood was lower and continued longer than that of CDDP on the market. Nausea, vomiting and fever were noted in most cases as adverse effects, but they were slight. These results suggest that CPL agents were very chemotherapeutic for unresectable HCC.
Subject(s)
Carcinoma, Hepatocellular/drug therapy , Cisplatin/administration & dosage , Iodized Oil/administration & dosage , Liver Neoplasms/drug therapy , Phosphatidylcholines/administration & dosage , Carcinoma, Hepatocellular/blood , Carcinoma, Hepatocellular/therapy , Cisplatin/blood , Cisplatin/therapeutic use , Drug Evaluation , Embolization, Therapeutic , Humans , Infusion Pumps , Infusions, Intra-Arterial/methods , Iodized Oil/therapeutic use , Liver Neoplasms/blood , Liver Neoplasms/therapy , Phosphatidylcholines/therapeutic use , Remission Induction , Suspensions , alpha-Fetoproteins/metabolismABSTRACT
A 52-year-old woman developed scleroderma, primary biliary cirrhosis, and Sjögren's syndrome after cosmetic silicone injections. These conditions may possibly represent 'human adjuvant disease'.
Subject(s)
Breast/surgery , Liver Cirrhosis, Biliary/etiology , Prostheses and Implants , Scleroderma, Systemic/etiology , Silicone Elastomers , Sjogren's Syndrome/etiology , Surgery, Plastic , Autoimmune Diseases/etiology , Female , Humans , Middle Aged , Postoperative ComplicationsABSTRACT
A new method for evaluating in vivo effect of mucolytic expectorants was devised. In this report, however, the focus is placed on methodology but not on pharmacology. Rabbits with subacute bronchitis were prepared by long-term exposure to minute amounts of SO2 gas. The sputum was quantitatively collected from the animal through a tracheal cannula at suitable time intervals according to the Perry and Boyd's method. As a preliminary study, changes in volume, viscosity and amounts of freeze-dried substance of sputa were examined after intraduodenal administration of water instead of mucolytic expectorants, the effects of which will be described in the subsequent paper, but no significant changes occurred with this test.