ABSTRACT
INTRODUCCIÓN: El magnesio es crítico para el funcionamiento del organismo. Los pacientes de edad avanzada tienen una predisposición multifactorial a la hipomagnesemia. OBJETIVO: Establecer una intervención para identificar pacientes de edad avanzada de atención primaria con hipomagnesemia, y estudiar las implicaciones financieras. MÉTODOS: De forma consensuada con atención primaria y endocrinología y nutrición, diseñamos y establecimos la intervención: el Sistema de Información de Laboratorio registra automáticamente el magnesio sérico en solicitudes de laboratorio de pacientes de atención primaria mayores de 65 años, si no medido en el año previo, ni solicitado en la petición actual, y disponibilidad de suero. Se contabilizó el número de nuevos casos identificados de hipomagnesemia y se calculó la tasa de detección mediante el número de pruebas de magnesio sérico medidas adicionalmente (N) y el de pacientes identificados con hipomagnesemia. Finalmente, se calculó el coste en reactivo por caso detectado, teniendo en cuenta el precio del reactivo por test (0.30 ) y número de pacientes identificados (N * 0.30 / Número de pacientes identificados). RESULTADOS: En seis meses, se midió magnesio sérico en 11749 pacientes de atención primaria mayores de 65 y se detectó hipomagnesemia en 1419, siendo en el 10% moderada o severa, y habiendo más casos a más edad. La tasa de detección fue mayor cuando vivían institucionalizados (16.0% vs 11.0%), y 2.5 o 1.9 el coste por caso detectado en función del lugar de residencia. CONCLUSIÓN: Los resultados de la intervención para detectar pacientes con hipomagnesemia mediante registro automático de magnesio sérico cuando solicitud de exploración analítica por cualquier motivo, sugieren que es eficiente, especialmente cuanto más mayores e institucionalizados
BACKGROUND: Magnesium is considered as an essential mineral for the functioning of the organism. Elderly patients have a multifactorial predisposition to hypomagnesemia. OBJECTIVE: To establish an intervention to identify elderly primary care patients with hypomagnesemia, and study the financial implications. METHODS: In consensus with general practitioners and endocrinology and nutrition specialists, we designed and established an intervention: the Laboratory Information System would automatically register serum magnesium in primary care laboratory requests from patients over 65, if not measured the previous year, or in the current request, and serum availability. The number of new identified cases of hypomagnesemia was counted and the detection rate was calculated by the number of additionally measured serum magnesium tests (N) and the number of identified patients with hypomagnesemia. Finally, the cost in reagent per detected case was calculated, taking into account the reagent price per test ( 0.30) and the number of identified patients (N * 0.30 / Number of identified patients). RESULTS: In the six months study period, serum magnesium was measured in 11,749 primary care patients older than 65 and hypomagnesemia was detected in 1,419, being 10% of the cases moderate or severe, identifying more cases with aging. The detection rate was higher when they lived institutionalized (16.0% vs. 11.0%), and the cost per detected case depended on the residence status (2.5 and 1.9 when at home and institutionalized respectively). CONCLUSION: The results of the intervention to identify patients with hypomagnesemia by means of an automatic registration of serum magnesium when lab testing request, suggest that it is efficient, especially in older population and when institutionalized
Subject(s)
Humans , Male , Female , Aged , Aged, 80 and over , Magnesium Deficiency/epidemiology , Diagnostic Tests, Routine/methods , Primary Health Care/statistics & numerical data , Evaluation of the Efficacy-Effectiveness of Interventions , Cost-Benefit Analysis , Colorimetry/methodsABSTRACT
INTRODUCTION: To study the pre-design and success of a strategy based on the addition of hemoglobin A1c (HbA1c) in the blood samples of certain primary care patients to detect new cases of type 2 diabetes. MATERIALS AND METHODS: In a first step, we retrospectively calculated the number of HbA1c that would have been measured in one year if HbA1c would have been processed, according to the guidelines of the American Diabetes Association (ADA). Based on those results we decided to prospectively measure HbA1c in every primary care patient above 45 years, with no HbA1c in the previous 3 years, and glucose concentration between 5.6-6.9 mmol/L, during an 18 months period. We calculated the number of HbA1c that were automatically added by the LIS based on our strategy, we evaluated the medical record of such subjects to confirm whether type 2 diabetes was finally confirmed, and we calculated the cost of our intervention. RESULTS: In a first stage, according to the guidelines, Hb1Ac should have been added to the blood samples of 13,085 patients, resulting in a cost of 14,973. In the prospective study, the laboratory added Hb1Ac to 2092 patients, leading to an expense of 2393. 314 patients had an HbA1c value ≥ 6.5% (48 mmol/mol). 82 were finally diagnosed as type 2 diabetes; 28 thanks to our strategy, with an individual cost of 85.4; and 54 due to the request of HbA1c by the general practitioners (GPs), with a cost of 47.5. CONCLUSION: The automatic laboratory-based strategy detected patients with type 2 diabetes in primary care, at a cost of 85.4 per new case.
Subject(s)
Clinical Laboratory Techniques/methods , Diabetes Mellitus, Type 2/diagnosis , Glycated Hemoglobin/metabolism , Primary Health Care/methods , Aged , Aged, 80 and over , Algorithms , Blood Glucose/metabolism , Clinical Laboratory Techniques/economics , Clinical Laboratory Techniques/standards , Cost-Benefit Analysis , Diabetes Mellitus, Type 2/blood , Humans , Middle Aged , Practice Guidelines as Topic , Primary Health Care/economics , Primary Health Care/standards , Prospective Studies , Reproducibility of Results , Retrospective Studies , Sensitivity and Specificity , United StatesABSTRACT
Antecedentes y objetivos: Las pruebas de laboratorio son cruciales en el diagnóstico y seguimiento de las disfunciones tiroideas, lo que hace necesario estudiar cuál es el patrón de su demanda y la variabilidad en la solicitud. Los objetivos del trabajo fueron comparar la variabilidad en la solicitud de pruebas de función y autoinmunidad tiroidea (tirotropina tiroxina libre [T4L], triyodotironina libre [T3L], anticuerpos frente a la tiroglobulina [TgAb] y anticuerpos antiperoxidasa [TPOAb]) por parte de los médicos de atención primaria entre diferentes departamentos de salud españoles, y calcular el potencial ahorro económico que supondría alcanzar las metas de algunos indicadores de adecuación descritos previamente en la literatura. Métodos: Se compararon el número de pruebas por 1.000 habitantes y las ratios de pruebas relacionadas (T4L/TSH, T3L/TSH, TgAb/TPOAb) entre los diferentes departamentos en función de su ubicación, tipo de gestión y la región. Se calculó el ahorro generado si cada departamento alcanzara la meta propuesta en la literatura para los indicadores T4L/TSH (0,25) y T3L/TSH (0,1). Resultados: Un total de 76 laboratorios que atienden a una población de 17.679.195 habitantes participaron en el estudio. La solicitud de TSH fue significativamente menor en los departamentos de salud rurales-urbanos y la solicitud de T3L, la ratio T3L/TSH y la ratio TgAb/TPOAb fueron mayores en los departamentos con gestión privada. El ahorro que se generaría si se cumplieran las especificaciones para las ratios de pruebas relacionadas ascendería a 937.260,5 €. Conclusiones: La elevada variabilidad descrita en la solicitud de pruebas de función y autoinmunidad tiroidea en España hace necesario implementar estrategias para mejorar esta solicitud (AU)
Background and objectives: Laboratory tests are crucial for diagnosis and monitoring of thyroid disorders. It is therefore necessary to study the pattern and variability in requests of thyroid function tests. The study objectives were to compare the inter-regional variability in the request of laboratory thyroid tests by general practitioners (GPs) in Spain, and to investigate the potential economic savings if the goals set for some suitability indicators were reached. Methods: Test requests per 1,000 inhabitants and test ratios (free thyroxine (FT4)/thyrotropin (TSH), free triiodothyronine (FT3)/TSH, thyroglobulin antibody (TgAb)/peroxidase antibody (TPOAb)) were compared between the different areas, according to their setting, location, and management. The resulting savings if each department achieved the goals for indicator (0.25 for FT4/TSH, 0.1 for FT3/TSH) were estimated. Results: Seventy-six laboratories covering a population of 17,679,195 inhabitants participated in the study. TSH was requested significantly less in urban-rural areas, and the requests for FT3/1,000 inhabitants, FT3/TSH, and TgAb/TPOAb were higher in departments with private management. The savings generated if specifications for the ratios of related tests were met would be 937,260.5 €. Conclusions: The high variability reported in requests for thyroid function and autoimmunity tests in Spain suggests the need for implementing strategies to improve use of such tests (AU)
Subject(s)
Humans , Thyroid Function Tests , Thyroid Diseases/epidemiology , Primary Health Care/statistics & numerical data , Reproducibility of Results , Reproducibility of Results , Laboratories, Hospital/statistics & numerical dataABSTRACT
BACKGROUND AND OBJECTIVES: Laboratory tests are crucial for diagnosis and monitoring of thyroid disorders. It is therefore necessary to study the pattern and variability in requests of thyroid function tests. The study objectives were to compare the inter-regional variability in the request of laboratory thyroid tests by general practitioners (GPs) in Spain, and to investigate the potential economic savings if the goals set for some suitability indicators were reached. METHODS: Test requests per 1,000 inhabitants and test ratios (free thyroxine (FT4)/thyrotropin (TSH), free triiodothyronine (FT3)/TSH, thyroglobulin antibody (TgAb)/peroxidase antibody (TPOAb)) were compared between the different areas, according to their setting, location, and management. The resulting savings if each department achieved the goals for indicator (0.25 for FT4/TSH, 0.1 for FT3/TSH) were estimated. RESULTS: Seventy-six laboratories covering a population of 17,679,195 inhabitants participated in the study. TSH was requested significantly less in urban-rural areas, and the requests for FT3/1,000 inhabitants, FT3/TSH, and TgAb/TPOAb were higher in departments with private management. The savings generated if specifications for the ratios of related tests were met would be 937,260.5 . CONCLUSIONS: The high variability reported in requests for thyroid function and autoimmunity tests in Spain suggests the need for implementing strategies to improve use of such tests.
Subject(s)
Thyroid Function Tests/statistics & numerical data , Thyroid Gland/physiopathology , Autoantibodies/analysis , Humans , Primary Health Care , Spain , Thyrotropin/analysis , Thyroxine/analysis , Triiodothyronine/analysisABSTRACT
This study aimed to assess the psychometric properties of the Spanish version of the Problem Areas in Diabetes (PAID-SP) Scale. Data were collected from patients with type 1 and type 2 diabetes. The findings suggested that the PAID-SP is a reliable and valid measure of diabetes-specific emotional problems.
Subject(s)
Diabetes Mellitus, Type 1/diagnosis , Diabetes Mellitus, Type 2/diagnosis , Emotions , Psychometrics/methods , Quality of Life , Stress, Psychological/psychology , Adolescent , Adult , Aged , Aged, 80 and over , Diabetes Mellitus, Type 1/psychology , Diabetes Mellitus, Type 2/psychology , Female , Humans , Male , Middle Aged , Reproducibility of Results , Spain , Young AdultABSTRACT
BACKGROUND: With the introduction of automated calcium measurements with multichannel continuous-flow analyzers in the 1970s, primary hyperparathyroidism (pHPT), the silent disease, began to be detected. Years later, with the first appearance of random access analyzers, laboratory tests were requested if the patient had clinical symptoms and pHPT was again overlooked. In this current scenario, serum calcium (s-Ca) is at risk of becoming a forgotten test. In consensus with endocrinologists and general practitioners (GPs), we implemented a strategy to detect asymptomatic pHPT patients. METHODS: During a 9 month period, the Laboratory Information System automatically added s-Ca to every sample of blood from primary care patients older than 45 years, without a s-Ca request in the previous three years. If hypercalcemia was detected (albumin-corrected s-Ca > 2.6 mmol/L), phosphate, 25-hydroxy vitamin D and parathyroid hormone (PTH) were automatically processed in the same sample. We reviewed the medical record of every patient with hypercalcemia. RESULTS: S-Ca was automatically added to 14,461 samples, with 79 hypercalcemia results. 14 hypercalcemia results with PTH levels in the reference range were clinically justified by causes other than pHPT. Of the remaining 65 patients, 41 were referred for evaluation by endocrinology. 34 resulted in a diagnosis of pHPT and 7 are currently in study. 24 patients were not followed by GPs to find out the primary cause of hypercalcemia. After a phone call from the laboratory, they are also currently being studied for hypercalcemia. Each case represented a cost of 110.4 US dollars. CONCLUSION: Our proposed opportunistic screening to discover pHPT seems cost-effective.
Subject(s)
Calcium/blood , Diagnostic Tests, Routine/methods , Hyperparathyroidism, Primary/blood , Hyperparathyroidism, Primary/diagnosis , Demography , Female , General Practitioners , Humans , Hypercalcemia/blood , Hypercalcemia/diagnosis , Hypercalcemia/etiology , Hyperparathyroidism, Primary/complications , Male , Middle AgedABSTRACT
A cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) was successfully isolated and characterized from the halophilic archaeon Haloferax mediterranei. The enzyme is a monomer with a molecular mass of 77 kDa and optimum activity at 55°C, pH 7.5 and 1.5 M NaCl. The enzyme displayed many activities related to the degradation and transformation of starch. Cyclization was found to be the predominant activity, yielding a mixture of cyclodextrins, mainly α-CD, followed by hydrolysis and to a lesser extent coupling and disproportionation activities. Gene encoding H. mediterranei CGTase was cloned and heterologously overexpressed. Sequence analysis revealed an open reading frame of 2142 bp that encodes a protein of 713 amino acids. The amino acid sequence displayed high homology with those belonging to the α-amylase family. The CGTase is secreted to the extracellular medium by the Tat pathway. Upstream of the CGTase gene, four maltose ABC transporter genes have been sequenced (malE, malF, malG, malK). The expression of the CGTase gene yielded a fully active CGTase with similar kinetic behavior to the wild-type enzyme. The H. mediterranei CGTase is the first halophilic archaeal CGTase characterized, sequenced and expressed.
Subject(s)
Glucosyltransferases/metabolism , Haloferax mediterranei/enzymology , Starch/metabolism , Chromatography, Liquid , Cyclization , Electrophoresis, Polyacrylamide Gel , Glucosyltransferases/chemistry , Haloferax mediterranei/metabolism , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Molecular Weight , Spectrometry, Mass, Electrospray Ionization , Substrate Specificity , TemperatureABSTRACT
Objetivo Mostrar el patrón de solicitud de hemoglobina glucosilada (HbA1c) en ocho departamentos de salud mediante el cálculo de indicadores de adecuación de la demanda. Métodos Estudio transversal en el que se recogen el número de HbA1c solicitadas desde Atención Primaria durante los años 2008 y 2009. Como indicador de adecuación se calculó el porcentaje de valores de HbA1c con resultado inferior a 6,5%. Las variables y los indicadores se recogieron y calcularon de forma automatizada. También se calculó en cada departamento de salud el número de determinaciones de HbA1c teóricas que deberían haber sido solicitadas según datos de prevalencia conocida. Resultados se observó un incremento progresivo de la demanda de determinaciones de HbA1c y, aproximadamente el 54% de los valores de HbA1c en siete de estos ocho departamentos fueron inferiores a 6,5%. El número de HbA1c teóricas que deberían haber sido solicitadas según la prevalencia de diabetes fue mayor que el número solicitado en todos los departamentos. Conclusión Los resultados parecen indicar la inadecuación en la solicitud de la HbA1c en los departamentos de salud estudiados, no sólo por la probable sobreutilización en pacientes no diabéticos sino por la infrautilización en pacientes que sí lo son (AU)
Objective To assess the pattern of glycosylated hemoglobin (HbA1c) requests by clinicians from eight health departments by calculating indicators of demand appropriateness. Methods A cross-sectional study of the number of HbA1c requests by primary care clinics in 2008 and 2009. The indicator of demand appropriateness was the proportion of HbA1c values lower than 6.5%. Variables were collected and indicators were automatically calculated. The number of HbA1c measurements that should theoretically have been requested according to known diabetes prevalence data was also calculated. Results A progressive increase was seen in demand for HbA1c measurements. Approximately 54% of HbA1c values obtained in seven of the eight departments studied were lower than 6.5%. The number of theoretical HbA1c requests that would have been expected based on the known prevalence of diabetes was higher than the number of HbA1c requests in all departments. Conclusion The results appear to suggest that HbA1c requests by the health departments studied were not always appropriate. HbA1c measurements were probably overused in patients without diabetes and underused in patients with diabetes (AU)
Subject(s)
Humans , Glycated Hemoglobin/analysis , Diabetes Mellitus, Type 2/prevention & control , Primary Health Care/methods , Evaluation of Results of Preventive Actions , Unnecessary Procedures/statistics & numerical dataABSTRACT
OBJECTIVE: To assess the pattern of glycosylated hemoglobin (HbA(1c)) requests by clinicians from eight health departments by calculating indicators of demand appropriateness. METHODS: A cross-sectional study of the number of HbA(1c) requests by primary care clinics in 2008 and 2009. The indicator of demand appropriateness was the proportion of HbA(1c) values lower than 6.5%. Variables were collected and indicators were automatically calculated. The number of HbA(1c) measurements that should theoretically have been requested according to known diabetes prevalence data was also calculated. RESULTS: A progressive increase was seen in demand for HbA(1c) measurements. Approximately 54% of HbA(1c) values obtained in seven of the eight departments studied were lower than 6.5%. The number of theoretical HbA(1c) requests that would have been expected based on the known prevalence of diabetes was higher than the number of HbA(1c) requests in all departments. CONCLUSION: The results appear to suggest that HbA(1c) requests by the health departments studied were not always appropriate. HbA(1c) measurements were probably overused in patients without diabetes and underused in patients with diabetes.
Subject(s)
Glycated Hemoglobin , Practice Patterns, Physicians' , Cross-Sectional Studies , Glycated Hemoglobin/analysis , Hematologic Tests/statistics & numerical data , Humans , Pilot Projects , Primary Health Care , SpainABSTRACT
NifS-like proteins are pyridoxal 5'-phosphate (PLP)-dependent enzymes involved in sulphur transfer metabolism. These enzymes have been catalogued as cysteine desulphurases (CDs) which catalyse the conversion of L-cysteine into L-alanine and an enzyme-bound persulphide radical. This reaction, assisted by different scaffold protein machineries, seems to be the main source of sulphur for the synthesis of essential cofactors of the [Fe-S] cluster. CDs genes have been detected in the tree domains of life, but, up until now, there has been no biochemical characterisation or study into the physiological role of this enzyme in haloarchaea. In this study, we have cloned, expressed and characterised a cysteine desulphurase (SufS) from Haloferax volcanii and demonstrated that this protein is able to reconstitute the [Fe-S] cluster of halophilic ferredoxin.
Subject(s)
Archaea/metabolism , Gene Expression Regulation, Bacterial , Haloferax/metabolism , Iron-Sulfur Proteins/chemistry , Lyases/genetics , Lyases/metabolism , DNA Primers/chemistry , Databases, Protein , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Hydrogen-Ion Concentration , Nucleotides/chemistry , Protein Folding , Recombinant Proteins/chemistry , Ultraviolet RaysABSTRACT
In order to identify genetic factors related to thyroid cancer susceptibility, we adopted a candidate gene approach. We studied tag- and putative functional SNPs in genes involved in thyroid cell differentiation and proliferation, and in genes found to be differentially expressed in thyroid carcinoma. A total of 768 SNPs in 97 genes were genotyped in a Spanish series of 615 cases and 525 controls, the former comprising the largest collection of patients with this pathology from a single population studied to date. SNPs in an LD block spanning the entire FOXE1 gene showed the strongest evidence of association with papillary thyroid carcinoma susceptibility. This association was validated in a second stage of the study that included an independent Italian series of 482 patients and 532 controls. The strongest association results were observed for rs1867277 (OR[per-allele] = 1.49; 95%CI = 1.30-1.70; P = 5.9x10(-9)). Functional assays of rs1867277 (NM_004473.3:c.-283G>A) within the FOXE1 5' UTR suggested that this variant affects FOXE1 transcription. DNA-binding assays demonstrated that, exclusively, the sequence containing the A allele recruited the USF1/USF2 transcription factors, while both alleles formed a complex in which DREAM/CREB/alphaCREM participated. Transfection studies showed an allele-dependent transcriptional regulation of FOXE1. We propose a FOXE1 regulation model dependent on the rs1867277 genotype, indicating that this SNP is a causal variant in thyroid cancer susceptibility. Our results constitute the first functional explanation for an association identified by a GWAS and thereby elucidate a mechanism of thyroid cancer susceptibility. They also attest to the efficacy of candidate gene approaches in the GWAS era.
Subject(s)
Forkhead Transcription Factors/genetics , Genetic Predisposition to Disease , Genetic Variation , Thyroid Neoplasms/metabolism , Upstream Stimulatory Factors/metabolism , Adult , Base Sequence , Binding Sites , Case-Control Studies , Female , Forkhead Transcription Factors/chemistry , Forkhead Transcription Factors/metabolism , Humans , Male , Middle Aged , Molecular Sequence Data , Polymorphism, Single Nucleotide , Promoter Regions, Genetic , Protein Binding , Spain , Thyroid Neoplasms/genetics , Upstream Stimulatory Factors/geneticsABSTRACT
Primary hyperaldosteronism (PHA) is thought to have a harmful effect on the cardiovascular system and, in recent years, the number of cases of hypertension due to PHA has been increasing. The aims of this study were to determine the prevalence of PHA and to assess cardiac damage associated with the condition in 183 consecutive hypertensive patients. A full secondary hypertension work-up was performed, and included ECG and echocardiography. In total, 11 (6%) patients were diagnosed with PHA. Compared with other hypertensives, those with PHA had higher systolic blood pressure, more frequently had evidence of left ventricular hypertrophy on ECG (45.5% vs 11.6%; P< .01), and had a larger left ventricular mass on echocardiography (145.5 g/m(2) vs 97.52 g/m(2); P< .0001). In conclusion, PHA is a significant contributor to the increasing prevalence of hypertension and its effect on the heart is greater than that of other causes of hypertension.
Subject(s)
Heart Diseases/etiology , Hyperaldosteronism/complications , Hyperaldosteronism/epidemiology , Hypertension/etiology , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Prevalence , Prospective StudiesABSTRACT
El hiperaldosteronismo primario (HAP) es una causa creciente de hipertensión arterial (HTA) en los últimos años, y se lo ha asociado a un efecto deletéreo cardiovascular. Con el objetivo de conocer la prevalencia real de HAP en nuestro medio y sus alteraciones cardiacas, hemos estudiado a 183 pacientes hipertensos consecutivos. Se realizó un completo cribado de HTA secundaria, ECG y ecocardiografía. Se estableció el diagnóstico de HAP en 11 (6%) pacientes. Éstos presentaban mayores cifras de presión arterial sistólica, mayor prevalencia de hipertrofia ventricular izquierda en el ECG (el 45,5 frente al 11,6%; p < 0,01) y mayor masa ventricular izquierda (145,5 y 97,5 g/m2; p < 0,0001) que el resto de los hipertensos. El HAP es una causa importante de elevación de las cifras de presión arterial y se asocia a una mayor afección cardiaca que otras causas de HTA
Primary hyperaldosteronism (PHA) is thought to have a harmful effect on the cardiovascular system and, in recent years, the number of cases of hypertension due to PHA has been increasing. The aims of this study were to determine the prevalence of PHA and to assess cardiac damage associated with the condition in 183 consecutive hypertensive patients. A full secondary hypertension work-up was performed, and included ECG and echocardiography. In total, 11 (6%) patients were diagnosed with PHA. Compared with other hypertensives, those with PHA had higher systolic blood pressure, more frequently had evidence of left ventricular hypertrophy on ECG (45.5% vs 11.6%; P<.01), and had a larger left ventricular mass on echocardiography (145.5 g/m2 vs 97.52 g/m2; P<.0001). In conclusion, PHA is a significant contributor to the increasing prevalence of hypertension and its effect on the heart is greater than that of other causes of hypertension
Subject(s)
Humans , Hyperaldosteronism/epidemiology , Hypertension/complications , Hypertrophy, Left Ventricular/epidemiology , Prospective Studies , Risk FactorsABSTRACT
The NAD-dependent glutamate dehydrogenase (GDH) gene from the halophilic archaeon Haloferax mediterranei has been cloned. The analysis of the nucleotide sequence revealed an open reading frame of 1323 bp that encodes a NAD-GDH. The amino acid sequence displayed high homology with those from other sources, especially the highly conserved residues involved in 2-oxoglutarate binding. The expression of this gene in Escherichia coli, the refolding and further characterization, yielded a fully active NAD-GDH with the same features than those found for the wild-type enzyme. This halophilic NAD-GDH showed a highly dependence on salts for both stability and activity, being essential for the refolding of the recombinant enzyme.
Subject(s)
Glutamate Dehydrogenase/chemistry , Haloferax mediterranei/enzymology , Haloferax mediterranei/genetics , Amino Acid Sequence , Archaeal Proteins/chemistry , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Cloning, Molecular , Gene Library , Glutamate Dehydrogenase/genetics , Glutamate Dehydrogenase/metabolism , Hydrogen-Ion Concentration , Molecular Sequence Data , Polymerase Chain Reaction , Protein Folding , Recombinant Proteins , Sequence Homology, Amino Acid , Substrate Specificity , TemperatureABSTRACT
Salinibacter ruber, an extremely halophilic member of the domain Bacteria, has two different cytoplasmic glutamate dehydrogenase activities, marked as GDHI and GDHII. GDHI showed a strong dependence on high salt concentrations for stability, but not for activity, displaying maximal activity in the absence of salts. GDHII depended on high salt concentrations for both activity and stability. It catalyzed amination of 2-oxoglutarate with optimal activity in 3 M KCl at pH 8. No activating effect was found when NaCl was replaced by KCl. Only GDHII displayed activity in the deamination reaction of glutamate with an optimal pH of 9.5. Both enzymes were activated by certain amino acids (L-leucine, L-histidine, L-phenylalanine) and by nucleotides such as ADP or ATP. A low-molecular-mass cytoplasmic fraction was found to be a highly effective activator of GDHII in the presence of high NaCl concentrations.
Subject(s)
Bacteroidetes/enzymology , Glutamate Dehydrogenase/isolation & purification , Glutamate Dehydrogenase/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Enzyme Activation , Enzyme Stability/drug effects , Glutamic Acid/metabolism , Histidine/metabolism , Hydrogen-Ion Concentration , Ketoglutaric Acids/metabolism , Leucine/metabolism , NAD/metabolism , NADP/metabolism , Phenylalanine/metabolism , Potassium Chloride , Sodium Chloride , Substrate SpecificityABSTRACT
Sulfolobus solfataricus secretes an acid-resistant alpha-amylase (amyA) during growth on starch as the sole carbon and energy source. Synthesis of this activity is subject to catabolite repression. To better understand alpha-amylase function and regulation, the structural gene was identified and disrupted and the resulting mutant was characterized. Internal alpha-amylase peptide sequences obtained by tandem mass spectroscopy were used to identify the amyA coding sequence. Anti-alpha-amylase antibodies raised against the purified protein immunoprecipitated secreted alpha-amylase activity and verified the enzymatic identity of the sequenced protein. A new gene replacement method was used to disrupt the amyA coding sequence by insertion of a modified allele of the S. solfataricus lacS gene. PCR and DNA sequence analysis were used to characterize the altered amyA locus in the recombinant strain. The amyA::lacS mutant lost the ability to grow on starch, glycogen, or pullulan as sole carbon and energy sources. During growth on a non-catabolite-repressing carbon source with added starch, the mutant produced no detectable secreted amylase activity as determined by enzyme assay, plate assay, or Western blot analysis. These results clarify the biological role of the alpha-amylase and provide additional methods for the directed genetic manipulation of the S. solfataricus genome.
Subject(s)
Gene Targeting , Hot Temperature , Mutation , Sulfolobus/enzymology , alpha-Amylases/genetics , alpha-Amylases/metabolism , Amino Acid Sequence , Culture Media , Gene Expression Regulation, Archaeal , Molecular Sequence Data , Recombination, Genetic , Spectrometry, Mass, Electrospray Ionization , Sulfolobus/genetics , Sulfolobus/growth & development , alpha-Amylases/chemistryABSTRACT
An electric water heater was modified for large-scale cultivation of aerobic acidophilic hyperthermophiles to enable recovery of secreted proteins. Critical changes included thermostat replacement, redesign of the temperature control circuit, and removal of the cathodic anticorrosion system. These alterations provided accurate temperature and pH control. The bioreactor was used to cultivate selected strains of the archaeon Sulfolobus solfataricus and other species within this genus. Reformulation of a basal salts medium facilitated preparation of large culture volumes and eliminated sterilization-induced precipitation of medium components. Substrate induction of synthesis of the S. solfataricus-secreted alpha-amylase during growth in a defined medium supported the utility of the bioreactor for studies of physiologically regulated processes. An improved purification strategy was developed by using strong cation-exchange chromatography for recovery of the alpha-amylase and the processing of large sample volumes of acidic culture supernatant. These findings should simplify efforts to study acidophilic hyperthermophilic microbes and their secreted proteins.
Subject(s)
Archaeal Proteins/metabolism , Bioreactors , Sulfolobus/growth & development , alpha-Amylases/metabolism , Culture Media , Gene Expression Regulation, Archaeal , Hot Temperature , Hydrogen-Ion Concentration , Sulfolobus/metabolismABSTRACT
BACKGROUND: Early diagnosis of familial medullary thyroid carcinoma (MTC) is currently done by genetic analysis. These techniques have replaced calcitonin stimulation testing, which was previously used for this purpose. Some studies suggest a relationship between MTC spread and calcitonin levels. The aim of this study was to assess whether the tumor burden of MTC associated with multiple endocrine neoplasia type 2A (MEN 2A) syndrome can be estimated from the plasma calcitonin values before surgery. STUDY DESIGN: We retrospectively studied the relationship of basal and peak calcitonin values before thyroidectomy with histopathologic findings in 53 patients with MEN 2A syndrome from 14 families. The MTC was classified according to TNM staging. Analysis of variance was used for statistical analysis complemented with equality contrasts for pairs of means by the least significant difference method with a Student's t-test and with the Bonferroni's adjustment. RESULTS: A positive association was found between tumor stage and basal and peak calcitonin levels. There were significant differences between the following: mean basal concentrations of patients with C cell hyperplasia (CCH) (34.3 pg/mL) and TNM stage II (1,097.4 pg/mL), p < 0.01; CCH and TNM stage III (2,940.8 pg/mL), p < 0.001; TNM stage I (165.3 pg/mL) and stage II (1,097.4 pg/mL), p < 0.01, and between TNM stages I and III, p < 0.001. Poststimulation mean concentrations were different between CCH (48.7 pg/mL) and TNM I (514.2 pg/mL), p < 0.001. CONCLUSIONS: Preoperative calcitonin testing may be useful for assessing tumor spread and should be considered when deciding the extent of surgery for MEN 2A MTC.
Subject(s)
Calcitonin/blood , Carcinoma, Medullary/blood , Carcinoma, Medullary/pathology , Multiple Endocrine Neoplasia Type 2a/blood , Preoperative Care , Thyroid Neoplasms/blood , Thyroid Neoplasms/pathology , Thyroidectomy , Adolescent , Adult , Aged , Biomarkers/blood , Carcinoma, Medullary/genetics , Carcinoma, Medullary/surgery , Child , Child, Preschool , Female , Genetic Predisposition to Disease , Humans , Male , Middle Aged , Multiple Endocrine Neoplasia Type 2a/pathology , Multiple Endocrine Neoplasia Type 2a/surgery , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplasm Staging , Retrospective Studies , Thyroid Neoplasms/genetics , Thyroid Neoplasms/surgeryABSTRACT
BACKGROUND: Genetic study of the RET proto-oncogene has modified the management, treatment, and prognosis of medullary thyroid carcinoma (MTC), multiple endocrine neoplasia 2A (MEN 2A), for patients with less advanced tumor stages. Classically, the diagnosis was based on an increase in basal and poststimulus peak calcitonin (bCT and pCT). Prophylactic thyroidectomy, based on results of genetic testing, may reduce recurrences in MTC. STUDY DESIGN: Of 82 MTC (MEN 2A) patients genetically diagnosed and surgically treated at our center, 22 received a prophylactic thyroidectomy (RET +, bCT and pCT with normal values and asymptomatic). We analyzed age, gender, phenotype, RET mutation, cervical ultrasound, laboratory tests (bCT, pCT, and CEA), surgery, histologic data, TNM, and followup. RESULTS: The 22 patients belonged to 8 families with MTC (MEN 2A). Mean age was 15.2 years (range 5 to 36 years). The RET mutation in 21 patients was Cys-->Tyr and in the remaining patient both in codon 634 in exon 11. The median values of bCT and pCT were 38 pg/mL (range < 15 to 75 pg/mL) and 148.5 pg/mL (range < 15 to 250 pg/mL), respectively. Total thyroidectomy was performed in 8 patients (age < or = 10 years) and associated central neck dissection in 14 patients (age> 10 years). Histologic study showed 7 C-cell hyperplasias and 15 MTCs (8 bilateral); the median size was 0.2 cm (range < 0.1 to 0.7cm); 1 patient had metastatic adenopathies. According to TNM, 7 were stage 0, 14 were stage I, and 1 was stage III. Postsurgery bCT and pCT values were normal in all patients, with a curative rate of 100%. MTC patients compared with C-cell hyperplasia patients were older on average, had higher mean bCT, mean pCT, and mean CEA. CONCLUSIONS: Prophylactic thyroidectomy based on genetic testing allows identification and treatment of patients at an early stage of the disease and decreases recurrence rates. pCT values above the upper limit of normal may be markers for the presence of MTC and should be considered in selecting operative procedures for these patients.