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P Lepage

Publications and source records attributed to P Lepage.

At least 19 recordsLinked to original sources
PubMed1996

The contribution of lysine-36 to catalysis by human myo-inositol monophosphatase.

The role of lysine residues in the catalytic mechanism of myo-inositol monophosphatase (EC 3.1.3.25) was investigated. The enzyme was completely inactivated by amidination with ethyl acetimidate or reductive methylation with formaldehyde and cyanoborohydride. Activity was retained when the active site was protected with Mg2+, Li+, and D,L-myo-inositol 1-phosphate. Using radiolabeling, peptide mapping, and sequence analysis, Lys-36 was shown to be the protected residue, which is responsible for inactivation. Replacing Lys-36 with glutamine produced a mutant protein, K36Q, with similar affinities for the substrate and the activator Mg2+, but a 50-fold lower turnover number as compared to the wild-type enzyme. Crystallographic studies did not indicate any gross structural changes in the mutant as compared to the native form. Initial velocity data were best described by a rapid equilibrium ordered mechanism with two Mg2+ binding before and a third one binding after the substrate. Inhibition by calcium was unaffected by the mutation, but inhibition by lithium was greatly reduced and became noncompetitive. The pH dependence of catalysis and the solvent isotope effect on kcat are altered in the mutant enzyme. D,L-myo-Inositol 1-phosphate, 4-nitrophenyl phosphate, and D-glucose 6-phosphate are cleaved at different rates by the wild-type enzyme, but with similar efficiency by K36Q. All data taken together are consistent with the hypothesis that modifying or replacing the lysine residue in position 36 decreases its polarizing effect on one of the catalytic metal ions and prevents the efficient deprotonation of the metal-bound water nucleophile.

Base Sequence↗
PubMed1995

Four years of natural history of HIV-1 infection in african women: a prospective cohort study in Kigali (Rwanda), 1988-1993.

Clinical features and mortality due to human immunodeficiency virus type-1 (HIV-1) infection in women are described as part of a prospective 4-year cohort study on perinatal transmission of HIV in Kigali, Rwanda. Two hundred fifteen HIV-seropositive (HIV+) and 216 HIV-seronegative (HIV-) pregnant women were enrolled at delivery between November 1988 and June 1989. Clinical information collected during systematic quarterly examinations was compared. HIV antibody tests were performed at delivery and CD4/CD8 lymphocyte counts at 15 days' postpartum. HIV--women who seroconverted during the follow-up period were excluded from the analysis of the comparison group starting at the date of seroconversion. At enrollment, all HIV+ women were asymptomatic for acquired immune deficiency syndrome (AIDS). Incidence of tuberculosis was 2.9 per 100 women-years (WY) after 4 years of follow-up in HIV+ women versus 0.2 per 100 WY among HIV- women (relative risk, 18.2; 95% confidence interval 2.4-137.0). Among HIV+ women, the incidence of AIDS (World Health Organization clinical AIDS definition) was 3.5 per 100 WY. The mortality rate was 4.4 per 100 WY among HIV+ women versus 0.5 per 100 WY among HIV- women. Clinical AIDS was present in only half of the fatalities. Tuberculosis was a major cause of morbidity and mortality in these HIV+ African women. An early diagnosis and an appropriate treatment or prevention of tuberculosis should improve the quality of life of HIV-infected patients in Africa.

Adult↗
PubMed1995

Activation of a MMTV/mdr3 fusion transcript from a cryptic viral promoter is stimulated by mdr-derived sequences located in intron I.

In P388/VCR-10 cells, resistance to cytotoxic drugs is caused by the overexpression of the mdr3 gene, in absence of gene amplification. The gene is transcriptionally activated following integration of a full-length mouse mammary tumor virus (MMTV) within intron I, upstream of the coding region of the gene. This integration results in the production of MMTV/mdr3 fusion transcripts that originate from the antisense 5'LTR of the provirus. The mechanism of mdr3 activation in these cells remains unclear since it cannot be accounted for either by activation from the normal MMTV promoter or by activation of the mdr3 promoter by MMTV enhancer sequences. Subcloning and sequence analysis of the genomic region encompassing the 5' LTR of the provirus with adjacent mdr3 sequences up to exon 2 showed that the LTR had not undergone small rearrangements or deletions. Transfections of fusion plasmids containing this genomic fragment and the reporter gene luciferase showed the presence of transcriptionally active sequences in that region. Deletions of 5' and 3' sequences from this fragment have shown that the antisense LTR itself has little contribution to the activation of the luciferase gene, whereas the mdr3 derived sequences that include part of intron I and the beginning of exon 2 strongly activated luciferase expression when inserted in either orientations upstream of the reporter gene. These results suggest the presence of an activator element within intron I of mdr3 capable of activating transcription from a cryptic start site present in the antisense MMTV LTR. Derepression of this activator sequence within intron I by a mechanism involving integration of a transposable element may be a prerequisite to transcriptional activation of the gene which is silent in the parental P388 cells. Further support for a derepression mechanism of activation in P388 cells is provided by the identification of independent genomic rearrangements in the 5' region of mdr3 in additional MDR P388 derivatives analyzed in this study.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Genomic structure, promoter sequence, and induction of expression of the mouse Nramp1 gene in macrophages.

A candidate gene for the mouse chromosome 1 host resistance locus Bcg/Ity/Lsh was recently cloned and designated Nramp (natural resistance-associated macrophage protein). Nramp is part of a small family of at least two genes, Nramp1 and Nramp2. Primer extension and cDNA cloning were used to isolate the complete 5' end of the Nramp1 mRNA. Analysis of genomic cosmid and bacteriophage clones overlapping the complete Nramp1 gene revealed that the gene was composed of 15 exons and spanned 11.5 kb of genomic DNA. Positioning of introns on the coding portion of the mRNA revealed a modular relationship between coding exons and predicted structural domains of the protein, with 8 of the 12 transmembrane (TM) domains encoded by individual exons. Northern blotting analysis indicated that Nramp1 expression was restricted to J774A.1 and RAW 264.7 macrophage lines and was dramatically increased by treatment with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). Primer extension and S1 nuclease mapping experiments were used to locate the transcription initiation site of Nramp1 and revealed the presence of one major and several minor initiation sites. Nucleotide sequencing of the corresponding region failed to detect classical TATA and CAAT elements, but identified two putative initiator sequences located near the major initiation site. Consensus sequences for binding of the macrophage and B-cell-specific transcription factor PU.1, as well as several LPS (NF-IL6) and IFN-gamma response elements, were also identified.

Amino Acid Sequence↗
PubMed1995

Rwanda.

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Homicide↗
PubMed1995

[Septic meningitis in children in Rwanda from 1983 to 1990. Retrospective study at the Kigali Hospital Center].

To assess septic meningitis in pediatric units in terms of the bacteriologic distribution, mortality, and groups at risk, we conducted a retrospective study in the pediatric department of the Kigali Hospital Center (Rwanda). Based on bacteriologic study of 1215 cerebrospinal fluid samples, there were 321 cases of septic meningitis due to identifiable germs and 68 involving cloudy fluid with no detectable germs, i.e. 1.5% of admissions to the Pediatric Unit of the Kigali Hospital Center. The most common organisms were pneumococcus (36.5%), Haemophilus influenzae (31%), salmonella (13%), and meningococcus (11.5%). Most of the children (75%) presenting septic meningitis were under the age of 5 years. Overall mortality was 38% with rates of 52% and 39% for cases involving pneumococcus and salmonella respectively. The predominant clinical symptoms of pneumococcus meningitis were coma (p:0.000055) and respiratory compromise (p:0.02). In contrast Haemophilus influenzae meningitis was associated with a lower incidence of coma (p:0.05) and malnutrition (p:0.017). Salmonella meningitis was characterized by a higher incidence of fever over 38.9 degrees C (p:0.025) and malnutrition (p:0.01). In patients with meningococcus meningitis, the incidence of convulsions appeared to be higher, at the threshold of statistical significance (p:0.052), whereas coma (p:0062) and respiratory distress (p:0.0024) were uncommon. Independently of etiology, no clinical symptom was associated with a statistically higher risk for death.

Adolescent↗
PubMed1994

P-glycoprotein homologues.

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ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Seroincidence of HIV-1 infection in African women of reproductive age: a prospective cohort study in Kigali, Rwanda, 1988-1992.

OBJECTIVE: To estimate the seroincidence of HIV-1 infection among women of reproductive age in Kigali, Rwanda. DESIGN: Fixed prospective cohort followed for 36 months between November 1988 and June 1992, as part of an ongoing study of mother-to-child transmission of HIV-1. SETTING: Centre Hospitalier, Kigali, Rwanda. SUBJECTS: A total of 216 HIV-seronegative women were enrolled at delivery between November 1988 and June 1989. METHODS: A blood sample was obtained at delivery to test for HIV antibodies (by enzyme-linked immunosorbent assay and Western blot). Serum was tested every 3 months during follow-up. Incidence density rates of HIV seroconversion were estimated. RESULTS: The follow-up rate after 3 years was 89%, assessed by the maximum person-years method. The seroincidence density rate was 3.5 per 100 women-years (95% confidence interval, 1.9-5.0). It decreased linearly from 7.6 during the first 6-months postpartum to 2.5 per 100 women-years during the last 6 months of the third year of follow-up. Maternal age did not affect HIV incidence rates. We examined the role of the cohort, counselling, and the first 6-month postpartum effects on this estimate. CONCLUSION: This fixed cohort provided an overall estimation of the HIV infection incidence rate and its dynamics. These figures could be used for programming future HIV preventive vaccine efficacy trials in Rwanda.

Adolescent↗
PubMed1994

Will robots ever replace attendants? Exploring the current capabilities and future potential of robots in education and rehabilitation.

This article describes the current capabilities and future potential of robots designed as supplements or replacements for human assistants or as tools for education and rehabilitation. A number of researchers have developed and tested a variety of robot assistants for individuals with disabilities. These robots can usually be classified as educational, vocational or independent-living assistants. Robots in all three categories are reviewed. A brief summary of the critical characteristics of human service robots is also presented and the limitations of current technology are discussed. Finally, current research aimed at addressing these limitations is described. White robots of all kinds have proven much more difficult to develop than people had expected, we conclude that it will eventually be possible to create an effective, reliable robot for human service tasks, but not as quickly as was predicted in the early 1980s.

Activities of Daily Living↗
PubMed1994

An assessment of the timing of mother-to-child transmission of human immunodeficiency virus type 1 by means of polymerase chain reaction.

To approximate the contributions of in utero, intrapartum, and postnatal transmission of human immunodeficiency virus type-1 (HIV-1) and to evaluate polymerase chain reaction (PCR) as a diagnostic tool for pediatric HIV infection, blood was collected at birth (cord blood), and at 3, 6-12, and 13-24 months in 218 children born to HIV-1-seropositive mothers in Kigali, Rwanda. Proviral DNA was detected by a double PCR using two sets of three primers (gag, pol, and env). Pediatric HIV-1 infection was defined according to serological and clinical criteria. The probability of having a positive PCR at a given time was calculated by a nonparametric method. Among children with unequivocal evidence of infection (n = 47), it was 30.5% on cord blood and 80.6% at 3 months. Thus, in children born to HIV-1-infected mothers, the estimated rate of transmission in the late postnatal period is 4.9%, and the rate of transmission in the intrapartum plus postnatal periods is 17.6%. Among 117 HIV-1-uninfected children born to HIV-1-infected mothers, six (5%) had a false-positive PCR on cord blood. These results should be taken into account in designing intervention trials aimed at reducing mother-to-child transmission of HIV-1.

Breast Feeding↗
PubMed1993

Recombinant technology as an alternative to chemical peptide synthesis: expression and characterization of HIV-1 Rev recombinant peptides.

Recombinant DNA technology has been widely used for the production of proteins in recent years. In this paper, we describe the expression and the purification of two specific peptides corresponding to parts of the human immunodeficiency virus Rev protein. The strategy of this method relies on the chemical synthesis of a pair of two complementary oligodeoxynucleotides corresponding to the coding region of the peptide of interest and the subsequent cloning into a prokaryotic expression vector. Transformation of Escherichia coli with these synthetic gene constructs yielded high production levels of recombinant protein in the bacteria. The recombinant protein was composed of two moieties, one corresponding to an "affinity handle" and the second corresponding to the peptide. Chemical cleavage of the fused protein followed by a combination of affinity chromatography and rp-HPLC led to rapid and convenient peptide purification. Peptide fused to the affinity handle as well as cleaved peptide were fully characterized by N-terminal microsequencing and mass spectrometry. The data presented demonstrate that although the major recombinant products had the expected amino acid composition, we detected unexpected processing such as alternative cleavage within the signal peptide, modified cysteines, and deamidations. These results emphasize the importance of the complete characterization of recombinant products by efficient analytical tools such as N-terminal microsequencing and mass spectrometry.

Amino Acid Sequence↗
PubMed1993

Infective and anti-infective properties of breastmilk from HIV-1-infected women.

Human immunodeficiency virus type 1 (HIV-1) is transmitted mainly by cell-to-cell contact. We postulated that transmission of HIV-1 through breastmilk could be favoured by the presence of infected cells, by deficiency of anti-infective substances in breastmilk, or both factors. 215 HIV-1-infected women were enrolled at delivery in Kigali, Rwanda; milk samples were collected 15 days, 6 months, and 18 months post partum. HIV-1 IgG, secretory IgA, and IgM were assayed by western blot, for the latter two after removal of IgG with protein G. In the 15-day and 6-month samples, we sought viral genome in milk cells by a double polymerase chain reaction with three sets of primers (gag, pol, and env). HIV-1 infection in the offspring was defined according to serological and clinical criteria. At 15 days, 6 months, and 18 months post partum, HIV-1 specific IgG was detected in 95%, 98%, and 97% of breastmilk samples, IgA in 23%, 28%, and 41%, and IgM in 66%, 78%, and 41%. In children who survived longer than 18 months, the probability of infection was associated with lack of persistence of IgM and IgA in their mothers' milk (adjusted chi 2 for trend, p = 0.01 for IgM and p = 0.05 for IgA). The presence of HIV-1-infected cells in the milk 15 days post partum was strongly predictive of HIV-1 infection in the child, by both univariate (p < 0.05) and multivariate analysis (p = 0.01). The combination of HIV-1-infected cells in breastmilk and a defective IgM response was the strongest predictor of infection. HIV-1 infection in breastfed children born to infected mothers is associated with the presence of integrated viral DNA in the mothers' milk cells. IgM and IgA anti-HIV-1 in breastmilk may protect against postnatal transmission of the virus.

Analysis of Variance↗
PubMed1993

Transcriptional activation of the mouse mdr3 gene coincides with the appearance of novel transcription initiation sites in multidrug-resistant P388 tumor cells.

In independently derived drug-resistant sublines of the mouse lymphoid tumor P388, multidrug resistance is associated with the exclusive overexpression of the mdr3 gene. In P388/VCR cells, mdr3 overexpression occurs in the absence of gene amplification, while in P388/ADM-2 cells overexpression is associated with mdr3 gene amplification. The mechanism underlying mdr3 overexpression in these cells was investigated. Measurement of the rate of transcription by nuclear "run-on" assays showed that increased mdr3 expression in P388/VCR cells was caused by transcriptional activation of the gene. Analysis of the 5' end of mdr3 mRNA transcripts by primer extension indicated that in P388/VCR cells, these mRNAs extended approximately 200 nucleotides upstream exon 2, about 60 nucleotides longer than their counterparts expressed in normal tissues from the known transcription start site of the gene (TS1). Northern blotting experiments using discrete exon and intron probes derived from the 5' end of the gene near TS1, together with ribonuclease protection using a complementary RNA probe from the same region, demonstrated that transcriptional activation in P388/VCR cells occurred from a novel transcription start site named TS3, located either upstream of TS1 or within intron 1 at a site immediately upstream a novel exon. In P388/ADM-2 cells, Northern blotting and ribonuclease protection identified overexpressed mdr3 mRNAs initiating near TS1 and a large partially spliced mdr3 mRNA species initiating upstream of TS1 at a novel initiation site designated TS2. Therefore, mdr3 overexpression in independently derived multidrug-resistant isolates of P388 cells is associated with the appearance of novel transcription start sites in the gene and novel sequences at the 5' end of the overexpressed mRNAs.

Animals↗
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

Sequence-specific deamidation: isolation and biochemical characterization of succinimide intermediates of recombinant hirudin.

Natural hirudin variant 2 with a lysine residue in position 47 (rHV2-Lys47) was produced in a genetically engineered strain of Saccharomyces cerevisiae as a secreted protein of 65 amino acids and purified to greater than 99% homogeneity. Only reversed-phase high-performance liquid chromatography (RP-HPLC) using very shallow acetonitrile gradients indicated the presence of a component in the final product (approximately 1% of total protein) with a slightly increased retention time. Using successive RP-HPLC purification steps, this hydrophobic impurity was isolated and separated into two constituents defined as components A1 and A2 which differed from the parent molecule by mass reductions of 17.2 Da (A1) and 17.6 Da (A2), respectively, as determined by electrospray mass spectrometry (ESMS). Proteolytic digestion with endoprotease Glu-C from Staphylococcus aureus (V8 protease) and analysis of the peptide mixture by ESMS showed that the mass difference between rHV2-Lys47 and component A1 was due to a modification between amino acids 1 and 43, while the corresponding mass difference with component A2 was the result of a modification within the peptide fragment comprising residues 50-61. Further analyses using amino acid sequencing and ESMS in combination with collision-activated dissociation (CAD) detected modifications at residues Asn33-Gly34 in component A1 and at Asn53-Gly54 in component A2. Both of these sites were previously shown to be susceptible to spontaneous deamidation under slightly basic pH conditions. Thus, the mass reductions of approximately 17 Da and the fact that both asparagines, Asn33 in component A1 and Asn53 in component A2, proved to be resistant to Edman degradation provided strong support for them being stable succinimide intermediates of the corresponding deamidation reactions. Both intermediates were shown to have inhibition constants for human alpha-thrombin on the order of 1 pM, identical to that of rHV2-Lys47. The isoelectric point of component A2 was determined to be within 0.01 pH unit of that of the parent molecule by isoelectric focusing in an immobilized pH gradient.

Amides↗
PubMed1993

Antimicrobial susceptibility and serotype distribution of Streptococcus pneumoniae from Rwanda, 1984-1990.

A total of 383 clinical isolates of Streptococcus pneumoniae, obtained from an equal number of patients in Kigali, Rwanda, was tested for resistance to penicillin G with a 1 microgram oxacillin disc. Of these isolates, 99 (25.8%) showed reduced zones of inhibition. By means of an agar dilution method, 21% all isolates were confirmed as relatively resistant (MIC > or = 0.12- < or = 1.0 mg/l) strains of Streptococcus pneumoniae (RRSP). A high degree of resistance to penicillin G (MIC > or = 2 mg/l) was not observed. Resistance to chloramphenicol (MIC > or = 8 mg/l) was found in 31% RRSP and in 6% penicillin susceptible strains (PSSP). Doxycycline resistance was common in both RRSP and PSSP strains. All isolates remained fully susceptible to erythromycin. Children more often harboured a strain giving a reduced inhibition zone than did adults (74/230 versus 25/153; P = 0.0005). A total of 32 serotypes or serogroups were identified, seven of them relating to 64.8% all isolates typed. Of all the isolates 84% belonged to a serotype represented in the 23-valent vaccine or to a cross-reacting serotype. Serotype 25, not included in the vaccine, accounted for 10.7% typed isolates from adults but only for 2.0% typed isolates from children. Results of susceptibility testing and clinical experience suggest that penicillin G, ampicillin and chloramphenicol should not be used alone as empirical treatment for pneumococcal meningitis in patients in Rwanda.

Adult↗
PubMed1993

Estimating the rate of mother-to-child transmission of HIV. Report of a workshop on methodological issues Ghent (Belgium), 17-20 February 1992. The Working Group on Mother-to-Child Transmission of HIV.

PURPOSE: In the last 8 years, numerous cohort studies have been conducted to estimate the rate of mother-to-child transmission (MTCT) of HIV. Many of these have faced problems in data collection and analysis, making it difficult to compare transmission rates between studies. This workshop on methodological aspects of the study of MTCT of HIV-1 was held in Ghent (Belgium) in February 1992. STUDY SELECTION AND DATA EXTRACTION: Fourteen teams of investigators participated, representing studies from Central (five) and Eastern Africa (three), Europe (two), Haiti (one) and the United States (three). A critical evaluation of the projects was carried out, under four headings: (1) enrollment and follow-up procedures, (2) diagnostic criteria and case definitions, (3) measurement and comparison of MTCT rates and (4) determinants of transmission. RESULTS OF DATA ANALYSIS: Reported transmission rates ranged from 13 to 32% in industrialized countries and from 25 to 48% in developing countries. However, no direct comparisons could be made because methods of calculation differed from study to study. Based on this review, a common methodology was developed. Agreement was reached on definitions of HIV-related signs/symptoms, paediatric AIDS and HIV-related deaths. A classification system of children born to HIV-1-infected mothers according to their probable HIV infection status during the first 15 months of life, allowed the elaboration of a direct method of computation of the transmission rate and of an indirect method for studies with a comparison group of children born to HIV-seronegative mothers. This standardized approach was subsequently applied to selected data sets. CONCLUSIONS: The methodology can now be applied to all studies with sufficient follow-up and comparisons made between transmission rates. This step is essential for assessing determinants of transmission and for the development of a common approach for the evaluation of interventions aimed at reducing or interrupting MTCT of HIV.

Adult↗