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J R Scott

Publications and source records attributed to J R Scott.

At least 55 records · Page 3Linked to original sources
PubMed1999

Binding site recognition by Rns, a virulence regulator in the AraC family.

The expression of CS1 pili by enterotoxigenic strains of Escherichia coli is regulated at the transcriptional level and requires the virulence regulator Rns, a member of the AraC family of regulatory proteins. Rns binds at two separate sites upstream of Pcoo (the promoter of CS1 pilin genes), which were identified in vitro with an MBP::Rns fusion protein in gel mobility and DNase I footprinting assays. At each site, Rns recognizes asymmetric nucleotide sequences in two regions of the major groove and binds along one face of the DNA helix. Both binding sites are required for activation of Pcoo in vivo, because mutagenesis of either site significantly reduced the level of expression from this promoter. Thus, Rns regulates the expression of CS1 pilin genes directly, not via a regulatory cascade. Analysis of Rns-nucleotide interactions at each site suggests that binding sites for Rns and related virulence regulators are not easily identified because they do not bind palindromic or repeated sequences. A strategy to identify asymmetric binding sites is presented and applied to locate potential binding sites upstream of other genes that Rns can activate, including those encoding the CS2 and CFA/I pili of enterotoxigenic E. coli and the global regulator virB of Shigella flexneri.

AraC Transcription Factor↗
PubMed1998

Transmission disequilibrium of maternally-inherited CTLA-4 microsatellite alleles in idiopathic recurrent miscarriage.

To elucidate the mechanisms that facilitate tolerance at the maternal-fetal interface, we are investigating the role of genes that are involved in peripheral self-tolerance in couples with idiopathic recurrent miscarriage. CTLA-4 is a negative regulator of T-cell proliferation and has been associated with human autoimmune disease. An AT(n) polymorphism in the 3'-untranslated region (UTR) of the human gene results in AT stretches that vary in length from 16 to 46 bp. We hypothesized that long stretches of AT repeats would result in mRNA instability, and reduced fetal survival in humans. We examined the transmission of AT(n) alleles in 60 couples with a history of > or = 3 unexplained spontaneous abortions to their 51liveborn children and 10 abortuses. The shorter allele was transmitted from heterozygous mothers to 26 of 35 liveborn children (chi2 = 8.3, P = 0.0040) and to three of nine aborted fetuses (chi2 = 1.0, P = 0.317). The shorter allele was transmitted from heterozygous fathers to 15 of 32 liveborn children (chi2 =0.12, P=0.726) and to five of eight aborted fetuses (chi2 = 0.5, P = 0.480). Furthermore, liveborn fetuses who inherited smaller alleles were more likely to represent the first successful pregnancy than liveborn fetuses who inherited larger maternal alleles (Pexact = 0.044) and fetuses of first pregnancies that inherited the smaller allele were significantly more likely to survive to term (Pexact = 0.0086). The preferential transmission of maternally-inherited shorter alleles to liveborn children, but random transmission of paternally-inherited alleles, suggests that CTLA-4 may be imprinted in humans and that this gene may play a role in inducing or maintaining tolerance at the maternal-fetal interface.

Abatacept↗
PubMed1998

Importance of matrix:analyte ratio for buffer tolerance using 2,5-dihydroxybenzoic acid as a matrix in matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry and matrix-assisted laser desorption/ionization-time of flight.

Many biological samples destined for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) contain buffers. The presence of these buffers often inhibits the ability to obtain spectra. Here, the results of a study of the effects of six different buffers on spectra of three representative small proteins are reported utilizing 2,5-dihydroxybenzoic acid as matrix. These proteins, bovine insulin, cytochrome c, and bovine albumin have masses from approximately 5000 to 66,000 Da. Three different sample preparation techniques were investigated: aerospray, dried-drop, and acetone redeposition. Both MALDI Fourier transform and time-of-flight mass spectrometry results show that buffer tolerance of MALDI-MS samples depends upon several factors, including the relative amount of the buffer in the MALDI matrix, as well as the identity of the specific buffer. Furthermore, the rate at which buffer tolerance decreases as buffer concentration is increased varies from buffer to buffer. The current results reveal that, at very high matrix:analyte ratios, buffer tolerance of MALDI is dramatically greater than concluded in previous literature reports.

Animals↗
PubMed1998

New tools in an old trade: CS1 pilus morphogenesis.

CS1 pili serve as the prototype for a large class of serologically distinct pili associated with enterotoxigenic Escherichia coli that cause diarrhoea in humans. The four genes essential for CS1 pilus morphogenesis, cooB, A, C and D, are arranged in an operon and encode structural and assembly proteins unlike those of other pilus systems commonly associated with Gram-negative bacteria. CS1 pili are composed primarily of the major pilin subunit, CooA, which determines the serological type of the pilus. The major pilin subunit is assembled into pili by the proteins CooB, CooC and CooD. CooD is both a minor component found at the pilus tip and an essential assembly protein, whereas CooC is an outer membrane protein thought to be involved in pilin transport. CooB is a novel periplasmic chaperone-like protein that forms intermolecular complexes with and stabilizes the major and minor pilins. Unlike other pilin chaperones, CooB also stabilizes the outer membrane component of the assembly system, CooC. The proteins of CS1 pili have no significant homology to those of the well-characterized Pap (pyelonephritis-associated) pili and related systems, although most of the features of pilus morphogenesis are similar. Therefore, these appear to be among the rare cases of convergent evolution. Thus, for CS1 pili, enterotoxigenic E. coli use new protein 'tools' in the old 'trade' of forming functional pili.

Bacterial Proteins↗
PubMed1998

Use of the lactococcal nisA promoter to regulate gene expression in gram-positive bacteria: comparison of induction level and promoter strength.

We characterized the regulated activity of the lactococcal nisA promoter in strains of the gram-positive species Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus faecalis, and Bacillus subtilis. nisA promoter activity was dependent on the proteins NisR and NisK, which constitute a two-component signal transduction system that responds to the extracellular inducer nisin. The nisin sensitivity and inducer concentration required for maximal induction varied among the strains. Significant induction of the nisA promoter (10- to 60-fold induction) was obtained in all of the species studied at a nisin concentration just below the concentration at which growth is inhibited. The efficiency of the nisA promoter was compared to the efficiencies of the Spac, xylA, and lacA promoters in B. subtilis and in S. pyogenes. Because nisA promoter-driven expression is regulated in many gram-positive bacteria, we expect it to be useful for genetic studies, especially studies with pathogenic streptococci in which no other regulated promoters have been described.

Bacillus subtilis↗
PubMed1997

CooB plays a chaperone-like role for the proteins involved in formation of CS1 pili of enterotoxigenic Escherichia coli.

CS1 pili serve as the prototype of a class of filamentous appendages found on the surface of strains of enterotoxigenic Escherichia coli. The four genes needed to synthesize functional CS1 pili in E. coli K12 are: cooA, which encodes the major pilin protein; cooD, which encodes a minor pilin protein found at the tip of the structure; cooC, which encodes a protein found in the outer membrane of piliated bacteria; and cooB. We show here that CooB, which is required for pilus assembly but is not part of the final structure, stabilizes CooA, CooC, and CooD. We previously reported that CooB is complexed with CooA in the periplasm and show here that CooB also is found complexed with CooD in the periplasm. CooB is associated with the membrane fraction only in the presence of CooC, suggesting that these two proteins also interact. This suggests that although it has no homology to known chaperone proteins, CooB serves a chaperone-like role for assembly of CS1.

Bacterial Proteins↗
PubMed1997

Excision of a conjugative transposon in vitro by the Int and Xis proteins of Tn916.

The roles of purified Int and Xis proteins of the conjugative transposon Tn 916 in excision of a deletion derivative of the closely related element Tn 1545 were investigated. At a low salt concentration (37.5 mM NaCl), Int alone was able to promote limited excision to produce a covalently closed circular form of the transposon, showing that Tn 916 Int can catalyze both DNA cleavage and strand exchange. This reaction was stimulated by Xis. At higher salt concentrations (150 mM NaCl), excision by Int alone was reduced to barely detectable levels and Xis was required for excision. The low salt, Xis-stimulated reaction was approximately 8-fold more efficient than the high salt, Xis-dependent reaction. These results reflect in vivo requirements for Int and Xis in excision.

Amino Acid Sequence↗
PubMed1997

Use of Tn917 to generate insertion mutations in the group A streptococcus.

The Enterococcus faecalis transposon Tn917 is functional in a broad range of bacteria, including both Gram-positive and Gram-negative species. We cloned Tn917-LTV3, a derivative carrying a promoterless lacZ (beta-galactosidase gene), into the thermosensitive shuttle replicon pG+host4 and assayed for chromosomal insertions in group A streptococcus (GAS). Tn917 transposed into the GAS chromosome at a frequency of (2.8 +/- 3.2) x 10(-5) per colony forming unit (cfu). Transposition products were predominantly simple insertions and no target site preference was detectable. Some transcriptional fusions were identified in which the promoterless lacZ of the transposon appeared to be expressed from an external promoter.

Chromosomes, Bacterial↗
PubMed1997

Subtotal hysterectomy in modern gynecology: a decision analysis.

OBJECTIVE: Our purpose was to compare the risks and benefits of subtotal (supracervical) hysterectomy with those of total hysterectomy in women at low risk for cervical cancer. STUDY DESIGN: A decision analysis was performed. Baseline probabilities for operative and postoperative morbidity, mortality, and long-term quality of life were established for subtotal and total hysterectomy. RESULTS: Operative complication rates and ranges for total abdominal hysterectomy were infection 3.0% (3.0% to 20.0%), hemorrhage 2.0% (2.0% to 15.4%), and adjacent organ injury 1.0% (0.7% to 2.0%). Those for subtotal hysterectomy were infection 1.4% (1.0% to 5.0%), hemorrhage 2.0% (0.7% to 4.0%), and adjacent organ injury 0.7% (0.6% to 1.0%). Operative mortality, the risk for development of cervicovaginal cancer, and long-term adverse effects on sexual or vesicourethral function were low in both groups. CONCLUSIONS: Recently proposed benefits from subtotal hysterectomy are not well proven. Total hysterectomy remains the procedure of choice for most women.

Decision Support Techniques↗
PubMed1997

Variable effects on murine pregnancy of immunoglobulin G fractions from women with antiphospholipid antibodies.

OBJECTIVE: Initial studies showed that passive immunization with human immunoglobulin G fractions containing antiphospholipid antibodies can result in murine fetal loss. We intended to use the murine model to study mechanisms of fetal loss associated with antiphospholipid antibodies. However, we have since found variable effects of antiphospholipid antibodies on murine pregnancy. The objective of this study was to determine the consistency of murine pregnancy loss from antiphospholipid antibody containing immunoglobulin G fraction. STUDY DESIGN: Pregnant C3H/HeN (mated with C57B1/6 males) and BALB/c (mated with BALB/c males) mice were passively immunized with antiphospholipid antibody containing human immunoglobulin G fraction from 20 women with antiphospholipid syndrome. The mice received either a single dose of 10 to 30 mg on day 12 of pregnancy or 10 mg per day on days 12 to 14 of gestation. Some mice receiving each dose of immunoglobulin G fraction were bled to confirm serum levels of anticardiolipin antibodies. Mice were killed on day 15 and the fetal status was determined. RESULTS: Overall, passive immunization with individual antiphospholipid antibody containing immunoglobulin G fractions resulted in 801 live pups (75%), 232 fetal deaths (22%), and 38 resorptions (3%) in 131 mice. The effect of immunoglobulin G fractions from individual patients was highly variable. Immunoglobulin G fraction from eight women resulted in high rates of fetal loss. However, in spite of high levels of anticardiolipin antibodies, fetal outcome was normal in mice immunized with immunoglobulin G fraction from the majority of women. The rate of fetal death did not uniformly increase with increasing doses of immunoglobulin G fraction and was unrelated to the donor's medical history. Fetal outcome was similar for both C3H/HeN and BALB/c mice. CONCLUSIONS: Human antiphospholipid antibodies have variable effects on murine pregnancy outcome. Characterization of antiphospholipid antibodies that do and do not cause murine fetal loss may provide insight into epitopes relevant to fetal loss associated with antiphospholipid syndrome.

Adult↗
PubMed1997

Pathogenic antibodies in women with obstetric features of antiphospholipid syndrome who have negative test results for lupus anticoagulant and anticardiolipin antibodies.

OBJECTIVE: Our goal was to determine whether women with clinical features of antiphospholipid syndrome but negative test results for lupus anticoagulant and anticardiolipin antibodies have pathogenic antibodies not identified by currently used methods. STUDY DESIGN: Sera were obtained from women with clinical features associated with antiphospholipid antibodies who had negative test results for lupus anticoagulant and anticardiolipin antibodies (antiphospholipid syndrome-like). We studied (1) the effect of passive immunization with their purified immunoglobulin G fraction on murine pregnancy (n = 35) and (2) the presence of antiphospholipid antibodies other than lupus anticoagulant or anticardiolipin antibodies (n = 39). Sera were also retested for anticardiolipin antibodies and lupus anticoagulant. RESULTS: Fetal loss occurred in 235 of 1088 (22%) pups in 137 mice immunized with immunoglobulin G fraction from antiphospholipid syndrome-like women compared with 23 of 402 (6%) pups in 53 control mice. Immunoglobulin G from 11 study patients resulted in the loss of at least one third of the exposed pups. Five women had positive levels of antiphosphatidylserine antibodies (>99th percentile). All levels were low positive, and three women also had low-positive levels of anticardiolipin antibodies on repeat testing. Five of the 11 (45%) women whose immunoglobulin G fractions caused at least 33% fetal loss also had positive test results for antiphospholipid antibodies. CONCLUSIONS: A subset of women with clinical disorders suspicious for antiphospholipid syndrome but who had negative test results for lupus anticoagulant and anticardiolipin antibodies by current methods have serum immunoglobulin G that is pathogenic to murine pregnancy. Testing for pathogenic immunoglobulin G may provide additional means to identify women with an as yet uncharacterized immune condition. The clinical relevance of low levels of antiphospholipid antibodies in these women remains unproved.

Abortion, Habitual↗
PubMed1997

Autoantibody screening for infertility: explaining the unexplained?

Several laboratories currently offer panels of serum autoantibody assays to screen women with unexplained infertility and those undergoing in vitro fertilization (IVF). Offering these tests implies that they have predictive value for the outcome of proposed infertility treatments such that the results of the testing would alter clinical management. Because screening for antiphospholipid antibodies adds expense to already costly procedures, it is an appropriate time to review the justification for the use of these panels.

Abortion, Habitual↗
PubMed1997

Intravenous immune globulin in the management of severe Rh D hemolytic disease.

Although intrauterine fetal transfusion has improved dramatically perinatal outcome in Rh D alloimmunization, in some cases the fetus is affected before transfusion is possible. Immune globulin (IVIG) administration is being increasingly used to successfully treat a variety of immune-mediated diseases, such as pregnancies affected by platelet alloimmunization. Although only a limited number of pregnancies have been reported, favorable outcomes with IVIG treatment of severe Rh disease have been described. We present a case report, review the published experience with IVIG treatment in severe, early-onset anti-D sensitization, and propose that IVIG may have an adjunctive role in the treatment of severe Rh isoimmunization.

Adult↗
PubMed1997

Role of putative virulence factors of Streptococcus pyogenes in mouse models of long-term throat colonization and pneumonia.

To investigate the role of putative virulence factors of Streptococcus pyogenes (group A streptococcus; GAS) in causing disease, we introduced specific mutations in GAS strain B514, a natural mouse pathogen, and tested the mutant strains in two models of infection. To study late stages of disease, we used our previously described mouse model (C3HeB/FeJ mice) in which pneumonia and systemic spread of the streptococcus follow intratracheal inoculation. To study the early stages of disease, we report here a model of long-term (at least 21 days) throat colonization following intranasal inoculation of C57BL/10SnJ mice. When the three emm family genes of GAS strain B514-Sm were deleted, the mutant showed no significant difference from the wild type in induction of long-term throat colonization or pneumonia. We inactivated the scpA gene, which encodes a complement C5a peptidase, by insertion of a nonreplicative plasmid and found no significant difference from the wild type in the incidence of throat colonization. However, there was a small but statistically significant decrease in the incidence of pneumonia caused by the scpA mutant. Finally, we demonstrated a very important effect of the hyaluronic acid capsule in both models. Following intranasal inoculation of mice with a mutant in which a nonreplicative plasmid was inserted into the hasA gene, which encodes hyaluronate synthase, we found that all bacteria recovered from the throats of the mice were encapsulated revertants. Following intratracheal inoculation with the hasA mutant, the incidence of pneumonia within 72 h was significantly reduced from that of the control strain (P = 0.006). These results indicate that the hyaluronic acid capsule of S. pyogenes B514 confers an important selective advantage for survival of the bacteria in the upper respiratory tract and is also an important determinant in induction of pneumonia in our model system.

Adhesins, Bacterial↗
PubMed1997

Role of mga in growth phase regulation of virulence genes of the group A streptococcus.

To determine whether growth phase affects the expression of mga and other virulence-associated genes in the group A streptococcus (GAS), total RNA was isolated from the serotype M6 GAS strain JRS4 at different phases of growth and transcript levels were quantitated by hybridization with radiolabeled DNA probes. Expression of mga (which encodes a multiple gene regulator) and the Mga-regulated genes emm (which encodes M protein) and scpA (which encodes a complement C5a peptidase) was found to be maximal in exponential phase and shut off as the bacteria entered stationary phase, while the housekeeping genes recA and rpsL showed constant transcript levels over the same period of growth. Expression of mga from a foreign phage promoter in a mga-deleted GAS strain (JRS519) altered the wild-type growth phase-dependent transcription profile seen for emm and scpA, as well as for mga. Therefore, the temporal control of mga expression requires its upstream promoter region, and the subsequent growth phase regulation of emm and scpA is Mga dependent. A number of putative virulence genes in JRS4 were shown not to require Mga for their expression, although several exhibited growth phase-dependent regulation that was similar to mga, i.e., slo (which encodes streptolysin O) and plr (encoding the plasmin receptor/glyceraldehyde-3-phosphate dehydrogenase). Still others showed a markedly different pattern of expression (the genes for the superantigen toxins MF and SpeC). These results suggest the existence of complex levels of global regulation sensitive to growth phase that directly control the expression of virulence genes and mga in GAS.

Adhesins, Bacterial↗