Biomedical subjects

A M Stevens

Publications and source records attributed to A M Stevens.

At least 37 records · Page 2Linked to original sources

PubMed1997

Quorum sensing in Vibrio fischeri: essential elements for activation of the luminescence genes.

LuxR is required for cell density-dependent activation of the Vibrio fischeri luminescence (lux) genes. It has not been possible to study full-length LuxR in vitro, but a polypeptide containing the C-terminal transcriptional-activator domain of LuxR (LuxRdeltaN) has been purified, and its binding to lux regulatory DNA has been investigated. By itself, LuxRdeltaN interacts with a region of lux regulatory DNA that is upstream of the lux box, which is a 20-bp element that is required for LuxR activation of the luminescence operon. Individually, neither the purified LuxRdeltaN nor RNA polymerase binds to the lux box region, but together the two proteins bind in synergy to the lux box-luxI promoter region. We show that binding of LuxRdeltaN to the upstream region is not a prerequisite for its synergistic binding with RNA polymerase to the lux box and the luxI promoter region. We also show that LuxRdeltaN and RNA polymerase are both required and sufficient for transcriptional activation of the lux operon. This argues against the hypothesis that LuxR functions to alleviate repression of the lux operon by another cellular factor. Rather, our data support the view that LuxR functions as an accessory factor that enables RNA polymerase to bind to and initiate transcription from the promoter of the lux operon.

Bacterial Proteins↗

PubMed1997

Adenocarcinoma of the cervix: a regional retrospective study.

A retrospective study using data from the Yorkshire Cancer Registry was undertaken to investigate the incidence, characteristics and prognosis of cases of cervical adenocarcinoma in the Yorkshire Region. Three hundred and twenty-six women resident in the Yorkshire Region formed the basis of the study. The overall five year survival for adenocarcinoma of the cervix is 53%. This was influenced by stage, lymph node involvement and differentiation. 37 out of 122 surgically treated patients received inappropriate treatment. There is a need for improved data collection in the treatment of these patients. This lends weight to the argument that cancer of the cervix should be managed in cancer units.

Adenocarcinoma↗

PubMed1996

Three-dimensional structure of human cytomegalovirus protease.

Herpesviruses encode a serine protease that specifically cleaves assembly protein. This protease is critical for replication, and represents a new target for antiviral drug design. Here we report the three-dimensional structure of the protease from human cytomegalovirus (hCMV) at 2.27 angstroms resolution. The structure reveals a unique fold and new catalytic strategy for cleavage. The monomer fold of the enzyme, a seven-stranded beta-barrel encircled by a chain of helices that form the carboxy terminus of the molecule, is unrelated to those observed in classic serine proteases such as chymotrypsin and subtilisin. The serine nucleophile at position 132 is activated by two juxtaposed histidine residues at positions 63 and 157. Dimerization, which seems to be necessary for activity, is observed in the crystals. Correlations of the structure with the sequences of herpesvirus proteases suggest that dimerization may confer specificity and recognition in substrate binding.

Binding Sites↗

PubMed1996

Hepatosplenic abscesses due to Brucella melitensis: report of a case involving a child and review of the literature.

Suppurative disease of the liver and/or spleen is a rare and serious complication of human brucellosis. In the English-language literature, only nine cases have been reported, all involving adults with chronic infection. We report the case of a young child in whom abscesses of the liver and spleen developed during acute brucellosis. Brucella melitensis was cultured from an aspirate of the liver and from the bone marrow. After percutaneous drainage of the liver abscess, the patient responded to a 56-day course of antimicrobial therapy. To our knowledge, this is the first reported case of hepatosplenic abscess due to a Brucella species in a child.

Abdominal Abscess↗

PubMed1996

[Illegal drug use by secondary school children in Belgium: north-south differences].

The study "Health Behaviour of Youngsters" regularly provides comparative data on health behaviour and related lifestyle of pupils from 11 to 18 years old. This study is based on repeated surveys among randomised representative samples of pupils, following the international standardised protocol from the WHO cross-national survey "Health Behaviour in School-aged Children". In Belgium, results from the 1990 survey (sample = 3528) show differences in illegal drugs use between the South (French-speaking) and the North (Flemish-speaking). For example, among the 17 year olds, 9.8% of the French-speaking pupils and 5.5% of the Flemish-speaking have already tried illegal drug(s) and in the same age groups, 2.9% and 1.5%, respectively declare to be regular users. The first hypotheses concerning those differences take into account the socio-economic and cultural environment as well as methodological problems inherent to these surveys.

Adolescent↗

PubMed1995

Using sampling techniques in protein crystallization.

The crystallization of homogeneous or highly purified macromolecules depends on many variables such as precipitant, pH, choice of buffer, protein concentration, temperature, the participation of different mono- and divalent ions, as well as the presence of minute amounts of detergent and organic molecules. Finding the best combination among these many parameters is a multi-variable optimization problem. This kind of problem can be treated mathematically by sampling techniques. We have used this technique for protein crystallization. The iterative procedure starts with random sampling, followed by quantitative evaluation and cycles with weighted sampling. A simple procedure, derived from this concept and called MON48, has been successfully applied to many protein crystallization problems.

Journal Article↗

PubMed1995

Conjugative transposons: an unusual and diverse set of integrated gene transfer elements.

Conjugative transposons are integrated DNA elements that excise themselves to form a covalently closed circular intermediate. This circular intermediate can either reintegrate in the same cell (intracellular transposition) or transfer by conjugation to a recipient and integrate into the recipient's genome (intercellular transposition). Conjugative transposons were first found in gram-positive cocci but are now known to be present in a variety of gram-positive and gram-negative bacteria also. Conjugative transposons have a surprisingly broad host range, and they probably contribute as much as plasmids to the spread of antibiotic resistance genes in some genera of disease-causing bacteria. Resistance genes need not be carried on the conjugative transposon to be transferred. Many conjugative transposons can mobilize coresident plasmids, and the Bacteroides conjugative transposons can even excise and mobilize unlinked integrated elements. The Bacteroides conjugative transposons are also unusual in that their transfer activities are regulated by tetracycline via a complex regulatory network.

Anti-Bacterial Agents↗

PubMed1995

Biphasic transcriptional regulation of the interferon regulatory factor-1 gene by prolactin: involvement of gamma-interferon-activated sequence and Stat-related proteins.

Stimulation of quiescent Nb2 T cells by PRL leads to the rapid transcriptional activation of a T cell activation gene, interferon regulatory factor-1 (IRF-1). IRF-1 is induced twice by PRL in a single cell cycle, first during G1 at 30-60 min and again over early S phase at 10-12 h. By nuclear run-on transcription analysis of IRF-1 promoter-chloramphenicol acetyl transferase (CAT) constructs, the -1.7 kilobase (kb) 5'-flanking IRF-1 DNA was shown to contain elements that mediate both G1 and S phase expression. The -200 bp IRF-1 promoter DNA contains elements that respond to G1 PRL stimulation in a protein synthesis independent manner, suggesting the involvement of pre-existing factors. Further promoter deletion analysis delineated a minimal PRL responsive region between -112 and -205 bp. Within this region is a Gamma Interferon Activated Sequence or GAS, consisting of two inverted GAAA motifs (-123/-113), which confers PRL-inducible expression to a reporter gene, suggesting that GAS can function as a PRL responsive element. Further, GAS exhibits binding with nuclear proteins in a PRL-inducible, cell cycle-dependent manner. One of these proteins appears to be related to the emerging family of Signal Transducer and Activator of Transcription or Stat factors. These studies suggest that the GAS site and Stat-like proteins participate in PRL receptor signal transduction to regulate the biphasic expression of the IRF-1 gene in PRL-stimulated T cells.

Animals↗

PubMed1994

Synergistic binding of the Vibrio fischeri LuxR transcriptional activator domain and RNA polymerase to the lux promoter region.

LuxR, the Vibrio fischeri luminescence gene (lux) activator, is the best-studied member of a family of bacterial transcription factors required for cell density-dependent expression of specific genes involved in associations with eukaryotic hosts. Neither LuxR nor any other LuxR homolog has been shown to bind DNA directly. We have purified the LuxR C-terminal transcriptional activator domain from extracts of recombinant Escherichia coli in which this polypeptide was expressed. The purified polypeptide by itself binds to lux regulatory DNA upstream of the lux box, a 20-bp palindrome that is required for LuxR activity in vivo, but it does not bind to the lux box. However, the LuxR C-terminal domain together with RNA polymerase protects a region including the lux box and the lux operon promoter from DNase I cleavage. There is very little protection of the lux operon promoter region from DNase I digestion in the presence of RNA polymerase alone. Apparently, there is a synergistic binding of the LuxR C-terminal domain and RNA polymerase to the promoter region. The upstream binding region for the purified polypeptide encompasses a binding site for cAMP receptor protein (CRP). Under some conditions, CRP binding can block the binding of the LuxR C-terminal domain to the upstream binding region, and it can also block the synergistic binding of the LuxR C-terminal domain and RNA polymerase to the lux box and luminescence gene promoter region. This description of DNA binding by the LuxR C-terminal domain should lead to an understanding of the molecular interactions of the LuxR family of transcriptional activators with regulatory DNA.

Bacterial Proteins↗

PubMed1994

Renin inhibitor SC-51106 complexed with human renin: discovery of a new binding site adjacent to P3.

SC-51106, a 'minimal-size' diol-based renin inhibitor lacking a P4 residue, has been co-crystallized with human renin and the structure of the complex determined by X-ray crystallography. This study defines the mode of binding of this important class of renin inhibitor, and in conjunction with molecular modeling, has led to the discovery of a new binding site adjacent to S3, which is termed the 'S3aux(iliary)' subsite.

Amino Acid Sequence↗

PubMed1994

Multiple prolactin-responsive elements mediate G1 and S phase expression of the interferon regulatory factor-1 gene.

The interferon regulatory factor-1 (IRF-1) gene is both an immediate-early G1 phase gene and an S phase gene inducible by PRL in rat Nb2 T lymphocytes. To understand the mechanism by which PRL regulates the biphasic expression of IRF-1, we cloned the rat IRF-1 gene and functionally characterized the IRF-1 promoter. Upon transfection into Nb2 T cells, 1.7 kilobases (kb) of IRF-1 5'-flanking DNA linked to a chloramphenicol acetyl transferase (CAT) reporter gene mediated a 30-fold induction of CAT enzyme activity in response to 24 h of PRL stimulation. Deletion mutants containing 1.3, 0.6, and 0.2 kb 5'-flanking DNA were incrementally less transcriptionally active, although 0.2 kb still mediated a 12-fold induction by PRL. The sequence between -1.7 and -0.2 kb linked to a heterologous thymidine kinase promoter failed to respond to PRL stimulation, suggesting that the activity of upstream PRL response elements may require an interaction with promoter-proximal elements. By assaying CAT enzyme activity across a 24-h PRL induction time course, we were able to assign G1 vs. S phase PRL responses of the IRF-1 gene to different regions of the IRF-1 5'-flanking and promoter DNA. The 0.2-kb IRF-CAT construct was induced by PRL stimulation during the G1 phase of the cell cycle. In contrast, the 1.7-kb IRF-CAT construct was inducible by PRL during both G1 and S phase of the cell cycle. Hence, the PRL-induced biphasic expression of the IRF-1 gene appears to be controlled by separate PRL-responsive elements: elements in the first 0.2 kb of the IRF-1 promoter region act during early activation, and elements between 0.2 and 1.7 kb act in concert with the proximal 0.2-kb region during S phase progression.

Animals↗

PubMed1993

Ophthalmic manifestations of infections with Cryptococcus neoformans in patients with the acquired immunodeficiency syndrome.

The present study was undertaken to determine the nature and the prevalence of ophthalmic manifestations of infections with Cryptococcus neoformans in human immunodeficiency virus seropositive patients and to analyze whether the presence or absence of ocular signs is associated with improved survival. Eighty human immunodeficiency virus seropositive patients with cryptococcal infection were enrolled. We observed papilledema in 26 of the 80 patients (32.5%). Visual loss and abducens nerve palsy occurred in seven patients (9%). Only two patients (2.5%) had optic atrophy. Visual loss caused by optic nerve involvement was less frequent among the 62 patients treated with oral conazoles exclusively than among the 18 patients who had received amphotericin B or a combination of amphotericin B and conazoles. Actual invasion of the intraocular structures with Cryptococcus neoformans was an uncommon complication in our series. In addition to the ocular manifestations attributable to cryptococcal disease, human immunodeficiency virus-related retinopathy was present in nearly half of the patients. Cytomegalovirus retinitis was diagnosed in four patients (5%). The 26 patients (32.5%) with papilledema had a median survival of 182 days vs 160 days for the patients without papilledema. The median survival for 18 patients (22.5%) with cotton-wool spots was 102 days vs 186 days for those without cotton-wool spots. The differences between these subgroups were not statistically significant.

AIDS-Related Opportunistic Infections↗

PubMed1993

Mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1) and its determinants: a cohort study in Kigali, Rwanda.

The authors report the results of the first 2 years of follow-up of a prospective cohort study on the mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1) and its determinants which started in November 1988 in Kigali, Rwanda. The study sample consists of 218 newborns of 215 HIV-1 seropositive women matched to 218 newborns of 216 HIV-1 seronegative women of the same age and parity. They were followed every 2 weeks during the first 2 years of follow-up. HIV-1 antibodies were detected by enzyme-linked immunoadsorbent assay and Western blot at 3-month intervals. Two methods of calculating the mother-to-child transmission rate were used: method 1 combines the information provided by the persistence of HIV-1 antibodies at 15 months of age in children born to HIV seropositive mothers and the excess mortality in this group compared with the cohort of children born to HIV seronegative mothers; method 2 is a case-by-case evaluation of all the children born to HIV seropositive mothers. A logistic regression model was used to study the determinants of transmission. The probability of survival at 24 months of age was 81% (95% confidence interval (CI) 75-86) in children born to seropositive mothers, compared with 95% (95% CI 92-98) in children born to seronegative mothers (p < 0.001). The mother-to-child transmission rate calculated with method 1 was 25.7% (95% CI 18.8-32.5). With method 2, the medium estimate was 24.7%. In the multivariate analysis, a CD4/CD8 ratio < 0.5 was the only maternal factor statistically associated with an increased risk of mother-to-child transmission of HIV-1 (odds ratio = 2.9, 95% CI 1.2-7.2). The authors' findings present evidence for a higher mother-to-child transmission rate of HIV-1 in children born in Rwanda than in industrialized countries.

Acquired Immunodeficiency Syndrome↗

PubMed1993

Tetracycline regulation of genes on Bacteroides conjugative transposons.

Human colonic Bacteroides species harbor a family of large conjugative transposons, called tetracycline resistance (Tcr) elements. Activities of these elements are enhanced by pregrowth of bacteria in medium containing tetracycline, indicating that at least some Tcr element genes are regulated by tetracycline. Previously, we identified a central regulatory locus on the Tcr elements that contained two genes, rteA and rteB, which appeared to encode a two-component regulatory system (A. M. Stevens, J. M. Sanders, N. B. Shoemaker, and A. A. Salyers, J. Bacteriol. 174:2935-2942, 1992). In the present study, we describe a gene which is located downstream of rteB in a separate transcriptional unit and which requires RteB for expression. Sequence analysis of this gene showed that it encoded a 217-amino-acid protein, which had no significant sequence similarity to any proteins in the GenBank or EMBL data base. An insertional disruption in the gene abolished self-transfer of the Tcr element to Bacteroides recipients, indicating that the gene was essential for self-transfer. The disruption also affected mobilization of coresident plasmids. Mobilization frequency was reduced 100- to 1,000-fold if the recipient was Escherichia coli but was not affected to the same extent if the recipient was an isogenic Bacteroides strain. The complex phenotype of the disruption mutant suggested that the newly identified gene, like rteA and rteB, had a regulatory function. Accordingly, it has been designated rteC. Our results indicate that regulation of Tc(r) element functions is unexpectedly complex and may involve a cascade of regulators, with RteA and RteB exerting central control over secondary regulators like RteC, which in turn control subsets of Tcr element structural genes.

Bacteroides↗

PubMed1993

Excision, transfer, and integration of NBU1, a mobilizable site-selective insertion element.

The Bacteroides species harbor a family of conjugative transposons called tetracycline resistance elements (Tcr elements) that transfer themselves from the chromosome of a donor to the chromosome of a recipient, mobilize coresident plasmids, and also mediate the excision and circularization of members of a family of 10- to 12-kbp insertion elements which share a small region of DNA homology and are called NBUs (for nonreplicating Bacteroides units). The NBUs are sometimes cotransferred with Tcr elements, and it was postulated previously that the excised circular forms of the NBUs were plasmidlike forms and were transferred like plasmids and then integrated into the recipient chromosome. We used chimeric plasmids containing one of the NBUs, NBU1, and a Bacteroides-Escherichia coli shuttle vector to show that this hypothesis is probably correct. NBU1 contained a region that allowed mobilization by both the Tcr elements and IncP plasmids, and we used these conjugal elements to allow us to estimate the frequencies of excision, mobilization, and integration of NBU1 in Bacteroides hosts to be approximately 10(-2), 10(-5) to 10(-4), and 10(-2), respectively. Although functions on the Tcr elements were required for the excision-circularization and mobilization of NBU1, no Tcr element functions were required for integration into the recipient chromosome. Analysis of the DNA sequences at the integration region of the circular form of NBU1, the primary insertion site in the Bacteroides thetaiotaomicron 5482 chromosome, and the resultant NBU1-chromosome junctions showed that NBU1 appeared to integrate into the primary insertion site by recombining within an identical 14-bp sequence present on both NBU1 and the target, thus leaving a copy of the 14-bp sequence at both junctions. The apparent integration mechanism and the target selection of NBU1 were different from those of both XBU4422, the only member of the conjugal Tcr elements for which these sequences are known, and Tn4399, a mobilizable Bacteroides transposon. The NBUs appear to be a distinct type of mobilizable insertion element.

Bacteroides↗

PubMed1992

Safety and immunogenicity of high-dose Edmonston-Zagreb measles vaccine in children with HIV-1 infection. A cohort study in Kigali, Rwanda.

OBJECTIVE: To compare the reactogenicity and immunogenicity of high-dose Edmonston-Zagreb (EZ) measles vaccine in children with and without human immunodeficiency virus, type 1 (HIV-1), infection. DESIGN: Prospective cohort study. SETTING: General pediatric clinic and home visits in Kigali, the capital of Rwanda. PARTICIPANTS: Infants born to HIV-1-seropositive and -seronegative mothers were vaccinated with a 10(5.0) 50% tissue culture infective dose of EZ measles vaccine at 6 months of age. Control visits were made 10 and 14 days later to monitor local and general reactions. Measles serum antibodies were measured by an enzyme-linked immunosorbent assay technique at birth and at 6 and 9 months of age. Three groups were compared: infected children (n = 43), uninfected children born to seropositive mothers (n = 135), and uninfected children born to seronegative mothers (n = 194). RESULTS: Three hundred twenty-three children (86.8%) were available for the reactogenicity study. No statistically significant difference between the three groups was found in the occurrence of minor adverse reactions. No severe adverse reaction was observed. One hundred ninety children (51.1%) were available for the immunogenicity study. The percentage of infants negative for measles antibody at 6 months was significantly higher (P = .021) in HIV-infected children (85%) and in uninfected children born to seropositive mothers (90%) than in uninfected children born to seronegative mothers (75%). The overall seroconversion rate at 9 months was 90% (95% confidence interval, 85.7% to 94.3%), without any statistically significant difference between the three groups. CONCLUSION: High-dose EZ vaccine administered at 6 months of age is safe and highly immunogenic in both HIV-infected and uninfected children.

Antibodies, Viral↗

PubMed1992

Interferon regulatory factor-1 is inducible by prolactin, interleukin-2 and concanavalin A in T cells.

Interferon regulatory factor-1 (IRF-1) gene expression is rapidly upregulated in the prolactin (PRL)-activated Nb2 rat T lymphoma cell line. To further elucidate its role as a T cell activation molecule, IRF-1 gene expression in response to various T cell stimuli was examined. In Nb2 T cells, PRL induced two peaks of IRF-1 gene expression: a rapid, transient peak at 1 h and a sustained peak at 12 h. PRL subsequently induced interferon-gamma (IFN-gamma) gene expression at 3-6 h. However, the early induction of IRF-1 and IFN-gamma does not appear to be interdependent. Interleukin-2 (IL-2) also induced IRF-1 gene expression in Nb2 T cells but only one broad peak at 10 h was observed. In primary mouse splenocytes, concanavalin A induced rapid and transient expression of the IRF-1 gene; maximal expression occurred by 6 h, and then returned to basal levels by 12-15 h. These results provide additional evidence for the importance of IRF-1 in T cell activation.

Animals↗

PubMed1992

Genes involved in production of plasmidlike forms by a Bacteroides conjugal chromosomal element share amino acid homology with two-component regulatory systems.

Many human colonic Bacteroides strains carry large (greater than 70-kbp) self-transmissible chromosomal tetracycline resistance (Tcr) elements. These Tcr elements can also mediate the excision and circularization of discrete nonadjacent segments of chromosomal DNA which are designated NBUs (nonreplicating Bacteroides units). We have localized a 6.5-kbp segment of Tcr element DNA that mediates NBU excision and circularization. Analysis of the DNA sequence of this region indicated that it contained three open reading frames, all transcribed in the same direction. The first gene was the Tcr gene, tetQ. The second two open reading frames exhibited amino acid similarity to known two-component regulatory systems. Complementation and gene fusion data supported the hypothesis that the three genes were organized in an operon. Transcription from the tetQ promoter region was inducible by tetracycline, as might be expected from the previous finding that NBU excision was detectable only in cells preexposed to tetracycline. The 6.5-kbp region appeared to be essential not only for NBU excision but also for self-transfer of the elements, another activity that is enhanced by preexposure to tetracycline. Accordingly, the two genes downstream of tetQ have been designated rteA and rteB (regulation of Tcr elements).

Amino Acid Sequence↗