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We carried out a prospective study including 554 vaginal swabs simultaneously tested for antenatal screening of Group B Streptococcus (GBS or Streptococcus agalactiae) using culture on the chromogenic medium Strepto B ID (Biomérieux, Marcy l'Etoile, France) and real time gene amplification on LightCycler (Roche Applied Science). We centrifuge the swabs with "SETS" device and separate centrifugates in 2 parts: one for the culture and the other one for molecular biology. First half of the centrifugate is inoculated onto Todd-Hewitt broth enriched with antibiotics. This broth is incubated to 35 degrees C during 24 hours and then subcultured on a Strepto B ID medium. This last one is incubated during 24 hours to 35 degrees C in capnophilic conditions before interpretation. DNA extraction for molecular biology is simply obtained by heating the microtubes to 95 degrees C in a water bath. The cfb gene is amplified, allowing a specific gene amplification of GBS even within a polymorphic flora. The concordance between both methods is 94.8%. The sensitivity and negative predictive values obtained are respectively 88.0 and 97.4% for real time PCR and 83.0 and 96.4% for culture on Strepto B ID. Both methods are thus concordant, with equal sensitivity and valid for detection of GBS colonization in pregnant women. However real time gene amplification allows reducing turn around time since molecular biology process (extraction+amplification) does not exceed 1 hour.

Agars and carrageenans are sulphated galactans which assemble in the red algal cell wall as a dense network of semi-crystalline fibers. These polysaccharides are degraded in heterogeneous phase by bacterial enzymes, namely agarases and carageenases. Crystallographic as well as enzymologic investigations of the sulphated galactans/galactanases systems highlight that the properties of these catalysts are well adapted to the degradation of solid polyanionic substrates. Indeed, as for cellulases or amylases, they are able to depolymerize their respective substrates according to a processive mode of action. However, at the molecular level, they are distinguished by the ionic nature of the interactions involved which do not allow the direct transposition of the processivity models developed for the degradation of neutral polysaccharides.

The protein encoded by ras and src protooncogenes are frequently activated in a constitutive state in human colorectal cancer. In this study, we investigated the effect of oncogenic p21ras and Py-MT/pp60c-src on the synthesis of syndecan-1, a membrane anchored proteoglycan playing a role in cell-matrix interaction and neoplastic growth control. To this end, we used Caco-2 cells transfected with an activated (Val-12) human Ha-ras gene or the polyoma middle T (Py-MT) oncogene, a constitutive activator of pp60c-src tyrosine kinase activity. As compared to control vector-transfected Caco-2 cells, both oncogene-transfected cells exhibited: (1) a decrease in syndecan-1 specific activity; (2) a decrease in size and sulfation of syndecan-1 ectodomain glycosaminoglycan side chains; and (3) an active heparanase specifically degrading the heparan sulfate chains. In conclusion, the tumorigenic progression induced by oncogenic p21ras or Py-MT/pp60c-src is associated with marked alterations of syndecan-1 at the post-translational level.

In order to determine the origin of cellulases in the gut of the earthworm Eisenia fetida andrei, the wall tissues of the different parts of the disinfected gut are cultured in vitro. Enzymatic activities (FPase: filter paper activity; CMCase: carboxymethycellulase, cellobiase) were measured both in the tissues and in the culture medium: the following activities were found, carboxymethycellulase (CMCase), filter paper activity (FPase). The first located in the crop, gizzard and midgut wall, the second in the foregut and midgut wall. The cellobiase was detected only in the crop and gizzard tissues, whereas it is present in each part of the gut of holoxenic worms. This fact indicates that this enzyme is mainly produced by microorganisms ingested. These results allow us to propose the hypothesis of the existence of synergy, for the cellulose hydrolysis between tissular enzymes and cellulolytic microorganisms.

A flocculating strain of Saccharomyces diastaticus NRRL2416 was isolated after ethidium bromide mutagenesis and density gradient separation. Flocculation did not change the general characteristics of the strain. Flocculation was better on maltodextrin-containing media than on others. It was found to be calcium-dependent and sensitive to growth conditions. The deflocculating effects of some sugars suggested that mannose may be involved in the mechanism of flocculation, as for strains of the FLO1 phenotypic group.

The zymograms of the digestive caeca and the intestine in seven species of Isopods are established. The glucidasic activities predominate. A relative proteasic poverty is noted as well as the absence of lipases however, the esterases exist. The enzymatic pattern of the gut suggests its participation in the alimentary digestion. The enzymes distribution allows us to establish relations with the food preference of the animals. Some particularities (trypsin, alpha and beta glucosidase) favour a comparison of the marine species with the supralittoral species on one hand and with terrestrial species on the other. This fact does not exclude however the systematic interest of the zymograms.

An alpha-L-rhamnosidase from the seeds of Fagopyrum esculentum (saracen corn) has previously been identified, and the effect of the enzyme on rhamnoisic bonds has been studied with various flavonoid glycosides. This alpha-L-rhamnosidase can be useful in structural studies, and a preliminary report of this study has appeared. The present paper describes the extensive purification of the enzyme and the determination of its properties. The purification involved extraction, ammonium sulfate fractionation and chromatography on Sephadex G 75, DEAE-Sephadex and Ultrogel AcA-44. The alpha-L-rhamnosidase was purified about 9600 fold and the final enzyme preparation was practically pure according to the criteria of disc electrophoresis. The molecular weight of this alpha-L-rhamnosidase, calculated from data obtained by disc gel electrophoresis and gel filtration, was about 70 000. Isoelectric focusing established the isoelectric point to be 3.7. The behaviour of the enzyme on a concanavalin-A-Sepharose column suggests the presence of residues resembling alpha-D-mannose or alpha-D-glucose in the protein. The various kinetic parameters, Kcat, Km and the Kcat/Km ratio have been determined at pH 5 on the following substrates: p-nitrophenyl-alpha-L-rhamnoside and rutinose (6-O-alpha-L-rhamnosyl-D-glucopyranose). All kinetics exhibit a Michaelian behaviour and the Km for the former substrate was 0.33 mM and for the latter, 2.2 mM. The Kcat/Km ratio corroborates the greater specificity of the enzyme for p-nitrophenyl-alpha-L-rhamnoside. L-Rhamnose, L-lyxose, 6-deoxy-D-glucose and methyl-alpha-D-mannoside were shown to behave strictly as competitive inhibitors of alpha-L-rhamnosidase activity; it seems that the methyl group of L-rhamnose is important for substrate binding to the enzyme.

In the genus Schizosaccharomyces intracellular osidases and nitrite and nitrate reductases are revealed; particularly all the species possessing invertase, alpha-glucosidase and alpha-galactosidase. These characters underline the homogeneity on the genus. On the basis of osidases, nitrite and nitrate reductases results, 2 groups can be distinguished in this genus.

Various enzyme activities can be detected in human seminal plasma; these are mainly hydrolases which traduct high proteolytic activities. Acid phosphatase constitutes the best prostatic marker, while alpha glucosidase characterizes well epididymal function. The enzymatic equipment of spermatozoa -excepted for acrosin- is very similar to that of seminal plasma, which suggests that most of sperm enzymes are incorporated from male genital secretions. Tight relationships exist between the enzymatic equipment of spermatozoa and their fertilizing capacity: motility is depending of enzymes contained in sperm mid piece and flagellum; acrosomal enzymes are necessary for penetration of oocytes. However we could only very rarely explain some case of male sterility by defect of semen enzymes.

Two strains of aerobic and mesophilic microorganisms were isolated from palm-tree plantation sand. They grew on insoluble polysaccharides: mannan, cellulose, chitin as only source of carbon. This lytic activity was used for the purification of the two strains. The morphology of the organisms and the presence of LL-diaminopimetic acid in their cell-wall are characteristic of the genus Streptomyces. Investigations lead to: 1) the characterization of their specific polysaccharidase activity toward insoluble and natural beta-and alpha-glycans (mannan, cellulose, chitin, pectine and starch) and the formation of soluble saccharides (mannobiose, cellobiose, beta-D-N-acetylglucosamine, galacturonic acid, and maltose); 2) the research for antagonist, or synergic, effect on pathogenic bacteria and certain phytopathogenic microorganisms; only in the case of these latter was a weak lytic activity exerted by the two Streptomyces isolates, but one of them was shown to stimulate Colletotrichum lindemuthianum and Phialophora cinerescens; 3) a study of antibiotic sensitivity; the two strains were sensitive to tetracycline and streptomycin, but they had native resistance to other aminosides (gentamicin, kanamycin), to erythromycin and to the beta-lactam antibiotics (penicillin and cephalosporin); they possessed a beta-lactamase bound to the cell membrane.

Glycosidase and peptidase of non-treated Mouse peritoneal macrophages were characterized. The enzymatic activities of Corynebacterium parvum stimulated Mouse macrophages were compared to those of macrophages obtained from non-treated animals. All the enzymatic activities, but beta-C-galactosidase, were higher in stimulated Mouse macrophages.

The digestive juice of Achatina balteata, a giant snail of the West African Coast catalyses the hydrolysis of several natural and synthetic compounds. Enzymatic activities on lactose, o- and p-nitrophenyl-beta-D-galactoside, p-nitrophenyl-beta-D-glucoside, p-nitrophenyl-beta-D (and alpha-L-) fucoside, o-nitrophenyl-beta-D-xyloside, p-nitrophenyl-N-acetyl-beta-D-glucosaminide and phenolphthalein-glucuronide have been shown to be present. The effect of pH and substrate concentration on these activities were studied. The galactosidase, glucosidase and fucosidase activities were studied with respect to temperature, heat inactivation, pH stability and incubation with trypsin. Kinetic experiments suggest the presence of several galactosidase activities. This hypothesis is confirmed by specific staining after polyacrylamide gel electrophoresis. These activities showed a broad specificity towards galactosides and glucosides. The digestive juice showed no action on acetyl-L-tyrosine and benzoyl-L-arginine ethyl esters. However a small protease activity was observed on hemoglobine. No lipase activity was found. Sulfatase content was low compared to that of Helix pomatia.

Extracts from the alimentary canal of Schistocerca gregaria Forsk allow hydrolysis of a remarkably wide range of oligosaccharides, heterosides and polysaccharides. Sucrose and starch are more rapidly and more intensely attacked, other osides much more slowly. The balance of released monosaccharides shows a clean predominance of glucose but some oses are released in appreciable amount only in case of delayed digestion.

Three bacterial species of different origin, Citrobacter freundii, Proteus mirabilis and Planococcus sp., formed glycocalyx in vitro and thereby showed that the phenomena does not only occur in vivo contrary to the opinion of many authors. The glycocalyx was produced in the attachment processes used or both extra- and intercellular attachment. The aggregates formed caused large errors in bacterial counts and led to questioning of the very principle of the counting procedures. The glycocalyx was not destroyed by the polyosidases utilized nor by ultrasound treatment. The glycocalyx structure differed only according to the bacterial species of origin and was unchanged by culture media or nutrient deprivation.

The main dietary carbohydrates are starch, cell wall polysaccharides (cellulose, hemicelluloses and pectins), some monosaccharides (glucose, fructose, galactose, etc.) and oligosaccharides (sucrose, lactose, alpha-galactosides, etc.). Recent analytical methods applied to these substances are described and criticized. A-type starches (cereals), cooked starches and some oligosaccharides are mainly digested in the small intestine of monogastric animals by enzymes of the salivary glands, pancreas and intestinal brush border. The total digestibility of these carbohydrates is almost 100%, whereas it is less than 70% for B-type starches (ex. potato). Cellulose, hemicelluloses, pectins and some oligosaccharides are partly digested by the microflora of the large intestine. Fiber total digestibility varies considerably and depends on the nature of the fiber and the animal species. It is less than 10% in chickens, whereas pigs seem to digest fibers as well as sheep. Hydrothermic treatments usually have no effect on starch digestibility but can be used for B-type starches. Some chemical treatments may improve fiber digestibility in monogastric animals.

We used a semiquantitative micromethod for detection of enzymatic activity (APIZYM) which allows a fast and simultaneous study of 67 hydrolase and dehydrogenase activities on dermo-epidermic skin samples. This wide investigated field led to the determination of the enzymatic profile (called zymogram) of the samples under study. This method was applied to normal skin and to psoriatic and uninvolved psoriatic skin; our results exhibited the good reproductibility of the method and the homogeneity of the studied samples. The enzymatic activities of the psoriatic skin are, in mean, twice those of normal skin. The uninvolved psoriatic skin showed reactions close to those of the normal skin. These observed variations of those different activities are perfectly similar to literature datas. The APIZYM method, thanks to its easy utilization and its fiability seems extremely interesting in the study of zymogram of cutaneous samples. The use of APIZYM in observations on naevus and malignant melanoma is to be exposed in a following publication.

The development of chemicall, physical and enzymatic methods lead to the determination of numerous structures of glycoprotein glycans and allowed to classify them into "structural families". On the basis of this knowledge, it has been possible, 1) to demonstrate that the oligosaccharides and glycoasparagines accumulating in tissues and urines of patients with diseases characterized by a lack in lysosomal glycosidases originate from glycoprotein glycans incompletely catabolized; 2) to propose a scheme for the normal and pathological catabolism of glycoproteins and 3) to elucidate the problem of the origin of lysosomal glycosidases. These latter are internalized into the lysosomes either through a mechanism of secretion-reuptake, or by following an intracellular traffic, or via the cell plasma membrane. In al cases, membrane receptors intervene which specifically recognize phosphorylated oligomannosidic structures carried by the acidic hydrolases.

The authors recall the enzymatic activity and transport activities which were localised in the glycocalyx, the microvillous membrane, the intracellular compartment and the basolateral membrane of the enterocyte. The relationships between the processes of digestion and absorption, on the one hand, active transport, energy metabolism and sodium and potassium dependent ATPase, on the other hand are considered. The application of these data to functional investigation of the enterocyte is discussed, emphasizing the complementary character of the biological tests used currently and the importance of sodium and potassium dependent ATPase in the phenomena of active transport.

After a screening performed on pectinolytic micro-organisms, a strain of B. subtilis was isolated. This strain has a pectic enzyme capable of beta-elimination on highly methylated pectin (degree of esterification = 85%), without the action of a pectinesterase. This activity corresponds to that of a pectin-lyase.

Like other solid tumors ovarian tumors have been intensely investigated these past years to isolate specific tumoral antigens. These antigens are now used by clinical teams for post therapeutic follow-up. However, out of the 25 major antigens actually described in ovarian carcinomas, the only one that can be used today is the carcinoembryonic antigen (CEA). Moreover, an international collaborative work has demonstrated cross reacting antigenicity between these major tumoral antigens. Cellular enzymes (galactosyl transferase and sialyl-transferase) and hormonal receptors for proteinic hormones such as steroid hormones) are being studied but cannot be used as screening markers at this time.

Amylase activity in blood and urine, lipase activity in blood, and amylase/creatinine clearance ratio have been prospectively compared in order to test these parameters against estimation of pancreatic isoamylase. Pancreatic isoamylase had been evaluated by inhibition methodology and not by electrophoresis. One hundred patients admitted for strictly sus-umbilical abdominal pain in emergency unit have been studied. Results show that we do not have in blood specific biochemical marker for acute pancreatitis. Lipase and P isoamylase activity evaluation have about the same specificity. In emergency situation the choice of routine investigation will be rather based on methodological simplicity and lower cost.

A new micromethod has been used to compare the enzymic profiles of two cell lines of Lepidoptera (Antheraea eucalypti and Malacosoma disstria). Differences are observed on alkaline phosphatase, on two aminopeptidases, on alpha and beta galactosidases and on alpha mannosidase. The whole intracellular activity of the enzymes revealed is detected without any previous treatment of the cells.

Two beta-D-glycosidases have been purified from the digestive juice of Achatina balteata by acetone fractionation, ion exchange chromatography through DEAE-Sephadex A-50, ammonium sulphate fractionation and gel chromatography through Sephadex G-200. The preparations are homogeneous by p/lyacrylamide gel electrophoresis. Both enzymes are highly specific for the beta-D-anomeric configuration of the glycosidic linkage. They hydrolyse lactose, cellobiose and synthetic beta-D-galactosides, -glucosides and -fucosides at a pH optimum of 5,2 to 5,6 and are inactive on alpha-glycosides. The hydrolyzed substrates are recognized by the same catalytic site as shown by mutual competition studies between substrates and competitive inhibition observed with aldonolactones and glycopyranoses such as D-galactose, D-glucose and D-fucose. The different substrates are not hydrolyzed at the same rate by the two enzymes. They also differ by their electrophoretic mobility, their behaviour in gel chromatography and their stability towards pH and heat. The most salient property is the important beta-D-fucosidase activity of the two purified enzymes.

Acid hydrolase activities were compared in human leucocytes, guinea pig and human alveolar macrophages. Several enzymes were characterized: N-acetyl-beta-D-glucosaminidase, N-acetyl-alpha- and beta-D-galactosaminidase, alpha and beta-D-galactosidase, alpha-D-mannosidase, alpha-L-fucosidase, beta-D-glucuronidase, neuraminidase, acid phosphatase and arylsulfatase. The enzymatic activities were lower in leucocytes than in alveolar macrophages, higher in human macrophages than in guinea pig macrophages, except for beta-D-glucuronidase, acid phosphatase and arylsulfatase activities.

A strain of Streptomyces hydrolysing the insoluble beta (1 leads to 4) mannan has been isolated from a soil of palm plantation. The first step in the degradation of the polysaccharide is a random hydrolysis by a beta mannanase, leading to mannotetra-, mannotri- and mannobiose. Liberation of free mannose is never observed. The hydrolysing pattern of oligomannosides and of their reduced homologues has been studied and a transfert reaction is postulated. This mannanase behaves as a true endopolysaccharidase.

An extract from living yeast forms of S. schenckii was prepared. The yeasts originated from a shake culture in B.H.I. broth (Difco) incubated for 3 days at 35 degrees C in darkness; they were harvested, washed and disrupted with glass beads in a model MSK Braun mechanical cell homogenizer; a freezing-thawing was added to improve the extract. After electrophoretic separation in agarose gel, the extract's components were characterized by their enzymic activity; with this technique, 30 bands were revealed. These enzymic activities were also investigated on the antigenic fractions of the extract revealed by a rabbit hyperimmunserum: 16 among 22 immunoprecipitates are identified by their catalytic properties. Study of the earliest precipitating antibodies (appearing-order and enzymic caracterization) in rabbits just immunized completes this work. How to ameliorate the quality of the extract by culture and extraction conditions is also specified.

The reaction pattern of the polygalacturonase from Helix pomatia juice has been studied. Hydrolysis of pectic acid occurs in a random manner. Oligogalacturonic acids are hydrolyzed in a sequential process, starting from the non-reducing end. This behavior is different from that of other endopolygalacturonases, which split these latter substrates both randomly and in a stepwise way from the reducing end.