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Biomedical subjects

G Humbert

Publications and source records attributed to G Humbert.

At least 19 recordsLinked to original sources
PubMed2005

Extracellular protease activity of different Pseudomonas strains: dependence of proteolytic activity on culture conditions.

AIMS: This study investigated the effect of growth conditions on proteolytic activity of a Pseudomonas strain, named Pseudomonas sp. LBSA1, isolated from bulk raw milk. It was compared with three Pseudomonas chlororaphis and one Pseudomonas fluorescens strain from culture collections. METHODS AND RESULTS: Bacteriae were grown in a minimal salt medium. For all the strains, addition of 1% (v/v) skim milk to the growth medium was sufficient to induce protease production in 48-h culture. Addition of 1 mmol l(-1) calcium chloride permitted the detection of proteolytic activity of four strains in 48-h cultures but not for Pseudomonas sp. LBSA1. The five strains presented two patterns of proteolytic activity when grown in the minimal salt medium supplemented with 2% (v/v) skim milk at various temperatures for 48 h. Two electrophoretic protease patterns were also obtained from the zymogram of extracellular medium for the five strains. CONCLUSIONS: The growth conditions permitting protease production are variable and do not depend on the genus of the producing strain. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time a study on proteolytic activity of P. chlororaphis strains is reported. Among the tested criteria, zymograms of extracellular medium were the only ones that permitted distinguishing the P. chlororaphis strains from the P. fluorescens strain.

Animals↗
PubMed2003

Characterization and proteolytic origins of specific peptides appearing during lipopolysaccharide experimental mastitis.

Based on the compositional change of the proteose peptone fraction, proteolysis was studied over time following lipopolysaccharide-induced experimental mastitis. Electrophoresis of the proteose peptone fraction revealed many degradation products. Five peptides were identified by amino-terminal sequencing as internal fragments of beta-, kappa-, alpha(s1)-, and alpha(s2)-casein that were generated by somatic cell proteases. Although kappa-casein is considered particularly resistant to endogenous proteolysis, a kappa-casein peptide was electrophoretically isolated in association with a beta-casein fragment. The in vitro kinetic studies of caseinate hydrolysis by elastase, one of the main polymorphonuclear neutrophil (PMN) proteases, suggested that the beta-casein peptide might be generated by elastase. In addition, elastase activity in milk PMN was higher during the inflammation of the mammary gland than prior to infusion.

Amino Acid Sequence↗
PubMed2001

Enzymatic assays for native plasmin, plasminogen and plasminogen activators in bovine milk.

Rapid and sensitive assays for plasmin, plasminogen and plasminogen activators (PA) were developed and applied to bovine milk. The reaction medium was clarified by addition of a dissolving agent after hydrolysis of a fluorescent substrate specific for plasmin. This final step enabled the use of larger sample amount with higher substrate concentration than other methods, and avoided previous sample preparation. The use of 4 g gelatin/l in buffers preserved plasmin activity, thus avoiding risks of overestimation of the assays results. Sensitivity, detection level, repeatability and analysis run time of plasmin and plasminogen assay were improved over previous enzymatic methods with synthetic substrates. The PA assay was assessed by measuring conversion of exogenous plasminogen into plasmin. A new kinetic approach was used to enable the direct determination of global PA activities on raw milk samples without interference from indigenous plasmin.

Animals↗
PubMed2000

Camel (camelus dromedarius) milk PP3: evidence for an insertion in the amino-terminal sequence of the camel milk whey protein.

The camel (camelus dromedarius) milk proteose peptone 3 (PP3) was purified successively by size exclusion fast protein liquid chromatography and reversed phase high performance liquid chromatography and then characterized by amino acid residue composition determination and chemical microsequencing after CNBr or trypsin cleavages. In comparison with the previously reported structure of camel milk whey protein, the camel PP3 contains an insertion in the N-terminal region which has approximately 24 residues, whereas the remaining C-terminal regions of these two homologous proteins are essentially identical. The camel PP3 seems to contain a potential O-glycosylation site localized in this insertion and 2 or 3 phosphorylated serine residues. PP3 belongs to the glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) family and could therefore play an immunological role in the camel or its suckling young.

Amino Acid Sequence↗
PubMed2000

Viscoelastic properties of oil-water interfaces covered by bovine beta-casein tryptic peptides.

A combination of proteolysis and dilational rheology has been used to study the behavior of films of beta-casein (beta-CN) and of peptides spread at the oil-water interface. Identification of the peptides produced by trypsin hydrolysis of beta-CN in emulsion at 37 degrees C provided information on the structure of beta-CN adsorbed at the oil-water interface. Good interface properties were observed for beta-CN or its peptides, probably because of the amphipathic nature of beta-CN or a synergistic effect between hydrophilic and hydrophobic peptides. Remarkable surface activity was found for the amphipathic peptide beta-CN (f114-169). Rheological studies had shown that interface films made with peptide fractions or with beta-CN were elastic rather than viscous. Film made with the purified peptide beta-CN (f114-169) was merely elastic at the triolein-water interface. A decrease of the viscoelastic modulus was observed for aging beta-CN film but not for aging peptide films; The beta-CN decrease was related to the flexibility of its structure. When the interface is increased by the dilation of an aqueous droplet plunged into oil, beta-CN may expose new polypeptide trains to cover the increased interface, unlike peptides with simpler structures.

Animals↗
PubMed2000

[Regenerative potential in the organ of Corti after otic intoxication].

The auditory sensory cells are sensitive to a variety of influences such as noise, ototoxic drugs and aging. In the cochlea of mammals, the destroyed sensory cells are not replaced by new sensory cells. That leads to cochlear deafness, a frequent disease in human. Unfortunately, such auditory impairment is out of reach of treatment. The development of new therapeutic strategies in this field requires a precise knowledge of the mechanisms involved in auditory sensory cells disappearance and in organ of Corti's degeneration. The aim of our study was to characterize cellular and molecular changes in the cochlea of rats which had been intoxicated with the ototoxic antibiotic amikacin. The animals were sacrificed at different survival times during and after the antibiotic treatment and their cochleas were investigated using transmission and scanning electron microscopy and using confocal microscopy after tissue labellings with different fluorescent probes. The results revealed the existence of three periods. The first one corresponds to the disappearance of the sensory cells which die by apoptosis. During the second period, the organ of Corti undergoes a scarring process; concomitantly, a contingent of nonsensory supporting cells attempts to transdifferentiate directly into sensory cells. This process however fails, and the supporting cells never reach the status of hair cells. A general process of dedifferentiation of all the epithelial cells of the organ of Corti followed by a massive apoptosis of numerous epithelial cells and of most ganglion cells occurs during the third period. After that, the organ of Corti is definitely reduced to a simple monolayered epithelium. On the basis of these data, experimental strategies aimed i) to protect the sensory cells against apoptosis and ii) to promote sensory cell regeneration are now under study. They might have important implications in human therapy.

Amikacin↗
PubMed1999

Sequential changes in anti-GAL-1 staining of the rat organ of Corti following amikacin exposure.

Hair cell loss and a non-functional epithelial reorganization appeared in the organ of Corti after acoustic or toxic damage. Moreover, in the drug damaged organ of Corti, transient atypical cells were recently described with characteristics of both immature hair cells and/or non-sensory epithelial cells. The phenotype of these atypical cells has been now investigated by using the galectine 1 (GAL-1) antibody. In the normal organ of Corti, this antibody recognizes all the epithelial cells except the sensory hair cells and their supporting cells. At PD 21, transient atypical cells were not stained by GAL-1 antibody, suggesting that they were originated from hair cells or their supporting cells. Later, the organ of Corti was substituted by an epithelial scare, GAL-1 stained. This study also emphasizes the particular resistance of the cochlear apex to degeneration after antibiotic intoxication.

Amikacin↗
PubMed1999

[Regenerative potential of the sensory epithelium of the mammalian organ of Corti after poisoning with ototoxic antibiotics].

Cochlear sensory hair cells are very sensitive to noxious influences such as ageing, noise and ototoxic drugs. Whereas lost auditory hair cells are replaced by new hair cells in the avian and lower vertebrates, cochlear sensory hair cells do not regenerate in mammals. Recently, we have observed transient atypical cells in the cochlea of amikacin intoxicated rats. We have related their presence to an attempt at sensory hair cell neodifferentiation. The aim of the present study was to i) investigate the mechanisms responsible for sensory hair cell losses during the intoxication and the subsequent appearance of atypical cells, ii) characterise the phenotype of these atypical cells and iii) evaluate the influence of cochlear immaturity in such an attempt at sensory hair cells neo-differentiation.

Amikacin↗
PubMed1998

Attempt at hair cell neodifferentiation in developing and adult amikacin intoxicated rat cochleae.

Recent studies have shown that an attempt at auditory hair cell neodifferentiation occurs in vivo in the rat organ of Corti after amikacin intoxication during the last stages of cochlear maturation. Atypical cells, with morphological characteristics reminiscent of very immature sensory hair cells, were transiently observed after outer hair cell losses. The aim of the present study was to assess (i) if this attempt at hair cell neodifferentiation was related to the degree of maturity of the organ of Corti and (ii) to characterise morphological and molecular changes in the scarring epithelium. We therefore investigated, using electron and confocal microscopy, morphological and molecular changes in cochleae from rats treated with amikacin at two different periods: from post natal day (PND) 1 to PND 8, when the organ of Corti is very immature; and from PND 30 to 37, when the organ of Corti is morphologically and functionally mature. In both groups, transient atypical cells were observed, attesting that the attempt at hair cell neodifferentiation is not strictly related to the immaturity of the cochlea. The results also suggest that Deiters cells are involved in the appearance of atypical cells, possibly through a transdifferentiation process. Finally, it appears that non-sensory epithelial cells from the outer spiral sulcus progressively colonize the region of pre-existing outer hair cells.

Amikacin↗
PubMed1998

Characterization of atypical cells in the juvenile rat organ of corti after aminoglycoside ototoxicity.

Hair cell regeneration is well documented in the inner ear sensory epithelia of lower vertebrates and birds and may occur in the vestibular organs of mammals. By contrast, hair cell loss in the mature mammalian cochlea is considered irreversible. However, recent reports have suggested that an attempt at hair cell regeneration could occur in vivo in aminoglycoside-lesioned cochleas from neonatal rats. After amikacin treatment, atypical cells with apical specialization reminiscent of early differentiating stereocilia are transiently present at the apex of the intoxicated cochleas but fail to differentiate as hair cells in later stages. In the present study, we used electronic microscopy, histochemistry, and confocal microscopy to investigate the cellular rearrangements in the amikacin-lesioned organ of Corti of rat pups. In addition, we used 5-bromo-2'-deoxyuridine immunocytochemistry to determine whether mitotic processes are involved in the formation of the atypical cells. The morphologic and molecular data suggest that atypical cells are not recovering hair cells, but share characteristics of immature hair cells and supporting cells. Proliferative cells were absent from the region occupied by atypical cells, suggesting that the latter did not arise through mitotic processes. Altogether, the present results support the hypothesis that atypical cells arise through direct transformation of some of the supporting cells that reorganize during hair cell degeneration.

Amikacin↗
PubMed1998

Amikacin intoxication induces apoptosis and cell proliferation in rat organ of Corti.

Recently, an attempt at cochlear hair cell neodifferentiation has been reported in amikacin-treated rats. In the present study, we aimed to ascertain whether hair cell losses are mediated by apoptosis and whether cell proliferation occurs in damaged intoxicated cochleas. The results show that apoptosis is responsible for hair cell losses and that cell proliferation occurs in the region of the outer spiral sulcus but not in the region of Deiters cells and pre-existing hair cells. We suggest that cell proliferation maintains a certain homeostasis in the number of non-sensory cells and participates in epithelial scar formation. Neodifferentiated cells therefore probably arise from direct transdifferentiation, which could be triggered by phagocytosis of apoptotic bodies.

Amikacin↗
PubMed1998

Conformational studies of a synthetic peptide from the putative lipid-binding domain of bovine milk component PP3.

In bovine milk, a glycosylated phosphoprotein, component PP3, is known for its remarkable emulsifying properties and its capability to inhibit lipolytic activities. The determination of its primary structure is not sufficient to explain these properties. Secondary structure predictions of component PP3 and of its homologous proteins were achieved using a combination of multiple predictive methods. Based on this study, the f 119-135 region of component PP3 was proposed to be likely to adopt an amphipathic helical conformation, which is a lipid-binding motif. The conformation of the synthetic peptide corresponding to the C-terminal f 119-135 part of bovine component PP3 was analyzed by circular dichroism experiments using various media. The circular dichroism data indicated that the peptide was able to form an amphipathic alpha-helix structure in trifluoroethanol as well as in the presence of sodium dodecyl sulfate or acidic and neutral lipids, but not in water. Moreover, the conformation of this peptide is solvent dependent because it was found to adopt a beta-sheet structure for low concentrations of sodium dodecyl sulfate or a low molar ratio of acidic lipid to peptide. Tensiometric measurements showed that the amphipathic C-terminal region of component PP3 is highly tensioactive and, thus, must be responsible for the particular behavior of the protein in emulsions.

Amino Acid Sequence↗
PubMed1997

Role of the O-phosphoserine clusters in the interaction of the bovine milk alpha s1-, beta-, kappa-caseins and the PP3 component with immobilized iron (III) ions.

alpha s1- and beta-Caseins have a sequence cluster -Ser(P)-Ser(P)-Ser(P)-Glu-Glu- which is not present in kappa-casein and the whey PP3 component. The affinity of these phosphoproteins for the iron(III)-iminodiacetic acid (IDA) complex immobilized on Sepharose was studied as a function of pH, urea concentration, calcium ion concentration, enzymatic dephosphorylation and temperature. The affinity of the three polyphosphorylated proteins (alpha s1- and beta-caseins, PP3) was similar. The sequence cluster was not a specific recognition pattern of the iron(III) ion. These three proteins presented a site of high affinity and a site of weak affinity. kappa-Casein, which had only one Ser(P) residue, presented only the site of weak affinity. Their primary site which was absent after dephosphorylation or calcium ion addition required the presence of at least two Ser(P) residues close in space. Their secondary site was sensitive to the presence of urea. It was sensitive to pH variation for PP3 and kappa-casein. The study of the affinity of a few free amino acids towards iron(III)-IDA showed that the secondary site involved tryptophan and tyrosine residues for alpha s1- and beta-caseins, histidine residues for PP3 and cysteine residues for kappa-casein.

Adsorption↗
PubMed1997

[Clinical and therapeutic aspects of spontaneous pneumothorax in human immunodeficiency virus infection: 9 cases].

Spontaneous pneumothorax in HIV infected patients are mostly due to a sub-pleural necrotizing pneumonitis most often related to Pneumocystis carinii pneumonia. From our experience of nine patients and a review of the literature, we describe the clinical characteristics and therapeutic management and confirm the frequent failure of simple chest tube drainage and the high morbidity and mortality rate despite treatment. An aggressive stepped-care management of thoracoscopic talc poudrage as initial therapy should be evaluated.

AIDS-Related Opportunistic Infections↗
PubMed1997

Impact of protease inhibitors on AIDS-defining events and hospitalizations in 10 French AIDS reference centres. Fédération National des Centres de Lutte contre le SIDA.

OBJECTIVE: To assess the clinical and economic consequences of the use of protease inhibitors in the treatment of HIV infection. DESIGN: Multicentric, observational, retrospective cohort study. SETTING: Ten AIDS reference centres in France. PATIENTS: All patients followed in each centre from September 1995 through October 1996. MAIN OUTCOME MEASURES: AIDS-defining events, death, health-care resources use, administration of antiretroviral therapy. RESULTS: Data from 7749 patients in 10 centres showed a drop in hospitalization days by 35%, new AIDS cases by 35%, and deaths by 46%. In the same period, the proportion of patients receiving antiretrovirals rose from 36 to 53% including highly active antiretroviral therapy (HAART), which rose from 0.3 to 18%. Overall cost evaluation showed a slight increase of monthly treatment cost of US$ 12 per patient. Comparison of the three centres that used HAART earliest to the three centres that used it latest showed a clear benefit to early HAART with a drop in hospitalization days by 41%, new AIDS cases by 41% and deaths by 69%. The proportion of patients with HAART rose to 27% and monthly health-care cost decreased by US$ 248852 (i.e., by US$ 101 per patient per month). Late prescribing centres experienced a less marked effect with a drop in hospitalization days by 22%, new AIDS cases by 31%, and deaths by 32.5%. Proportion of patients with HAART rose to 12% and monthly health-care costs increased by US$ 113578 (i.e., by US$ 38 per patient per month). CONCLUSIONS: This study supports the extensive use of HAART in HIV-infected patients.

Acquired Immunodeficiency Syndrome↗