A comparative study of the thermal denaturation parameters of lysozyme in the dissolved and crystalline states.

Differential scanning calorimetry was used for investigating the conformational changes of lysozyme resulting from the combined actions of temperature and of denaturants at various concentrations. The transition temperatures, for the protein in the dissolved and in the crystalline states (tetragonal crystals, crosslinked by glutaraldehyde), were thus investigated in a variety of environmental conditions. The effect of a wide range of alcohols demonstrates that lysozyme, whether in solution or crystalline, displays structural features which are on the whole strikingly similar. By contrast, in the case of urea this similarity becomes apparent only for concentrations higher than 4 M. Molecular interpretation of the data, as discussed in the text, is entirely consistent with information from X-ray studies.

A relationship between anion transport and a structural transition of the human erythrocyte membrane.

Scanning microcalorimetry was employed as an aid in examining some structural features of the anion transport system in red blood cell vesicles. Two structural transitions were previously shown to be sensitive to several covalent and non-covalent inhibitors of anion transport in red cells. In this study, these transitions were selectively removed, either thermally or enzymatically, and the subsequent effect on 35SO2- 4 efflux in red cell vesicles was determined. It is shown that removal of one of these transitions (B2) has a negligible inhibitory effect on anion transport. Cytoplasmic, intermolecular disulfide linkages between band 3 dimers are known to form during the B2 transition. The integrity of the 4,4-diisothiocyanostilbene-2,2-disulfonate-sensitive C transition, on the other hand, is shown to be a requirement for anion transport. The localized region of the membrane giving rise to this transition contains the transmembrane segment of band 3, as well as membrane phospholipids. The calorimetric results suggest a structure of band 3 which involves independent structural domains, and are consistent with the transmembrane segment playing a direct role in the transport process.

Purification and characterization of papaya glutamine cyclotransferase, a plant enzyme highly resistant to chemical, acid and thermal denaturation.

Papaya glutamine cyclotransferase (PQC), present in the laticiferous cells of the tropical species Carica papaya, was purified near to homogeneity. Starting from the soluble fraction of the collected plant latex, a combination of ion-exchange chromatography on SP-Sepharose Fast Flow, hydrophobic interaction chromatography on Fractogel TSK Butyl-650 and affinity chromatography on immobilized trypsin provided a purification factor of 279 with an overall yield of 80%. In the course of the purification procedure, the two solvent accessible thiol functions located on the hydrophobic surface of the enzyme were converted into their S-methylthioderivatives. Papaya QC, a glycoprotein with a molecular mass of 33000 Da, contains a unique and highly basic polypeptide chain devoid of disulfide bridges as well as of covalently attached phosphate groups. Its absorption spectrum is dominated by the chromophores tyrosine which, nonetheless, do not contribute to the fluorescence emission of the plant enzyme. With a lambdamax of emission at 338 nm and a moderate susceptibility to be quenched by acrylamide, most of the tryptophyl residues of papaya QC appear to be sterically shielded by surrounding protein atoms. Fluorescence can thus be used to monitor unfolding of this enzyme. Preliminary experiments show that papaya QC is exceptionally resistant to chemical (guanidinium hydrochloride), acid and thermal denaturation. At first sight also, this enzyme exhibits high resistance to proteolysis by the papaya cysteine proteinases, yet present in great excess (around 100 mol of proteinases per mol of PQC) in the plant latex. Altogether, these results awaken much curiosity and interest to further investigate how the structure of this plant enzyme is specified.

Preparation and preliminary characterization of poly(ethylene glycol)-pepstatin conjugate.

The carboxyl function of pepstatin has been coupled, through an amide bond, to methoxypoly(ethylene glycol) (5 kDa), to which an amino function had been previously grafted. The mPEG-pepstatin conjugate inhibits hog pepsin (aspartic proteinase) in vitro as pepstatin itself, however, with a 400 times higher apparent Ki. The conjugate apparently does not inhibit proteinases belonging to other proteinase families such as serine (trypsin, carboxypeptidase Y), cysteine (Papaya proteinase III), or metallo (collagenase) proteinases.

Reversible modification of thiol-containing polypeptides with poly (ethylene glycol) through formation of mixed disulfide bonds. The case of papaya proteinase III.

Papaya proteinase III (PPIII) was purified, as the S-methylthio derivative from the latex of Carica papaya L., by ion-exchange chromatography. Separation of reactivable PPIII from the irreversibly oxidized molecular species of this enzyme was readily achieved after a selective conversion of the reactivated proteinase into the S-monomethoxypoly(ethylene glycol)thio derivative (S-mPEG thio PPIII). From this derivative, a PPIII preparation titrating 1 mol of thiol/mol of enzyme was regenerated. From the physicochemical properties of S-mPEG thio PPIII that were investigated, it is concluded that interactions between the mPEG and the PPIII chains occur only to a limited extent. In addition to its usefulness for purifying thiol-containing enzymes, the mPEG modification resulting from mixed disulfide bond formation may find other practical applications.

[Familial primary empty sella turcica. Apropos of a family with 3 cases]. / Selle turcique vide primitive familiale. A propos d'une famille comportant 3 cas.

Three members of a family, the father and two children, present a primary empty sella. The only clinical symptom is headache. The ophthalmologic examen, and the pituitary function are quite normal. No other anomaly is associated. This type of cases has never been published.

[Primperan and the dyspeptic syndrome]. / Primpéran et syndrome dyspeptique.

Primperan modifies the behaviour of the antro-pyloro-duodenal functional unit and is therefore a logical treatment for dyspeptic disorders. In a double blind study, the oral administration, before meals, of 30 mg of Primperan per day for 15 days was found to be significantly superior to a placebo in the correction of dyspeptic symptoms.

The alteration of the functional properties of human haemoglobin by spectrin.

Kinetic investigation by means of stopped flow techniques showed the rate of deoxygenation of haemoglobin to be slower in the presence of spectrin. At pH 7.15, the kinetic constant was 27.2 sec-1 in presence of spectrin instead of 34.3 sec-1 for haemoglobin alone. Also, equilibrium studies have revealed that the oxygen pressure for half-saturated haemoglobin decreased when spectrin was added to the reaction medium. At pH 7.35, the log (pO2)1/2 was 0.88 for haemoglobin in presence of spectrin instead of 0.93 for haemoglobin alone. From these results, an interaction between spectrin and haemoglobin may be suspected.

The influence of experimental conditions on the spectrin-hemoglobin interaction.

Human spectrin, when isolated, purified and stored in such conditions that preserve its tetrameric form, is able to associate with human hemoglobin as it is clearly shown by gel filtration. However, this hemoglobin-spectrin association does not seem to have a significant effect on hemoglobin oxygenation as indicated by equilibrium and rapid kinetics measurements.

[Treatment of intestinal nematode infections with albendazole. Preliminary results (author's transl)]. / Traitement des nématodoses intestinales par l'albendazole. Résultats préliminaires.

A randomized double-blind study, in order to evaluate effectiveness and tolerance of albendazole, was carried out in 186 african subjects presenting intestinal nematode infections in Mali and Senegal. The drug, well tolerated, given as a single dose of 0.4 g (adolescents and adults) or 0.2 g (children) is curative in more than 90% for ascariasis. For ancylostomiasis and trichuriasis, the effectiveness is less satisfactory in children, probably due to insufficient dosage in this age group.

[A left pseudo-pleurisy syndrome manifesting as a liver abscess complicating malignant localized amebic colitis]. / Syndrome pseudo-pleurétique gauch révélateur d'un abcès du foie complaiquant une amibiase colique maligne localisée.

The authors report on a case of localised bipolar malignant colic amebiasis in a 34 year old man, as a complication of undiagnosed benin colic amebiasis; the initial signs consisted of left basi-thoracic symptoms for which a sub phrenic abcess secondary to rupture of two abcesses of the left lobe of the liver was responsible. The surgical intervention consisted of a right hemicolectomy without anastomosis with a double derivation by ileostomy colostomy and drainage of the abcesses. The evolution was rapidly fatal by an irreversible state of shock.

Quantitative determination of polyethylene glycol based upon its salting out and partitioning of a dye into the resulting aqueous two-phase system.

A method is described that allows quantitative determination of polyethylene glycol (PEG) concentrations by spectrophotometric measurement of fluorescein dye absorbance after its partitioning into an aqueous two-phase system containing mPEG (M(r) 5 kDa) in the upper phase and ammonium sulfate in the lower phase. The absorbance decrease of fluorescein in the lower phase is directly proportional to the mPEG concentration, with two proportionality constants equal to 4.42 x 10(5) and 2.84 x 10(5) M-1 cm-1 in the range of 0-0.4 and 0.4-1 microM, respectively. This experimental technique can be extended to PEGs of other molecular weights by means of calibration curves that give for each size of PEG the adequate proportionality constants. The results indicate that the quantitative determination is not affected by the presence of many substances such as proteins, reducing agents, and salts, at the usual concentrations.

Revisiting the enzymes stored in the laticifers of Carica papaya in the context of their possible participation in the plant defence mechanism.

In the tropical species Carica papaya, the articulated and anastomosing laticifers form a dense network of vessels displayed in all aerial parts of the plant. Damaging the papaya tree inevitably severs its laticifers, eliciting an abrupt release of latex. Besides the well-known cysteine proteinases, papain, chymopapain, caricain and glycyl endopeptidase, papaya latex is also a rich source of other enzymes. Together, these enzymes could provide an important contribution to plant defence mechanisms by sanitising and sealing the wounded areas on the tree.

Preliminary characterization of bovine beta-lactoglobulin after its conjugation to polyethylene glycol.

The major component of the whey fraction of bovine milk, beta-lactoglobulin (betaLG), has been transformed by grafting polyethylene glycol chains either on the thiol group (free and after reduction of the S-S bridges) of the cysteine residues, or on the amino group of the lysine residues and/or of the N-terminal amino acid. Acylation of the protein was achieved at a controlled pH of 7.0 using increasing ratios of activated PEG to betaLG. Transformation of the dimeric form into the monomer occurred at least for the fully pegylated adduct. The number of polymer chains fixed per mole of protein was determined by dosage of the free amino functions still present after reaction. The incidence of pegylation on the secondary structure of betaLG was evaluated using the Fourier Transform Infrared Spectroscopy (FTIR). Denaturation studies with guanidinium hydrochloride (Gu-HCl) by means of spectrofluorimetric measurements, showed an identical behavior of native as well as of pegylated betaLG.The antigenicity of the fully pegylated adduct was examined through antigenic competition towards native betaLG. The pegylated protein exhibited less than 1/100 of the native betaLG inhibition capacity, that could moreover never be complete. This is thus demonstrating the loss of accessibility for at least multiple conformational epitopes through pegylation procedure.Spectrofluorimetric measurements showed that betaLG-N-PEG(7) was still able to bind retinol while no effect on the intrinsic fluorescence could be detected by adding palmitic acid. Whether this last ligand binds or not to pegylated betaLG is discussed.

Comparative study on two kidney graft rinsing and preservation solutions in terms of the post-transplantation risk of delayed graft function and cost.

OBJECTIVE: To determine whether Belzer solution (Viaspan, Bristol-Myers Squibb, Brussels, Belgium), which is more expensive than Eurocollins solution, was better at preventing delayed graft function (DGF) and whether it was cost-effective as it could potentially reduce post-transplantation complications. METHOD: The risk of occurrence of complications associated with the use of these two rinsing and preserving solutions was estimated from a survey of 106 patients undergoing renal transplantation between 1 January 1993 and 31 March 1998. Both efficacy and adverse outcomes were recorded along with the costs directly associated with the transplantation procedure in the hospital setting: hospitalization, rinsing and preserving solutions, medical and technical interventions and diagnostic tests. RESULTS: For the 45 kidney grafts rinsed and preserved with Eurocollins (strategy S1: n1 = 45) the cost/graft was estimated at 40 euros. With Viaspan (strategy S2: n2 = 61) the corresponding cost/graft was 424 euros. Logistic regression analysis showed that Viaspan was better than Eurocollins solution (ebeta = 0.437; P = 0.05) in preventing DGF. Overall, S2 was less expensive than S1, from the hospital's perspective. The mean difference per patient was 278 euros, which amounts to a saving of 2% of the total cost per renal transplantation. For rinsing and preserving kidney grafts Belzer solution is therefore preferable to Eurocollins solution.

Under proper control, oxidation of proteins with known chemical structure provides an accurate and absolute method for the determination of their molar concentration.

Oxidation at 120 degrees C of inorganic and organic (including amino acids, di- and tripeptides) model compounds by K(2)Cr(2)O(7) in the presence of H(2)SO(4) (mass fraction: 0.572), Ag(2)SO(4) (catalyst), and HgSO(4) results in the quantitative conversion of their C-atoms into CO(2) within 24 h or less. Under these stressed, well-defined conditions, the S-atoms present in cysteine and cystine residues are oxidized into SO(3) while, interestingly, the oxidation states of all the other (including the N-) atoms normally present in a protein do remain quite unchanged. When the chemical structure of a given protein is available, the total number of electrons the protein is able to transfer to K(2)Cr(2)O(7) and thereof, the total number of moles of Cr(3+) ions which the protein is able to generate upon oxidation can be accurately calculated. In such cases, unknown protein molar concentrations can thus be determined through straightforward spectrophotometric measurements of Cr(3+) concentrations. The values of molar absorption coefficients for several well-characterized proteins have been redetermined on this basis and observed to be in excellent agreement with the most precise values reported in the literature, which fully assesses the validity of the method. When applied to highly purified proteins of known chemical structure (more generally of known atomic composition), this method is absolute and accurate (+/-1%). Furthermore, it is well adapted to series measurements since available commercial kits for chemical oxygen demand (COD) measurements can readily be adapted to work under the experimental conditions recommended here for the protein assay.