Biomedical subjects

R Jin

Publications and source records attributed to R Jin.

At least 19 recordsLinked to original sources

PubMed2000

Immunosuppressive activity of the Chinese medicinal plant Tripterygium wilfordii. I. Prolongation of rat cardiac and renal allograft survival by the PG27 extract and immunosuppressive synergy in combination therapy with cyclosporine.

BACKGROUND: PG27 is an immunosuppressive fraction purified from an extract of a Chinese medicinal plant, Tripterygium wilfordii. We tested PG27 in rat cardiac and renal allotransplantation, and we examined the immunosuppressive interaction with cyclosporine (CsA). METHODS: Brown Norway (BN) rat heart or kidney allografts were transplanted into the abdomen of Lewis rats, which were treated by the intraperitoneal or oral route with PG27, CsA, or both. RESULTS: PG27 administered intraperitoneally to Lewis recipients for 16 days at 10-30 mg/kg/day significantly increased the median survival time of BN heart allografts from 7 to 18-22 days. Oral administration was effective, with cardiac allograft survival prolonged to > 100 days with 52 days of treatment. PG27 at 20-30 mg/kg/day significantly extended the median survival time of BN kidney allograft recipients from 9 to 36.5-77 days, and 30 mg/kg/day for 52 days extended survival beyond 200 days. PG27 combined with CsA significantly enhanced heart and kidney allograft survival, even at doses of CsA ineffective when administered alone. The addition of 5 or 10 mg/kg/day PG27 reduced by 50-75% the CsA dose needed for 100% kidney allograft survival. The combination index was less than 1.0, indicating synergy of PG27 with CsA in prolonging cardiac and renal allograft survival. Furthermore, the PG27/CsA combination exerted a positive influence on renal allograft function. PG490 (triptolide, a constituent of PG27) and PG490-88 (a semisynthetic derivative of PG490) suppressed rejection of cardiac and renal allografts. CONCLUSIONS: The PG27 herbal extract demonstrated immunosuppressive activity by prolonging heart and kidney allograft survival, displaying synergy in the immunosuppressive interaction with CsA, and improving renal allograft function in combination with CsA. PG490 and PG490-88 compounds were also effective.

Animals↗

PubMed2000

Immunosuppressive activity of the Chinese medicinal plant Tripterygium wilfordii. II. Prolongation of hamster-to-rat cardiac xenograft survival by combination therapy with the PG27 extract and cyclosporine.

BACKGROUND: PG27 is an immunosuppressive fraction purified from an extract of a Chinese medicinal plant Tripterygium wilfordii, which we investigated alone and in combination with cyclosporine (CsA) in a concordant, hamster-to-rat cardiac xenotransplantation model. METHODS: Golden Syrian hamster hearts were heterotopically transplanted into the abdomen of Lewis rat recipients, which were treated intraperitoneally or orally with PG27, CsA, or both. RESULTS: Combination therapy with 30 mg/kg(day of PG27 and CsA at 10 mg/kg/day successfully suppressed acute hamster-to-rat cardiac xenograft rejection. Treatment with PG27 or CsA alone was ineffective. Among several effective combinations, the best regimen involved PG27 at 30 mg/kg/day and CsA at 5 mg/ kg/day from days 8 to 35 and then CsA at 5 mg/kg/day from days 36 to 100, which produced 100% survival beyond 100 days. CsA suppressed the heterospecific lymphocytotoxic antibody response and inhibited IgG but not IgM xenoantibody production (which led to xenograft rejection), whereas PG27 alone did not prevent antibody production. The PG27/CsA combination blocked the lymphocytotoxic antibody response and IgG and IgM xenoantibody production induced by cardiac xenotransplantation. CONCLUSIONS: PG27 combined with CsA substantially prolonged hamster-to-rat cardiac xenograft survival, as well as completely inhibiting xenoantibody production.

Animals↗

PubMed2000

Metallothionein 1E mRNA is highly expressed in oestrogen receptor-negative human invasive ductal breast cancer.

Metallothioneins (MTs), a group of ubiquitous metalloproteins, comprise isoforms encoded by ten functional genes in humans. Different MT isoforms possibly play different functional roles during development or under various physiological conditions. The MT-1E isoform mRNA has been recently shown to be differentially expressed in oestrogen receptor (OR)-positive and OR-negative breast cancer cell lines. In this study, we evaluated MT-1E mRNA expression via semi-quantitative RT-PCR in 51 primary invasive ductal breast cancer tissues, concurrently with OR-positive and progesterone receptor (PR)-positive MCF7 cells, OR-negative and PR-negative MDA-MB-231 cells and PR-transfected MDA-MB-231 breast cancer cells (ABC28). We demonstrated significantly higher MT-1E mRNA expression in OR-negative compared with OR-positive breast cancer tissues (P = 0.026). MCF7 cells lacked MT-1E mRNA expression, while both OR- and PR-negative MDA-MD-231 cells exhibited a high level of MT-1E mRNA expression. The level of MT-1E mRNA expression in progesterone-treated and -untreated ABC28 cells remained similar as the parental cell line MDA-MB-231-C2 cells. The results suggest that MT-1E may have specific and functional roles in OR-negative invasive ductal breast cancers, possibly mediated via effector genes downstream of the oestrogen receptor, but not through the PR pathway.

Biomarkers, Tumor↗

PubMed2000

Immune regulatory effects of central nervous system antigens in culture.

Evidence from several different experimental systems suggests that regulatory cells specific for self-antigens exist in the normal immune repertoire, and that these cells are necessary for maintenance of self-tolerance and prevention of autoimmune disease. We attempted to demonstrate the existence of regulatory cells specific for central nervous system (CNS) antigens in normal mice. We tested the effects of myelin basic protein (MBP), glial fibrillary acidic protein (GFAP) and a mixture of soluble brain proteins (SBP) on cultured splenocytes. MBP at 50 microg/ml inhibited antigen-driven proliferation and this suppressive effect could be partially blocked by neutralizing antibodies to transforming growth factor (TGF)-beta. MBP decreased expression of mRNA for the cytokines IL-2 and IFN-gamma, and slightly increased mRNA expression for TGF-beta. These effects did not appear to be mediated by regulatory cells specific for MBP, since MBP also suppressed proliferation in MBP-deficient shiverer mice and the suppressive effect could not be reproduced with selected MBP peptides. SBP at 250 microg/ml also inhibited antigen-driven proliferation, but this effect could not be blocked by neutralizing antibodies against IL-4, IL-10 or TGF-beta. SBP reduced expression of mRNA for IL-2, IL-10 and TGF-beta. These results are more consistent with the presence of a soluble inhibitory factor than with the action of SBP-specific regulatory cells. GFAP had no significant effect on proliferation. These results do not support the existence of regulatory cells specific for CNS antigens. Further investigation into non-antigen-specific mechanisms will be important in defining how autoimmune damage in the CNS is prevented.

Adjuvants, Immunologic↗

PubMed1999

Frequency of the fragile X syndrome in Chinese mentally retarded populations is similar to that in Caucasians.

Fragile X syndrome is recognized as the most common inherited cause of mental retardation in western countries. The prevalence of the fragile X syndrome in Asian populations is uncertain. We report a multi-institutional collaborative study of molecular screening for the fragile X syndrome from 1,127 Chinese mentally retarded (MR) individuals. We found that 2.8% of the Chinese MR population screened by DNA analysis had the fragile X full mutation. Our screening indicated that the fragile X syndrome prevalence was very close to that of Caucasian subjects. In addition, we found that 62.5% of fragile X chromosomes had a single haplotype for DXS548-FRAXAC1 (21-18 repeats) which was present in only 9.7% of controls. This unique distribution of microsatellite markers flanking the FMR1 CGG repeats suggests that the fragile X syndrome in Chinese populations, as in the Caucasian, may also be derived from founder chromosomes.

Alleles↗

PubMed1999

Shared and unique determinants of the erythropoietin (EPO) receptor are important for binding EPO and EPO mimetic peptide.

We have shown previously that Phe93 in the extracellular domain of the erythropoietin (EPO) receptor (EPOR) is crucial for binding EPO. Substitution of Phe93 with alanine resulted in a dramatic decrease in EPO binding to the Escherichia coli-expressed extracellular domain of the EPOR (EPO-binding protein or EBP) and no detectable binding to full-length mutant receptor expressed in COS cells. Remarkably, Phe93 forms extensive contacts with a peptide ligand in the crystal structure of the EBP bound to an EPO-mimetic peptide (EMP1), suggesting that Phe93 is also important for EMP1 binding. We used alanine substitution of EBP residues that contact EMP1 in the crystal structure to investigate the function of these residues in both EMP1 and EPO binding. The three largest hydrophobic contacts at Phe93, Met150, and Phe205 and a hydrogen bonding interaction at Thr151 were examined. Our results indicate that Phe93 and Phe205 are important for both EPO and EMP1 binding, Met150 is not important for EPO binding but is critical for EMP1 binding, and Thr151 is not important for binding either ligand. Thus, Phe93 and Phe205 are important binding determinants for both EPO and EMP1, even though these ligands share no sequence or structural homology, suggesting that these residues may represent a minimum epitope on the EPOR for productive ligand binding.

Circular Dichroism↗

PubMed1999

Ingested interferon alpha induces Mx mRNA.

We have demonstrated that ingested murine interferon alpha (IFN-alpha) suppressed clinical relapse in chronic relapsing experimental autoimmune encephalomyelitis (CR-EAE), decreased inflammation and suppressed the adoptive transfer of EAE, and is a biological response modifier in patients with multiple sclerosis. We examined the relative levels of the Mx mRNA signal using semiquantitative reverse transcription-polymerase chain reaction analysis on splenocytes from mice and peripheral blood mononuclear cells from man after IFN-alpha ingestion. Both mice and man demonstrated inducible levels of Mx mRNA after ingesting IFN-alpha. Murine spleen T cells and CD8(+)T cells also demonstrated upregulation of Mx mRNA. Murine whole splenocytes demonstrated upregulation of Mx mRNA after IFN-alpha ingestion of 10 and 100 U, but not after 0, 1000, 5000 U. Ingested IFN-alpha acts via established pathways of type 1 IFN signalling.

Administration, Oral↗

PubMed1999

The organizational context of non-lethal workplace violence: its interpersonal, temporal, and spatial correlates.

This article examines 993 violent incidents involving faculty, students, and staff that occurred at a highly ranked teaching and research university and its affiliated medical center. Violent incidents were included in the sample if they involved faculty, students, or staff, regardless of their specific location or context (i.e., whether they occurred on campus and/or off-campus and whether they occurred within the context of work or some other activity). The theoretical goal of the project was to compare work-related incidents with non-work-related incidents of interpersonal violence occurring in a single, multifunctioning, and professionally hierarchical organization. Data were collected over a 7-year period from three police departments (city, county, and university), university records, and criminal history records obtained from the state police. The coding protocol was developed to capture crime-scene information pertinent to each of the incidents. This included information about the victim, the perpetrator, the relationship between the victim and perpetrator, and the violent incident. The data were examined using nonparametric statistics and logistic regression to model predictive differences between the workplace and non-workplace incidents. The results suggest that the workplace incidents of violence differ from the non-workplace incidents according to their time, victim age, degree of victim injury, and whether the workplace is a medical location. The authors conclude that these differences are better explained by the movement of people in and out of the workplace who bring societal violence with them, rather than by a category or type of workplace violence.

Adult↗

PubMed1999

The effect of electroacupuncture on auditory P300 potential in mongolism cases.

In treating children with mongolism (Down's syndrome) with electroacupuncture, the latent period of P3 is shortened, which shows that the cerebral reaction to external stimulus in classification, encoding and recognition is accelerated. Along with the elevated P3 amplitude, the cerebrum is more capable of processing the external information, showing improvement of the mental state.

Acupuncture Therapy↗

PubMed1998

Role of human CYP4F2 in hepatic catabolism of the proinflammatory agent leukotriene B4.

Leukotriene B4 (LTB4), an arachidonic acid derivative, is a potent proinflammatory agent whose actions are terminated by catabolism via a microsomal omega-hydroxylation pathway. Although the liver serves as the principal site for LTB4 clearance from the systemic circulation, the attributes of hepatic LTB4 metabolism are ill defined in humans. Thus, we examined metabolism of LTB4 to its omega-hydroxylated metabolite 20-hydroxyleukotriene B4 (20-OH LTB4) by human liver microsomes and also purified the hepatic P450 enzyme underlying this reaction. Liver microsomes from 10 different subjects converted LTB4 to 20-OH LTB4 at similar rates (1.06 +/- 0.3 nmol/min/nmol P450; 0.25 +/- 0.1 nmol/min/mg protein). Analysis of the microsomal LTB4 20-hydroxylation reaction revealed kinetic parameters (apparent Km of 74.8 microM with a VMAX of 2.42 nmol/min/nmol P450) consistent with catalysis by a single P450 enzyme. Conventional chromatography combined with immunochemical screening with rat CYP4A1 antibodies was then used to isolate a P450 enzyme from human liver microsomes with a molecular weight of 57,000 and an NH2-terminal amino acid sequence 94% homologous (12Trp --> 12Gly) over the first 17 residues with the human CYP4F2 cDNA-derived sequence. Upon reconstitution with P450 reductase and phospholipid, CYP4F2 converted LTB4 to 20-OH LTB4 at a turnover rate of 392 pmol/min/nmol P450, whereas the other human liver P450s tested, including CYP4A11, exhibited neglible LTB4 omega-hydroxylase activity. Polyclonal antibodies to CYP4F2 were found to markedly inhibit (91.9 +/- 5%; n = 5) LTB4 20-hydroxylation by human liver microsomes. Microsomal 20-OH LTB4 formation was also inhibited 30% by arachidonic acid, a known CYP4F2 substrate, and 50% by prostaglandin A1 but was unaffected by lauric acid, palmitic acid, and PGF2alpha. Finally, a strong correlation (r = 0.86; P < 0.002; n = 10) was observed between CYP4F2 content and LTB4 20-hydroxylase activity in the human liver samples. Our results indicate that CYP4F2 is the principle LTB4 omega-hydroxylating enzyme expressed in human liver and, as such, may play an important role in regulating circulating as well as hepatic levels of this powerful proinflammatory eicosanoid.

Amino Acid Sequence↗

PubMed1998

Probing the ligand binding domain of the GluR2 receptor by proteolysis and deletion mutagenesis defines domain boundaries and yields a crystallizable construct.

Ionotropic glutamate receptors constitute an important family of ligand-gated ion channels for which there is little biochemical or structural data. Here we probe the domain structure and boundaries of the ligand binding domain of the AMPA-sensitive GluR2 receptor by limited proteolysis and deletion mutagenesis. To identify the proteolytic fragments, Maldi mass spectrometry and N-terminal amino acid sequencing were employed. Trypsin digestion of HS1S2 (Chen GQ, Gouaux E. 1997. Proc Natl Acad Sci USA 94:13431-13436) in the presence and absence of glutamate showed that the ligand stabilized the S1 and S2 fragments against complete digestion. Using limited proteolysis and multiple sequence alignments of glutamate receptors as guides, nine constructs were made, folded, and screened for ligand binding activity. From this screen, the S1S21 construct proved to be trypsin- and chymotrypsin-resistant, stable to storage at 4 degrees C, and amenable to three-dimensional crystal formation. The HS1S21 variant was readily prepared on a large scale, the His tag was easily removed by trypsin, and crystals were produced that diffracted to beyond 1.5 A resolution. These experiments, for the first time, pave the way to economical overproduction of the ligand binding domains of glutamate receptors and more accurately map the boundaries of the ligand binding domain.

Amino Acid Sequence↗

PubMed1998

Anticarcinogenic effects of shikaron, a preparation of eight Chinese herbs in mice treated with a carcinogen, N-butyl-N'-butanolnitrosoamine.

We studied the effects of Shikaron, which is composed of 8 Chinese herb extracts, on carcinogenesis and the cytotoxic activity and cytokine production of lymphocytes in mice treated with a carcinogen, N-butyl-N'-butanolnitrosoamine (BBN). We found a significantly lower incidence of urinary bladder carcinoma in mice treated with BBN plus Shikaron 200 mg/kg/day (5 of 20 mice, 25%), than in mice treated with BBN alone (16 of 20 mice, 80%). Shikaron protected the cytotoxic activity of lymphocytes against YAC-1 cells from suppression by BBN. Cytotoxic activity against P-815 cells significantly increased in mice treated with BBN plus Shikaron, as compared with normal mice and BBN-treated mice. Shikaron also protected production of interleukin-2 and interferon-gamma by lymphocytes from suppression by BBN. These findings strongly suggest that Shikaron exerted an anticarcinogenic effect in carcinogen-treated mice through activation of NK and LAK cells and cytokine production by T lymphocytes.

Animals↗

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↗