[Congenital afibrinogenemia: a case report]. / Afibrinogénémie congénitale: à propos d'une nouvelle observation.

Afibrinogenemia is a rare autosomal recessive disease. Its clinical manifestations vary in severity, ranging from minimal bleeding to cataclysmic hemorrhage, and can begin at birth or, sometimes, later. We report a case of a female infant, 10 months of age, hospitalized in the pediatrics department because of a postvaccination hematoma. Biologic exploration found congenital afibrinogenemia. Through this case, we review the clinical features of this disease and its management.

Removal kinetic of Escherichia coli and enterococci in a laboratory pilot scale wastewater maturation pond.

During the last 15 years several authors studied the disinfection in waste stabilisation pond (WSP) and several empirical models were developed. There are huge differences between the models describing this process and there is really a need to improve the design of ponds for better disinfection. This paper addresses the Escherichia coli and enterococci disinfection in a laboratory pilot scale maturation pond (1.5 l) with light intensity (0, 12 and 25 W/m(2)) under controlled pH, temperature and dissolved oxygen (DO) conditions. The aim of this study is to improve modelling for a better design of disinfection in maturation ponds (MP) and to identify the key parameters influencing the process. It was found that kinetic coefficients K values for E. coli and enterococci are closely dependent on physicochemical parameters. K values increase with increasing pH, I, T and DO. E. coli disinfection depends closely on the pH and the DO and increases strongly when the pH is above 8.5. The enterococci disinfection depends essentially on DO. Two equations are suggested to calculate the kinetic coefficient K related to the environmental average state variables.

Mutagenesis of the bovSERPINA3-3 demonstrates the requirement of aspartate-371 for intermolecular interaction and formation of dimers.

The family of serpins is known to fold into a metastable state that is required for the proteinase inhibition mechanism. One of the consequences of this conformational flexibility is the tendency of some mutated serpins to form polymers, which occur through the insertion of the reactive center loop of one serpin molecule into the A-sheet of another. This "A-sheet polymerization" has remained an attractive explanation for the molecular mechanism of serpinopathies. Polymerization of serpins can also take place in vitro under certain conditions (e.g., pH or temperature). Surprisingly, on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, bovSERPINA3-3 extracted from skeletal muscle or expressed in Escherichia coli was mainly observed as a homodimer. Here, in this report, by site-directed mutagenesis of recombinant bovSERPINA3-3, with substitution D371A, we demonstrate the importance of D371 for the intermolecular linkage observed in denaturing and reducing conditions. This residue influences the electrophoretic and conformational properties of bovSERPINA3-3. By structural modeling of mature bovSERPINA3-3, we propose a new "non-A-sheet swap" model of serpin homodimer in which D371 is involved at the molecular interface.

Removal improvement of bacteria (Escherichia coli and enterococci) in maturation ponds using baffles.

Korba wastewater treatment plant is a conventional activated sludge followed by three maturation ponds (MP1, MP2, MP3) in series acting as a tertiary treatment. The first study of wastewater treatment plants showed that the effluent concentration of Escherichia coli and enterococci at the outlet of the (MP3) varies between 10(3) and 10(4)CFU/100 ml. After the hydrodynamic study conducted by Rhodamine WT which showed short-circuiting in the MP1, two baffles were introduced in the first maturation pond (MP1) to improve the hydrodynamic and the sanitary performances. The second hydraulic study showed that the dispersion number 'd' was reduced from 1.45 to 0.43 by this engineering intervention and the Peclet number was raised from 0.69 to 2.32. The hydraulic retention time was increased by 14 h. Because of well-designed baffles, the removal efficiency of E. coli and enterococci was raised between 0.2 and 0.7 log units for the first maturation pond.

Comparative analysis of existing disinfection models.

For a long time Marais's model has been the main tool for disinfection prediction in waste stabilization ponds (WSPs), although various authors have developed other disinfection models. Some ten other empirical models have been listed over the past fifteen years. Unfortunately, their predictions of disinfection in a given pond are very different. The existing models are too empirical to give reliable predictions: often their explanatory variables were chosen arbitrarily. In this work, we try to demonstrate that if influent variables have daily variations, the use of their average values in simulations may overestimate the disinfection effect. New methods are thus needed to provide better fittings of the models. Better knowledge of the mechanisms involved is needed to improve disinfection models.

Bovine muscle 20S proteasome: I. Simple purification procedure and enzymatic characterization in relation with postmortem conditions.

Over the last decade, several sets of evidence support a possible contribution of the 20S proteasome to the meat tenderizing process. This assumption was emphasized by recent investigations demonstrating that the 20S proteasome was active in the absence of activators and exhibited endo- and exoproteolytic activities, a status often strongly debated before. In the present work, we developed a new rapid and simple purification procedure for muscle 20S proteasome and revisited the physicochemical properties of this complex in relation with the postmortem muscle environmental conditions, i.e. temperature, pH, osmolarity, etc. From a crude extract obtained from freshly excised muscle tissue, reasonable amounts of highly pure proteasome were prepared within a maximum of 4 days using only three chromatography steps. This purified proteasome was used to investigate the effect of pH, temperature, ionic strength and sodium dodecyl sulphate (SDS) on the major hydrolytic activities of this complex, i.e. trypsin-like (TL), chymotrypsin-like (CL) and peptidylglutamyl peptide hydrolase (PGPH) activities. Taken together, the data obtained suggest that the 20S proteasome constitutes a high hydrolytic potential in postmortem muscle conditions. To attest this finding, the 20S proteasome was further quantified by ELISA in at death and postmortem muscles including Longissimus, Rectus abdominis, Diaphragma pedialis and Tensor fascia latae bovine muscles. The primary conclusion was that time course changes in proteasome concentrations were not dependent on the kinetics of the pH fall. Secondly, the proteasome concentration in conditioned meat was in good agreement with previously reported proteolytic activity. Furthermore, the decrease in the muscle proteasome concentration can be considered as slow and this is particularly true in type 1 muscles for which the decrease in the amount of this complex did not exceed 7% during the first three days postmortem. This would suggest that the 20S proteasome was relatively stable during meat conditioning, a feature supporting a potential role in the meat tenderizing process.

Bovine muscle 20S proteasome. II: Contribution of the 20S proteasome to meat tenderization as revealed by an ultrastructural approach.

The role of the 20S proteasome proteolytic effects was revisited using an ultrastructural approach with the aim to explain some particular structural changes identified in type I muscles and in high pH meat. In both types of meat, major changes observed after ageing are an increase in the thickness of the Z-line followed by the appearance of an amorphous protein structure spreading out over the I-band. This was followed by a total degradation of this amorphous structure and of the Z-line. Partial transversal fragmentation of the myofibrils within the I-band can also be detected. The data reported clearly demonstrate that the 20S proteasome was able to mimic these sequential structural changes, a feature never obtained with either calpains or cathepsins. It is the first time that a direct implication of this complex in postmortem muscle is postulated.

Bovine muscle 20S proteasome. III: Quantification in tissue crude extracts using ELISA and radial immunodiffusion techniques and practical applications.

The 20S proteasome is a large complex (700kDa) that exhibits endo- and exo-peptidase activities with wide specificity. In postmortem muscles, several sets of evidence suggest a possible significant contribution of proteasome to meat tenderisation. Hence, an accurate and rapid quantification procedure is needed to attest that new function during the ageing of meat. In the present work, we developed an ELISA test enabling the quantification of nM concentrations of the 20S proteasome. We further tested the radial immunodiffusion (RID) technique described as a more simple method that can quantitatively determine the concentration of an antigen in a complex mixture. The ELISA test allowed us to quantify the 20S protesome in tissue homogenates and fluids with a recovery of 100%, a coefficient of variation lower than 5% and a detection limit of 9ng/ml. Quantification of the 20S proteasome in various bovine tissue by ELISA showed the highest concentration in liver followed by spleen and kidney, with muscles exhibiting the lowest concentrations. In addition, measurement of the proteasome concentration in eight different bovine muscles with various metabolic profiles led to the conclusion that the relationship between muscle metabolic properties and proteasome concentration is rather complex. Nevertheless, heart muscle exhibited the highest proteasome content (331µg/g wet tissue) whereas the lowest values were found for M. Tensor Fascia Latae (213µg/g wet tissue), a fast twitch white muscle, M. Supraspinatus (209µg/g wet tissue), a slow twitch red muscle and M. Pectoralis profondus (203µg/g wet tissue), an intermediate muscle. As compared to other endogenous peptidases, muscle tissue contains relatively high amounts of proteasome. Hence this complex can be quantified using the RID, which allows quantification of protein in the µg range. Plotting the concentration values determined with both methods for all bovine tissues tested gave a straight line with a correlation coefficient of 0.99.

Serine peptidase inhibitors, the best predictor of beef ageing amongst a large set of quantitative variables.

For consumers, tenderness is the most important sensory attribute of beef meat and, though to a lesser extent, of pork. Tenderness is therefore by far the most common cause of its unacceptability. The major challenge for the beef industry is to evaluate the toughness of the meat as soon as possible after death. In this context, the aim of the present work was to develop an equation to predict the myofibrillar ultimate resistance of raw meat. The study was done on the Longissimus muscle from twenty three 19 months-old Charolais bulls grown in the same INRA farm. Muscles excised within 1h post-mortem were vacuum packed and stored at 15°C during 24h and then transferred to 4°C until used. The activities of lactate dehydrogenase, citrate synthase and myofibrillar Mg-Ca dependent ATPase, the levels of lactate dehydrogenase enzyme, myoglobin, myosin types I, IIa and IIb, cysteine and serine peptidase inhibitors, the pH, the osmolarity, the expressible juice, µ-calpain, m-calpain and calpastatin and meat toughness were measured. According to the physical method used here, the force measured on raw meat represents the resistance of the myofibillar structure. Stepwise linear regression was used to determine the best equation (p<0.05) for predicting toughness at 6 days post-mortem. A 6-variables predictive equation including serine peptidase inhibitors (partial R(2)=0.4), the rate (partial R(2)=0.25), and the extent of pH decline (partial R(2)=0.03), the at death LDH activity (partial R(2)=0.24), the extent of increase in osmotic pressure (partial R(2)=0.13), and the rate of µ-calpain activity loss (partial R(2)=0.09), explained 70% of the variability in meat toughness at 6 days post-mortem. This equation was developed from 20 animals and the other 3 animals, chosen randomly, were used to validate it. The absolute need for a predictive model of meat toughness and the nature of the serine peptidase inhibitors together with their potential target enzymes are discussed.

Purification of bovine cathepsin B: proteomic characterization of the different forms and production of specific antibodies.

Cathepsin B (EC 3.4.22.1) has been highly purified (14,225 fold) from bovine kidney by a rapid procedure that included the preparation of an enriched lysosomal extract, a selective fractionation with ammonium sulphate, size-exclusion chromatography, two cation-exchange chromatographies, and anion-exchange chromatography on diethylaminoethyl-Sephacel. After the last purification step, two hydrolytic peaks against Z-Phe-Arg-AMC were separated from each other, a minor peak corresponding to the cathepsin B single-chain form and a major one representing the double-chain form of cathepsin B. The single-chain form showed a molecular mass of 31 kDa on sodium dodecyl sulphate - polyacrylamide gel electrphoresis (PAGE) under reducing conditions, whereas the heavy chain of the double-chain form appeared as a doublet with molecular masses of 23.4 and 25 kDa, respectively. The identity of the different cathepsin B isoforms and the quality of the final enzyme preparation were confirmed by using two types of antibodies, one against a synthetic peptide sequence and one against purified cathepsin B. The proteomic study confirmed the identity of the different SDS-PAGE protein bands as cathepsin B isoforms and allowed the comparison and study of some structural differences between them at the level of their primary structures.

Plasma proteasome level is a potential marker in patients with solid tumors and hemopoietic malignancies.

BACKGROUND: Proteasomes are nonlysosomal proteolytic structures that have been implicated in cell growth and differentiation. Abnormal expression levels of proteasomes have been described in tumor cells, and proteasomes can be detected and measured in plasma. The objective of this study was to characterize differences in proteasome levels between normal, healthy donors and patients with neoplastic disease and to correlate the findings with clinical status and other biologic markers of disease spread. METHODS: Plasma proteasome levels were measured using a sandwich enzyme-linked immunosorbent assay in normal donors (n = 73 donors) and in patients with solid tumors (n = 20 patients), acute leukemia (n = 35 patients), myeloproliferative (n = 37 patients) and myelodysplastic (n = 19 patients) syndromes, chronic lymphocytic leukemia (n = 44 patients), non-Hodgkin lymphoma (n =104 patients), Hodgkin disease (n = 14 patients), other lymphoid disorders (n = 17 patients), and multiple myeloma (n = 27 patients). RESULTS: In the normal donors, the plasma proteasome concentration was 2356 ng/mL +/- 127 ng/mL. Patients with solid tumors exhibited a significantly higher value (7589 ng/mL +/- 2124 ng/mL), similar to the patients with myeloproliferative (4099 ng/mL +/- 498 ng/mL) and myelodysplastic (2922 ng/mL +/- 322 ng/mL) syndromes. Patients with lymphoproliferative disorders, in contrast, had significantly lower values than normal donors (1751 ng/mL +/- 107 ng/mL), except those in aggressive phase of the disease. This low level persisted in patients who were in complete remission. Proteasome levels decreased during the initial phase of treatment. Although there was a significant correlation with serum lactic dehydrogenase levels, frequent discrepancies were noted. There was no correlation with C-reactive protein or beta2-microglobulin levels, even in the group of patients with multiple myeloma. CONCLUSIONS: The plasma proteasome level is a potential new tool for the monitoring of patients with neoplastic disease. It is not correlated solely with cell lysis and may be involved in the pathophysiology of disease progression.

Myoglobin inhibition of most protease activities measured with fluorescent substrates is an artifact!

Myoglobin has been suggested to be a potential inhibitor of endogenous muscle proteases as different as cathepsin B, cathepsin L, cathepsin H and calpains all being supposed to be important in post-mortem muscle. The present work aimed at verifying the ability of myoglobin and its prosthetic group, hemin, to inhibit a series of endopeptidases including papain, cathepsin B, trypsin, calpains as well as two activities of the 20S proteasome. The conclusion of the present work was that inhibition of proteolytic activities of endopeptidases by myoglobin is an artifact. This was based on the following evidences: (1) a similar extent of inhibition was observed for all proteases tested whether myoglobin or hemin were added before starting the reaction or after having stopped it; (2) a quenching of the probes fluorescence by myoglobin and hemin; (3) no inhibition of calpains were found when assayed with non labeled casein as substrate and the activity expressed as the increase in the absorbency at 280 nm of the TCA soluble protein fragments.(1).

Purification and characterization of a new potential in vivo inhibitor of cathepsin L from bovine skeletal muscle.

Four papain-inhibiting peaks, labeled F-I, F-II, F-III, and F-IV, were fractionated from a crude bovine muscle extract by gel filtration chromatography on Sephadex G100, and the F-III fraction was analyzed. From F-III, a cysteine proteinase inhibitor was purified by two successive anionic exchange chromatography steps on Q-Sepharose and Mono-Q columns. This inhibitor has a molecular weight of about 30 kDa. Regarding its specificity toward different proteinases, the purified 30 kDa inhibitor was inactive against serine (trypsin and chymotrypsin) and aspartyl (pepsin) families. In contrast, cathepsin L, H, B, and papain, four enzymes of the cysteine class were strongly inhibited suggesting that this inhibitor was specific to the cysteine proteinase group. However, no inhibitory activity was shown against calpains. Kinetic parameters, including inhibition constants (Ki), rate constant for association (kass) and time required for almost complete inhibition of proteinase in vivo were determined. The values are consistent with a possible physiological function for this inhibitor protein in controlling in vivo cathepsin L activity.

pH is regulated differently by glucose in skeletal muscle from fed and starved rats: a study using 31P-NMR spectroscopy.

The aim of this study was to determine whether exogenous glucose metabolism influences the pH in superfused EDL muscle from growing rats fed or starved for 48 h (body weight 55 and 45 g, respectively). Energy state and intracellular pH of muscle were repeatedly monitored by 31P-nuclear magnetic resonance spectroscopy (31P-NMRS); glycogen and other energy metabolites were assayed enzymatically in muscle extracts at the end of the experiment. In EDL muscles from starved rats superfused with glucose for 4 h, intracellular pH was elevated (7-7.3), lactate concentration low, glycogen repletion very intense and citrate synthase activity high. We conclude that glucose was routed mainly toward both oxidative phosphorylation and glycogen synthesis in EDL muscles after food deprivation of rats. In contrast, the major pathway in muscles from fed rats may be glycolysis because the glycogen pool remained constant throughout the experiment. The additional and minor pH component (in the range of 6.5 to 6.8) seen in muscles from fed rats, even in the presence of exogenous glucose, might be due to impaired glucose utilization because this component appears also in muscles from starved rats superfused without glucose or with a nonmetabolizable analog of glucose. Consequently, direct pH measurement by 31P-NMR may be considered to be a precise criterion for evaluation of differences in metabolic potentialities of muscle studied ex vivo in relation to the nutritional state of rats.

Fish muscle cytoskeleton integrity is not dependent on intact thin filaments.

Striated muscle cytoskeleton was studied by ultrastructure and electrophoresis. Treatment of sea bass white muscle myofibrils and glycerinated fibres with calpain caused disruption of costameres, intermediate filaments, and Z-line, without altering sarcomeres. V8 protease also caused loss of costameres and Z-line, and disrupted sarcomeres without affecting the intermediate filaments. Recombinant lipase caused loss of Z-lines and also sarcolemma detachment, without changing sarcomeres or intermediate filaments. DNase-1 removed thin filaments and partially removed Z-lines while leaving intact the sarcolemma attachments and intermediate filaments. Calpain, V8 protease, lipase and DNase-1 treatments induced extensive loss of alpha-actinin from the Z-line, which could be related to titin cleavage (calpain, V8), phosphoinositide hydrolysis (lipase), and actin depolymerisation (DNase-1). These results show that the cytoskeletal components are independent of intact thin filaments.

Tissue distribution and characterization of a 30-kDa cysteine proteinase inhibitor from bovine skeletal muscle.

Gel chromatography on a Sephadex G100 column of a crude extract obtained from bovine diaphragma muscle separated four fractions (F-I, F-II, F-III and F-IV) in the range of 12-70 kDa that were active against either papain, trypsin or both. From the F-III fraction, a cysteine proteinase inhibitor was purified by two successive anionic exchange chromatographies on Q-Sepharose and Mono Q columns. The pooled active fraction had a Mw of approximately 30 kDa, and isoelectrofocusing revealed one band with a pI of 6.7. The papain-inhibiting activity was unaffected by dithiothreital or 2-mercaptoethanol treatment, and only one band was obtained after SDS-poly acrylamide gel electrophoresis under both reducing and non-reducing conditions. These results suggest that the 30-kDa muscle cysteine proteinase inhibitor did not contain disulphide bonds essential for activity and the protein was a monomer. This proteinase inhibitor is stable between 40 and 80 degrees C and pH 5-12. Furthermore, the 30-kDa inhibitor is stable to papain proteolysis. The tissue distribution of this inhibitor was investigated using double immunodiffusion and Western blot techniques that provided evidence for its presence in bovine heart, spleen, liver and lung and its absence in bovine plasma.

Comparison between two statistical models for prediction of turkey breast meat colour.

The aims of the present study were: (1) to determine the relevant objective measurements which could express visual assessment of turkey meat colour; and (2) to use these variables for the early prediction of the colour development of turkey breast meat. The colour of the meat was assessed subjectively by an expert at a processing plant at 24 hr post mortem, using a four-category scale (score a: light-pale meat, score b: light pink meat or normal meat, score c: dark meat, score d: very dark meat). Objective measurements included meat pH, temperature, dielectric loss factor, pigment concentration, L(∗) (lightness), a(∗) (redness) and b(∗) (yellowness) colour coordinates determined at different times post mortem. Colour coordinates and pH were chosen as relevant variables when measured at 1 and 4 hr post mortem and were used in prediction models. Linear analysis (canonical discriminant analysis) showed that the efficiency of prediction was 15%. A non-linear analysis (neural network) gave better prediction; the colour of the meat being correctly predicted for 70% of the muscles.

Predicting variability of ageing and toughness in beef M. Longissimus lumborum et thoracis.

The object of this study was to determine muscle characteristics which might predict meat toughness. Eleven Charolais cattle were slaughtered at approximately 26 months of age and the Longissimus lumborum et thoracis muscle was taken 1 hr post mortem and stored at 12 °C for 24 hr and then at 4 °C. The average half-life for ageing in these raw muscles was 4.6 days but the toughness varied widely between the animals. Toughness varied 3-fold and the rate of ageing varied 20-fold between animals. Correlations were done to determine which characteristics might explain this variability. Toughness was correlated positively with increase in oxidative status of muscle and the initial levels of calpastatin. Toughness was correlated negatively with the initial levels of µ- and m-calpains and cysteine and serine proteinase inhibitors, the initial pH values and the rates of their decline. The rates of ageing were highly correlated positively with the initial levels of proteinase inhibitors and the rates of decline of calpastatin and negatively with the ultimate amounts of expressible juice. There was a wide variability in tenderness in M. Longissimus lumborum et thoracis from similar animals. Variations in metabolism and enzyme activity controlled by inhibitors and calpains appear to be largely responsible for this variability.

Proteolytic activity of proteasome on myofibrillar structures.

The physiologic function of proteasome remains unclear. Evidence suggests a role in degradation of ubiquitin-protein conjugates, MHC antigen presentation, and some specificity of substrate within certain cell types. To explore further the properties of proteasome we have examined its effect on a well defined structure, the myofibril. We find that despite its large size (20S) proteasome is able to degrade myofibrils and intact, permeabilized muscle fibrils. The proteins degraded showed some specificity because actin, myosin and desmin were degraded faster than alpha-actinin, troponin T and tropomyosin. Changes in ultrastructure were slow and included a general loss of structure with Z and I bands effected before the M band and costameres.