Role of betaine:CoA ligase (CaiC) in the activation of betaines and the transfer of coenzyme A in Escherichia coli.

AIMS: Characterization of the role of CaiC in the biotransformation of trimethylammonium compounds into l(-)-carnitine in Escherichia coli. METHODS AND RESULTS: The caiC gene was cloned and overexpressed in E. coli and its effect on the production of l(-)-carnitine was analysed. Betaine:CoA ligase and CoA transferase activities were analysed in cell free extracts and products were studied by electrospray mass spectrometry (ESI-MS). Substrate specificity of the caiC gene product was high, reflecting the high specialization of the carnitine pathway. Although CoA-transferase activity was also detected in vitro, the main in vivo role of CaiC was found to be the synthesis of betainyl-CoAs. Overexpression of CaiC allowed the biotransformation of crotonobetaine to l(-)-carnitine to be enhanced nearly 20-fold, the yield reaching up to 30% (with growing cells). Higher yields were obtained using resting cells (up to 60%), even when d(+)-carnitine was used as substrate. CONCLUSIONS: The expression of CaiC is a control step in the biotransformation of trimethylammonium compounds in E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: A bacterial betaine:CoA ligase has been characterized for the first time, underlining its important role for the production of l-carnitine with Escherichia coli.

Impairing and monitoring glucose catabolite repression in L-carnitine biosynthesis.

Signal transduction pathways are usually avoided when optimizing a biotransformation process because they require complex mathematical formulations. The aim of this work was to use a Systems Biology approach to optimize and monitor the biotransformation of L-carnitine using signal transduction pathways. To this end, a dynamic model was constructed, integrating the metabolic pathways of L-carnitine biosynthesis as well as the expression of this metabolism by means of its regulation by transcription factors such as cAMP-CRP and CaiF. The model was validated using different C-sources as well as different reactor feeding approaches. A linear relationship between the external cellular cAMP and the L-carnitine production levels was predicted before being experimentally confirmed in several scenarios. Moreover, results of the model simulations and subsequent experimental findings demonstrated that the addition of exogenous cAMP was able to restore the L-carnitine production when glucose was used as C-source. Additionally, a way to monitor the L-carnitine biosynthesis by using the level of cAMP as a marker of the biotransformation state was in silico and experimentally demonstrated.

Artroplastia de cadera / Hip arthroplasty

La artroplastia total de cadera (ATC) es una intervención coste-efectiva para disminuir el dolor, mejorar la función y la calidad de vida de los pacientes con patología degenerativa o inflamatoria de la cadera. La mayoría de sistemas disponibles actualmente son modulares y la fijación de los componentes protésicos al hueso puede conseguirse mediante dos técnicas: la cementación y la osteointegración. Tanto los vástagos cementados como los no cementados pueden ser considerados el patrón oro para la supervivencia a largo plazo; sin embargo, las técnicas no cementadas son hoy en día el método preferido para la mayoría de reemplazos acetabulares. Actualmente la superficie de carga con par de fricción metal-polietileno entrelazado es la más utilizada. La mayoría de complicaciones después de una ATC son infrecuentes, y pueden ser prevenidas y tratadas fácilmente. El objetivo inmediato de la rehabilitación en la fase aguda se centra en reducir el dolor, mejorar la movilidad, restaurar la función e identificar y prevenir las complicaciones postoperatorias inmediatas. Las guías clínicas para los pacientes intervenidos de una prótesis de cadera, y más específicamente los protocolos de ejercicios terapéuticos, varían dependiendo de las instituciones u hospitales y del ámbito de actuación. Una adecuada valoración de los resultados de la artroplastia requiere el uso de instrumentos genéricos de medición de la calidad de vida, siendo el más utilizado el Medical Outcomes Study-Short Form 36, y de instrumentos específicos que sean lo suficientemente sensibles para detectar los cambios clínicos de interés, como la escala de cadera de Harris (Harris Hip Score). Los resultados publicados demuestran una mejoría excelente tanto clínica, como funcional y radiográfica después de la ATC y aproximadamente el 90 % de las ATC tienen éxito, en términos de no dolor ni complicaciones a los 10-15 años de la cirugía

Total hip arthroplasty (THA) is a cost-effective intervention to decrease pain, improve function and quality of life of the patients with degenerative or inflammatory disease of the hip. Most of the currently available systems are modular and fixation of the prosthetic components to the bone may be achieved with two techniques: cementation and osteointegration. Both the cemented stems and non-cemented ones may be considered the gold standard for long term survival. However, the non-cemented techniques are presently the preferred method for most of cetabular replacements. Currently friction of metal on polyethylene bearing surface is used most. Most of the complications after a THA are uncommon and may be easily prevented and treated. The immediate objective of rehabilitation in the acute phase is focused on reducing pain, improving mobility, restoring function and identifying and preventing immediately postoperative complications. Clinical guidelines for the patients operated on for a hip prosthesis and more specifically the therapeutic exercise protocols, vary according to the institutions or hospitals and action setting. An adequate assessment of the results of the arthroplasty requires the use of generic instruments of measurement of quality of life. That used most is the Medical Outcomes Study-Short Form 36. It is also necessary to use specific instruments that are sufficiently sensitive to detect clinical changes of interest, such as the Harris Hip Score. The results published show excellent clinical, functional and radiological improvement after the THA. Approximately 90 % of the THAs are successful in terms of no pain or complications at 10 to 15 years of the surgery (AU)

Salt stress effects on the central and carnitine metabolisms of Escherichia coli.

The aim was to understand how interaction of the central carbon and the secondary carnitine metabolisms is affected under salt stress and its effect on the production of L-carnitine by Escherichia coli. The biotransformation of crotonobetaine into L-carnitine by resting cells of E. coli O44 K74 was improved by salt stress, a yield of nearly twofold that for the control being obtained with 0.5 M NaCl. Crotonobetaine and the L-carnitine formed acted as an osmoprotectant during cell growth and biotransformation in the presence of NaCl. The enzyme activities involved in the biotransformation process (crotonobetaine hydration reaction and crotonobetaine reduction reaction), in the synthesis of acetyl-CoA/acetate (pyruvate dehydrogenase, acetyl-CoA synthetase [ACS] and ATP/acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid cycle (isocitrate dehydrogenase [ICDH]) and glyoxylate shunt (isocitrate lyase [ICL]) were followed in batch with resting cells both in the presence and absence of NaCl and in perturbation experiments performed on growing cells in a high density cell recycle membrane reactor. Further, the levels of carnitine, crotonobetaine, gamma-butyrobetaine and ATP and the NADH/NAD(+) ratio were measured in order to know how the metabolic state was modified and coenzyme pools redistributed as a result of NaCl's effect on the energy content of the cell. The results provided the first experimental evidence of the important role played by salt stress during resting and growing cell biotransformation (0.5 M NaCl increased the L-carnitine production in nearly 85%), and the need for high levels of ATP to maintain metabolite transport and biotransformation. Moreover, the main metabolic pathways and carbon flow operating during cell biotransformation was that controlled by the ICDH/ICL ratio, which decreased from 8.0 to 2.5, and the phosphotransferase/ACS ratio, which increased from 2.1 to 5.2, after a NaCl pulse fivefold the steady-state level. Resting E. coli cells were seen to be made up of heterogeneous populations consisting of several types of subpopulation (intact, depolarized, and permeabilized cells) differing in viability and metabolic activity as biotransformation run-time and the NaCl concentration increased. The results are discussed in relation with the general stress response of E. coli, which alters the NADH/NAD(+) ratio, ATP content, and central carbon enzyme activities.

Artroplastia de cadera / Hip arthroplasty

La artroplastia total de cadera (ATC) es una intervención coste-efectiva para disminuir el dolor, mejorar la función y la calidad de vida de los pacientes con patología degenerativa o inflamatoria de la cadera. La mayoría de sistemas disponibles actualmente son modulares y la fijación de los componentes protésicos al hueso puede conseguirse mediante dos técnicas: la cementación y la osteointegración. Tanto los vástagos cementados como los no cementados pueden ser considerados el patrón oro para la supervivencia a largo plazo; sin embargo, las técnicas no cementadas son hoy en día el método preferido para la mayoría de reemplazos acetabulares. Actualmente la superficie de carga con par de fricción metal-polietileno entrelazado es la más utilizada. La mayoría de complicaciones después de una ATC son infrecuentes, y pueden ser prevenidas y tratadas fácilmente. El objetivo inmediato de la rehabilitación en la fase aguda se centra en reducir el dolor, mejorar la movilidad, restaurarla función e identificar y prevenir las complicaciones postoperatorias inmediatas. Las guías clínicas para los pacientes intervenidos de una prótesis de cadera, y más específicamente los protocolos de ejercicios terapéuticos, varían dependiendo de las instituciones u hospitales y del ámbito de actuación. Una adecuada valoración de los resultados de la artroplastia requiere el uso de instrumentos genéricos de medición dela calidad de vida, siendo el más utilizado el Medical Outcomes Study-Short Form 36, y de instrumentos específicos quesean lo suficientemente sensibles para detectar los cambios clínicos de interés, como la escala de cadera de Harris (Harris Hip Score).Los resultados publicados demuestran una mejoría excelente tanto clínica, como funcional y radiográfica después dela ATC y aproximadamente el 90 % de las ATC tienen éxito, en términos de no dolor ni complicaciones a los 10-15 años de la cirugía (AU)

Total hip arthroplasty (THA) is a cost-effective intervention to decrease pain, improve function and quality of life of the patients with degenerative or inflammatory disease of the hip. Most of the currently available systems are modular and fixation of the prosthetic components to the bone may beachieved with two techniques: cementation and osteointegration. Both the cemented stems and non-cemented ones may be considered the gold standard for long term survival. However, the non-cemented techniques are presently the preferred method for most of cetabular replacements. Currently friction of metal on polyethylene bearing surface is used most. Most of the complications after a THA are uncommon and may be easily prevented and treated. The immediate objective of rehabilitation in the acute phase is focused on reducing pain, improving mobility, restoring function and identifying and preventing immediately postoperative complications. Clinical guidelines for the patients operated on for a hip prosthesis and more specifically the therapeutic exercise protocols, vary according to the institutions or hospitals and action setting. An adequate assessment of the results of the arthroplasty requires the use of generic instruments of measurement of quality of life. That used most is the Medical Outcomes Study-Short Form 36. It is also necessary to use specific instruments that are sufficiently sensitive to detect clinical changes of interest, such as the Harris Hip Score. The results published show excellent clinical, functional and radiological improvement after the THA. Approximately 90 % of the THAs are successful in terms of no pain or complications at 10 to 15 years of the surgery (AU)

Role of energetic coenzyme pools in the production of L-carnitine by Escherichia coli.

The aim of this work was to understand the steps controlling the biotransformation of trimethylammonium compounds into L(-)-carnitine by Escherichia coli. The high-cell density reactor steady-state levels of carbon source (glycerol), biotransformation substrate (crotonobetaine), acetate (anaerobiosis product) and fumarate (as an electron acceptor) were pulsed by increasing them fivefold. Following the pulse, the evolution of the enzyme activities involved in the biotransformation process of crotonobetaine into L(-)-carnitine (crotonobetaine hydration), in the synthesis of acetyl-CoA (ACS: acetyl-CoA synthetase and PTA: ATP: acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid (ICDH: isocitrate dehydrogenase) and glyoxylate (ICL: isocitrate lyase) cycles was monitored. In addition, the levels of carnitine, the cell ATP content and the NADH/NAD(+) ratio were measured in order to assess the importance and participation of these energetic coenzymes in the catabolic system. The results provided an experimental demonstration of the important role of the glyoxylate shunt during biotransformation and the need for high levels of ATP to maintain metabolite transport and biotransformation. Moreover, the results obtained for the NADH/NAD(+) pool indicated that it is correlated with the biotransformation process at the NAD(+) regeneration and ATP production level in anaerobiosis. More importantly, a linear correlation between the NADH/NAD(+) ratio and the levels of the ICDH and ICL (carbon and electron flows) and the PTA and ACS (acetate and ATP production and acetyl-CoA synthesis) activity levels was assessed. The main metabolic pathway operating during cell metabolic perturbation with a pulse of glycerol and acetate in the high-cell density membrane reactor was that related to ICDH and ICL, both regulating the carbon metabolism, together with PTA and ACS enzymes (regulating ATP production).

Estudio de la hormona del crecimiento / Study of growth hormone

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Design of metabolic engineering strategies for maximizing L-(-)-carnitine production by Escherichia coli. Integration of the metabolic and bioreactor levels.

In this work metabolic engineering strategies for maximizing L-(-)-carnitine production by Escherichia coli based on the Biochemical System Theory and the Indirect Optimization Method are presented. The model integrates the metabolic and the bioreactor levels using power-law formalism. Based on the S-system model, the Indirect Optimization Method was applied, leading to profiles of parameter values that are compatible with both the physiology of the cells and the bioreactor system operating conditions. This guarantees their viability and fitness and yields higher rates of L-(-)-carnitine production. Experimental results using a high cell density reactor were compared with optimized predictions from the Indirect Optimization Method. When two parameters (the dilution rate and the initial crotonobetaine concentration) were directly changed in the real experimental system to the prescribed optimum values, the system showed better performance in L-(-)-carnitine production (74% increase in production rate), in close agreement with the model's predictions. The model shows control points at macroscopic (reactor operation) and microscopic (molecular) levels where conversion and productivity can be increased. In accordance with the optimized solution, the next logical step to improve the L-(-)-carnitine production rate will involve metabolic engineering of the E. coli strain by overexpressing the carnitine transferase, CaiB, activity and the protein carrier, CaiT, responsible for substrate and product transport in and out of the cell. By this means it is predicted production may be enhanced by up to three times the original value.

Prótesis total de rodilla estándar. Análisis de supervivencia del implante a los 5 años / Standard total knee replacement. Analysis of 5-year implant survival rates

El objetivo de este estudio es realizar un análisis de supervivencia del implante en las artroplastias totales de rodilla estándar a los 5 años de la intervención, así como el estudio de los factores relacionados con dicha supervivencia. Pacientes y métodos. Se ha realizado un estudio prospectivo que incluye 271 pacientes que fueron intervenidos de prótesis total de rodilla estándar en el período comprendido entre 1988 y 1993 y posteriormente trasladados al servicio de Rehabilitación. Para la recogida de datos utilizamos el cuestionario modificado de la British Orthopaedic Association. Para el análisis de supervivencia hemos utilizado la función de Kaplan-Meier y consideramos como evento o acontecimiento final para la supervivencia la retirada de la prótesis. Resultados. El porcentaje de supervivencia de esta serie a los 5 años es del 94,98 por ciento. Los factores asociados significativamente con la supervivencia a los 5 años han sido: valgo al alta hospitalaria (p < 0,001), luxación de rótula (p < 0,001; supervivencia 70,59 por ciento), infección (p < 0,001; supervivencia 28,57 por ciento), descementación (p < 0,001; supervivencia 50 por ciento), inestabilidad (p < 0,001; supervivencia 25 por ciento) y dolor de etiología desconocida (p < 0,001; supervivencia 92 por ciento). Los resultados funcionales y de impresión subjetiva a los 5 años han sido excelentes o buenos en el 72,2 por ciento de los casos. Conclusiones. En esta serie los resultados de las prótesis totales de rodilla estándar a los 5 años de la cirugía son predecibles, constatándose una alta supervivencia del implante y unos resultados funcionales y de impresión subjetiva excelentes o buenos en la gran mayoría de los casos (AU)

Link between primary and secondary metabolism in the biotransformation of trimethylammonium compounds by escherichia coli.

The aim of this work was to understand the steps controlling the process of biotransformation of trimethylamonium compounds into L(-)-carnitine by Escherichia coli and the link between the central carbon or primary and the secondary metabolism expressed. Thus, the enzyme activities involved in the biotransformation process of crotonobetaine into L(-)-carnitine (crotonobetaine hydration reaction and crotonobetaine reduction reaction), in the synthesis of acetyl-CoA (pyruvate dehydrogenase, acetyl-CoA synthetase, and ATP:acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid (isocitrate dehydrogenase) and glyoxylate (isocitrate lyase) cycles, were followed in batch with both growing and resting cells and during continuous cell growth in stirred-tank and high-cell-density membrane reactors. In addition, the levels of carnitine, crotonobetaine, gamma-butyrobetaine, ATP, NADH/NAD(+), and acetyl-CoA/CoA ratios were measured to determine how metabolic fluxes were distributed in the catabolic system. The results provide the first experimental evidence demonstrating the important role of the glyoxylate shunt during biotransformation of resting cells and the need for high levels of ATP to maintain metabolite transport and biotransformation (2.1 to 16.0 mmol L cellular/mmol ATP L reactor h). Moreover, the results obtained for the pool of acetyl-CoA/CoA indicate that it also correlated with the biotransformation process. The main metabolic pathway operating during cell growth in the high cell-density membrane reactor was that related to isocitrate dehydrogenase (during start-up) and isocitrate lyase (during steady-state operation), together with phosphotransacetylase and acetyl-CoA synthetase. More importantly, the link between central carbon and L(-)-carnitine metabolism at the level of the ATP pool was also confirmed.

Erythrocyte aggregability and disaggregability in thalassemia trait carriers analyzed by a laser backscattering technique.

Erythrocyte aggregability was determined by a laser backscattering light technique in 23 beta minor thalassemia carriers and in 36 age and sex matched controls. The aggregation time (Ta) was statistically higher in cases than in controls (2.8 +/- 1.0 vs 2.3 +/- 0.4, p < 0.05) and the aggregation index at 10 sec (AI10) was statistically lower (25.1 +/- 5.7 vs 28.2 +/- 3.8, p < 0.05), suggesting both parameters a statistically lower erythrocyte aggregability tendency. However, the total disaggregation threshold (gammaD) was statistically higher in cases than in controls (134.4 +/- 34.1 vs 105.1 +/- 33.1, p < 0.05), indicating that once aggregates are formed a higher shear rate is needed to break them up. No differences were observed in plasmatic factors, i.e., fibrinogen, total cholesterol and triglycerides, that could have influenced erythrocyte aggregation. A negative statistically significant correlation was found between erythrocyte indexes and the total disaggregation threshold. The lower erythrocyte aggregation found in minor thalassemia carriers could be attributed in part to the morphological alterations, although others mechanisms such as modifications in the membrane structure of the RBC can not be ruled out.

Prótesis total de rodilla. Estudio de los factores determinantes del alta hospitalaria en rehabilitación / Total knee prosthesis. Study of the determining factors of hospital discharge in rehabilitation

Introducción: El objetivo de este estudio es analizar qué factores son determinantes del alta hospitalaria en la rehabilitación de los pacientes intervenidos de prótesis total de rodilla. Pacientes y métodos: Se ha realizado un estudio prospectivo que incluye 162 pacientes que fueron intervenidos consecutivamente de prótesis total de rodilla en el período de tiempo comprendido entre junio de 1999 y junio de 2000 y posteriormente trasladados al Servicio de Rehabilitación para realizar tratamiento médico rehabilitador en régimen de hospitalización. Para la recogida de datos utilizamos el cuestionario modificado de la British Orthopaedic Association. Se ha realizado un análisis de regresión lineal múltiple, para estudiar qué variables están asociadas con la estancia hospitalaria en rehabilitación (variable dependiente), ajustando por el efecto de las demás. Resultados: En el análisis bivariable han sido factores asociados con una menor estancia hospitalaria en rehabilitación: la edad avanzada (p<0,05), el índice de masa corporal elevado (p<0,0002), la no presencia de complicaciones quirúrgicas (p<0,01), el mayor balance articular en flexión a la semana del postoperatorio (p<0,004) y la menor media de días de movilización pasiva continua (p<0,02); pero en el análisis multivariable, los dos únicos factores determinantes del alta hospitalaria con correlación inversa, son la edad (p<0,001; IC 95 por ciento 1,86-2,06) y el balance articular en flexión a la semana del postoperatorio (p<0,01; IC 95 por ciento 1,92-1,99).Conclusiones: La edad y el balance articular en flexión a la primera semana son las dos únicas variables determinantes del alta hospitalaria (AU)

Modeling of the biotransformation of crotonobetaine into L-(-)-carnitine by Escherichia coli strains.

A simple unstructured model, which includes carbon source as the limiting and essential substrate and oxygen as an enhancing substrate for cell growth, has been implemented to depict cell population evolution of two Escherichia coli strains and the expression of their trimethylammonium metabolism in batch and continuous reactors. Although the model is applied to represent the trans-crotonobetaine to L-(-)-carnitine biotransformation, it is also useful for understanding the complete metabolic flow of trimethylammonium compounds in E. coli. Cell growth and biotransformation were studied in both anaerobic and aerobic conditions. For this reason we derived equations to modify the specific growth rate, mu, and the cell yield on the carbon source (glycerol), Y(xg), as oxygen increased the rate of growth. Inhibition functions representing an excess of the glycerol and oxygen were included to depict cell evolution during extreme conditions. As a result, the model fitted experimental data for various growth conditions, including different carbon source concentrations, initial oxygen levels, and the existence of a certain degree of cell death. Moreover, the production of enzymes involved within the E. coli trimethylammonium metabolism and related to trans-crotonobetaine biotransformation was also modeled as a function of both the cell and oxygen concentrations within the system. The model describes all the activities of the different enzymes within the transformed and wild strains, able to produce L-(-)-carnitine from trans-crotonobetaine under both anaerobic and aerobic conditions. Crotonobetaine reductase inhibition by either oxygen or the addition of fumarate as well as its non-reversible catalytic action was taken into consideration. The proposed model was useful for describing the whole set of variables under both growing and resting conditions. Both E. coli strains within membrane high-density reactors were well represented by the model as results matched the experimental data.

Comparación de la determinación de homocisteina total en plasma o suero / Comparison of the determination of total homocysteine in plasma or serum

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Stabilization of alpha-chymotrypsin by ionic liquids in transesterification reactions.

Five different ionic liquids, based on dialkylimidazolium and quaternary ammonium cations associated with perfluorinated and bis (trifluoromethyl) sulfonyl amide anions, were used as reaction media to synthesize N-acetyl-L-tyrosine propyl ester by transesterification with alpha-chymotrypsin at 2% (v/v) water content at 50 degrees C. The synthetic activity was reduced by the increase in alkyl chains length of cations and by increases in anion size, which was related to the decrease in polarity. Incubation of the enzyme (with and without substrate) in ionic liquids exhibited first-order deactivation kinetics at 50 degrees C, allowing determination of deactivation rate constants and half-life times (1-3 h). Ionic liquids showed a clear relative stabilization effect on the enzyme, which was improved by increased chain length of the alkyl substituents on the imidazolium ring cations and the anion size. This effect was 10-times enhanced by the presence of substrate. For example, 1-butyl-3-methylimidazolium hexafluorophosphate increased the alpha-chymotrypsin half-life by 200 times in the presence of substrate with respect to the 1-propanol medium. These results show that ionic liquids are excellent enzyme-stabilizing agents and reaction media for clean biocatalysis in non-conventional conditions.

Evaluation of an automated method for the quantification of von Willebrand factor antigen. Its application in the study of vascular dysfunction.

BACKGROUND AND OBJECTIVES: Abnormally high levels of von Willebrand factor (VWF) have been described as a response to physiologic and chronic alterations. It is therefore of great interest to have sensitive, accurate, fast and easy to use assays to quantify VWF. The aim of this study was to evaluate the performance characteristics of the immunoturbidimetric assay IL Test VWF:Ag and the use of this determination in the study of vascular dysfunction. DESIGN AND METHODS: The reproducibility, accuracy and linearity of the method were determined. A method comparison study was performed using an ELISA as the reference test. The assay reference range and its age-dependence were established. To evaluate the utility of the assay in vascular dysfunction, a cohort of 30 type 1 diabetes mellitus (DM) patients were analyzed, 11 of whom showed microvascular complications. RESULTS: The linearity range found was 10-150 IU/dL. Reported values above 150 IU/dL should be diluted and reanalyzed, expanding the range up to 600 IU/dL. The coefficients of variation were below 5% and inaccuracies below +/-15% within the linearity range. The comparison with the reference ELISA showed a good correlation coefficient (r = 0.985, VWF values from 5-680 IU/dl). The reference range was found to be 40.8 - 158.1 IU/dL. Significant differences between the DM type 1 patients with microvascular complications and a sex-age matched control group were observed. This difference was not observed for the DM patients not showing vascular alterations. INTERPRETATION AND CONCLUSIONS: The method is reproducible, accurate and linear. The assay correlates well with the reference ELISA. There is a relationship between patients showing microvascular diabetic complications and high levels of VWF:Ag. The assay is easy to perform, fully automated, and suitable for analyzing a small number of samples.

L(-)-carnitine production using a recombinant Escherichia coli strain.

The L(-)-carnitine production by biotransformation using the recombinant strain Escherichia coli pT7-5KE32 has been studied and optimized with crotonobetaine and D(+)-carnitine as substrates. A resting rather than a growing cells system for L(-)-carnitine production was chosen, crotonobetaine being the best substrate. High biocatalytic activity was obtained after growing the cells under anaerobic conditions at 37 degrees C and with crotonobetaine or L(-)-carnitine as inducer. The growth incubation temperature (37 degrees C) was high enough as to activate the heat-inducible lambdap(L) promoter inserted in the plasmid pGP1-2. The best biotransformation conditions were with resting cells, under aerobiosis, with 4 g l(-1) and 100 mM biomass and substrate concentrations respectively. Under these conditions the biotransformation time (1 h) was shorter and the L(-)-carnitine yield (70%) higher than previously reported. Consequently productivity value (11.3 g l(-1)h(-1)) was highly improved when comparing with other published works. The resting cells could be reused until eight times maintaining product yield levels well over 50% that meant to increase ten times the L(-)-carnitine obtained per gram of biomass.

[Mid-term behavior of the bone fixation in non-cemented Miller-Galante 1 total knee arthroplasty]. / Comportement à moyen terme de la fixation osseuse de prothèses totales de genou de Miller-Galante 1 implantées sans ciment.

PURPOSE OF THE STUDY: This retrospective work was conducted to analyze the quality of the bone-implant interface at mid-term in 45 cases treated with a non-cemented Miller-Galante 1 total knee arthroplasty. MATERIAL AND METHODS: All the protheses reviewed were implanted without cement. A patellar implant was cemented in 31.1 p. 100 of the cases. Female sex predominated in this series (77.5 p. 100) and the mean age at operation was 67 +/- 6 years. Pre- and postoperative assessment was based on the HSS score. Mean follow-up was 8.3 years (range 7-11 years). We used the method advocated by the International Knee Society to analyze lucent lines on tibial and femoral implants. Non-parametric tests were used for the statistical analysis with a significance level set at 5 p. 100. RESULTS: The mean HSS score rose from 55 +/- 12 preoperatively to 80 +/- 13 postoperatively, with 62.2 p. 100 good or excellent results at last follow-up. One re-operation was required for aseptic loosening. The femoral implant presented a lucent line in 24.4 p. 100 of the cases at the first follow-up examination only. The tibial implant presented an anterior lucent line at the second follow-up examination then a medial line at the last follow-up in 22.2 p. 100 and 26.6 p. 100 of the cases respectively. Presence of a lucent line (tibial or femoral) on at least one view was significantly correlated with activity (p=0.01) and tibial slope (p=0.0087). DISCUSSION: The disappearance of the lucent lines seen on the femoral component at the second follow-up examination was the expression of its secondary integration. Inversely, we observed an evolution in the lines observed on the tibia. This was probably the result of posterolateral impaction and anteromedial ascension micromovements of the tibial component. An excessive tibial slope was statistically related to development of lucent lines. It increased tibial translation on weight bearing and probably induced an abnormal alteration of the polyethylene. We did not observe any case of massive osteolysis of the tibial metaphysis as described in the literature for non-cemented knee arthroplasties. The screws of the Miller-Galante 1 prosthesis do not protrude from the tibial implant (which would risk generating polyethylene debris) and the pieces used (screws and implants) are all made of the same metal. CONCLUSION: The quality of primary fixation of the non-cemented Miller-Galante 1 tibial implant was not totally satisfactory at mid-term. Inversely, simple impaction of the femoral component was sufficient to ensure stable positioning.