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P Pothier

Publications and source records attributed to P Pothier.

At least 55 records · Page 3Linked to original sources
PubMed1993

Epitope mapping of the major inner capsid protein of group A rotavirus using peptide synthesis.

Three hundred and ninety-one consecutive heptapeptides derived from the VP6 protein of bovine rotavirus (397 AA) were synthesized using the "pepscan" method and were assayed on the synthesis pins with monoclonal antibodies to VP6. Heptapeptides reactive with MAbs were located in four main regions: regions AA 32-64, AA 155-167, AA 208-274, and a fourth region at the C-terminal, from AA 380 to AA 397. Among these regions, two sequences were also reactive with the MAbs when longer peptides were assayed. The sequence located between AA 58 and AA 62 (NWNFD), recognized by MAbs RV-1026, RV-50, and RV-443, was previously reported. A new site was defined in the region essential for trimerization, between AA 159 and AA 165 (PYSASFT), which was recognized by MAbs RV-133 and RV-138.

Amino Acid Sequence↗
PubMed1993

In vitro reconstitution of rotavirus transcriptional activity using viral cores and recombinant baculovirus expressed VP6.

Purified baculovirus-expressed group A rotavirus VP6 polypeptide was shown to be active in the recovery of the transcriptase activity associated with the reconstitution of the single-shelled rotavirus particle. Recombinant VP6 polypeptide was able to restore the transcriptional activity in purified viral cores from both SA-11 and RF rotavirus strains. Recombinant group C VP 6 (Cowden strain) is capable of binding as a trimer to group A viral core particles but unable to restore the transcriptase activity, suggesting that the binding of the polypeptide to cores is not the only requirement to restore the transcriptase activity. The VP 6 group A polypeptide was shown to bind as a monomer to viral cores, indicating that trimerization of VP 6 may be not required for reconstitution of the polymerase activity.

Antigens, Viral↗
PubMed1993

An intestinal secretory protein is found in most glands associated with the gastrointestinal tract: von Ebner's and salivary glands, gallbladder, and pancreas.

We have previously shown that monoclonal antibodies (MAb) prepared against the duodenal mucosa of 4-day-old mice disclosed the presence of two antigens associated with the formation of intestinal crypts. One of these, MIM-1/39, was found in the apical cytoplasm of undifferentiated epithelial crypt cells of the duodenum and colon. We report here the immunolocalization of MIM-1/39 in different glands associated with the gastrointestinal tract, using a polyclonal antibody produced against antigen MIM-1/39. By indirect immunofluorescence on 1-micron thick Lowicryl K4M sections, MIM-1/39 was detected in secretory granules of serous cells in lingual (von Ebner's gland), sublingual, submandibular and parotid glands, and in pancreas; it was also found in epithelial cells of the gallbladder and in the secretory granules of chief cells of gastric glands. Liver, kidney, and Brunner's glands were not immunoreactive. Immunocytochemistry revealed the presence of antigen MIM-1/39 in small secretory granules of the gallbladder, duodenum, colon, and in the large secretory granules in serous cells of lingual and parotid glands, in pancreas, and in gastric chief cells. In Western blotting, the MIM-1/39 MAb revealed two bands (330 and 350 KD) in adult mouse duodenal mucosa, gallbladder and stomach, whereas only one (330 KD) was disclosed in pancreatic juice. However, two bands (330 and 350 KD) were detected in pancreatic juice with the polyclonal antibody. The distribution of MIM-1/39 was different from that reported for IgA, bound and free secretory components, cryptdin, and Tamm-Horsfall protein. Therefore, MIM-1/39 appears to be a unique protein. Its exact role remains to be elucidated.

Animals↗
PubMed1992

Localization of group-specific epitopes on the major capsid protein of group A rotavirus.

Chemical cleavage of the VP6 protein of bovine rotavirus showed that VP6-specific monoclonal antibodies (MAbs) reacted with the amino acid sequence between glycine 48 and asparagine 107. Furthermore, three synthetic peptides (amino acids 48 to 64, 60 to 75 and 91 to 108) containing part of this sequence and 22 consecutive overlapping heptapeptides corresponding to the region between amino acids 48 and 75 were analysed for their immunoreactivity using group-specific MAbs. The MAbs recognized peptides 48-64 and/or 60-75, and a set of overlapping heptapeptides located between residues 53 (asparagine) and 67 (glycine), which have two short sequences in common: IRNW (residues 56 to 59), recognized by MAb RV-133, and (NW)NFD (residues 58/60 to 62), recognized by MAbs RV-50, -1026 and -443. These results indicate that the sequence between amino acid residues 48 and 75 is present in one of the immunodominant sites of VP6.

Amino Acid Sequence↗
PubMed1991

Radioautographic localization of epidermal growth factor receptors in human fetal gut.

The present investigation was undertaken to study the localization and accessibility of epidermal growth factor binding sites in the human fetal gut (15-19 weeks of gestation) using light-microscopic and quantitative autoradiography. Exposure of colonic explants to 5 nmol/L 125I-labeled epidermal growth factor for 60 minutes at 22 degrees C revealed extensive accumulation at binding sites in undifferentiated cells of the crypts and at the base of the villus, as well as in the inner circular layer of the muscularis externa bordering the submucosa. Some labeling was also present in the mesenchymal and vascular elements of the lamina propria. Labeling was virtually absent on the brush border at all levels of the epithelium. Quantitative analysis revealed a distinct gradient in grain density along the various compartments of the crypt-villus axis. Epithelial cells in the deep portions of the crypts showed the highest grain density (9.2 grains/microns 2) with values gradually decreasing to 6.5 in upper crypt and 3.9 in lower villus cells. The upper third of the villus showed very little labeling (0.4 grains/microns 2). Cellular distribution of silver grains in lower villous cells revealed a polarization of labeling in the basolateral infranuclear region. Experiments performed at 4 degrees C and at various incubation times showed similar results. Using isolated loops of intact colon and jejunum, segments in which labeled epidermal growth factor was only accessible on the serosal side showed extensive labeling and distribution similar to that found in explanted tissue. On the other hand, labeled epidermal growth factor could not access these same receptor sites when infused into the lumen, either at 22 degrees C or 4 degrees C. These results show that in the human fetal gut (a) the greatest concentration of epidermal growth factor binding sites is found in regions of high proliferative activity and (b) binding sites are absent from the brush border, suggesting that, under normal circumstances, systemic but not luminal epidermal growth factor has free access to its specific receptor.

Autoradiography↗
PubMed1991

Use of synthetic peptides to locate neutralizing antigenic domains on the fusion protein of respiratory syncytial virus.

Chemical and enzymic cleavages of the F1 subunit of the fusion (F) protein of respiratory syncytial (RS) virus showed that the sequence 184-Gly to 314-Trp reacted with neutralizing monoclonal antibodies (MAbs). Twelve synthetic peptides covering a part of this sequence were analysed for their immunoreactivity with neutralizing MAbs and anti-RS virus rabbit serum. Two sequential antigenic domains corresponding to amino acids 200 to 225 and 255 to 278 were defined with anti-RS virus rabbit serum. The peptides 205-225 and 259-278, belonging to these antigenic domains, inhibited binding to the F protein and the neutralizing activity of the anti-RS virus rabbit serum. One MAb (RS-348) reacted with peptides containing amino acids 200 to 225. Moreover, the peptide 205-225 induced an anti-peptide rabbit serum neutralizing RS virus in vitro. These results indicate that the sequence from residues 200 to 225 was present in one of the immunodominant sites of the F protein.

Amino Acid Sequence↗
PubMed1991

Prevalence of respiratory syncytial virus subgroups A and B in France from 1982 to 1990.

A fluorescence antibody test with monoclonal antibodies was used to determine the subgroup (A or B) of respiratory syncytial virus from infants hospitalized in Caen, France, over eight consecutive epidemics from 1982 to 1990. From 1982 to 1985, 27 (30%) out of 90 frozen nasal slides were classified as subgroup A strains and 63 (70%) were classified as subgroup B. B strains predominated over A in 1983-1984 and 1984-1985. From 1985 to 1990, 284 respiratory syncytial virus field strains were reisolated from frozen materials; 115 (40.5%) were typed as subgroup A and 169 (59.5%) were typed as subgroup B. In 1985-1986, 1986-1987, and 1988-1989, both subgroups were present in almost equal numbers; subgroup A (88.3%) predominated in 1987-1988, and subgroup B (84.5%) predominated in 1989-1990. In conclusion, both subgroups occur together each year, and one subgroup rarely predominates, e.g., subgroup A in 1987-1988 and subgroup B in 1983-1984 and 1989-1990. Therefore, there is a gradual change of the predominant subgroup into another over a period of about 5 years; the relative frequency of subgroup A strains increased from 1983 to 1988, whereas the percentage of subgroup B decreased during the same period.

Antibodies, Monoclonal↗
PubMed1990

Presence and binding characteristics of calcitriol receptors in human fetal gut.

In the present study, we show for the first time the presence of calcitriol-specific binding sites in hypertonic extracts of cells isolated from human fetal small intestine and colon from 13-21 weeks of gestation. Woolf plot analysis of the binding characteristics revealed the presence of a single class of high affinity receptors. The presence of specific receptors for calcitriol in fetal intestine and colon opens interesting possibilities as to the role of this hormone in human gut development.

Calcitriol↗
PubMed1990

Migration of fetal intestinal intervillous cells in neonatal mice.

The migration of intestinal intervillous epithelial cells labeled in the fetus was followed in neonatal mice. At 17 days of gestation, a first group of pregnant mice received three intraperitoneal injections of 3H-thymidine (150 microCi/injection) administered at 30 min intervals. Two mothers were sacrificed 3 hours after the first injection. Mice from different litters were also sacrificed on days 0, 2, 4, 8, 12, 14, and 16 after birth. A second group of pregnant mice was injected at 18 1/2 days of gestation and offspring were sacrificed on days 6, 8, 10, 12, 14, and 16 after birth. Segments of duodenum and ileum were fixed in glutaraldehyde, postfixed in osmium tetroxide, dehydrated, and embedded in Epon. Sections were stained with aldehyde fuchsin and processed for radioautography. By following the leading front and trailing edge of labeled cells in the longest villi of the duodenum and ileum, we observed that 1) extrusion zones become active immediately after birth and 2) the longest villi do not elongate until 10 days after birth in the duodenum and 14 days in the ileum, that is, when all labeled epithelial cells originally present in the fetus have been extruded. Moreover, by measuring the distance between the internal limit of the inner circular layer of smooth muscle and the intervillous epithelium at 17 days of gestation (12.95 +/- 1.18 microns) or the bottom of the crypts at day 3 (14.81 +/- 0.91 microns), we propose that crypts do not develop as downgrowths: rather the intervillous epithelium is reshaped and the crypt-villus junction moves upward, away from the muscularis externa.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Multicentre evaluation of a new commercial latex agglutination test using a monoclonal antibody for rotavirus detection.

A new commercial latex-agglutination test using a monoclonal antibody for detection of rotavirus (Slidex Rota-Kit 2) was compared with three other tests (Slidex Rota-Kit, Rotalex, Rotazyme II) using immunoelectron microscopy and a monoclonal enzyme immunoassay as reference tests. Slidex Rota-Kit 2 was more sensitive and specific than the other tests, and would thus appear to be a practical and accurate rotavirus assay for use in routine laboratory work.

Antibodies, Monoclonal↗
PubMed1989

[Value of rapid diagnostic methods in rotavirus infection in the neonatal period].

One hundred and forty eight faecal specimens were collected from 40 newborn infants during an outbreak of rotavirus infections in a neonatal unit. Rotavirus was sought every 3 days by immunoelectron microscopy (IEM) and was found in 12 newborn babies. Rotavirus excretion persisted for up to 6 days after healing of the diarrhoea. No significant difference was found between infected (n = 12) and uninfected (n = 28) infants with regard to gestational age (35.2 +/- 3.7 vs 36.9 +/- 2.6 weeks), birth weight (2,064 +/- 693 vs 2,197 +/- 529 g), and postnatal ages at the time of the outbreak. The 148 stool specimens were analysed by IEM considered the reference technique and compared with two ELISA methods, and three Latex methods. Rotavirus was identified by IEM in 20 stools. Equivocal results were observed with Slidex Rotakit Polyclonal (16.9%), Slidex Rotakit Monoclonal (2.7%) and Rotalex (13.8%). The positive and negative predictive values were respectively 87.5% and 96.9% for the Slidex Rotakit Monoclonal test, 86.7% and 94.7% for the Rotazyme II test, 100% and 95.5% for the ELISA monoclonal test.

Carrier State↗
PubMed1988

Presence and characteristics of epidermal growth factor receptors in human fetal small intestine and colon.

In the present study, we demonstrate for the first time the presence of important concentrations of EGF binding sites in isolated epithelial cells of both human fetal small intestine and colon as early as 12 weeks gestation. The pattern of EGF binding in the small intestine between 12 and 17 weeks show that binding was significantly higher (2.5-fold) in younger fetuses than in older fetuses. Moreover, the fetal colon exhibited a much higher binding capacity (1.5-2.5 times) than corresponding intestinal cells for all age groups studied. Analysis of Scatchard representations reveal that the concentration of high- and low-affinity binding sites in colonic epithelial cells are twice the values observed in corresponding intestinal cells. The present data raise interesting possibilities as to the role of this growth factor in human fetal gut development.

Binding Sites↗
PubMed1988

Biologic effects of epidermal growth factor in human fetal jejunum.

The influence of epidermal growth factor (EGF) on the differentiation and proliferation of human fetal jejunum was studied in organ cultures. Fetal intestine (11-14-wk gestation) was cultured for 5 days at 37 degrees C in serum-free Leibovitz L-15 medium alone or supplemented with 25, 50, and 100 ng EGF/ml culture medium. The addition of hormone did not modify the morphology of the intestinal explants. Biochemical studies revealed that lactase activity was significantly increased with the addition of 50 and 100 ng EGF/ml culture medium. On the other hand, the increase in sucrase, trehalase, and glucoamylase activities that normally occurs during the culture was repressed in the presence of increasing concentrations of EGF. Deoxyribonucleic acid synthesis was significantly decreased after 5 days of culture even in the presence of the lowest EGF concentration used. Concomitantly, the labeling index of the epithelial cells dropped drastically in the presence of EGF. The EGF-induced variation in DNA synthesis was already evident within 24 h of culture, whereas enzymic modifications occurred only between the third and fifth day of culture. The simultaneous addition of EGF and hydrocortisone (50 ng/ml) did not reveal any synergistic action between the two hormones on the hydrolytic activities of the brush border. However, EGF did inhibit hydrocortisone-stimulated DNA synthesis. The present work provides for the first time some basic data on the influence of EGF on brush border hydrolytic activities and on epithelial cell proliferation of human fetal jejunum. These observations strongly suggest that EGF plays an important role in the fetal development of the human gastrointestinal tract.

Cell Division↗
PubMed1987

Loss of microtubules and alteration of glycoprotein migration in organ cultures of mouse intestine exposed to nocodazole or colchicine.

Explants from mouse jejunum were cultured for 3-7 h in the absence (control) or presence of colchicine (100 micrograms/ml) or nocodazole (10 micrograms/ml). In recovery experiments, explants were cultured in fresh medium for an additional period. To label glycoproteins, 3H-fucose was added during the last 3 or 6 h of the initial culture or recovery period. Subcellular fractionation studies revealed that colchicine and nocodazole inhibited migration of labelled glycoproteins to the brush border (P2) by 40-45%. Radioautographic studies of absorptive cells showed that colchicine and nocodazole inhibited labelling of the microvillous border by 67% and 87%, while labelling of the basolateral plasma membrane increased by 114% and 275%. Immunocytochemical studies revealed that both colchicine and nocodazole caused the virtual disappearance of the microtubular network in the absorptive cells. It is possible that some glycoproteins normally destined for the microvillous border are rerouted to the basolateral membrane. The observed loss of microtubules after drug treatment suggests that microtubules may play a role in the intracellular migration of membrane glycoproteins. Additional support for this concept is provided by the fact that in recovery experiments the distribution of label returned to control values after the microtubular network became re-established.

Animals↗
PubMed1987

Effect of monensin on cell ultrastructure and glycoprotein migration in adult mouse jejunal epithelium in organ culture.

Explants from adult mouse jejunum were cultured for 3 h in a medium which contained both 3H-fucose (10 or 25 microCi/ml) and monensin (100 microM) or 3H-fucose only (control). Radiochemical analysis of cell fractions showed that 3H-fucose labelling of the brush border fraction decreased 42% in monensin-treated explants, suggesting that in absorptive cells the intracellular transport of newly synthesized glycoproteins to the apical plasma membrane had been inhibited. Electron-microscopic examination of treated explants revealed a variation in response to the drug from region to region. In some areas, both absorptive and goblet cells exhibited little alteration. In others, the Golgi cisternae of both absorptive and goblet cells were entirely replaced by large vacuoles, and in the latter cell type, the cisternae of the rough endoplasmic reticulum were greatly distended. Electron-microscopic radioautographic analysis showed that in absorptive and goblet cells exhibiting little morphological change, intracellular transport of newly synthesized glycoproteins was similar to that in controls. In regions where absorptive cells exhibited extensive Golgi modifications, intracellular transport remained normal in some cases; more often-however, there was a marked inhibition (over 70%) of transport of labelled glycoproteins to the apical surface. Transport to the basolateral membrane was never affected. In goblet cells exhibiting modifications of the Golgi apparatus and rough endoplasmic reticulum, no incorporation of 3H-fucose label in the Golgi apparatus occurred, suggesting a block of intracellular transport proximal to the site at which 3H-fucose is added. In absorptive cells, this does not appear to be the case, since the level of 3H-fucose incorporation in all treated cells remained similar to that in controls.

Animals↗