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Biomedical subjects

P Thibault

Publications and source records attributed to P Thibault.

At least 37 records · Page 2Linked to original sources
PubMed2001

Early detection of pancreatic cancer in patients with chronic pancreatitis: diagnostic utility of a K-ras point mutation in the pancreatic juice.

OBJECTIVE: Point mutations of the K-ras oncogene at codon 12 have been described several months before the onset of pancreatic cancer in isolated cases of chronic pancreatitis (CP). The aim of this study was to evaluate the interest of a prospective follow-up of patients with CP and K-ras mutations at codon 12 in the detection of early pancreatic cancer. METHODS: From February 1996 to March 1998, 36 patients (mean age 52.6 yr, 31 men, five women) with CP (alcoholic: 61.1%, pancreas divisum: 5.6%, autoimmune: 5.6%, unknown origin: 27.7%) were included and then prospectively monitored (median duration of 22 months) for detection of pancreatic carcinoma. K-ras point mutations were examined by two-step polymerase chain reaction combined with restriction enzyme digestion in pancreatic juice collected during endoscopic retrograde pancreatography. RESULTS: Ten patients (27.8%) were positive for K-ras mutation. Patients with and without the mutation were not different with respect to age and sex ratio. K-ras mutations were homogeneously distributed according to the etiology (alcoholic vs nonalcoholic) and morphological characteristics (ductal stricture or mass vs none) of CP. A pancreatic carcinoma was discovered at an invasive stage in two patients, respectively at 7 and 17 months after disclosure of a K-ras mutation, versus none in patients without the mutation (p < 0.02). CONCLUSIONS: Presence of a K-ras gene mutation is not rare in patients with CP and represents an increased risk of developing pancreatic cancer. However, its utility for the detection of early pancreatic cancer remains doubtful in clinical practice.

Adenocarcinoma↗
PubMed2001

Transcription factor HIF-1 is a necessary mediator of the pasteur effect in mammalian cells.

The ability to respond to differential levels of oxygen is important to all respiring cells. The response to oxygen deficiency, or hypoxia, takes many forms and ranges from systemic adaptations to those that are cell autonomous. Perhaps the most ancient of the cell-autonomous adaptations to hypoxia is a metabolic one: the Pasteur effect, which includes decreased oxidative phosphorylation and an increase in anaerobic fermentation. Because anaerobic fermentation produces far less ATP than oxidative phosphorylation per molecule of glucose, increased activity of the glycolytic pathway is necessary to maintain free ATP levels in the hypoxic cell. Here, we present genetic and biochemical evidence that, in mammalian cells, this metabolic switch is regulated by the transcription factor HIF-1. As a result, cells lacking HIF-1alpha exhibit decreased growth rates during hypoxia, as well as decreased levels of lactic acid production and decreased acidosis. We show that this decrease in glycolytic capacity results in dramatically lowered free ATP levels in HIF-1alpha-deficient hypoxic cells. Thus, HIF-1 activation is an essential control element of the metabolic state during hypoxia; this requirement has important implications for the regulation of cell growth during development, angiogenesis, and vascular injury.

Adaptation, Physiological↗
PubMed2001

Immunofluorometric assay of human kallikrein 10 and its identification in biological fluids and tissues.

BACKGROUND: The human kallikrein 10 gene [KLK10, also known as normal epithelial cell-specific 1 gene (NES1)] is a member of the human kallikrein gene family. The KLK10 gene encodes for a secreted serine protease (hK10). We hypothesize that hK10 is secreted into various biological fluids and that its concentration changes in some disease states. The aim of this study was to develop a sensitive and specific immunoassay for hK10. METHODS: Recombinant hK10 protein was produced and purified using a Pichia pastoris yeast expression system. The protein was used as an immunogen to generate mouse and rabbit polyclonal anti-hK10 antisera. A sandwich-type immunofluorometric assay was then developed using these antibodies. RESULTS: The hK10 immunoassay has a detection limit of 0.05 microg/L. The assay is specific for hK10 and has no detectable cross-reactivity with other homologous kallikrein proteins, such as prostate-specific antigen (hK3), human glandular kallikrein 2 (hK2), and human kallikrein 6 (hK6). The assay was linear from 0 to 20 microg/L with within- and between-run CVs <10%. hK10 is expressed in many tissues, including the salivary glands, skin, and colon and is also detectable in biological fluids, including breast milk, seminal plasma, cerebrospinal fluid, amniotic fluid, and serum. CONCLUSIONS: We report development of the first immunofluorometric assay for hK10 and describe the distribution of hK10 in biological fluids and tissue extracts. This assay can be used to examine the value of hK10 as a disease biomarker.

Amino Acid Sequence↗
PubMed2001

[Metastasis to trocar site after lymphadenectomy with retroperitoneal laparoscopy].

The authors report a case of trocar site metastasis, one year after laparoscopic retroperitoneal lymphadenectomy. This is the seventh published case of trocar site metastasis in laparoscopic urological surgery. The lymph node resection procedure and the histological type of the tumour appear to be risk factors for the development of wall metastases.

Carcinoma, Squamous Cell↗
PubMed2001

[Treatment of stress urinary incontinence with percutaneous colposuspension: unsatisfactory technique].

OBJECTIVE: The objective of this study was to evaluate the results of a percutaneous needle colposuspension technique with bone fixation (Vesica system) in the treatment of female urinary stress incontinence (USI) of the woman, with a minimum follow-up of one-year. MATERIAL AND METHODS: 34 women aged 35 to 86 years (mean: 62 years) were treated for USI due to bladder neck hypermobility according to the Vesica technique. All patients had a positive preoperative Bonney manoeuvre without sphincter incompetence on urethral pressure profile. RESULTS: Overall, 1 year postoperatively, 9 patients (26%) were completely dry and did not wear any protection, 19 (56%) were improved, but still presented occasional leaks and 6 (17%) were considered to be failures. Physical examination revealed postoperative leaks in 24 patients with recurrence of hypermobility and positive Bonney manoeuvre in every case. There was one subcutaneous abscess and 2 cases of persistent pain at the bone implant site. No cases of bone infection or screw explantation were observed. No cases of retention beyond the 3rd postoperative day were observed. CONCLUSION: The percutaneous colposuspension technique gives disappointing results at one year due to recurrence of urethral hypermobility in every case.

Adult↗
PubMed2001

[Hemorrhagic cystitis: etiology and treatment].

Haemorrhagic cystitis consists of acute or insidious diffuse bleeding from the bladder mucosa. Although the aetiology of haemorrhagic cystitis is varied, the two predominant causes are chemical and irradiation. The chemical compounds most frequently responsible for haemorrhagic cystitis are oxaphosphorines (cyclophosphamide, ifosfamide). Haemorrhagic cystitis may also be due to toxic or infectious causes (bacterial, fungal, parasitic or viral). Finally, haemorrhagic cystitis can occur in the context of a systemic disease or may be isolated and idiopathic. Prevention of haemorrhagic cystitis, based on general measures and specific measures, is essential, but is not always effective. In the case of proven haemorrhagic cystitis, various treatment options are available, including and can associate clot extraction, continuous bladder irrigation, bladder instillations of haemostatic factors, formalin, hyperbaric oxygen therapy, arterial embolization or salvage surgery. Although therapeutic management is usually effective, it can sometimes be difficult due to the severity of the bleeding and its repercussions on the patient's general state. Deaths are not exceptional, emphasizing the seriousness of haemorrhagic cystitis. Based on a review of the literature, the authors review the aetiology and treatment of haemorrhagic cystitis.

Cystitis↗
PubMed2001

Galectin-3-mediated adherence of Proteus mirabilis to Madin-Darby canine kidney cells.

Proteus mirabilis is an important cause of urinary tract infections (UTIs) and can result in acute pyelonephritis. Proteus mirabilis expresses several, morphologically distinct, fimbrial species, and previous studies have shown that the nonagglutinating fimbriae (NAF) mediate bacterial adherence to a number of cell lines, including Madin-Darby canine kidney (MDCK) cells. Immunoblot overlay analysis of the plasma membrane fraction from MDCK cells with purified NAF revealed a 34-kDa band, which has been analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Database search identified galectin-3 as a potential protein candidate. Immunocytochemical assay of MDCK cells with a galectin-3-specific monoclonal antibody, anti-Mac-2, confirmed its presence on the plasma membrane extracellular surface. Preincubation of P. mirabilis with anti-Mac-2 monoclonal antibodies, specific for galectin-3, resulted in the inhibition of bacterial binding to MDCK cells. These data suggest a role for galectin-3, interacting with appropriately glycosylated surface receptors and P. mirabilis fimbriae, as a mediator of bacterial adherence in vitro.

Animals↗
PubMed2001

The impact on clinical practice of endoscopic ultrasonography used for the diagnosis and staging of pancreatic adenocarcinoma.

CONTEXT: Endoscopic ultrasonography is considered a highly accurate procedure for diagnosing small pancreatic tumors and assessing their locoregional extension. OBJECTIVE: To evaluate the impact of endoscopic ultrasonography on the management of pancreatic adenocarcinoma in clinical practice. PATIENTS: Sixty-four consecutive patients (mean age 70.5 plus/minus 11.9 years) hospitalized for staging or diagnosis of pancreatic adenocarcinoma were retrospectively (from January 1995 to November 1997) or prospectively studied (from December 1997 to August 1999). SETTING: Group 1 consisted of 52 patients with pancreatic adenocarcinoma which was discovered using computerized tomography scanning and/or ultrasound. Endoscopic ultrasonography was utilized for staging purposes only in patients who were considered to be operable and the tumor to be resectable based on computerized tomography scanning criteria. Group 2 consisted of 12 patients who were diagnosed as having a pancreatic adenocarcinoma using endoscopic ultrasonography whereas computerized tomography scanning and ultrasound was negative. MAIN OUTCOME MEASURES: The impact of endoscopic ultrasonography was analyzed on the basis of the number of patients requiring endoscopic ultrasonography as a staging procedure (Group 1) and by evaluating the performance of endoscopic ultrasonography in determining resectability (Groups 1 and 2) based on the surgical and anatomopathological results. RESULTS: Endoscopic ultrasonography was performed in 20 out of 64 patients (31.3%): 8/52 in Group 1 (15.4%) and all 12 patients of Group 2. Endoscopic ultrasonography correctly assessed an absolute contraindication to resection in 11 cases. Resection was confirmed in 8 of the 9 cases selected by endoscopic ultrasonography. The positive predictive value, negative predictive value and overall accuracy of endoscopic ultrasonography for determining resection were 89%, 100%, and 95%, respectively. CONCLUSIONS: The impact of endoscopic ultrasonography seems especially relevant for the detection of pancreatic tumors after negative computerized tomography scanning, and for the prevention of unnecessary laparotomies as complementary staging after ultrasonography and computerized tomography scanning.

Adenocarcinoma↗
PubMed2000

Chemical and serological characterization of the Bordetella hinzii lipopolysaccharides.

Bordetella hinzii has recently been isolated from immunocompromised human hosts. The polysaccharides isolated from its endotoxin (lipopolysaccharide, LPS) were investigated using chemical analyses, NMR, gas-liquid chromatography/mass spectrometry and mass spectrometry by plasma desorption, matrix-assisted laser desorption/ionization and electrospray. The following structure for the O-chain-free LPS was deduced from the experimental results: carbohydrate structure [see text] Mass spectrometry and serology revealed that the O-chains were different from the homopolymer common to Bordetella bronchiseptica and Bordetella parapertussis strains and were composed of a trisaccharide repeating unit. Masses up to 8 kDa were obtained for native LPS molecular species.

Antibodies, Monoclonal↗
PubMed2000

Occurrence of sulfate-reducing bacteria under a wide range of physico-chemical conditions in Au and Cu-Zn mine tailings.

Sulfate-reducing bacteria (SRB) have been observed in mining environments, but their presence has not been linked to specific physico-chemical and mineralogical factors. The present study was undertaken to assess the presence of SRB in several Au and Cu-Zn mine tailings located near Timmins, Ont., Canada, and determine the factors responsible for their presence. Vegetated and non-vegetated mine tailings were sampled for SRB enumeration, pH, Eh, water content, total carbon content and sequential chemical extraction. Results first showed that SRB populations were present at all sites and that their distribution varied with depth. Populations were recovered from neutral pH and slightly anoxic tailings and from highly acidic (pH 2) and oxic tailings. The total carbon content of the tailings was generally low and not related to the presence of vegetation. In addition, the carbon content did not affect SRB population distribution and appeared to be more related to the type of tailings, i.e., oxidized and acidic Cu-Zn tailings contained on average more carbon than Au tailings. Results also indicated that the water content of the tailings varied greatly with depth and was not related to the presence of SRB populations. The sequential chemical extraction showed that the pyrite content of the tailings was lower in Au tailings than in Cu-Zn tailings, and that some oxidized Cu-Zn sites were depleted in pyrite due to microbial and chemical oxidation. Our results indicate that SRB could be cultured from a variety of sites and sample types, and that factors such as pH, Eh, water content and carbon content at the collection sites did not exert control on their presence.

Journal Article↗
PubMed2000

Separation and identification of peptides from gel-isolated membrane proteins using a microfabricated device for combined capillary electrophoresis/nanoelectrospray mass spectrometry.

The coupling of microfabricated devices to nanoelectrospray mass spectrometers using both a triple quadrupole and a quadrupole time-of-flight mass spectrometer (QqTOF MS) is presented for the analysis of trace-level membrane proteins. Short disposable nanoelectrospray emitters were directly coupled to the chip device via a low dead volume connection. The analytical performance of this integrated device in terms of sensitivity and reproducibility was evaluated for standard peptide mixtures. A concentration detection limit ranging from 3.2 to 43.5 nM for different peptides was achieved in selected ion monitoring, thus representing a 10-fold improvement in sensitivity compared to that of microelectrospray using the same chip/mass spectrometer. Replicate injections indicated that reproducibility of migration time was typically less than 3.1% RSD whereas RSD values of 6-13% were observed on peak areas. Although complete resolution of individual components is not typically achieved for complex digests, the present chip capillary electrophoresis (chip-CE) device enabled proper sample cleanup and partial separation of multicomponent samples prior to mass spectral identification. Analyses of protein digests were typically achieved in less than 1.5 min with peak widths of 1.8-2.5 s (half-height definition) as indicated from individual reconstructed ion electropherograms. The application of this chip-CE/QqTOF MS system is further demonstrated for the identification of membrane proteins which form a subset of the Haemophilus influenzae proteome. Bands first separated by 1D-gel electrophoresis were excised and digested, and extracted tryptic peptides were loaded on the chip without any further sample cleanup or on-line adsorption preconcentration. Accurate molecular mass determination (< 5 ppm) in peptide-mapping experiments was obtained by introducing an internal standard via a postseparation channel. The analytical potential of this integrated device for the identification of trace-level proteins from different strains of H. influenzae is demonstrated using both peptide mass-fingerprint database searching and on-line tandem mass spectrometry.

Amino Acid Sequence↗
PubMed2000

Post-translational modification of p53 protein in response to ionizing radiation analyzed by mass spectrometry.

The p53 tumor suppressor protein promotes cell cycle arrest or apoptosis in response to DNA damage and other forms of stress. p53 protein functions as a transcription factor by binding to specific DNA sequences and regulating the transcription of target genes. This activity of p53 is reported to be regulated by phosphorylation and acetylation occuring at various sites on the molecule. Here, we have used a direct and non-radioactive approach involving mass spectrometric analysis of p53 protein to identify sites that are covalently modified in vivo, either constitutively or in response to ionizing radiation. Following partial purification by immuno-affinity chromatography and enzymatic in-gel digestion, the resulting p53 peptides were analyzed by MALDI-TOF and nanoelectrospray mass spectrometry. Mass spectrometry analyses identified four sites at the N terminus that were phosphorylated in response to irradiation, a single constitutive phosphorylation site at serine 315 and several acetylation sites.

Amino Acid Sequence↗
PubMed2000

Phosphorylation of caldesmon by p21-activated kinase. Implications for the Ca(2+) sensitivity of smooth muscle contraction.

We have previously shown that p21-activated kinase, PAK, induces Ca(2+)-independent contraction of Triton-skinned smooth muscle with concomitant increase in phosphorylation of caldesmon and desmin but not myosin-regulatory light chain (Van Eyk, J. E., Arrell, D. K., Foster, D. B., Strauss, J. D., Heinonen, T. Y., Furmaniak-Kazmierczak, E., Cote, G. P., and Mak, A. S. (1998) J. Biol. Chem. 273, 23433-23439). In this study, we provide biochemical evidence implicating a role for PAK in Ca(2+)-independent contraction of smooth muscle via phosphorylation of caldesmon. Mass spectroscopy data show that stoichiometric phosphorylation occurs at Ser(657) and Ser(687) abutting the calmodulin-binding sites A and B of chicken gizzard caldesmon, respectively. Phosphorylation of Ser(657) and Ser(687) has an important functional impact on caldesmon. PAK-phosphorylation reduces binding of caldesmon to calmodulin by about 10-fold whereas binding of calmodulin to caldesmon partially inhibits PAK phosphorylation. Phosphorylated caldesmon displays a modest reduction in affinity for actin-tropomyosin but is significantly less effective in inhibiting actin-activated S1 ATPase activity in the presence of tropomyosin. We conclude that PAK-phosphorylation of caldesmon at the calmodulin-binding sites modulates caldesmon inhibition of actin-myosin ATPase activity and may, in concert with the actions of Rho-kinase, contribute to the regulation of Ca(2+) sensitivity of smooth muscle contraction.

Actins↗
PubMed2000

Rapid and sensitive separation of trace level protein digests using microfabricated devices coupled to a quadrupole--time-of-flight mass spectrometer.

The application of microfabricated devices coupled to a quadrupole time-of-flight mass spectrometer (Qq-TOF-MS) is presented for the analysis of trace level digests of gel-isolated proteins. In order to enhance the sample loading for proteomics analyses, two different on-chip sample preconcentration techniques were evaluated. First, a sample stacking procedure that used polarity switching to remove the sample buffer prior to zone electrophoresis was easily integrated on the microfabricated devices. With the present chip design, this preconcentration technique provided up to 70 nL sample injection with sub-nM detection limits for most peptide standards. For applications requiring larger sample loading, a disposable adsorption preconcentrator using a C18 membrane is incorporated outside the chip. This preconcentration method yielded lower peptide recoveries than that obtainable with sample stacking, and provided a convenient means of injecting several microL of sample with detection limits of typically 2.5 nM for hydrophobic peptides. The analytical merits of both sample enrichment approaches are described for the identification of bands isolated from two-dimensional (2-D) gel separation of protein extracts from Haemophilus influenzae. Accurate molecular mass measurements (< 5 ppm) in peptide mapping experiments is obtained by introducing an internal standard via a post-separation channel. Rapid identification of trace level peptides is also demonstrated using on-line tandem mass spectrometry and database searching with peptide sequence tags.

Animals↗
PubMed2000

Integration of immobilized trypsin bead beds for protein digestion within a microfluidic chip incorporating capillary electrophoresis separations and an electrospray mass spectrometry interface.

A microfluidic device is described in which an electrospray interface to a mass spectrometer is integrated with a capillary electrophoresis channel, an injector and a protein digestion bed on a monolithic substrate. A large channel, 800 microm wide, 150 microm deep and 15 mm long, was created to act as a reactor bed for trypsin immobilized on 40-60 microm diameter beads. Separation was performed in channels etched 10 microm deep, 30 microm wide and about 45 mm long, feeding into a capillary, attached to the chip with a low dead volume coupling, that was 30 mm in length, with a 50 microm i.d. and 180 microm o.d. Sample was pumped through the reactor bed at flow rates between 0.5 and 60 microL/min. The application of this device for rapid digestion, separation and identification of proteins is demonstrated for melittin, cytochrome c and bovine serum albumin (BSA). The rate and efficiency of digestion was related to the flow rate of the substrate solution through the reactor bed. A flow rate of 1 or 0.5 microL/min was found adequate for complete consumption of cytochrome c or BSA, corresponding to a digestion time of 3-6 min at room temperature. Coverage of the amino acid sequence ranged from 92% for cytochrome c to 71% for BSA, with some missed cleavages observed. Melittin was consumed within 5 s. In contrast, a similar extent of digestion of melittin in a cuvet took 10-15 min. The kinetic limitations associated with the rapid digestion of low picomole levels of substrate were minimized using an integrated digestion bed with hydrodynamic flow to provide an increased ratio of trypsin to sample. This chip design thus provides a convenient platform for automated sample processing in proteomics applications.

Animals↗
PubMed2000

Structural characterization of the outer core and the O-chain linkage region of lipopolysaccharide from Pseudomonas aeruginosa serotype O5.

The point of attachment of the O-chain in the outer core region of Pseudomonas aeruginosa serotype O5 lipopolysaccharide (LPS) was determined following a detailed analysis of the extended core oligosaccharide, containing one trisaccharide O-chain repeating unit, present in both the wild-type strain PAO1 and O-chain deficient mutant strains AK1401 and PAO-rfc. The structure of the extended core oligosaccharide was determined by various mass spectrometric methods as well as one-dimensional and two-dimensional NMR spectroscopy. Furthermore, the one-dimensional analogues of NOESY and TOCSY experiments were applied to confirm the structure of the outer core region in the O-chain polysaccharide. In both the extended core oligosaccharide and the core of the smooth LPS, a loss of one of the beta-glucosyl residues and the translocation of the alpha-rhamnosyl residue, followed by the attachment of the first O-chain repeating unit was observed. This process is complicated and could involve two distinct rhamnosyltransferases, one with alpha-1, 6-linkage specificity and another with alpha-1,3-linkage specificity. It is also plausible that an alpha-1,3 rhamnosyltransferase facilitates the addition of the 'new' alpha-rhamnosyl residue that will act as a receptor for the attachment of the single O-antigen repeating unit in the LPS of the semi-rough mutant. The 2-amino-2-deoxy-fucosyl residue of the first O-chain repeating unit directly attached to the core was found to have a beta-anomeric configuration instead of an alpha configuration, characteristic for this residue as a component of the O-chain polysaccharide. The results of this study provide the first example of the mechanistic implications of the structure of the outer core region in a fully assembled O-chain containing LPS, differing from the O-chain deficient rough LPS.

Bacterial Capsules↗