Biomedical subjects

F Gauthier

Publications and source records attributed to F Gauthier.

At least 19 recordsLinked to original sources

PubMed2001

Reversible inhibition of cathepsin L-like proteases by 4-mer pseudopeptides.

A library of 121 pseudopeptides was designed to develop reversible inhibitors of trypanosomal enzymes (cruzain from Trypanosoma cruzi and congopain from Trypanosoma congolense). The peptides share the framework: Cha-X1-X2-Pro (Cha=cyclohexyl-alanine, X1 and X2 were phenylalanyl analogs), based on a previous report [Lecaille, F., Authié, E., Moreau, T., Serveau, C., Gauthier, F. and Lalmanach, G. (2001) Eur. J. Biochem. 268, 2733-2741]. Five peptides containing a nitro-substituted aromatic residue (Tyr/Phe) and one a 4-chloro-phenylalanine at the X1 position, and 3-(2-naphthyl)-alanine, homocyclohexylalanine or 3-nitro-tyrosine (3-NO(2)-Tyr) at the X2 position, were selected. They inhibited congopain more effectively than cruzain, except Cha-4-NO(2)-Phe-3-NO(2)-Tyr-Pro which bound the two parasitic enzymes similarly. Among this series, Cha-3-NO(2)-Tyr-HoCha-Pro and Cha-4-NO(2)-Phe-3-NO(2)-Tyr-Pro are the most selective for congopain relative to host cathepsins. No hydrolysis occurred upon prolonged incubation time with purified enzymes. In addition introduction of non-proteogenic residues in the peptidyl backbone greatly enhanced resistance to proteolysis by mammalian sera.

Cathepsin B↗

PubMed2001

Infantile Alexander disease: spectrum of GFAP mutations and genotype-phenotype correlation.

Heterozygous, de novo mutations in the glial fibrillary acidic protein (GFAP) gene have recently been reported in 12 patients affected by neuropathologically proved Alexander disease. We searched for GFAP mutations in a series of patients who had heterogeneous clinical symptoms but were candidates for Alexander disease on the basis of suggestive neuroimaging abnormalities. Missense, heterozygous, de novo GFAP mutations were found in exons 1 or 4 for 14 of the 15 patients analyzed, including patients without macrocephaly. Nine patients carried arginine mutations (four had R79H; four had R239C; and one had R239H) that have been described elsewhere, whereas the other five had one of four novel mutations, of which two affect arginine (2R88C and 1R88S) and two affect nonarginine residues (1L76F and 1N77Y). All mutations were located in the rod domain of GFAP, and there is a correlation between clinical severity and the affected amino acid. These results confirm that GFAP mutations are a reliable molecular marker for the diagnosis of infantile Alexander disease, and they also form a basis for the recommendation of GFAP analysis for prenatal diagnosis to detect potential cases of germinal mosaicism.

Adolescent↗

PubMed2001

Sim1 haploinsufficiency causes hyperphagia, obesity and reduction of the paraventricular nucleus of the hypothalamus.

The bHLH-PAS transcription factor SIM1 is required for the development of the paraventricular nucleus (PVN) of the hypothalamus. Mice homozygous for a null allele of Sim1 (Sim1(-/-)) lack a PVN and die perinatally. In contrast, we show here that Sim1 heterozygous mice are viable but develop early-onset obesity, with increased linear growth, hyperinsulinemia and hyperleptinemia. Sim1(+/-) mice are hyperphagic but their energy expenditure is not decreased, distinguishing them from other mouse models of early-onset obesity such as deficiencies in leptin and melanocortin receptor 4. Quantitative histological comparison with normal littermates showed that the PVN of Sim1(+/-) mice contains on average 24% fewer cells without a selective loss of any identifiable major cell type. Since acquired lesions in the PVN also induce increased appetite without a decrease in energy expenditure, we propose that abnormalities of PVN development cause the obesity of Sim1(+/-) mice. Severe obesity was described recently in a patient with a balanced translocation disrupting SIM1. Pathways controlling the development of the PVN thus have the potential to cause obesity in both mice and humans.

Adipose Tissue↗

PubMed2001

Purification and characterization of active recombinant rat kallikrein rK9.

The rat tissue kallikrein rK9 is most abundant in the submandibular gland and the prostate. It has been successfully expressed in the Pichia pastoris yeast expression system. A full-length cDNA coding for the mature rK9 was fused in frame with yeast alpha-factor cDNA. The fusion protein was secreted into the medium with high yield without being processed by the yeast KEX2 signal peptidase. Mature rK9 was efficiently released from the fusion protein by trypsin and was purified to homogeneity by one-step affinity chromatography using soya bean trypsin inhibitor (SBTI) as affinity ligand. The identity of the recombinant enzyme was checked by N-terminal amino acid sequencing, Western blot analysis and kinetic studies. The dual trypsin- and chymotrypsin-like enzymatic specificity of rK9 was assessed by determining specificity constants (k(cat)/K(m)) for the hydrolysis of fluorogenic substrates, the peptide sequences of which were derived from proparathyroid hormone (pro-PTH) and from semenogelin-I. Our results confirmed the presence of an extended binding site in the rK9 active site. We also identified a far more sensitive substrate of this enzyme than those previously described, Abz-VKKRSARQ-EDDnp, which was hydrolysed with a catalytic efficiency k(cat)/K(m) of 420000 M(-1)s(-1). Finally, we showed that four of the five major proteins contained in secretions of rat seminal vesicles were rapidly degraded by recombinant rK9.

Amino Acid Sequence↗

PubMed2001

Long-term evaluation of the beneficial effect of subtotal splenectomy for management of hereditary spherocytosis.

Clinical manifestations of hereditary spherocytosis (HS) can be abrogated by splenectomy. However, concerns exist regarding exposure of patients to a lifelong risk for overwhelming infections and, to a lesser extent, to vascular complications after total splenectomy. In the search for alternative treatment modalities, we assessed, in a previous pilot study, the potential usefulness of subtotal splenectomy in a small population of patients. During a mean follow-up period of 3.5 years, subtotal splenectomy was shown to be effective in decreasing the hemolytic rate, while maintaining the phagocytic function of the spleen. In the current study, we evaluated the clinical and biologic features of 40 patients with HS who underwent subtotal splenectomy and were monitored for periods ranging from 1 to 14 years. The beneficial effect of subtotal splenectomy included a sustained decrease in hemolytic rate and a continued maintenance of phagocytic function of the splenic remnant. However, mild-to-moderate hemolysis was persistent and accounted for secondary gallstone formation and aplastic crisis in a small subset of patients. Surprisingly, regrowth of the remnant spleen did not seem to have a major impact on the beneficial outcomes of these individuals. Our results suggest that subtotal splenectomy appears to be a reasonable treatment option for management of patients with HS, especially young children.

Adolescent↗

PubMed2001

Immunisation of cattle with cysteine proteinases of Trypanosoma congolense: targetting the disease rather than the parasite.

In order to test the hypothesis that trypanosome cysteine proteinases (CPs) contribute to pathology of trypanosomosis, cattle were immunised with CP1 and/or CP2, the major CPs of Trypanosoma congolense, and subsequently challenged with T. congolense. Immunisation had no effect on the establishment of infection and the development of acute anaemia. However, immunised cattle, unlike control cattle, maintained or gained weight during infection. Their haematocrit and leukocyte counts showed a tendency to recovery after 2-3 months of infection. Cattle immunised with CP2 mounted early and prominent IgG responses to CPs and to the variable surface glycoprotein following challenge. Thus trypanosome CPs may play a role in anaemia and immunosuppression; conversely, anti-CP antibody may modulate the trypanosome-induced pathology.

Animals↗

PubMed2001

Functional expression of the catalytic domains of two cysteine proteinases from Trypanosoma congolense.

The catalytic domains of two closely related cysteine proteinases (CP1 and CP2) from Trypanosoma congolense, referred to as C1 and C2, were expressed as proforms in Escherichia coli (C1) and in the baculovirus system (C1 and C2). While the bacterial expression system did not allow recovery of active C1, the baculovirus system led to secretion of inactive zymogens which could be processed at acidic pH into mature enzymes. Active C1 and C2 were purified from serum-free culture supernatants by anion-exchange chromatography and characterised. Their kinetic parameters and pH activity profiles confirmed the relatedness between C2 and native CP2 (congopain). These properties also underline major functional differences between C1 and C2, that appear to relate to discrete but essential sequence differences. It is likely that these two enzymes perform distinct roles in vivo, in the parasite and/or in the host-parasite relationships.

Amino Acid Sequence↗

PubMed2001

The ecology of "fecal indicator" bacteria commonly found in pulp and paper mill water systems.

Coliform bacteria have long been used to indicate fecal contamination of water and thus a health hazard. In this study, the in-mill water and external effluent treatment systems of seven typical Canadian pulp and paper mills were all shown to support the growth of numerous coliforms, especially Klebsiella Spp., Escherichia coli. Enterobacter spp., and Citrobacter spp. In all mills and most sampled locations, klebsiellas were the predominant coliforms. Although all but one of the mills had no sewage input and most disinfected their feed (input) water, all contained the most typical fecal indicator bacterium, E. coli. Many of the mill coliforms were classified as fecal coliforms by standard "MPN" and metabolic tests, but this was shown to be due to their thermotolerance, not their origin. Mill coliforms were shown not to be just simple transients from feedwater or furnish (wood), but to be continuously growing, especially in some of the primary clarifiers. Isolated mill coliforms grew very well on a sterilized raw combined mill effluent. The fecal streptococci (enterococci), alternative indicators of fecal health hazards, were common in all mills in the absence of sewage. Ten strains of E. coli isolated from four mills were all shown to be non-toxigenic strains of harmless serotypes. No salmonellas were found. Therefore, the use of total coliform, fecal coliform, enterococci, or E. coli counts as indicators of fecal contamination, and thus of health hazard in pulp and paper mill effluents or biosolids (sludges) known to be free of fecal input is invalid.

Bioreactors↗

PubMed2001

Modification of bovine beta-lactoglobulin by glycation in a powdered state or in aqueous solution: adsorption at the air-water interface.

The adsorption at the air-water interface of native and various glycated forms of beta-Lactoglobulin B (beta-LG), prepared under two different experimental conditions, was investigated by ellipsometry, surface tension and shear elastic constant measurements. The measurements were performed in 0.1 M phosphate buffer, 0.1 M NaCl, pH 6.8. It was found that the interfacial properties of beta-LG were more affected when the glycation was performed in solution than in the dry-way system. Dry-way glycated beta-LG, despite a higher glycation extent, affected slightly its interfacial behaviour. Solution glycated beta-LG exhibited a higher adsorption and more rigid interface as expressed by shear elastic constant measurement at saturation (16.5 mN/m against 8.7 and 11.5 mN/m for native and control treated beta-LG, respectively). These results were attributed to the specific molecular species induced during glycation in solution, which includes monomers and unfolded covalent homodimers of beta-LG molecules with a high tendency to self-association via non-covalent interactions.

Journal Article↗

PubMed2001

Subsite specificity of trypanosomal cathepsin L-like cysteine proteases. Probing the S2 pocket with phenylalanine-derived amino acids.

The S2 subsite of mammalian cysteine proteinases of the papain family is essential for specificity. Among natural amino acids, all these enzymes prefer bulky hydrophobic residues such as phenylalanine at P2. This holds true for their trypanosomal counterparts: cruzain from Trypanosoma cruzi and congopain from T. congolense. A detailed analysis of the S2 specificity of parasitic proteases was performed to gain information that might be of interest for the design of more selective pseudopeptidyl inhibitors. Nonproteogenic phenylalanyl analogs (Xaa) have been introduced into position P2 of fluorogenic substrates dansyl-Xaa-Arg-Ala-Pro-Trp, and their kinetic constants (Km, kcat/Km) have been determined with congopain and cruzain, and related host cathepsins B and L. Trypanosomal cysteine proteases are poorly stereoselective towards D/L-Phe, the inversion of chirality modifying the efficiency of the reaction but not the Km. Congopain binds cyclohexylalanine better than aromatic Phe derivatives. Another characteristic feature of congopain compared to cruzain and cathepsins B and L was that it could accomodate a phenylglycyl residue (kcat/Km = 1300 mM-1.s-1), while lengthening of the side chain by a methylene group only slightly impaired the specificity constant towards trypanosomal cysteine proteases. Mono- and di-halogenation or nitration of Phe did not affect Km for cathepsin L-like enzymes, but the presence of constrained Phe derivatives prevented a correct fitting into the S2 subsite. A model of congopain has been built to study the fit of Phe analogs within the S2 pocket. Phe analogs adopted a positioning within the S2 pocket similar to that of the Tyr of the cruzain/Z-Tyr-Ala-fluoromethylketone complex. However, cyclohexylalanine has an energetically favorable chair-like conformation and can penetrate deeper into the subsite. Fitting of modeled Phe analogs were in good agreement with kinetic parameters. Furthermore, a linear relationship could be established with logP, supporting the suggestion that fitting into the S2 pocket of trypanosomal cysteine proteases depends on the hydrophobicity of Phe analogs.

Animals↗

PubMed2001

Insulin-like growth factor binding proteins (IGFBPs) as potential physiological substrates for human kallikreins hK2 and hK3.

Insulin-like growth factors (IGFs) are important growth regulators of both normal and malignant prostate cells. Their action is regulated by six insulin-like growth factor binding proteins (IGFBPs). The proteolytic cleavage of IGFBPs by various proteases decreases dramatically their affinity for their ligands and therefore enhances the bioavailability of IGFs. To elucidate the putative biological role of prostatic kallikreins hK2 and hK3 (prostate-specific antigen) in tumour progression, we analyzed the degradation of IGFBP-2, -3, -4 and -5 by these two tissue kallikreins. We found that hK3, already characterized as an IGFBP-3 degrading protease, cleaved IGFBP-4 but not IGFBP-2 and -5, whereas hK2 cleaved all of the IGFBPs much more effectively, and at concentrations far lower than those reported for other IGFBP-degrading proteases. The proteolytic patterns after cleavage of IGFBPs by hK2 and hK3 were similar and were not modified in the presence of IGF-I. Heparin, but not other glycosaminoglycans, enhanced dramatically the ability of hK3 but not hK2 to degrade IGFBP-3 and IGFBP-4. More importantly, the IGFBP fragments generated by hK2 and hK3 had no IGF-binding capacity, as assessed by Western ligand blotting. Our results suggest that the prostatic kallikreins hK2 and hK3 may influence specifically the tumoral growth of prostate cells through the degradation of IGFBPs, to increase IGF bioavailability.

Binding Sites↗

PubMed2001

Characterization of PSA-RP2, a protein related to prostate-specific antigen and encoded by alternative hKLK3 transcripts.

Despite the wide use of prostate-specific antigen (PSA) as a marker of prostate cancer, analysis of its gene products has not yet been completed. The structure of two alternative mRNAs (0.9 and 1.65 kb) of the hKLK3 gene that retain the third intron is reported here. These partially spliced transcripts were detected by hybridization or RT-PCR in normal prostate tissue, benign prostate hyperplasia (BPH) and cancerous prostate tissues, and also in the prostate LNCaP cell line. Insertion of the unspliced intron creates an in-frame stop codon and results in a truncated prepro PSA variant of 180 amino-acid residues. This novel variant, designated PSA-RP2, has an alternate C-terminal tail and lacks the serine residue essential for the catalytic activity of PSA. Prepro PSA-RP2 was transiently produced in COS-7 cells and detected in the spent medium using an anti-PSA serum. Secreted PSA-RP2 was glycosylated with an apparent molecular mass of 25 kDa. Our findings suggest that PSA-RP2 contributes to the molecular heterogeneity of free-PSA in the serum of patients with benign or malignant prostate tumors.

Alternative Splicing↗

PubMed2001

Primary nephrectomy for emergency: a rare event in the International Society of Paediatric Oncology Nephroblastoma Trial and Study no. 9.

Experience of the International Society of Paediatric Oncology (SIOP) Trials and Studies indicates that the preoperative chemotherapy in Wilms' tumour improves stage distribution, decreases complication rate and reduces postoperative treatment. However, some situations may lead to prompt primary surgery. The aim of the study is to assess reasons leading to primary emergency nephrectomy. Records of 720 patients with non-metastatic unilateral nephroblastoma who were registered in the SIOP Trial and Study 9 were reviewed. Twenty-four (3%) cases of primary emergency nephrectomy were identified. Reasons leading to emergency nephrectomy were massive bleedings from ruptured tumours in 13 patients, suspicion of an "acute abdomen" in 7, bowel occlusion in 2 and other in 2. Postoperative treatment included radiotherapy in 71% of cases and anthracyclines in 92%. Complications were frequent and happened in 25% of patients, the outcome however, was favourable and 22 of 24 patients are alive (from 9 to 79 months). The 7 patients with a suspicion of an "acute abdomen" probably constitute the group which could have been markedly reduced if adequately diagnosed and observed prior to surgery.

Child↗

PubMed2001

Acute liver failure in infancy: a 14-year experience of a pediatric liver transplantation center.

Clinical charts of 80 infants younger than 1 year who presented over a 14-year period (1986 to 2000) with acute liver failure (ALF), defined as prolonged prothrombin time greater than 17 seconds and decrease of clotting factor V plasma level below 50% of normal, were reviewed retrospectively. The main causes of ALF were inherited metabolic disorders in 42.5% of cases, including mitochondrial respiratory chain disorders in 17, type I hereditary tyrosinemia in 12, and urea cycle disorders in 2; neonatal hemochromatosis in 16% of cases; and acute viral hepatitis in 15% of cases (hepatitis B in 6, herpes virus type 6 in 4, and herpes simplex virus type 1 in 2). The cause of ALF remained undetermined in 16% of cases. A total of 19 (24%) infants survived without orthotopic liver transplantation; 38 (47%) infants died from sepsis, multiple organ failure, or because the underlying disease contraindicated orthotopic liver transplantation (12 [15%] infants), and 23 (29%) infants underwent orthotopic liver transplantation within 12 months from onset, 12 of whom are alive with a mean follow-up period of 5.2 years from orthotopic liver transplantation. We conclude that ALF during the first year of life is a severe condition with poor prognosis, despite the advent of liver transplantation.

Hospitals, Pediatric↗

PubMed2001

Cathepsin L, but not cathepsin B, is a potential kininogenase.

Although papain-like enzymes are strongly inhibited by their natural tight-binding inhibitors of the cystatin superfamily, cathepsins B and L may still retain some residual proteolytic activity toward Z-Phe-Arg-AMC in the presence of an excess of kininogen. This activity is abolished by adding E-64 or chicken cystatin. Cathepsins B and L show a single band of gelatinolytic activity when subjected to gelatin-SDS-PAGE. Adding high Mr kininogen, low Mr kininogen, T-kininogen, or chicken cystatin to cathepsin L results in additional intense bands of enzyme activity corresponding to the protease-inhibitor complexes. Cathepsin B does not produce these additional bands. This gelatinolytic activity was inhibited by E-64, but not by EDTA, PMSF or Pefabloc. Cathepsin L also specifically generated kinins from high and low molecular weight kininogens in vitro, but cathepsin B did not. T-kininogen did not release any immunoreactive kinins when complexed with cathepsin L, as previously observed using tissue kallikreins. The ability of cathepsin L to generate vasoactive peptides raises the question of the physiological significance of this mechanism during inflammation.

Animals↗

PubMed2001

[Update on kidney tumors in children: role of the pediatric surgeon].

The surgical treatment of malignant renal tumours in children (nephroblastomas) is part of a multimodal therapeutic strategy, defined by the International Society of Paediatric Oncology protocol. Resection of the primary tumour and determination of the post-operative stage determine subsequent treatment. The modalities of the surgical procedure are described together with the particular cases concerning infants under the age of 6 months, extension to the renal vein and inferior vena cava, emergency surgery, bilateral, tumours or tumour in a solitary kidney, lymph node involvement, metastatic nephroblastomas and complications of surgery.

General Surgery↗

PubMed2000

[Congenital disorders of the biliary ducts].

Congenital disorders of the bile ducts manifesting as neonatal cholestasis, are: biliary atresia, Alagille syndrome and neonatal sclerosing cholangitis. Biliary atresia must be considered as a neonatal surgical emergency: diagnosis and therapeutic intervention should be completed before one month of age. Survival at 10 years is about 70%, 14% without and 56% with liver transplantation. The cause of the disease is unknown. Alagille syndrome is defined by the association of bile duct paucity, pulmonary stenosis, butterfly-like vertebrae, peculiar facies and posterior embryotoxon. JAGGEDI gene mutations have been identified allowing prenatal diagnosis. The diagnosis of neonatal sclerosing cholangitis can only be performed by cholangiography.

Adult↗

PubMed2000

Postshunt encephalopathy in liver transplanted children with portal vein thrombosis.

BACKGROUND: Surgical portosystemic shunting has been reported to alleviate successfully portal hypertension in liver transplanted recipients with portal vein thrombosis. METHODS: We report two liver transplanted children with portal vein thrombosis who developed post-shunt acute encephalopathy. In one child, a mesocaval H-type shunt was created surgically because of bleeding related to Roux-en-Y loop varices at 3 months posttransplantation; in the other, a large spontaneous splenorenal shunt was discovered at the time of diagnosis of portal vein thrombosis on day 34 posttransplantation and was preserved. RESULTS: Post-shunt encephalopathy developed 6 months and 2.7 years after transplantation, causing death in one child. CONCLUSIONS: This report illustrates the risk and the possible dismal outcome of post-shunt encephalopathy in liver transplanted children. Therapeutic procedures other than portosystemic shunting that will restore an hepatopetal portal flow to the liver graft should be considered in liver-transplanted children with portal vein thrombosis.

Brain Diseases↗