Biomedical subjects

J Content

Publications and source records attributed to J Content.

At least 55 records · Page 3Linked to original sources

PubMed1996

Immunogenicity and protective efficacy of a tuberculosis DNA vaccine.

Tuberculosis is the most widespread and lethal infectious disease affecting humans. Immunization of mice with plasmid DNA constructs encoding one of the secreted components of Mycobacterium tuberculosis, antigen 85 (Ag85), induced substantial humoral and cell-mediated immune responses and conferred significant protection against challenge with live M. tuberculosis and M. bovis bacille Calmette-Guérin (BCG). These results indicate that immunization with DNA encoding a mycobacterial antigen provides an efficient and simple method for generating protective immunity and that this technique may be useful for defining the protective antigens of M. tuberculosis, leading to the development of a more effective vaccine.

Animals↗

PubMed1995

In vitro NCp7 enhancement of ribozyme-mediated cleavage of full-length human IL-6 mRNA.

We have previously shown that a ribozyme directed against human interleukin-6 (IL-6) mRNA is efficient in vivo, despite its poor activity in vitro on full-length IL-6 mRNA. We compared the effect of the nucleocapsid protein of HIV-1 (NCp7) on the ribozyme cleavage reaction of a long (1041 nt) and a short (19 nt) substrate IL-6 RNA in vitro. At a one to five molar ratio of the long substrate to ribozyme, almost no cleavage is observed after 30 min at 37 degrees C. The NCp7 protein significantly increases the catalytic activity of the ribozyme on this substrate (from 0 to 53% after 7 min at 37 degrees C), but not on the short one. A kinetic analysis of single turnover reactions performed with ribozyme in at least fivefold molar excess over substrate also lead to a stimulation (70-fold) of the reaction rate with long substrate, but not with the shorter one. Preferential increases of the catalytic activity on the long substrate suggests that the NCp7 protein prevents misfolding of RNAs.

Base Sequence↗

PubMed1995

Catalytic activity and stability of hammerhead ribozymes containing 2'-acetamido-2'-deoxyribonucleosides.

Hammerhead ribozymes may be of great therapeutic importance, since they can, in theory, be developed to cleave any undesired target RNA. In order to design ribozymes which are stable against endogenous RNases, we incorporated 2'-acetamido-2'-deoxynucleosides in the catalytic core of a hammerhead ribozyme. The 2'-acetamido function has, like the hydroxyl group, both proton donor and proton acceptor capacities, which seem to be crucial for efficient catalytic activity. However, the presence of 2'-acetamido-2'-deoxypyrimidine residues in the catalytic core caused a considerable drop in cleavage rate. Replacement of the purine residues led to complete loss of the catalytic activity. Surprisingly, these 2'-modifications showed no beneficial influence on the stability of the ribozymes against RNases present in cell culture supernatant containing 10% fetal calf serum.

Acetamides↗

PubMed1995

Analysis of the Mycobacterium tuberculosis 85A antigen promoter region.

A mycobacterial expression-secretion vector was constructed in which the Escherichia coli alkaline phosphatase (phoA) reporter gene was placed under the control of the Mycobacterium tuberculosis 85A promoter and secretion signal sequences. In recombinant Mycobacterium smegmatis and Mycobacterium bovis BCG, PhoA activity could readily be detected on the mycobacterial cell surface and in the culture supernatant, indicating that the 85A signals can drive heterologous expression and secretion in both species. In contrast to the mycobacteria, the 85A promoter did not function in E. coli. We mapped the promoter region by progressive deletions using BAL 31 exonuclease and by primer extension analysis. Insertion and deletion mutations within the promoter region indicated that, unlike most E. coli promoters but similar to Streptomyces promoters, the position of the putative -35 region was not critical for efficient promoter activity. In addition, we investigated the ability of the identified signals to drive the production and secretion in BCG of recombinant Schistosoma mansoni glutathione S-transferase (Sm28GST), a protective antigen against schistosomiasis. BALB/c mice immunized with the recombinant BCG by a single dose exhibited a weak but specific T-cell response to Sm28GST.

Alkaline Phosphatase↗

PubMed1995

Cloning and chromosomal localization of a pseudogene corresponding to a mRNA for a soluble IL-6 receptor.

A polymerase chain reaction assay (Lust J.A. et al. (1992). Cytokine 4:96-100) was used to detect a mRNA coding for a soluble IL-6 receptor in human hepatoma cells. In addition to the expected amplification product, we found a sequence (SR4) which could be aligned to it with 78% identity. After cloning and sequencing a genomic 2.5-2.7 kB EcoRI fragment containing SR4, this sequence turned out to be part of a pseudogene corresponding to the transmembrane domain deleted soluble IL-6 receptor. Screening of a panel of interspecies hybrids revealed that it maps to chromosome 9.

Antigens, CD↗

PubMed1994

Construction of a ribozyme directed against human interleukin-6 mRNA: evaluation of its catalytic activity in vitro and in vivo.

We have designed a ribozyme (Rz) that cleaves human interleukin-6 (IL-6) mRNA in vivo. This Rz was tested in vitro, and was found to give expected size fragments. It was then incorporated into a mammalian expression vector containing the constitutive cytomegalovirus (CMV) immediate early promoter and transfected into human U amniotic cells (UAC). Cell clones that stably express this catalytic RNA have been obtained. Some of them displayed a marked reduction of tumor necrosis factor (TNF)-induced IL-6 production. Their reduced ability to express IL-6 was related to the amount of Rz they produced and to the extent of IL-6 mRNA cleavage as observed by a ribonuclease protection assay. These data provide a method to study further the role of IL-6 production in various biologic situations, and suggest the feasibility of developing Rzs directed against various cytokines to study their biologic role and mechanism of action.

Base Sequence↗

PubMed1994

Induction of metallothionein and stomatin by interleukin-6 and glucocorticoids in a human amniotic cell line.

Interleukin 6 (IL-6) is an important mediator of various kinds of inflammatory and immune responses. The human amniotic cell line UAC has an increased number of IL-6 receptors after treatment by glucocorticoids. To find a possible activity of IL-6 on these cells, a cDNA library of IL-6- and dexamethasone-treated cells was screened with cDNA probes from both induced and non-induced cells. Two cDNAs showed a differential hybridization signal. The first one corresponds to metallothionein, a group of small cysteine-rich proteins thought to participate in the metabolism and storage of zinc and to protect cells against oxidative damage. A second cDNA corresponds to the recently cloned cDNA of band 7 integral membrane protein also called stomatin. In hereditary stomatocytosis, absence of this protein in erythrocyte membranes is associated with high Na+ and low K+ intracellular concentrations [Stewart, G. W., Hepworth-Jones, B. E., Keen, J. N., Dash, B. C. J., Argent, A. C. & Casimir, C. M. (1992) Blood 79, 1593-1601]. In UAC cells both metallothionein and stomatin are induced by dexamethasone and IL-6 in a more than additive manner. Western blot analysis shows that stomatin protein is induced in a similar way as its mRNA. IL-6 and dexamethasone induce a state of resistance against hydrogen peroxide toxicity in UAC cells. Metallothionein induction might be partly responsible for this cytoprotection against oxidative stress.

Amnion↗

PubMed1994

Development of a human interleukin-6 receptor antagonist.

Neutralizing monoclonal antibodies specific for human interleukin-6 (IL-6) bind two distinct sites on the IL-6 protein (sites I and II). Their interference with IL-6 receptor binding suggested that site I is a receptor-binding site of IL-6, whereas site II is important for signal transduction. Mutagenesis of site II could therefore result in the isolation of IL-6 receptor antagonists. To test this hypothesis, a panel of IL-6 mutant proteins was constructed that did not bind to a site II-specific monoclonal antibody. One such site II mutant protein (with double substitution of Gln-160 with Glu and Thr-163 with Pro) was found to be an antagonist of human IL-6. It was inactive on human CESS cells, weakly active on human HepG2 cells, but active on mouse B9 cells. It could specifically antagonize the activity of wild-type IL-6 on CESS and HepG2 cells. The binding affinity of this variant for the 80-kDa IL-6 receptor was similar to that of wild-type IL-6. High affinity binding to CESS cells, however, was abolished, suggesting that the mutant protein is inactive because the complex of the 80-kDa IL-6 receptor and the mutant protein cannot associate with the signal transducer gp130. The human IL-6 antagonist protein may be potentially useful as a therapeutic agent.

Animals↗

PubMed1994

Mutagenesis of the human interleukin-6 fourth predicted alpha-helix: involvement of the Arg168 in the binding site.

Random substitutions of amino acid 161-184 of human interleukin-6 (hIL-6) have been generated at the cDNA level using oligonucleotide-directed mutagenesis. Among the majority of the mutant proteins showing a reduced biological activity on murine hybridoma cells, only those having a substitution of Met161, Arg168, Arg179 or Met184, retained a tertiary structure similar to the IL-6 folding. These residues are thus probably involved in the interaction with the IL-6 receptor. However, the contacts established by Arg168 and Arg179 seem far more important for the biological activity. According to Bazan's model of cytokine folding and the receptor binding site on the fourth alpha-helix, based on growth hormone similarity, we propose that Arg168 and Arg179 are located on the exposed surface of this presumed helix.

Amino Acid Sequence↗

PubMed1994

Rapid diagnosis of cytomegalovirus lung infection by DNA amplification in bronchoalveolar lavages.

We used polymerase chain reaction (PCR) to detect cytomegalovirus (CMV) deoxyribonucleic acid (DNA) in the bronchoalveolar lavages (BAL) of 16 CMV-infected patients with active disease. We also tested PCR on a control group of 20 patients including latently infected patients without evidence of active CMV infection. Results were compared with those of CMV culture and of a rapid method of diagnosis which detects CMV in BAL cells by nucleic acid hybridization. PCR allowed the diagnosis in 93% of the actively infected patients compared to 73% for the CMV culture. Among the 20 control patients without evidence of active CMV infection, PCR was negative in all the 24 BAL tested. Hybridization on BAL cells with the CMV probe detected nine out of 10 actively infected patients, but the specificity of the test was only 68.5%. In our experience, PCR appears to be at least as sensitive as CMV culture, it provides results faster and it performs better than the detection of the virus by hybridization on BAL cells. Only active CMV infection was detected with the PCR conditions used in our study. This suggests that the PCR can be applied to bronchoalveolar lavage fluids as a rapid method to detect CMV lung infection.

AIDS-Related Opportunistic Infections↗

PubMed1994

Regulation of lymphocyte calcitonin receptors by interleukin-1 and interleukin-6.

We have reported the existence of specific and high-affinity calcitonin (CT) receptors on normal human T-lymphocytes. Because of the increasingly recognized importance of interleukin-1 (IL-1) and IL-6 in the control of bone metabolism, we have examined their influence on the binding parameters of labeled salmon calcitonin (sCT) on lymphocytes. After a 24-hour incubation, IL-1 at 100-5000 U/ml and IL-6, at 1-1000 U/ml, decreased the apparent number of CT binding sites (Bmax) on T-lymphocytes. The effects of IL-6 on purified T-lymphocytes were dose related and 100 U of IL-6/ml reduced sCT binding to 57 +/- 16% (mean +/- SD) of the control values (n = 6). There was no significant change in CT binding affinity (Kd, 0.71 +/- 0.54 x 10(-10) M for controls versus 0.90 +/- 0.55 x 10(-10) M after IL-6) and the decrease in Bmax was reversible after 48 hours. The effects of IL-1 appeared to be mediated through an increased production of IL-6 as they were neutralized by a polyclonal antiserum against IL-6. Added alone, the antiserum caused a slight increase in the apparent number of CT binding sites on T-lymphocytes to 115 +/- 5% of control values (n = 3). In summary, IL-1 and IL-6 can induce a marked apparent loss of CT binding sites on normal T-lymphocytes at concentrations known to be active on bone metabolism. The contributions of our observations to the osteolytic activity of these cytokines deserve further investigation.

Animals↗

PubMed1994

Detection by polymerase chain reaction of genes encoding aminoglycoside-modifying enzymes in methicillin-resistant Staphylococcus aureus isolates of epidemic phage types. Belgian Study Group of Hospital Infections (GDEPIH/GOSPIZ).

The polymerase chain reaction (PCR) was used to identify the aacA-aphD, aphA3 and aadC genes, encoding the aminoglycoside-modifying enzymes AAC(6')-APH(2"), APH(3')III and ANT(4'4"), respectively, and the methicillin resistance determinant mecA, in epidemic aminoglycoside and methicillin-resistant isolates of Staphylococcus aureus. In total, 37 isolates collected in the period 1980-1985 and 81 isolates from the period 1991-1992 were obtained from 10 different Belgian hospitals. Epidemic isolates from the earlier period were characterised by phage type C (6/47/54/75) of phage group III, whereas two other epidemic phage types of group III-types A (77) and B (47/54/75/77/84/85)--were commonest in isolates from the second period. The bifunctional AAC(6')-APH(2") was the enzyme encountered most frequently. The prevalence of APH(3')III decreased significantly in the 1991-1992 period, while ANT(4',4") was found solely in isolates from this period. Resistance mechanisms were more complex in isolates from the 1991-1992 period and the mecA gene was detected in all isolates. The PCR results corresponded well with those obtained in the radiochemical phosphocellulose paper binding assay. Isolates from the 1991-1992 period were shown to express significantly higher levels of acetyltransferase activity than isolates from the 1980s.

Acetylation↗

PubMed1994

Structure of the Mycobacterium tuberculosis antigen 88, a protein related to the Escherichia coli PstA periplasmic phosphate permease subunit.

We report the cloning and sequencing of the gene coding for antigen 88 from Mycobacterium tuberculosis by using monoclonal antibodies to screen an expression library in lambda gt11. The gene encodes a 403-amino-acid-residue protein with a calculated molecular mass of 43,790 Da which contains seven putative transmembrane alpha-helical domains and presents a significant homology to the PstA protein of Escherichia coli. In its N-terminal region, it contains a 61-amino-acid region highly homologous to the fifth transmembrane helix of E. coli PstC. PstA and PstC are the two hydrophobic subunits of an E. coli periplasmic phosphate permease. Since the phosphate-binding subunit of this putative permease in M. tuberculosis has previously been characterized, i.e., the 38-kDa mycobacterial protein (also called protein antigen b, Ag 5, and Ag 78) homologous to PstS of E. coli, it seems likely that functional permeases analogous to the periplasmic permeases of gram-negative bacteria also exist in mycobacteria.

ATP-Binding Cassette Transporters↗

PubMed1994

Nucleotide sequence of the 85B-protein gene of Mycobacterium bovis BCG and Mycobacterium tuberculosis.

We have cloned and sequenced the genes coding for the 85B antigen from M. bovis BCG and M. tuberculosis. Within this gene, the only difference in sequence between M. bovis BCG and M. tuberculosis corresponds, respectively, to a C-->T yielding a Leu-->Phe replacement at position 100 of the mature 85B protein. Therefore as we described previously for the 85A gene, there is also very little variation between these two species within the 85B gene.

Acyltransferases↗

PubMed1993

Involvement of the Arg179 in the active site of human IL-6.

Three internal-amino acid deletions of amino acids 171-179 of human interleukin 6 (IL-6) were introduced at the cDNA level. While all deletion proteins were biologically inactive, immunoprecipitations with a set of conformation-specific anti-(IL-6) monoclonal antibodies showed that only mutant delta 177-179 does not present major alterations in folding. This finding, together with the observation that delta 177-179 is not able to compete with IL-6 for binding to the soluble human IL-6 receptor, suggested that some or all of these three residues participate to the composition of the receptor-binding site of human IL-6. A large number of single-amino-acid-substitution mutants were generated in residues 177, 178 and 179. Their detailed analysis revealed that Arg179 is crucial for activity in mouse cells, because all amino acid substitutions in this position cause a dramatic drop of biological activity on murine hybridoma cells without affecting the overall protein folding. The only substitution which preserved some residual activity was the conservative Arg to Lys change. This demonstrates the absolute requirement for a positive charge in position 179 for the interaction of human IL-6 with its receptor.

Arginine↗

PubMed1993

Use of the polymerase chain reaction (PCR) for the detection of aacA genes encoding aminoglycoside-6'-N-acetyltransferases in reference strains and gram-negative clinical isolates from two Belgium hospitals.

Genes encoding aminoglycoside 6'-N-acetyltransferases, were identified using the polymerase chain reaction (PCR). Four sets of primers delineating DNA fragments of 209 bp, 250 bp, 260 bp and 347 bp, specific for the four known aacA genes, and probes within these fragments, were constructed based on the nucleotide sequences of the aacA genes. The specificity of the primers was evaluated using reference strains encoding various aminoglycoside-modifying enzymes. The primers reacted with their corresponding aacA genes and did not cross-react with genes coding for other aminoglycoside-modifying enzymes. One hundred and sixty-one aminoglycoside resistant clinical isolates showing AAC(6')I activity were tested using the PCR assays. The gene described by Tran Van Nhieu & Collatz (1987) was the most frequently identified aacA gene. One strain of Citrobacter freundii contained two distinct aacA genes. However, in 46% of the strains, the majority being Serratia spp. and Acinetobacter spp. none of the specific amplified DNA fragments for any of the known aacA genes could be detected.

Acetyltransferases↗

PubMed1993

Prenatal diagnosis of 52 pregnancies at risk for congenital cytomegalovirus infection.

OBJECTIVE: To determine the feasibility of prenatal diagnosis of fetal cytomegalovirus (CMV) infection. METHODS: Fifty-two pregnant women were investigated in our unit between October 1985 and July 1992. The diagnostic procedures included ultrasound examination, amniocentesis, and fetal blood sampling. Specific tests for CMV infection included specific immunoglobulin (Ig) M antibodies, viral culture, and amplification of CMV DNA by polymerase chain reaction. Nonspecific tests included white blood cell count, hemoglobin, hematocrit, platelets, and gamma-glutamyl transferase determination. RESULTS: The combination of tests allowed an antenatal diagnosis of CMV in 13 of the 16 infected fetuses (sensitivity 81%). Amniocentesis allowed the diagnosis in 12 of the 13 antenatally diagnosed cases. The sensitivity of CMV IgM antibody detection in fetal blood was 69%. The culture of fetal blood was never positive. Thrombocytopenia was present in six cases, and ultrasound was abnormal in five. CONCLUSIONS: Amniotic fluid is the best sample to diagnose CMV infection, and fetal blood sampling and sonography are important to assess the fetal condition. Our experience underscores the importance of repetitive sampling.

Amniocentesis↗

PubMed1992

Cloning and sequence analysis of the gene encoding an NADP-dependent alcohol dehydrogenase in Mycobacterium bovis BCG.

The nucleotide sequence of a 1619-bp fragment of Mycobacterium bovis BCG containing the gene that encodes an alcohol dehydrogenase (ADH) has been determined. The M(r) calculated from the deduced amino acid (aa) sequence, as well as the N terminus, are in good accordance with those determined for the ADH purified from M. bovis BCG extracts. The M. bovis BCG cloned adh gene was expressed in Escherichia coli by its own promoter and the synthesized product shows ADH activity in the butane-1-ol-NADP system. Based on comparison of the aa sequence, this enzyme belongs to the zinc-containing, long-chain alcohol/polyol dehydrogenase family, which has been primarily described in eukaryotes. Of the 22 strictly conserved residues in this group, 19 are also conserved in M. bovis BCG ADH (BCGADH).

Alcohol Dehydrogenase↗