Paule Boussard

Purification and characterization of a wound-inducible thaumatin-like protein from the latex of Carica papaya

A 22.137 kDa protein constituent of fresh latex was isolated both from the latex of regularly damaged papaya trees and from a commercially available papain preparation. The protein was purified up to apparent homogeneity and was shown to be absent in the latex of papaya trees that had never been previously mechanically injured. This suggests that the protein belongs to pathogenesis-related protein family, as expected for several other protein constituents of papaya latex. The protein was identified as a thaumatin-like protein (class 5 of the pathogenesis-related proteins) on the basis of its partial amino acid sequence. By sequence analysis of the Carica genome, three different forms of thaumatin-like protein were identified, where the latex constituent belongs to a well-known form, allowing the molecular modeling of its spatial structure. The papaya latex thaumatin-like protein was further characterized. The protein appears to be stable in the pH interval from 2 to 10 and resistant to chemical denaturation by guanidium chloride, with a DeltaG(water)(0) of 15.2 kcal/mol and to proteolysis by the four papaya cysteine proteinases. The physiological role of this protein is discussed.

Relationship between slime production, antibiotic sensitivity and the phagetype of coagulase-negative staphylococci

Three hundred and three strains of coagulase–negative staphylococci (CNS) were collected from the fingers of healthy donors (289) and from blood cultures (14). Twelve different species were identified (5 5. auricularis, 45 S. capitis, 15 S. cohnii, 86 S. epidermidis, 23 S. haemolyticus, 37 S. hominis, 1 S. lentus, 5 S. saprophyticus, 7 S. sciuri, 6 S. simulans, 54 5. xylosus and 19 5. warneri). Amongst these CNS strains, 151 were slime producers, 112 were phage–typable and 188, 133, 126 and 91 were, respectively, resistant to penicillin, teicopiamn, erythromicin and kanamycin. Slime–producing strains were resistant to at least seven antibiotics with a probability of 001 < P < 005. Non–slime–producing strains were sensitive to all the tested antibiotics with a probability of 0001 < P < 001. There was no direct relationship between antibiotic sensitivity and phage type, although a non–typable strain was more often resistant to seven or more antibiotics than a typable one (005 < P <01).

Study on the activation of phospholipases A2 by purinergic agonists in rat submandibular ductal cells

Extracellular ATP and benzoyl-ATP (Bz-ATP) increased the release of [3H]arachidonic acid ([3H]AA) from prelabeled rat submandibular gland (RSMG) ductal cells respectively two- and threefold. Both agonists also increased the release of [3H]AA from acini but at a lower level (+50% and +100% respectively). Carbachol had no significant effect on either cellular population. In ductal cells phorbol myristate acetate, an activator of protein kinase C, slightly increased the basal release of [3H]AA but did not affect the release of [3H]AA in response to ATP. Staurosporine, an inhibitor of protein kinases, inhibited the response to the purines. The removal of calcium from the extracellular medium decreased the response to ATP and Bz-ATP. Only barium could partly substitute for calcium to restore the purinergic response. Zinc inhibited the release of [3H]AA. Permeabilization of the cells with streptolysin O (SLO) activated the calcium-independent phospholipase A2 activity (iPLA2). The iPLA2, not the calcium-dependent PLA2 (cPLA2), released [3H]oleic acid ([3H]OA) from RSMG ductal cells. It is concluded that RSMG ducts have a higher PLA2 activity when compared to acini. This activity is accounted for by iPLA2 and cPLA2. Both enzymes are activated by P2X agonists by a staurosporine-sensitive mechanism. Cells permeabilized with SLO or membranes from Escherichia coli as a substrate are not good models to study the regulation of these enzymes. In intact RSMG ductal cells the two activities can be distinguished by rather specific inhibitors, by different ionic conditions and also by the fatty acid used to label the cells.

Structural characterization of two papaya chitinases, a family GH19 of glycosyl hydrolases

Abstract.Two chitinases, able to use tetra-N-acetylglucosamine, chitin and chitosan as substrates, were characterized after purification from Carica papaya latex. The complete amino acid sequence of the major form and about 40% of the minor one were determined through proteolytic digestions and mass spectroscopy analysis. Sequencing demonstrated that both papaya chitinases are members of the family 19 of glycosyl hydrolases (GH19). Based on the known 3-D structures of other members of family GH19, it was expected that papaya chitinases would adopt all-alpha structures. However, circular dichroism and infrared spectroscopy indicated, for the papaya chitinases, a content of 15–20% of extended structures besides the expected 40% of alpha helices. Since the fully sequenced papaya chitinase contains a large number of proline residues the possibility that papaya chitinase contains polyproline II stretches was examined in the context of their resistance against proteolytic degradation.

Prevention of the adhesion of Pseudomonas aeruginosa to human buccal epithelial cells

Abstract The influence of protamine on the adhesion of Pseudomonas aeruginosa to human buccal cells was investigated using 2 collection strains and 5 other strains isolated from the hospital environment. The serotype and pyocin type of the strains was established and their antibiotic sensitivity determined. For concentrations lower than 100 μg/ml there was a decrease in adhesion correlating with protamine concentration. Maximum inhibition was reached at about 100 μg/ml.

Difference of Pseudomonas aeruginosa sensitivity to chloroxylenol according to the culture medium

The authors recorded notable difference of sensitivity of Pseudomonas aeruginosa to chloroxylenol according to the growth medium; the amount of magnesium of the culture medium and the growth phase were not major factors. This difference, which can be extended to various strains, is due to a difference of permeability of the outer membrane. It is suggested that adsorption of medium components on the surface of the bacteria could participate in the phenomenon. Similar results were obtained with phenol and crystal violet.

Adhesion of Klebsiellapneumoniae to human epithelial cells and influence of protamine

Bacterial adhesion is an obligatory step leading to colonisation of the host. In vitro adhesion of Klebsiella pneumoniae, at 10 8 and 10 9 bacteria ml -1 , to buccal cells from three non-smoking donors showed that at 10 9 bacteria ml -1 the number of adherent microorganisms is more statistically significant (p<0.01, ANOVA) THAN AT 10 8 bacteria ml -1 . With protamine at a concentration of 100 μg ml -1 , under our experimental conditions this basic protein abolished 60% of the adhesion on buccal and urinary cells. For urinary cells from one donor, the number of adherent bacteria was generally greater when cells were collected in the second part of the menstrual cycle

In vitro modification of antimicrobial efficacy by protamine

Abstract The influence of serum and protamine on the in vitro effectiveness of some preservatives has been studied. The products tested were formaldehyde, benzalkonium chloride, esters of parahydroxybenzoic acid, benzoic and sorbic acids. Serum only strongly depleted the activity of benzalkonium chloride. In the case of protamine, this basic protein markedly enhanced the bactericidal effect of benzoic and sorbic acids and allowed a reduction in the concentration by a factor of four while still maintaining the same bactericidal power on Pseudomonas aeruginosa .

Influence of protamine on the susceptibility of Pseudomonas aeruginosa to cefotaxime, sulphadimethoxine, polymyxin B and some β-lactam antibiotics

Protamine, a basic protein, was shown to influence the activity of antibacterial agents. When added to tryptic soy broth it enhanced the bactericidal activity of cefotaxime, sulphadimethoxine, ticarcillin, piperacillin and carbenicillin against Pseudomonas aeruginosa. In contrast, the activity of polymyxin B against this species was markedly reduced in the presence of protamine.

Influence of protamine on the in vitro sensitivity of Pseudomonas aeruginosa to antibiotics

We have studied the influence of protamine on the sensitivity of three strains of Pseudomonas aeruginosa to various antibiotics: streptomycin, gentamicin, polymyxin B, and beta-lactams. While protamine enhanced the antibacterial action of beta-lactams towards P. aeruginosa, it did not alter the effect of aminoglycosides. The antibacterial power of polymyxin B, on the other hand, was drastically reduced. The observed changes in this strains sensitivity to antibiotics could be due to changes in the permeability of the outer membrane.