Role of calpains in postmortem proteolysis in chicken muscle.

Tenderness is governed by postmortem biochemical processes, particularly proteolysis. In mammals, the calpain system is generally accepted as the main system involved in postmortem proteolysis. In poultry, the 2 calpains (mu and mu/m--a form only found in bird tissue) have greater calcium sensitivity. In this study, we quantified by zymography the changes in postmortem calpain system activity. The mu/m-calpain activity remained steady, whereas the mu-calpain activity had disappeared by 6 h after postmortem, showing an activation by calcium. Changes in the electrophoretic pattern of sarcoplasmic and myofibrillar proteins are observed in the first postmortem hours concomitantly to the decrease in mu-calpain activity. The 30-kDa protein, considered as a good marker of postmortem aging in cattle, appeared from 6 h and then steadily increased. In chicken muscle, the rapid maximum tenderness reached could be explained by a greater activation of the calpain system.

Proteins dealing with N-ethylmaleimide inhibition of pig heart mitochondrial phosphate transport system.

Our data clearly demonstrate that protective effect of phosphate and protective effect of mersalyl against NEM-inhibition of phosphate transport act at the level of two kinds of proteins. (1)Two major components are phosphate and nigericin NEM sensitive. According to our previous data [13] it has been also demonstrated that these two proteins components are valinomycin NEM sensitive (results not shown here) suggesting a relationship between these proteins and the energy linked proton translocation process. Relationships between these proteins and the phosphate translocation process are not evident and are under further investigations. (2) Two other insoluble major components localised at the level of the subparticular fraction are mersalyl NEM sensitive. We can suggest that these proteins are implicated in the translocation of phosphate in pig heart mitochondria.

Effect of phosphate and ionophores on (14C)-NEM incorporation in mitochondrial membranes and relationships with phosphate carrier system.

Phosphate transport in mitochondria was investigated with respect to its inhibition by NEM. The reactivity of the Pi carrier SH groups was influenced by phosphate or ionophores during preincubation before the addition of NEM. Furthermore in order to obtain some mitochondrial protein fractions where the typical effects of phosphate and ionophores on [14C]-NEM fixations were observed, mitochondria were submitted to hypotonic treatment and sonication. The following results were obtained: 1. -- Phosphate and grisorixin (a new ionophore of the nigericin group) decreased the inhibition of phosphate transport by NEM. The same effect was observed for [14C]-NEM incorporation. 2. -- Valinomycin increased [14C]-NEM incorporation. The valinomycin effect was abolished by phosphate. ClCCP alone affected [14C]-NEM incorporation slightly. Valinomycin plus ClCCP decreased NEM inhibition of phosphate transport and [14C]-NEM incorporation like grisorixin. 3. -- The variability of SH group reactivity can be interpreted by a control of SH group accessibility by transmembrane delta pH as previously suggested. 4. -- Typical effects of phosphate or ionophores were observed in whole pig heart and rat liver mitochondria. These effects were enhanced in the same supernatant protein fraction resulting from sonication in pig heart mitochondria : phosphate decreased [14C]-NEM incorporation by 1,50 nmoles/mg protein, grisorixin by 0.95 nmoles, whereas valinomycin increased it by 0.75 nmoles. For rat liver mitochondria the phosphate effect and the valinomycin increased it by 0.75 nmoles. For rat liver mitochondria the phosphate effect valinomycin effect on [14C]-NEM incorporation were observed in the subparticular fraction obtained after sonification.

Phosphate transport and proteins with SH groups in rat liver mitochondria.

Phosphate transport in rat liver mitochondria was studied by following [32P] phosphate uptake within physiological concentrations. Transport inhibition due to mersalyl and protection by mersalyl against N-ethylmaleimide measured in those conditions corresponded to earlier results obtained by the swelling technique. When mitochondria were incubated with [3H] N-ethylmaleimide in the presence of mersalyl, the radioactive labeling in proteins of particles obtained after sonication was decreased in all fractions, but three proteins were both highly alkylated and also highly protected by mersalyl (M.W. 48,000 - 36,000 - 31,000). Two of these (M.W. 36,000 and 31,000) were partially purified by ultrogel chromatography in the presence of sodium dodecyl sulfate. Furthermore, it was shown that both phosphate and nigericin diminished labeling by N-ethylmaleimide in the final supernatant fraction. Two proteins (M.W. 98,000 and 31,000) were significantly alkylated by [3H] N-ethylmaleimide and protected by phosphate and nigericin.

Glycosylation and deglycosylation of proteasomes (prosomes) from calf-liver cells: high abundance of neuraminic acid.

Proteasomes (prosomes) of calf-liver cells were probed with three different biotinylated lectins: Limulus polyphemus agglutinin (LPA), specific for neuraminic acid; Solanum tuberosum agglutinin (STA), specific for GlcNac; and concanavalin A (Con A), specific for Man/Glc. While only one proteasomal protein reacted with STA, most of the proteasomal proteins reacted with LPA and several with Con A. Deglycosylation with N-glycosidase F showed that the detected glycan residues were asparagine-linked. Finally we demonstrate an alternative method for the isolation of proteasomes based on the affinity of certain proteasomal proteins to Con A.

Replicon typing of 71 multiresistant Serratia marcescens strains.

Replicon typing is the identification of plasmids by hybridization with specific DNA probes which contain the genes involved in plasmid maintenance. This new method has been used to classify plasmids into replicon (rep) groups which can often be correlated with incompatibility (Inc) groups. We studied 71 multiresistant Serratia marcescens strains with 19 rep probes constructed from reference plasmid replicons belonging to known Inc groups. These probes are known to react with enteric bacterial plasmids. However, they did not represent the totality of the thirty known Inc groups. For 52% of the studied strains, plasmids were identified and classified into groups FIB, FIC, FIIA, HI2, L/M, N, B/O, P, W, Y and Com9. Most (79%) of the plasmid preparations hybridized with a single rep probe, and 21% hybridized with two different probes. Electrophoretic analysis of DNA suggested that double hybridization could result from the presence of either two different Inc plasmids in the same strain (e.g. S37) or one single plasmid with a multireplicon (e.g. S113).

Use of pulsed-field gel electrophoresis as an epidemiologic tool during an outbreak of Pseudomonas aeruginosa lung infections in an intensive care unit.

OBJECTIVE: A retrospective study was performed to evaluate the use of DNA polymorphism analysis by pulsed-field gel electrophoresis (PFGE) in assessing the rate of exogenous contamination during an outbreak of Pseudomonas aeruginosa lung infections in an intensive care unit ICU. Another goal was to determine the risk factors, involved in the outbreak. DESIGN: Rectal swabs and tracheal secretions were cultured from all patients upon admission and thereafter once a week throughout their stay in the ICU. Resistance patterns were determined in all P. aeruginosa isolates. We determined the serotypes, pyocin types, plasmid profiles and total DNA macrorestriction patterns for isolates. The restriction fragment length polymorphism (RFLP) of Dra I total DNA digest was studied by PFGE. A retrospective case-control study was performed to determine the risk factors for P. aeruginosa bronchopulmonary colonization. SETTING: The study was carried out in the medical ICU of Besancon University Hospital (France). RESULTS: The typability, stability and reproducibility of phenotypic markers were not completely satisfactory. Only the RFLP profile satisfied all the criteria for a good typing technique. In four of the 17 patients, P. aeruginosa strains with the same DNA pattern were found. Among the previously reported risk factors for hospital-acquired bronchopulmonary infections, only invasive procedures were determined by multivariate analysis to be significant in our study group. The oropharynx and the bronchial tract are the most likely endogenous sources. CONCLUSION: PFGE-RFLP is a valuable tool for the epidemiologic study of P. aeruginosa. This typing method revealed that exogenous contamination is not always the major source of P. aeruginosa lung infections in mechanically ventilated patients in ICUs.

Discriminatory power and usefulness of pulsed-field gel electrophoresis in epidemiological studies of Pseudomonas aeruginosa.

We assessed the discriminatory power of pulsed-field gel electrophoresis (PFGE) for the analysis of DNA restriction fragment length polymorphism (RFLP) in Pseudomonas aeruginosa. We determined DraI PFGE-RFLP of DNA of unrelated clinical and environmental strains, and clinical strains isolated from two intensive care units of the Besançon University Hospital. The typeability and reproducibility was 100%. The discriminatory index was 0.998, and the DNA patterns were stable in vitro and in vivo. There was a very low correlation between PFGE-RFLP and traditional typing methods. The typeability, reproducibility, the high discriminatory power and the stability of PFGE-RFLP make this a valuable method to be used in conjunction with serotyping. Further standardization and quantitative interpretation are possible and should lead to this technique becoming a library typing system.

Nasal carriage of Staphylococcus aureus and cross-contamination in a surgical intensive care unit: efficacy of mupirocin ointment.

A six month prospective study was carried out in a surgical intensive care unit (SICU) of a university hospital to assess the incidence and routes of exogenous colonization by Staphylococcus aureus. A total of 157 patients were included in the study. One thousand one hundred and eleven specimens (nasal, surgical wound swabs, tracheal secretions obtained on admission and once a week thereafter, and all clinical specimens) were collected over a four month period from patients without nasal decontamination (A). They were compared with 729 specimens collected over a two month period from patients treated with nasal mupirocin ointment (B). All S. aureus strains were typed by restriction fragment length polymorphism (RFLP) pulsed-field gel electrophoresis after SmaI macrorestriction. The nasal colonization rates on admission were 25.5 and 32.7% in groups A and B, respectively. Thirty-one untreated patients (31.3%) and three patients (5.1%) treated with nasal ointment, acquired the nasal S. aureus in the SICU (P = 0.00027). Nasal carriers were more frequently colonized in the bronchopulmonary tract (Bp) and surgical wound (Sw) (62%) than patients who were not nasal carriers (14%) (P < 0.00001). The patterns were identical for nasal, Bp and Sw strains from the same patient. RFLP analysis characterized seven epidemic strains of methicillin-resistant S. aureus (MRSA) which colonized 60% of group A and 9% of group B patients (P < 0.00001). The bronchopulmonary tract infection rate was reduced in group B (P = 0.032). In conclusion, in an SICU, nasal carriage of S. aureus appeared to be the source of endogenous and cross-colonization. The use of nasal mupirocin ointment reduced the incidence of Bp and Sw colonization, as well as the MRSA infection rate.

Typing of hospital strains of Xanthomonas maltophilia by pulsed-field gel electrophoresis.

We report the use of pulsed-field gel electrophoresis (PFGE) to characterize Xanthomonas maltophilia isolates from an incident of hospital-acquired infection over a 12-day period in a haematological unit. Ten isolates from five patients (from throat, urine, stool and blood) and two isolates from environmental sites in the unit were compared with 10 epidemiologically unrelated clinical strains, isolated over a 3-month period from several units in two hospitals, by PFGE of DraI digests of chromosomal DNA. The profiles obtained were stable, reproducible and discriminatory. The 10 unrelated strains had different DNA profiles. Each of the five patients in the unit was colonized by a different strain and the isolates from a water faucet and a shower pommel had the same DNA profile as the strains of two patients who had used these fittings. A case-controlled study showed that the only factor that correlated with colonization was the origin of the patient from another unit (P = 0.0122). We conclude that PFGE is a rapid and discriminatory technique for the typing of X. maltophilia where a common origin of infection is suspected.

The effect of proteasome on myofibrillar structures in bovine skeletal muscle.

When bovine myofibrils are incubated with the 20S proteasome their structure is rapidly damaged with loss of material, particularly from the Z discs and I bands. After 24 hr of incubation the myofibrils rupture and debris appears. Certain myofibrillar proteins, including nebulin, myosin, actin and tropomyosin, are hydrolysed during the incubation; others are solubilised (α-actinin). The 20S proteasome completely and rapidly hydrolyses purified myofibrillar proteins in an energy-independent manner. This shows that the 20S proteasome probably plays a role in the postmortem transformation of muscle and more generally in the hydrolysis of cellular proteins.(1).

Activities of metabolic and contractile enzymes in 18 bovine muscles.

Contractile and metabolic characteristics of 18 bovine muscles were studied using 10 animals of various ages (2-10 years), sexes (females, males and castrated males) and types (dairy, meat or crossbred). Contractile type was assessed by measuring myofibrillar Ca-Mg-activated ATPase activity. Metabolic type was appraised by determining phosphorylase, glycogen synthetase, hexokinase, haem iron as well as glycolytic and mitochondrial oxidative activities. 'Glycolytic potential' and buffer capacity (as change in lactate concentration as a function of change in pH) were measured. The results showed that myofibrillar ATPase activity was positively related to glycolytic activities and negatively related to oxidative activities. Ultimate pH was negatively correlated with myofibrillar ATPase activity and had little if any relationship to the glycolytic potential. Metabolic and contractile activities were essentially unaffected by sex, age or breed type.

[Organ culture preservation of the human cornea at +31 degrees C and risk of infection]. / Conservation en culture d'organe à +31 degrees C de la cornée humaine et risque infectieux.

PURPOSE: In order to reduce the risk of infection, we analyzed each stage of conservation of human cornea in organ culture at +31 degrees C. METHODS: This epidomiological study was conducted in 266 human corneas preserved in organ culture between January 1991 and December 1993. There were 3 stages: In the period of preservation (analysis of the contaminated medium), Before clinical use of the graft (analysis of the preservation medium), After the penetrating keratoplasty (analysis of the corneo-scleral rim and the transportation medium). The bacteriological media used were thioglycolate broth, trypticase soja and Sabouraud. RESULTS: In 266 storage media, 42 (15.7%) cultures are positive. The most commonly found organism was Staphylococcus aureus (21.4%). At the end of the conservation procedure, all of the cultures of the media were sterile (n = 165). After penetrating keratoplasty, 8 cultures were positive for the transportation medium and the corneo-scleral rim (5.1%), 3 cultures were positive for the corneo-scleral rim only (1.9%) and 5 cultures were positive (3.2%) for the transportation medium without contamination of the corneo-scleral rim. CONCLUSION: Preservation at +31 degrees C in organ culture of human corneas allows elimination of the contaminated or potentially contaminant corneas before an eventual transplantation. In our experience, the risk of infection is especially situated in the period of preservation which shows the insuffiency of the decontamination procedures or the antibiotical content of the medium and probably the virulence of the organisms in donors hospitalized for long period.

[Listeriosis in patients with malignant hemopathy. 3 cases in the same hospital ward]. / Listériose chez des malades ayant une hémopathie maligne. Trois observations dans un même service.

During the listeriosis epidemic which occurred in France in the summer 1992, three patients with malignant haematopathies hospitalized in our service contracted the disease. Although the retrospective investigations were hindered by the variable incubation period and the impossibility of examining the foods eaten at the time of infection, there was a high probability that two of the patients had been infected by cooked ham and dairy products at home. The third patient was apparently infected in hospital with well-cooked food found to be contaminated. The hypothesis of coinfection has been raised.

[Pseudomonas aeruginosa septicemia. Host-related risk factors in 82 episodes]. / Septicémies à Pseudomonas aeruginosa. Facteurs de risque liés à l'hôte au cours de 82 épisodes.

OBJECTIVES: The prognosis of septicaemia due to Pseudomonas aeruginosa is severe with mortality ranging from 32 to 73%. We retrospectively studied 82 episodes in order to determine whether risk factors could be identified. METHODS: Eighty-two episodes of Pseudomonas aeruginosa septicaemia, observed between 1986 and 1991, were analyzed. Risk of death within 2 days of the first positive blood culture (mortality = 19.5%) were assessed with univariate and multivariate analyses. RESULTS: Patient age ranged from 1 to 92 years. Most had been hospitalized in medical wards (49%) or intensive care units (28%) (NS). The type of septicaemia (several bacteria in 21%), the source of the infection (nosocomial in 78%), portal, predisposing factors (cancer, haematologic disease: 54%) and MacCabe index were not significantly correlated with risk of death at two days following first positive blood culture. With univariate analysis body temperature below 38,5 degrees C was significant (p = 0.007) for death at day 2 and appropriate antibiotic treatment after diagnosis was significant (p < 0.001) for absence of death on day 2. For multivariate analysis, chemotherapy and shock syndrome were significant (p = 0.005 and 0.09 respectively) for death at day 2 and appropriate antibiotic treatment was significant (p = 0.005) for absence of death on day 2. CONCLUSION: Antibiotic prescription appears to be the most easily controlled significant factor predictive of outcome in Pseudomonas aeruginosa septicaemia.

Muscle differentiation in the bovine fetus: a histological and histochemical approach.

The chronology of muscle fiber differentiation was analysed in 37 fetal calves of 69 to 266 days of age. Semitendinosus muscle weight was measured throughout the experimental period and biochemical, histological and histochemical investigations were made to determine respectively the protein and DNA content of the muscle, the size and the number of the fibers and their ATPase and SDH activity. The relative growth of all the quantitative characteristics (muscle weight, protein and DNA content) was much greater in the early stages of gestation than in the new-born animal. In the younger fetuses DNA relative growth was faster than protein relative growth, whereas at the end of gestation the reverse progression was observed. Before 90 days, the muscle tissue was composed of myotube-like cells without any clear organization. The organization of muscle tissue into clear bundles occurred around 120 days of age, and about 30 days later the large myotubes transformed into myofibers. The myotubes reacted positively for acid-ATPase activity, whereas the large population of smaller cells which developed in parallel did not. The number of muscle cells increased up to 240 days of age, as did the percentage of fibers positive for acid-ATPase activity. Finally, oxidative differentiation occurred around 260 days of age, with the appearance of a population of cells characterized by increased SDH activity. A comparison of these results with previous findings suggests that the muscular tissue differentiates through similar stages in various species, but over different lengths of time. The percentage of mature weight might provide a better inter-species time scale than chronological age.

[Pseudomonas aeruginosa infection in cystic fibrosis: predominance of a single strain and the influence of antibiotic therapy and the environment]. / Infection à bacille pyocyanique au cours de la mucoviscidose: prédominance d'un clone, influence des antibiotiques et de l'environnement.

We analysed the implantation pattern and persistence of Pseudomonas aeruginosa in the tracheobronchial tracts of patients with cystic fibrosis, and investigated the relation of this bacterium with the environment and antibiotic therapy. We used four different techniques to ensure the precise and detailed identification of isolates. In particular, chromosomal differences were assessed by pulsed field electrophoresis (pulsotype determination). Sputum samples were collected from 8 patients, from 6 to 22 years-old, over a period of 19 to 24 months. Only a single strain was isolated from samples from each five patients taken at different times, and there was a predominant strain in the samples from two others. Patient 8, aged 14, was free of infection throughout the study. None of the infections was eradicated by antibiotic therapy (an association of two antibiotics for 15 days at one or two months interval). The strains isolated from two patients became resistant to imipenem: 3 out of the 4 resistant strains were the result of mutation in the resident, susceptible strain. Swabs were taken from the environments of infected patients and were tested for P. aeruginosa: this bacteria was found in three sites, and two of these contained an isolate with the same pulsotype as the strain responsible for the infection, whereas no P. aeruginosa was detected in the environment of an uninfected patient. The detailed and accurate identification of the isolates (by pulsotyping) enabled us--to show that each infected patient was infected by a single or predominant strain,--to investigate the relationship of these strains with those in the environment and the effects of antibiotic therapy.

Glucose lowering therapeutic strategies for type 2 diabetic patients with chronic kidney disease in primary care setting in france: a cross-sectional study.

Aim. To understand glucose lowering therapeutic strategies of French general practitioners (GPs) in the management of type 2 diabetes mellitus (T2DM) patients with chronic kidney disease (CKD). Methods. A multicenter cross-sectional study was conducted from March to June 2011 among a sample of French GPs who contribute to the IMS Lifelink Disease Analyzer database. Eligible patients were those with T2DM and moderate-to-severe CKD who visited their GPs at least once during the study period. Data were collected through electronic medical records and an additional questionnaire. Results. 116 GPs included 297 patients: 86 with stage 3a (Group 1, GFR = 45-60 mL/min/1.73 m(2)) and 211 with stages 3b, 4, or 5 (Group 2, GFR < 45 mL/min/1.73 m(2)). Patients' mean age was approximately 75 years. Insulin was used in 19% of patients, and was predominant in those with severe CKD. More than two-thirds of patients were treated with glucose lowering agents which were either contraindicated or not recommended for CKD. Conclusion Physicians most commonly considered the severity of diabetes and not CKD in their therapeutic decision making, exposing patients to potential iatrogenic risks. The recent patient oriented approach and individualization of glycemic objectives according to patient profile rather than standard HbA1c would improve this situation.

Chronic kidney disease in type 2 diabetes patients in France: prevalence, influence of glycaemic control and implications for the pharmacological management of diabetes.

AIM: Type 2 diabetes mellitus (T2DM) is often associated with chronic kidney disease. For this reason, this article reviews the relationship between treatment of T2DM and renal disease. METHOD: The review presents the recent French data on the management of diabetes in patients with renal impairment, and discusses the implications of renal disease for the treatment of such patients. Prescribing data are presented for various antidiabetic treatments, and the use of the more commonly prescribed medications is discussed with reference to T2DM patients with renal disease. RESULTS: In France, it is estimated that 4-5% of the general population has T2DM and that almost 40% of patients with end-stage renal failure have diabetes. Diabetes and renal disease are both risk factors for cardiovascular morbidity and mortality. Glycaemic control is pivotal in T2DM patients for minimizing the risk of vascular complications and hypoglycaemic episodes, particularly in patients with renal disease who also have a higher risk of hypoglycaemia. Whereas poorly controlled glycaemia increases the risk of renal disease and its progression, the risk is diminished in patients treated intensively for diabetes and in those who achieve stable glycaemic control. Intensive multitargeted treatment can also help to decrease cardiovascular morbidity and mortality, especially if started early in patients who have not yet developed macrovascular complications. CONCLUSION: In recent years, considerable improvement has been observed in France regarding the follow-up of diabetic patients. Less extensive, but nonetheless significant, improvement has also been observed in glycaemic control. However, even though treatment decisions generally take renal function into account, some at-risk treatments are often still being used in patients with renal insufficiency.