Biomedical subjects

J R Scott

Publications and source records attributed to J R Scott.

At least 37 records · Page 2Linked to original sources

PubMed2000

Rns, a virulence regulator within the AraC family, requires binding sites upstream and downstream of its own promoter to function as an activator.

Strains of enterotoxigenic Escherichia coli that express CS1 and CS2 pili require the transcriptional activator Rns, a member of the AraC family, for the expression of the pilin genes. Rns is also an activator of its own expression. However, the arrangement of its binding sites near its own promoter is unusual for a prokaryotic activator. Most activators have at least one binding site 30-80 nucleotides upstream of the transcription start site, but Rns has a single upstream binding site centred at -227. Rns also has two binding sites downstream of the transcription start site centred at +43 and +82, a region generally thought to be reserved for repressors. In vitro, the binding of a MBP::Rns fusion protein to each of these sites facilitates the binding of RNA polymerase to the rns promoter and the formation of an open complex. In vivo, the upstream binding site and one downstream site are required for Rns-dependent activation of its promoter despite the atypical location of these binding sites for an activator. This suggests that Rns may represent a new class of prokaryotic activators.

ATP-Binding Cassette Transporters↗

PubMed2000

Alloimmune causes of recurrent pregnancy loss.

Recurrent pregnancy loss is a vexing problem facing many couples. Some authorities have suggested that idiopathic recurrent pregnancy loss is alloimmune in nature. Suggested mechanisms include the presence of cytotoxic antibodies, absence of maternal blocking antibodies, inappropriate sharing of human leukocyte antigens, and disturbances in natural killer cell function and distribution. Proposed therapies for so-called alloimmune-related pregnancy loss include leukocyte immunization and intravenous immune globulin. Unfortunately, neither has been successful in well-designed trials and both are expensive. Although alloimmune disturbances may be responsible for some idiopathic recurrent pregnancy loss, the pathophysiology is far from certain and there is currently no clinical indication for immunotherapy.

Abortion, Habitual↗

PubMed2000

Is vaginal birth after cesarean less expensive than repeat cesarean delivery?

OBJECTIVE: This study was undertaken to compare total medical costs of trial of labor after cesarean with those of elective repeat cesarean without labor, with both short- and long-term neonatal costs associated with such procedures taken into account. STUDY DESIGN: Costs associated with All Patient Refined diagnosis-related groups and Current Procedural Terminology for a large not-for-profit health care system were applied to an algorithm describing maternal and neonatal outcomes of trial of labor. Perinatal morbidity rates and cost estimates for long-term neurologic damage associated with uterine rupture were derived from published literature. RESULTS: If a 70% vaginal birth rate for women undergoing a trial of labor and delivery in a tertiary center with a mean uterine rupture to delivery time of 13 minutes is assumed, the net cost differential ranged from a saving of $149 to a loss of $217, depending on morbidity assumptions. For vaginal birth after cesarean success rates <70%, trial of labor in the presence of two previous scars, and institutional factors increasing the perinatal morbidity rate by just 4% with respect to that seen in tertiary centers, trial of labor resulted in a net financial loss to the health care system regardless of all other assumptions made. CONCLUSIONS: When costs as opposed to charges are considered and the cost of long-term care for neurologically injured infants is taken into account, trial of labor after previous cesarean is unlikely to be associated with a significant cost saving for the health care system. Recent government-mandated length-of-stay requirements are likely to make the economic benefit of vaginal birth after cesarean even less favorable. Factors other than cost must govern decisions regarding trial of labor or repeat cesarean.

Algorithms↗

PubMed2000

Neonatal alloimmune thrombocytopenia: antenatal management.

OBJECTIVE: The optimal management of pregnancies at risk for neonatal alloimmune thrombocytopenia is debated. Proposed management includes the administration of intravenous immunoglobulin and serial determination of the fetal platelet count. The aims of our study were to determine the effectiveness and likely mechanism of action of intravenous immunoglobulin and to evaluate the safety of cordocentesis in cases of neonatal alloimmune thrombocytopenia. STUDY DESIGN: Eighteen mother-infant pairs were studied. All were at risk for neonatal alloimmune thrombocytopenia on the basis of delivery of a previously affected infant and confirmation of specific maternal antiplatelet antibodies. The pertinent antigen was HPA-1a in 13 cases, HPA-3a in 2 cases, and undetermined in 3 cases. Serial cordocenteses were used to determine fetal platelet counts. If the platelet count was <50,000/microL before 37 weeks' gestation, treatment was initiated with intravenous immunoglobulin administered to either the fetus (n = 2) or the mother (n = 8). In 3 cases fetal and maternal immunoglobulin G levels were determined before and after treatment. RESULTS: Seven (39%) fetuses had adequate platelet counts, were not treated, and were delivered of infants with normal platelet counts. Eleven (61%) fetuses were thrombocytopenic. Eight thrombocytopenic infants were treated with maternally administered intravenous immunoglobulin. In 6 (75%) of 8 cases the fetal platelet count increased after administration of intravenous immunoglobulin, but 2 fetuses remained severely thrombocytopenic. Two thrombocytopenic fetuses were treated with intravenous immunoglobulin infusion directly into the umbilical vein; both remained thrombocytopenic. Moreover, fetal immunoglobulin G levels did not correlate well with the response to intravenous immunoglobulin. Two (5.3%) of 38 cordocenteses were complicated by hemorrhagic complications, necessitating immediate cesarean delivery despite the use of prophylactic platelet transfusion in one case. CONCLUSION: Severe fetal alloimmune thrombocytopenia does not always occur in subsequent fetuses. Thus either fetal antigen status or platelet counts or both of these are necessary to determine whether treatment is needed. The effect of intravenous immunoglobulin on raising the fetal platelet count is inconsistent and appears to be caused by maternal or placental factors rather than a direct inhibition of fetal platelet destruction by immunoglobulin. The risk of hemorrhagic complications from cordocentesis in pregnancies complicated by neonatal alloimmune thrombocytopenia is higher than generally appreciated and is not always avoided by platelet transfusion at the time of the procedure.

Antigens, Human Platelet↗

PubMed2000

Role of streptolysin O in a mouse model of invasive group A streptococcal disease.

Many of the virulence factors that have been characterized for group A streptococci (GAS) are not expressed in all clinical isolates. One putative virulence factor that is present among most is streptolysin O (Slo), a protein with well-characterized cytolytic activity for many eukaryotic cells types. In other bacterial pathogens, proteins homologous to Slo have been shown to be essential for virulence, but the role of Slo in GAS had not been previously examined. To investigate the role of Slo in GAS virulence, we examined both revertible and stable slo mutants in a mouse model of invasive disease. When the revertible slo mutant was used to infect mice, the reversion frequency of bacteria isolated from the wounds and spleens of infected animals was more than 100 times that of the inoculum, indicating that there was selective pressure in the animal for Slo(+) GAS. Experiments with the stable slo mutant demonstrated that Slo was not necessary for the formation of necrotic lesions, nor was it necessary for escape from the lesion to cause disseminated infection. Bacteria were present in the spleens of 50% of the mice that survived infection with the stable slo mutant, indicating that dissemination of Slo(-) GAS does not always cause disease. Finally, mice infected with the stable slo mutant exhibited a significant decrease in mortality rates compared to mice infected with wild-type GAS (P < 0.05), indicating that Slo plays an important role in GAS virulence.

Animals↗

PubMed2000

Phenytoin metabolism by human cytochrome P450: involvement of P450 3A and 2C forms in secondary metabolism and drug-protein adduct formation.

The anticonvulsant phenytoin (5,5-diphenylhydantoin) provokes a skin rash in 5 to 10% of patients, which heralds the start of an idiosyncratic reaction that may result from covalent modification of normal self proteins by reactive drug metabolites. Phenytoin is metabolized by cytochrome P450 (P450) enzymes primarily to 5-(p-hydroxyphenyl-),5-phenylhydantoin (HPPH), which may be further metabolized to a catechol that spontaneously oxidizes to semiquinone and quinone species that covalently modify proteins. The aim of this study was to determine which P450s catalyze HPPH metabolism to the catechol, proposed to be the final enzymatic step in phenytoin bioactivation. Recombinant human P450s were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. Novel bicistronic expression vectors were constructed for P450 2C19 and the three major variants of P450 2C9, i.e., 2C9*1, 2C9*2, and 2C9*3. HPPH metabolism and covalent adduct formation were assessed in parallel. P450 2C19 was the most effective catalyst of HPPH oxidation to the catechol metabolite and was also associated with the highest levels of covalent adduct formation. P450 3A4, 3A5, 3A7, 2C9*1, and 2C9*2 also catalyzed bioactivation of HPPH, but to a lesser extent. Fluorographic analysis showed that the major targets of adduct formation in bacterial membranes were the catalytic P450 forms, as suggested from experiments with human liver microsomes. These results suggest that P450 2C19 and other forms from the 2C and 3A subfamilies may be targets as well as catalysts of drug-protein adduct formation from phenytoin.

Anticonvulsants↗

PubMed1999

A conserved residue in the tip proteins of CS1 and CFA/I pili of enterotoxigenic Escherichia coli that is essential for adherence.

Enterotoxigenic Escherichia coli associated with human diarrheal disease utilize any of a limited group of serologically distinguishable pili for attachment to intestinal cells. These include CS1 and CFA/I pili. We show here that chemical modification of arginyl residues in CS1 pili abolishes CS1-mediated agglutination of bovine erythrocytes, which serves as a model system for attachment. Alanine substitution of the single arginyl residue in CooA, the major pilin, had no effect on the assembly of pili or on hemagglutination. In contrast, substitution of alanine for R181 in CooD, the minor pilin associated with the pilus tip, abolished hemagglutination, and substitution of R20 reduced hemagglutination. Neither of these substitutions affected CS1 pilus assembly. This shows that CooD is essential for CS1-mediated attachment and identifies specific residues that are involved in receptor binding but not in pilus assembly. In addition to mediating agglutination of bovine erythrocytes, CFA/I also mediates agglutination of human erythrocytes. Substitution of R181 by alanine in the CooD homolog, CfaE, abolished both of these reactions. We conclude that the same region of the pilus tip protein is involved in adherence of CS1 and CFA/I pili, although their receptor specificities differ. This suggests that the region of the pilus tip adhesin protein that includes R181 might be an appropriate target for therapeutic intervention or for a vaccine to protect against human diarrhea caused by enterotoxigenic E. coli strains that have serologically different pili.

Amino Acid Sequence↗

PubMed1999

Mononuclear-cell immunisation in prevention of recurrent miscarriages: a randomised trial.

BACKGROUND: Couples with unexplained recurrent miscarriage may have an alloimmune abnormality that prevents the mother from developing immune responses essential for the survival of the genetically foreign conceptus. Immunisation with paternal mononuclear cells is used as a treatment for such alloimmune-mediated pregnancy losses. However, the published results on this treatment are conflicting. In this study (the Recurrent Miscarriage [REMIS] Study), we investigated whether paternal mononuclear cell immunisation improves the rate of successful pregnancies. METHODS: Women who had had three or more spontaneous abortions of unknown cause were enrolled in a double-blind, multicentre, randomised clinical trial. 91 were assigned immunisation with paternal mononuclear cells (treatment) and 92 immunisation with sterile saline (control). The primary outcomes were the inability to achieve pregnancy within 12 months of randomisation, or a pregnancy which terminated before 28 weeks of gestation (failure); and pregnancy of 28 or more weeks of gestation (success). Two analyses were done: one included all women (intention to treat), and the other included only those who became pregnant. FINDINGS: Two women in each group received no treatment, and eight (three treatment, five control) were censored after an interim analysis. In the analysis of all randomised women who completed the trial, the success rate was 31/86 (36%) in the treatment group and 41/85 (48%) in the control group (odds ratio 0.60 [95% CI 0.33-1.12], p=0.108). In the analysis of pregnant women only, the corresponding success rates were 31/68 (46%) and 41/63 (65%; odds ratio 0.45 [0.22-0.91], p=0.026). The results were unchanged after adjustment for maternal age, number of previous miscarriages, and whether or not the couple had had a previous viable pregnancy. Similar results were obtained in a subgroup analysis of 133 couples with no previous livebirth. INTERPRETATION: Immunisation with paternal mononuclear cells does not improve pregnancy outcome in women with unexplained recurrent miscarriage. This therapy should not be offered as a treatment for pregnancy loss.

Abortion, Habitual↗

PubMed1999

Cloning and kinetic characterization of the Trypanosoma cruzi S-adenosylmethionine decarboxylase.

The gene for S-adenosylmethionine decarboxylase (AdoMetDC), a rate-limiting enzyme in the biosynthesis of polyamines, has been cloned from a Trypanosoma cruz cDNA library. The cDNA clone contains a 1.1 kb open reading frame predicted to encode a 42 kDa protein that shares 31% sequence identity to the human proenzyme. T. cruzi AdoMetDC expressed and purified from E. coli is auto-catalytically processed into two subunits of 32 kDa (alpha) and 10 kDa (beta). The catalytic activity of the purified recombinant enzyme is activated by the addition of putrescine to the reaction. To determine the effect of putrescine on the kinetics of the reaction, the velocity data collected at various substrate and putrescine concentrations were fit to the rate equation describing a non-essential activator. In the presence of fully saturating putrescine, k(cat) increases by 9-fold over the unactivated rate to 0.06 s(-1). The model derived Km for AdoMet is 0.05 mM in the absence of putrescine and the model-derived Kd for putrescine binding to free enzyme is 2.5 mM. The Km for AdoMet increases by alpha 2-fold when the enzyme is fully saturated with putrescine. Unlike human AdoMetDC, cadaverine activates the T. cruzi enzyme to a similar extent as putrescine.

Adenosylmethionine Decarboxylase↗

PubMed1999

Fragmentation of an alkali metal-attached peptide probed by collision-induced dissociation fourier transform mass spectrometry and computational methodology.

Collision-induced dissociation of metal-cationized N-CBZ-Gly-Pro-Gly-Pro-Ala was studied by Fourier transform mass spectrometry. Lithium-, sodium-, potassium- and rubidium-cationized peptide species were generated by matrix-assisted laser desorption/ionization (MALDI) using 2,5-dihydroxybenzoic acid as matrix, together with appropriate metal salts. The experimental mass spectrometric results were interpreted with the aid of Monte Carlo conformational searches using the Amber(*) force field, together with ab initio molecular orbital calculations with Gaussian-94 for the singly lithium- and potassium-cationized peptides. It is concluded that metal coordination plays a key role in guiding the gas-phase fragmentation of the cationized peptide. In contrast to lithium and sodium, potassium and rubidium apparently do not coordinate to the C-terminal carbonyl. When the peptide is cationized with the two smaller alkali metals, losses corresponding to alanine and CBZ are observed, while the coordination of potassium and rubidium results in only CBZ loss upon dissociation.

Amino Acid Sequence↗

PubMed1999

Anti-beta2-glycoprotein I antibodies in women with recurrent spontaneous abortion, unexplained fetal death, and antiphospholipid syndrome.

OBJECTIVE: Studies in rheumatologic and hematologic populations suggest that anti-beta(2)-glycoprotein I antibodies are more specific for the clinical manifestations of antiphospholipid syndrome than anticardiolipin antibodies. However, the association between anti-beta(2)-glycoprotein I and pregnancy loss remains uncertain. We sought to determine whether anti-beta(2)-glycoprotein I is associated with the obstetric features of antiphospholipid syndrome. STUDY DESIGN: Sera from 4 groups of women were studied: (1) 152 healthy fertile control subjects, (2) 141 subjects with unexplained recurrent spontaneous abortions, (3) 58 subjects with unexplained fetal deaths, and (4) 73 subjects with well-characterized antiphospholipid syndrome. Serum anticardiolipin and anti-beta(2)-glycoprotein I levels were determined by enzyme-linked immunoassay. RESULTS: Patients with antiphospholipid syndrome had significantly higher levels of immunoglobulin G and immunoglobulin M anticardiolipin and anti-beta(2)-glycoprotein I than the other 3 groups (P <.0001). However, women in the recurrent spontaneous abortion, fetal death, and fertile control groups had similar levels of each antibody. Similarly, there were no differences in the proportion of women with positive test results for each autoantibody in these 3 groups. Linear regression analysis showed significant correlation between anticardiolipin immunoglobulin G and beta(2)-glycoprotein I immunoglobulin G (R (2) = 0.544786, P =.0001) and anticardiolipin immunoglobulin M and beta(2)-glycoprotein I immunoglobulin M (R (2) = 0.525048, P =.0001). CONCLUSION: Both anticardiolipin and anti-beta(2)-glycoprotein I are associated with antiphospholipid syndrome. However, testing for anti-beta(2)-glycoprotein I does not identify additional patients with either recurrent spontaneous abortions or unexplained fetal deaths who initially have negative test responses for anticardiolipin. This is likely because of the strong correlation between the 2 autoantibodies. Our data do not support routine testing for anti-beta(2)-glycoprotein I in addition to testing for antiphospholipid antibodies in women with recurrent pregnancy loss and unexplained fetal death.

Abortion, Habitual↗

PubMed1999

Regulation of excision of the conjugative transposon Tn916.

Excision from the donor DNA molecule is the first step in conjugative transposition of Tn916 and is followed by circularization of the transposon and its transfer to a new host. We have demonstrated that, in Gram-positive hosts, the Xis protein, as well as the site-specific recombinase Int, is required for the excision of Tn916. Using assays for closure of the excised covalently closed transposon and for repair of the donor DNA molecule, we found that neither protein alone is rate limiting for excision, but overexpression of Int and Xis together results in increased excision. After excision, the frequency of Tn916 circle formation was found to be the same as the frequency of repair of the donor DNA molecule. This suggests that a single reaction results in the closure of both molecules. We have also identified two transcripts that encode Int, one of which also encodes Xis and one of which does not, suggesting that there are steps in conjugative transposition of Tn916 that require Int without Xis.

Bacillus subtilis↗

PubMed1999

Excision of the conjugative transposon Tn916 in Lactococcus lactis.

In Lactococcus lactis excision of Tn916 is limited by the concentration of integrase and is increased by providing more excisionase. However, even with increased excision of Tn916 in L. lactis, no conjugative transfer is detectable. This suggests that L. lactis is deficient in a host factor(s) required for conjugative transposition.

Conjugation, Genetic↗

PubMed1999

Attenuated expression of the mga virulence regulon in an M serotype 50 mouse-virulent group A streptococcal strain.

The attenuated expression of virulence genes found in a group A streptococcal strain that is naturally pathogenic for mice was postulated to result from a defect in the strain's multigene regulator, Mga. The sequence of the mga gene reveals three amino acid changes in the gene product that might affect protein function. The defect in the mga gene was complemented by providing either the closely similar mga4 allele or a more divergent mga1 allele in trans. Complementation increased the amount of emm50 transcript and the quantity of surface-extractable M protein, restoring virulence function.

Amino Acid Sequence↗

PubMed1999

A response regulator that represses transcription of several virulence operons in the group A streptococcus.

A search for homologs of the Bacillus subtilis PhoP response regulator in the group A streptococcus (GAS) genome revealed three good candidates. Inactivation of one of these, recently identified as csrR (J. C. Levin and M. R. Wessels, Mol. Microbiol. 30:209-219, 1998), caused the strain to produce mucoid colonies and to increase transcription of hasA, the first gene in the operon for capsule synthesis. We report here that a nonpolar insertion in this gene also increased transcription of ska (encoding streptokinase), sagA (streptolysin S), and speMF (mitogenic factor) but did not affect transcription of slo (streptolysin O), mga (multiple gene regulator of GAS), emm (M protein), scpA (complement C5a peptidase), or speB or speC (pyrogenic exotoxins B and C). The amounts of streptokinase, streptolysin S, and capsule paralleled the levels of transcription of their genes in all cases. Because CsrR represses genes unrelated to those for capsule synthesis, and because CsrA-CsrB is a global regulatory system in Escherichia coli whose mechanism is unrelated to that of these genes in GAS, the locus has been renamed covR, for "control of virulence genes" in GAS. Transcription of the covR operon was also increased in the nonpolar insertion mutant, indicating that CovR represses its own synthesis as well. All phenotypes of the covR nonpolar insertion mutant were complemented by the covR gene on a plasmid. CovR acts on operons expressed both in exponential and in stationary phase, demonstrating that the CovR-CovS pathway is separate from growth phase-dependent regulation in GAS. Therefore, CovR is the first multiple-gene repressor of virulence factors described for this important human pathogen.

Adhesins, Bacterial↗

PubMed1999

Regulation of mga transcription in the group A streptococcus: specific binding of mga within its own promoter and evidence for a negative regulator.

Transcription of mga, encoding the multiple virulence gene regulator of the group A streptococcus, is positively autoregulated. This regulation requires a DNA region (Pmga) that contains both a promoter proximal to mga (P2) and a promoter located further upstream (P1). To determine if Mga has a direct role in this process, its ability to bind to specific sequences within Pmga was tested. A purified fusion of Mga to the C-terminal end of maltose-binding protein (MBP-Mga), encoded by malE-mga, was shown previously to bind to the promoter regions of Mga-regulated genes, including scpA and emm. We report here that MBP-Mga can function in vivo to regulate emm and mga. Electrophoretic mobility shift assays and DNase I footprinting were used to demonstrate specific binding of MBP-Mga to two ca. 59-bp binding sites in Pmga centered around bases -108 and -180 from the major P2 start of transcription. Mga binding sites from Pemm and PscpA were shown to compete for binding at the two Pmga sites, suggesting that the same domain of Mga interacts at all of these promoter targets. Deletion of the distal Pmga binding site (site I) in vivo resulted in loss of Mga-dependent transcription from the P2 start. However, the same lesion resulted in an increase in P1 transcription that was independent of Mga. This suggests the existence of a repressor of mga transcription with a binding site overlapping those of Mga.

Antigens, Bacterial↗

PubMed1999

The frequency of conjugative transposition of Tn916 is not determined by the frequency of excision.

Excision and formation of a covalently closed circular transposon molecule are required for conjugative transposition of Tn916 but are not the only factors that limit the frequency of conjugative transposition from one host to another. We found that in gram-positive bacteria, an increase in the frequency of excision and circularization of Tn916 caused by expression of integrase (Int) and excisionase (Xis) from a xylose-inducible promoter does not lead to an increase in the frequency of conjugative transposition. We also found that the concentration of Int and Xis in the recipient cell does not limit the frequency of conjugative transposition and that increased excision does not result in increased expression of transfer functions required to mobilize a plasmid containing the Tn916 origin of transfer. We conclude that in gram-positive hosts in which the Tn916 functions Int and Xis are overexpressed, the frequency of conjugative transposition is limited by the availability of transfer functions.

Bacillus subtilis↗

PubMed1999

The level of expression of the minor pilin subunit, CooD, determines the number of CS1 pili assembled on the cell surface of Escherichia coli.

CooD, the minor subunit of CS1 pili of enterotoxigenic Escherichia coli, is essential for the assembly of stable, functional pili. We previously proposed that CooD is a rate-limiting initiator of CS1 pilus assembly and predicted that the level of CooD expression should therefore determine the number of CS1 pili assembled on the cell surface. In this study, we confirm that CooD is required for the initiation of pilus assembly rather than for the stabilization of pili after they are assembled by demonstrating that specific modulation of cooD expression also modulates the number of CS1 pili on bacterial cells.

Bacterial Proteins↗