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Biomedical subjects

R Jin

Publications and source records attributed to R Jin.

At least 73 records · Page 4Linked to original sources
PubMed1998

Metabolism of arachidonic acid to 20-hydroxy-5,8,11, 14-eicosatetraenoic acid by P450 enzymes in human liver: involvement of CYP4F2 and CYP4A11.

20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) is a principal arachidonic acid (AA) metabolite formed via P450-dependent oxidation in hepatic and renal microsomes. Although 20-HETE plays an important role in the regulation of cell and/or organ physiology, the P450 enzyme(s) catalyzing its formation in humans remain undefined. In this study, we have characterized AA omega-hydroxylation to 20-HETE by human hepatic microsomes and identified the underlying P450s. Analysis of microsomal AA omega-hydroxylation revealed biphasic kinetics (KM1 and VMAX1 = 23 microM and 5.5 min-1; KM2 and VMAX2 = 144 microM and 18.8 min-1) consistent with catalysis by at least two enzymes. Of the human P450s examined, CYP4A11 and CYP4F2 were both potent AA omega-hydroxylases, exhibiting rates of 15.6 and 6.8 nmol 20-HETE formed/min/nmol P450, respectively. Kinetic parameters of 20-HETE formation by CYP4F2 (KM = 24 microM; VMAX = 7.4 min-1) and CYP4A11 (KM = 228 microM; VMAX = 49.1 min-1) resembled the low and high KM components, respectively, found in liver microsomes. Antibodies to CYP4F2 markedly inhibited (93.4 +/- 6%; n = 5) formation of 20-HETE by hepatic microsomes, whereas antibodies to CYP4A11 were much less inhibitory (13.0 +/- 9%; n = 5). Moreover, a strong correlation (r = 0.78; P < .02) was found between microsomal CYP4F2 content and AA omega-hydroxylation among nine subjects. The correlation (r = 0.76; P < .02) also noted between CYP4A11 content and 20-HETE formation stemmed from the relationship (r = 0.83; P < . 02) between hepatic CYP4A11 and CYP4F2 levels in the subjects. Finally, immunoblot analysis revealed that in addition to liver, both P450s also were expressed in human kidney. Our results indicate that AA omega-hydroxylation in human liver is catalyzed by two enzymes of the CYP4 gene family, namely CYP4F2 and CYP4A11, and that CYP4F2 underlies most 20-HETE formation occurring at relevant AA concentrations.

Arachidonic Acid↗
PubMed1997

Why is the initiation nick site of an AT-rich rolling circle plasmid at the tip of a GC-rich cruciform?

pT181 and other closely related rolling circle plasmids have the nicking site for initiation of replication between the arms of a GC-rich inverted repeat sequence adjacent to the binding site for the dimeric initiator protein. Replication is initiated by the initiator-induced extrusion of this sequence as a cruciform, creating a single-stranded region for nicking by the protein. Nicking is followed by assembly of the replisome without relaxation of the secondary structure. Following termination, the initiator protein is released with a short oligonucleotide attached to one subunit, which prevents it from being recycled, a necessary feature of the plasmid's replication control system. The modified initiator can cleave single-stranded substrates and can nick and relax supercoiled plasmid DNA weakly. Although it can bind to its recognition sequence in the leading strand origin, the modified protein cannot induce cruciform extrusion, and it is proposed that this inability is the key to understanding the biological rationale for having the nicking site at the tip of a cruciform: the need to provide the functional initiator with a catalytic advantage over the modified one sufficient to offset the numerical advantage and metabolic stability of the latter.

Bacterial Proteins↗
PubMed1997

Formation, solvolysis, and transcarbamoylation reactions of bis(S-glutathionyl) adducts of 2,4- and 2,6-diisocyanatotoluene.

During our ongoing studies of the reactions of toluene diisocyanate (2,4- and 2,6-diisocyanatotoluene, TDI) in vivo, it became apparent that reactive form(s) of these diisocyanates reach(es) the circulatory system after passage through the respiratory system. Based on recent work by others regarding the transcarbamoylation reactions of monoisocyanates, we hypothesized that the reactive form could be masked as an S-thiocarbamoylglutathione adduct of one or more of the isocyanato moieties. In this study, the glutathione adducts of 2,4- and 2,6-diisocyanatotoluene were synthesized under physiological conditions. Bis adducts were the major products when near-equimolar amounts of glutathione and the individual diisocyanato compounds were mixed at physiological pH, and were formed in high yield. Little to no mono adducts formed under these reaction conditions. The masses of the bis adducts were confirmed by electrospray mass spectrometry (MS), and 1H NMR analysis strongly suggested that the thiol of the cysteine residue of glutathione was the nucleophile in each case. The rates of solvolysis of the two bis adducts in aqueous buffer under conditions of physiological temperature and pH were determined, and electrospray MS analysis showed that the corresponding mono(glutathionyl)-TDIs were formed in these reactions. Incubation in vitro of each of the bis(glutathionyl)-TDI adducts with a 12 amino acid peptide (Thr-Cys-Val-Glu-Trp-Leu-Arg-Arg-Tyr-Leu-Lys-Asn) at pH 7.5 resulted in transfer of one mono(glutathionyl)-toluylisocyanato moiety to the peptide as detected by HPLC and on-line electrospray MS analyses. In both the solvolysis and transfer experiments, the 2,4-TDI-derived bis(glutathionyl) adduct reacted most quickly, while both the bis(glutathionyl)-2,6-TDI adduct and its transfer product with the peptide were more stable than their 2,4-TDI-derived counterparts. The results indicate high stoichiometry in formation and ready transfer to nucleophilic sites of protein, and suggest that the isocyanato moiety of both 2,4- and 2,6-TDI may be regenerated in vivo from their bis(glutathionyl) adducts. As a consequence, the thiol status of particular tissues may be a contributing factor to individual TDI toxicity susceptibility, and a mechanism by which toxicity at sites distant to the initial point of contact may be proposed.

Amino Acid Sequence↗
PubMed1997

Virucidal treatment of blood protein products with UVC radiation.

The virus safety of blood derivatives continues to be of concern, especially with respect to nonenveloped and/or heat-stable viruses. Previously, we demonstrated that treatment of whole plasma, AHF concentrate or fibrinogen with short wavelength ultraviolet light (UVC) results in the inactivation of > or = 10(6) infectious doses (ID) of encephalomyocarditis virus (EMCV), hepatitis A virus (HAV) and porcine parvovirus (PPV), each of which is nonenveloped. Protein recovery was enhanced greatly by inclusion of the flavonoid, rutin, added prior to UVC exposure to quench reactive oxygen species. We now report on the treatment of albumin and intravenous immune globulin (IVIG) isolated by a previously described, integrated chromatographic method. Albumin was treated with either 0.1 or 0.2 J/cm2 UVC in the presence of 0.8 or 1.6 mM rutin; IVIG was treated with either 0.05 or 0.1 J/cm2 UVC in the presence of 0.5 or 1.0 mM rutin. Our results show that > or = 10(6.9) ID of EMCV and PPV were inactivated under each of the conditions studied except the treatment of albumin with 0.1 J/cm2 UVC in the presence of 1.6 mM rutin where 10(4.3) ID of EMCV and > or = 10(6.9) ID of PPV were killed. It appears that the sensitivity of PPV to UVC exceeds that of EMCV and that virus kill with UVC is higher in IVIG than in albumin. In the absence of rutin, UVC increased the extent of aggregation of both albumin and IVIG by two- to three-fold. With rutin present, the increase in albumin aggregation was reduced, and it was virtually eliminated by subsequent processing on Sephacryl S-200, a step in the existing procedure designed to remove aggregates. The increase in aggregation of IVIG appeared to be eliminated on inclusion of either 0.5 mM or 1 mM rutin. We conclude that both albumin and IVIG can be treated with UVC to inactivate > or = 10(6) ID of nonenveloped viruses. The inclusion of rutin during treatment helps protect against protein aggregation.

Blood Proteins↗
PubMed1997

In vitro inhibitory activity of RepC/C*, the inactivated form of the pT181 plasmid initiation protein, RepC.

pT181 is a Staphylococcus aureus rolling circle plasmid that regulates its replication by controlling the synthesis of its dimeric initiator protein RepC/C and by inactivating the protein following its use in replication (A. Rasooly and R. P. Novick, Science 262:1048-1050, 1993). This inactivation consists of the addition of an oligonucleotide, representing several nucleotides immediately 3' to the initiation nick site, to the active site tyrosine of one of the two subunits, generating a heterodimer, RepC/C*. Previous results suggested that the inactive form was metabolically stable and was present at a much higher level than the active form (A. Rasooly and R. P. Novick, Science 262:1048-1050, 1993). In the present study we have measured total RepC antigen as a function of plasmid copy number and have analyzed the interaction of the two forms. We find that pT181-containing staphylococci contain approximately one RepC dimer per plasmid copy over a 50-fold range of copy numbers. This is consistent with previous measurements of the rate of RepC synthesis, which suggested that one RepC dimer is synthesized per replication event (J. Bargonetti, P.-Z. Wang and R. P. Novick, EMBO J. 12:3659-3667, 1993). The RepC/C* heterodimer, which is inactive for replication, is a competitive inhibitor of the replication and the topoisomerase-like and cruciform-enhancing activities of the native protein. These results suggest that the inactive form may have a specific regulatory role in vivo. Since the known plasmid-determined controls, which maintain a constant plasmid copy number, are designed to ensure the synthesis of one RepC/C dimer per plasmid replication event, it is difficult to envision any role for yet another negative regulator of replication. Conceivably, under conditions where the initiator is overproduced, such as in the absence of the normal antisense regulation of initiator production, RepC/C* could serve as a fail-safe means of preventing autocatalytic replication.

Bacterial Proteins↗
PubMed1997

Ingested IFN-alpha has biological effects in humans with relapsing-remitting multiple sclerosis.

Parenterally administered human recombinant type I interferons (hrIFN) in relapsing-remitting multiple sclerosis (RRMS) decrease relapses and spontaneous in vitro IFN-gamma production, reduce clinical progression, and decrease magnetic resonance imaging (MRI)-defined disease activity and lesions. Parenterally administered type I IFN use is limited by clinical and chemical toxicities, and the induction of antibodies that abrogate their activity in vivo correlated with the loss of clinical benefit. Therefore, we determined whether ingested IFN-alpha was non-toxic and had biological effects in humans. Ingested hrIFN-alpha showed no toxicity in normal volunteers or patients with RRMS at doses ranging from 300 to 100,000 units. In subjects with RRMS, a significant decrease in Con A-mediated proliferation and serum soluble intercellular adhesion molecule-1 (sICAM-1), a surrogate measure for disease activity in MS, was found after ingesting 10,000 and 30,000 units IFN-alpha The RRMS subjects also showed decreased IL-2 secretion after ingesting 10,000 units IFN-alpha and decreased IFN-gamma, TGF-beta and IL-10 production after ingesting 30,000 units IFN-alpha. The decreased secretion of IFN-gamma and IL-2 by ingested IFN-alpha suggests that oral IFN-alpha may cause a functional inhibition of Th J-like T helper cells in RRMS, a potential site of intervention at the level of effector T cells in MS. Our studies support the oral use of human IFN-alpha as a biological response modifier in humans.

Administration, Oral↗
PubMed1997

[Nucleotide sequence of the Rhizobium huakuii common nodution genes nodA and nodBC].

An another clone pRaN109 which contains nod genes was isolated and identified from the clone bank of R. huakuii 159 DNA using the 32P-labeled 2.3 kb R. meliloti nod DNA fragment as a hybridization probe. Analysis of homologous DNA-DNA hybridization and nucleotide sequence indicated that the 9 kb EcoRI fragment of pRaN109 DNA carried the nodD1BC genes and the 18 kb EcoRI fragment of pRaN109 DNA contained nodD2A genes. The common nodA gene was separated by 6.7 kb from the nodBC genes. Both the nodA and nodB-nodC cistrons showed nod box upstream of these genes. Nucleotide sequence analysis of the genes describes a obviously different organization of common nod genes in R. huakuii 159 compared with that reported for most of the strains from different genera described up to now.

Acyltransferases↗
PubMed1996

The inactive pT181 initiator heterodimer, RepC/C, binds but fails to induce melting of the plasmid replication origin.

Staphylococcus aureus plasmid pT181 replicates via a rolling circle mechanism. The synthesis of the pT181 initiator protein (RepC) is regulated by antisense RNAs, and RepC is inactivated after usage by the attachment of an oligonucleotide to one of its subunits. The inactivated heterodimeric RepC/C* has been shown be unable to initiate replication in vitro (Rasooly, A., and Novick, R. P. (1993) Science 262, 1048-1050). The inactive RepC/C* has been found to be very stable and constitute about 90-95% of the total RepC antigen inside the cell. We studied the specific interaction of the RepC/C and RepC/C* complex with the pT181 double strand origin. The results indicated that RepC/C and RepC/C* footprint supercoiled DNA differently although their footprints on linear DNA are similar; we also find that RepC/C is able to enhance cruciform extrusion while RepC/C* cannot. RepC/C* binds and bends the double strand origin much more weakly than does RepC/C. These results suggest that the attached oligonucleotide induces a conformational change in the RepC/C* molecule that is responsible for its lack of activity.

Bacterial Proteins↗
PubMed1996

Identification of a critical ligand binding determinant of the human erythropoietin receptor. Evidence for common ligand binding motifs in the cytokine receptor family.

The erythropoietin receptor (EPOR) is a member of a family of cytokine and growth factor receptors that share conserved features in their extracellular and cytoplasmic domains. We have used site-specific mutagenesis within the extracellular domain of the EPOR to search for amino acid residues involved in erythropoietin (EPO) binding. Mutant proteins were expressed in bacteria as soluble EPO binding proteins (EBP) and characterized for EPO binding activity in a number of different assays. Substitution of phenylalanine at position 93 (Phe93) with alanine (F93A mutation) resulted in a drastic reduction in EPO binding in the EBP. More conservative tyrosine or tryptophan substitutions at Phe93 resulted in much less dramatic effects on EPO binding. Biophysical studies indicated that the F93A mutation does not result in gross structural alterations in the EBP. Furthermore, the F93A mutation in full-length EPOR expressed in COS cells abolished detectable EPO binding. This was not a result of processing or transport defects, since mutant receptor was present on the surface of the cells. Mutations in the region immediately around Phe93 and in residues homologous to other reported ligand binding determinants of the cytokine receptor family had small to moderate effects on EPO binding. These data indicate that Phe93 is a critical EPO binding determinant of the EPOR. Furthermore, since Phe93 aligns with critical ligand binding determinants in other receptors of the cytokine receptor family, these data suggest that receptors of this family may use common structural motifs to bind their cognate ligands.

Alanine↗
PubMed1996

Molecular anatomy of the beta' subunit of the E. coli RNA polymerase: identification of regions involved in polymerase assembly.

BACKGROUND: The E. coli RNA polymerase is a multisubunit enzyme, which is present in two different forms: the catalytic competent core enzyme (alpha2beta beta') and the promoter selective holoenzyme (alpha2beta beta' sigma). Correct assembly of individual subunits into core or holoenzyme is essential for the function of this enzyme. RESULTS: Mutant beta' proteins truncated near the centre or at the C-terminus were able to form stable core enzyme-like complexes under reconstitution conditions. Mutant beta' proteins lacking the region between amino acids 201-477 failed to form holoenzyme complexes while retaining the ability to form core enzyme complexes. Furthermore, free beta' subunit interacted with free sigma subunit to form a stable beta' sigma subassembly. Removal of amino acids 201-477 from the beta' subunit strongly interfered with this interaction. CONCLUSION: Our results suggest that the N-terminal region of the beta' subunit is involved in the assembly of core enzyme. The region between amino acids 201 and 477 on beta' may be directly or indirectly involved in the interaction between the beta' subunit and the sigma subunit.

Amino Acid Sequence↗
PubMed1996

A search for persistent rubella virus infection in persons with chronic symptoms after rubella and rubella immunization and in patients with juvenile rheumatoid arthritis.

Peripheral blood polymorphonuclear leukocytes, mononuclear cells, and plasma and nasopharyngeal specimens were obtained from 6 subjects with persistent symptoms following rubella immunization, 1 subject with persistent symptoms following rubella, 11 children with juvenile rheumatoid arthritis, 17 recently immunized control subjects, and 1 control subject with acute clinical rubella. Rubella virus was isolated from the blood or nasopharynx of four of the 18 control subjects. In contrast, rubella virus was not recovered from any specimens from the seven subjects with persistent symptoms following immunization or natural infection or from the 11 children with juvenile rheumatoid arthritis. A polymerase chain reaction assay detected rubella virus in the blood from three of 14 control subjects but not in the blood from two subjects with persistent symptoms following rubella immunization or in that from three children with juvenile rheumatoid arthritis. We have not been able to confirm the findings of others who have reportedly recovered rubella virus from lymphocytes of persons with persistent symptoms following rubella or rubella immunization.

Adolescent↗
PubMed1996

Effect of shi-ka-ron on cytokine production of lymphocytes in mice treated with cyclophosphamide.

The effect of Shi-ka-ron (CX) on cytokine production of lymphocytes in mice treated with cyclophosphamide was investigated. Shi-ka-ron, a traditional Chinese prescription, consists of 8 crude Chinese herbal extracts. Its main efficacy is to strengthen the body resistance. We observed that each CX component stimulated interleukine-2 and interferon-gamma production of murine splenic lymphocytes both in vitro and in vivo. IL-2 and IFN-gamma production of splenic lymphocytes were also examined in mice treated with CX combined with cyclophosphamide (CY) in vivo. We found that CX not only could increase IL-2 and IFN-gamma production in vitro, but also in vivo at suboptimal concentration.

Animals↗
PubMed1996

Effects of Chinese herbs on macrophage functions in N-butyl-N-butanolnitrosoamine treated mice.

We investigated the effect of Chinese herbs Lithospermi radix, Astragali radix and Cnidii rhizoma on the functions of macrophages obtained from mice treated with the carcinogen N-butyl-N-butanolnitrosoamine (BBN). The chemotactic activity of murine macrophages was significantly decreased by 17 weeks of treatment with BBN compared with controls. Production of IL-1 and TNF was also markedly reduced. Treatment with Lithospermi radix, Astragali radix, and Cnidii rhizoma significantly inhibited BBN-induced suppression chemotactic activity and production of IL-1 and TNF-alpha by macrophages. Moreover, we found that Astragali radix treated macrophage chemotaxis, it or Cnidii rhizoma induced productions of TNF-alpha were in excess of control.

Animals↗
PubMed1995

Virucidal short wavelength ultraviolet light treatment of plasma and factor VIII concentrate: protection of proteins by antioxidants.

The use of solvent/detergent mixtures and various forms of heat treatment to inactivate viruses has become widespread in the preparation of blood derivatives. Because viruses that lack lipid envelopes and/or are heat resistant, eg, hepatitis A virus (HAV) or parvovirus B19 may be present, the use of two methods of virus elimination that operate by different mechanisms has been advocated. We now report on short wavelength ultraviolet light (UVC) irradiation for virus inactivation and enhancement of its compatibility with proteins by quenchers of reactive oxygen species (ROS). Treatment of an antihemophilic factor (AHF) concentrate or whole plasma with 0.1 J/cm2 inactivated 10(5) to > or = 10(6) infectious doses (ID) of encephalomyocarditis virus (EMCV), HAV, bacteriophage M13, vesicular stomatitis virus (VSV), and porcine parvovirus. However, the recovery of factor VIII was 30% or lower on treatment of an AHF concentrate and 60% on treatment of plasma. Factor VIII recovery could be increased with little or no effect on virus kill by addition of rutin, a flavonoid known to quench both type I and type II ROS. On treatment of plasma in the presence of rutin, the recovery of several other coagulation factors was also enhanced by rutin addition and typically exceeded 75%. Electrophoretic analysis of treated AHF concentrate confirmed the advantage of rutin presence; UVC irradiation of plasma did not cause discernible changes in electrophoretic banding patterns, even in the absence of rutin. We conclude that addition of UVC treatment to existing processes used in the manufacture of blood derivatives will provide an added margin of safety, especially for nonenveloped or heat-stable viruses.

Animals↗
PubMed1995

Evidence for contact between the cyclic AMP receptor protein and the delta 70 subunit of Escherichia coli RNA polymerase.

The loop at the 52-position of the cAMP receptor protein (CRP) has been suggested as a potential site for contacting RNA polymerase on Class II promoters where the CRP binding site is located at position -41.5 (Bell, A., Gaston, K., Williams, R., Chapman, K., Kolb, A., Buc, H., Minchin, S., Williams, J., and Busby, S. (1990) Nucleic Acids Res. 18, 7243-7250). Using protein-protein photo-cross-linking, evidence is presented showing that the 52-loop of CRP is in physical proximity to the delta subunit of RNA polymerase holoenzyme. This interaction required the presence of a functional preinitiation complex. The CRP suppressor mutation, K52N, increased the efficiency of cross-linking, indicating an improved physical interaction between the CRP 52-loop and the delta subunit. Evidence for direct interaction between the CRP 156-162 loop and delta subunit of RNA polymerase on both gal and lac promoters are also provided. The data indicate that CRP bound to the gal promoter contacts both the alpha and delta 70 subunits of RNA polymerase.

Cyclic AMP Receptor Protein↗