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Biomedical subjects

J Laporte

Publications and source records attributed to J Laporte.

At least 73 records · Page 4Linked to original sources
PubMed1994

X linked myotubular myopathy (MTM1) maps between DXS304 and DXS305, closely linked to the DXS455 VNTR and a new, highly informative microsatellite marker (DXS1684).

The locus for X linked recessive myotubular myopathy (MTM1) has previously been mapped to Xq28 by linkage analysis. We report two new families that show recombination between MTM1 and either DXS304 or DXS52. These families and a third previously described recombinant family were analysed with two highly polymorphic markers in the DXS304-DXS52 interval, the DXS455 VNTR and a newly characterised microsatellite, DXS1684 (82% heterozygosity). These markers did not recombine with MTM1 in the three families. Together with the recent mapping of an interstitial X chromosome deletion in a female patient with moderate signs of myotubular myopathy, our data suggest the following order of loci in Xq28: cen-DXS304-(DXS455, MTM1)-DXS1684-DXS305-DXS52-tel. This considerably refined localisation of the MTM1 locus should facilitate positional cloning of the gene. The availability of highly polymorphic and very closely linked markers will markedly improve carrier and prenatal diagnosis of MTM1.

Base Sequence↗
PubMed1994

[Long-term retrograde cerebral perfusion in surgery of the aortic arch. Apropos of 2 cases].

The authors present two cases of aortic arch replacement for aortic dissection: one in a male patient 58 years old and the other in a female patient 78 years old. Cerebral protection during repair of the aortic arch was performed with retrograde cerebral perfusion (RCP). Durations of RCP were 75 and 120 minutes respectively. Good neurological recovery in both patients appeared to confirm the efficacy of RCP with respect to cerebral protection during surgery of the aortic arch.

Aged↗
PubMed1993

Identification, expression in E. coli and insect cells of the non-structural protein NS2 encoded by mRNA2 of bovine coronavirus (BCV).

The coding part of mRNA 2 (ORF2) of BCV (F15 strain) was cloned and sequenced. The comparison of our sequence data with the sequence of the same ORF of BCV Quebec strain previously published revealed a major difference in the length of the C-terminal part of the NS2 protein. In vitro transcription and translation of ORF2 resulted in the synthesis of a single protein migrating with a Mr of 31 kDa. The ORF2 was fused in frame with the glutathione S transferase gene (GSH) in the pGEX vector. The fusion protein was synthesized as inclusion bodies which were concentrated and used to raise a monospecific antiserum. Alternatively the fusion protein was solubilized, purified by affinity chromatography and cleaved with Factor Xa to yield pure recombinant NS2. The ORF2 was also expressed in the baculovirus system and the recombinant proteins expressed in pro- and eukaryotic systems were compared on the basis of their size and immunoreactivity. Immunoprecipitation performed with the monospecific antiserum allowed us to identify NS2 in HRT18 infected cells, to follow its kinetic of synthesis, and to ascertain that NS2 was not incorporated in the virion as a minor structural component.

Animals↗
PubMed1992

Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization.

Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG78), selected in the cDNA library, including a large portion of the nucleocapsid (N), matrix (M) and peplomeric (S) protein genes, were used as probes for a slot blot hybridization assay. Two probe labelling techniques were compared, radiolabelling with 32P and enzymatic labelling through covalent linkage to peroxidase and chemiluminescence detection. The radioactive probe 174 detected as little as 1 to 3 pg of viral RNA, while the less sensitive enzymatic probe could not reveal more than 100 pg of RNA. No significant detection amplification was achieved when a mixture of the three probes was used. Probe 174 allowed specific identification for BECV. No hybridization was noticed either with rotaviruses or even with other antigenically unrelated members of the family Coronaviridae such as transmissible gastroenteritis virus. The test proved valid for detection of BECV in the supernatant of infected HRT-18 cells: genomic RNA could be detected after direct spotting of samples, but prior nucleic acid extraction after proteinase K treatment improved virus detection. BECV diagnosis in faecal samples using enzymatic probe was compared with conventional diagnostic methods.

Animals↗
PubMed1992

Bovine coronavirus spike glycoprotein: localization of an immunodominant region at the amino-terminal end of S2.

We have identified the binding site of monoclonal antibodies (MAbs) against the S2 subunit of the bovine coronavirus spike (S) glycoprotein. The location of this site was first investigated by using prokaryotic expression of DNA restriction fragments covering the entire S gene. The amino acid sequence containing the antibody binding site was shortened from 70 to 20 amino acids by digestion of plasmid DNA with exonuclease III, followed by sequencing of the smallest digestion product encoding an immunoreactive fusion protein. Finally we synthesized a set of nonapeptides covering the 20 amino acid sequence extending from the N-terminal residue of the S2 subunit (Ala 769 to Tyr 798). MAbs reacted mainly with six consecutive overlapping peptides with the sequence TTGYRFTNFEPFTV. Polyclonal antibodies from hyperimmunized or convalescent animals reacted only with the recombinant proteins identified by MAbs, and the hyperimmune serum bound to the same set of peptides. This suggests that this highly conserved linear antigenic determinant corresponds to an immunodominant region. This region resembles both in location and immunodominance the linear determinant defined on the infectious bronchitis virus S2 subunit. The presence of similar regions in the N-terminal region of the S2 subunit of other coronaviruses is discussed.

Amino Acid Sequence↗
PubMed1992

Bovine coronavirus peplomer glycoproteins: detailed antigenic analyses of S1, S2 and HE.

Forty-four monoclonal antibodies (MAbs) to the G110 isolate of bovine enteric coronavirus were used for the characterization of the peplomer proteins S and HE. Fourteen of these MAbs reacted with HE and the remaining 30 with the products of the S gene, S1 (19 MAbs), S2 (six MAbs) and gp200 (five MAbs). S1 and HE were found to carry major neutralization determinants, and S1 appeared to elicit the production of the MAbs displaying the highest neutralizing activity. The topography of the epitopes was assessed by means of a competitive binding assay; the 44 MAbs defined four independent antigenic domains on S1, two on S2, one on gp200 and two on HE. All the neutralizing anti-S1 MAbs mapped in antigenic sites A and B and all the neutralizing anti-HE MAbs in HE-B. Antigenic site S1-B was further subdivided into four subsites. Functional mapping was performed by testing a library of neutralization-resistant mutants against the neutralizing MAbs. Analysis of their reactivity in a neutralization test confirmed the overall distribution of epitopes in S1-B and HE-B.

Animals↗
PubMed1991

Metabolism of arachidonic acid by guinea pig Clara cells.

A method for the isolation of non-ciliated bronchiolar epithelial (Clara) cells from the guinea pig is described. Following digestion of the lung tissue with Type XXIV protease, the isolated lung cells showed a viability greater than 90% and contained 3% of Clara cells. Several cell populations were then separated on the basis of size using 2 centrifugal elutriations. The macrophages and endothelial cells were removed from the Clara cells enriched fractions by differential adherence on Petri dishes. The Clara cell-rich suspension was then further purified by centrifugation on Percoll non-continuous density gradients consisting of 48-52-55% Percoll solution. The lower interface and the pellet of the non-continuous gradient consisted of approximately 80% Clara cells. Identification of isolated Clara cells was confirmed by light microscopic observations after nitroblue tetrazolium staining and by ultrastructural characteristic features as observed by electron microscopy. The metabolism of arachidonic acid into prostaglandins and TxB2 by purified Clara cells was examined by enzyme immunoassay (EIA) and leukotriene formation was investigated by reverse phase high performance liquid chromatography (RP-HPLC). Enriched guinea pig Clara cells incubated with arachidonic acid released TxB2, PGE2 and 6-keto PGF1 alpha, but did not produce leukotrienes. These cells could however transform exogenous leukotriene A4 into leukotriene B4. These results suggest that guinea pig Clara cells possess the enzymes of the cyclooxygenase pathway required for TxB2, PGE2 and 6-keto-PGF1 alpha synthesis. Clara cells do not possess the 5-lipoxygenase enzyme but show some leukotriene A4 hydrolase activity since they can produce leukotriene B4 upon incubation with leukotriene A4.

6-Ketoprostaglandin F1 alpha↗
PubMed1991

Purification of natural killer-like Kurloff cells and arachidonic acid metabolism.

Pulmonary and splenic Kurloff cells have been purified from estrogen-treated guinea pig. Enzymatic digestion of lung tissue and mechanical dispersion of cells yielded about 650 x 10(6) viable cells. After centrifugal elutriation and centrifugation on continuous Percoll gradient, a population of high-density (1,100 g/ml) pulmonary Kurloff cells were obtained with high viability (approximately 99%) and purity (approximately 99%). Splenic Kurloff cells have been isolated by disruption of spleen tissue and centrifugation on continuous Percoll gradient. High-density splenic Kurloff cells (150 x 10(6) cells per spleen) were also obtained with high purity (approximately 99%) and viability (approximately 99%). Pulmonary and splenic Kurloff cells were incubated with various concentrations of arachidonic acid (10, 30 and 100 microM) in the absence or presence of 2 microM ionophore A23187. With 10 microM arachidonic acid the relative production of cyclooxygenase products was the following: TxB2 greater than PGE2 approximately PGI2. For an arachidonic acid concentration superior to 10 microM, the profile of release was PGE2 much greater than TxB2 greater than PGI2. Arachidonic acid metabolism through the 5-lipoxygenase pathway was also studied by incubating pulmonary or splenic Kurloff cells with 10 microM arachidonic acid in the absence or presence of 2 microM ionophore A23187, or in some experiments, with 2.5 microM leukotriene A4. Reverse phase HPLC profiles clearly indicated that high-density Kurloff cells did not express 5-lipoxygenase activity. However, these cells showed the ability to convert exogenous leukotriene A4 into leukotriene B4 suggesting the presence of LTA4 hydrolase activity. These data have been confirmed by a sensitive RIA method. This study constitutes the first report on the purification of pulmonary Kurloff cells and on arachidonic acid metabolism by these cells. The possible implications of Kurloff cells in various biological events are discussed.

Animals↗
PubMed1991

Synthesis of eicosanoids by isolated guinea pig tracheocytes.

Epithelial cells from the guinea pig trachea (tracheocytes) have been separated from the lamina propria by incubation with EDTA. Contaminating eosinophils were removed by plating on guinea pig immunoglobulin G (IgG)-coated plates. Purity of the cell suspension was assessed by electron microscopy. Enzyme immunoassay analyses of supernatants of guinea pig tracheocytes incubated with 10 microns arachidonic acid (AA) in the presence or absence of 2 microns ionophore A23187 showed that the cells released up to eight times more thromboxane, prostaglandin E2, and 6-ketoprostaglandin F1 alpha than control cells. Ionophore A23187 by itself stimulated to a smaller extent the release of these eicosanoids and did not potentiate the effect of AA. The release of cyclooxygenase products by these cells was highly modified by cell densities in the incubation media. The release of leukotrienes (LT) by stimulated epithelial cells was also investigated using reverse-phase high-performance liquid chromatography. Our results showed that the cells could not release LT on incubation with ionophore A23187 and/or AA. However, the cells released LTB4 in the medium on incubation with 2 microns LTA4. Peptido-leukotrienes were not detected. These results indicated that guinea pig tracheocytes have the cyclooxygenase and the LTA4 hydrolase but not the 5-lipoxygenase. It is postulated that tracheocytes may participate in transcellular metabolism of LTA4 generated by other cell types.

Animals↗
PubMed1990

Topological and functional analysis of epitopes on the S(E2) and HE(E3) glycoproteins of bovine enteric coronavirus.

Monoclonal antibodies (Mabs) were selected which reacted with bovine enteric coronavirus S and HE. Mabs to S were used to identify 2 cleavage products of S, S/gp105 and S/gp90. Monoclonals to S/gp105 and HE neutralised the virus; only Mabs to the latter inhibited haemagglutination and acetyl-esterase activity. Topological distribution of epitopes was studied on these 3 glycoproteins by means of competition binding experiments. Two independent epitopes were characterised on HE, 4 on S/gp105, and 2 on S/gp90. Neutralising Mabs defined one major site on both S/gp105 and HE; however a minor neutralisation epitope was also delineated on S/gp105. Functional mapping using neutralisation-resistant mutants confirmed the topological distribution of epitopes on S/gp105.

Animals↗
PubMed1990

Release of prostaglandins and thromboxanes by guinea pig isolated type II pneumocytes.

Lung cells have been isolated by enzymatic digestion of guinea pig lungs and mechanical dispersion to obtain a suspension of viable cells (approximately 500 X 10(6) cells). Type II pneumocytes have been purified to approximately 92% by centrifugal elutriation (2000 rpm, 15 ml/min) followed by a plating in plastic dishes coated with guinea pig IgG (500 micrograms/ml). We have investigated the arachidonic acid metabolism through the cyclooxygenase pathway in this freshly isolated type II cells (2 x 10(6) cells/ml). Purified type II pneumocytes produced thromboxane B2 (TxB2) predominantly and to a smaller extent the 6-keto prostaglandin PGF1 alpha (6-keto-PGF1 alpha) and prostaglandin E2 (PGE2) after incubation with 10 microM arachidonic acid. The stimulation of pneumocytes with 2 microM calcium ionophore A23187 released less eicosanoids than were produced when cells were incubated with 10 microM arachidonic acid. There was no additive effect when the cells were treated with both arachidonic acid and the ionophore A23187. Guinea pig type II pneumocytes failed to release significant amounts of TxB2, 6-keto-PGF1 alpha and PGE2 after stimulation with 10 nM leukotriene B4, 10 nM leukotriene D4, 10 nM platelet-activating factor, 5 microM formyl-methionyl-leucyl-phenylalanine, 0.2 microM bradykinin and 10 nM phorbol myristate acetate. Our findings indicate that guinea pig type II pneunomocytes possess the enzymatic machinery necessary to convert arachidonic acid to specific cyclooxygenase products, which may suggest a role for these cells in lung inflammatory processes.

6-Ketoprostaglandin F1 alpha↗
PubMed1990

Nucleotide sequence of the glycoprotein S gene of bovine enteric coronavirus and comparison with the S proteins of two mouse hepatitis virus strains.

The gene encoding the spike glycoprotein (S) of bovine enteric coronavirus (BECV) was cloned and its complete sequence of 4092 nucleotides was determined. This sequence contained a single long open reading frame with a coding capacity of 1363 amino acids (Mr 150,747). The predicted protein had 19 N-glycosylation sites. A signal sequence comprising 17 amino acids was observed starting from the first methionine residue. A potential peptidase cleavage site was located between amino acids 763 and 767. These cleavage explain the maturation of the primary product of the S gene to S1 (Mr 104,692) and S2 (Mr 84,175) spike structural proteins. Two amphipathic alpha-helices (amino acids 1007 to 1077 and 1269 to 1294) which may constitute the 12 nm stalk of the viral spike were also observed; another alpha-helix (amino acids 1305 to 1335) may be involved in the anchorage of the spike in the viral membrane. Comparison of this protein sequence to the described homologous mouse hepatitis (MHV) strain A59 and MHV-JHM S protein sequences led us to suggest that MHV-A59 and MHV-JHM S genes could be derived from a deletion of the BECV S gene.

Amino Acid Sequence↗
PubMed1989

KIN, a mammalian nuclear protein immunologically related to E. coli RecA protein.

A polypeptide of about 120 kDa, called KIN, has been identified in rat FR 3T3 cells by immunoblotting using affinity-purified antibodies against the RecA protein of Escherichia coli (38 kDa). The KIN protein as shown by fluorescent light microscopy and electron microscopy is essentially concentrated in the nucleus. Its level is higher in proliferating than in quiescent cells. Cell treatment with mitomycin C increases the level of the KIN protein. We sought similar proteins in other mammalian cells. Proteins with the same electrophoretic mobility were detected in mouse, monkey and human cell lines as well as in rat and mouse embryos.

Animals↗