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1.
Appl Microbiol Biotechnol ; 58(3): 322-9, 2002 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-11935183

RESUMEN

The Serratia marcescens extracellular lipase (LipA) is an enzyme applicable to enantioselective hydrolysis of racemic substrates. The enzyme is secreted through an ATP-binding cassette (ABC) exporter, the Lip system, encoded by the lipBCD genes. The S. marcescens recombinant carrying pLIPE121, which encodes the lipA gene in pUC19, exhibited a higher LipA production level than the wild-type strain. However, the level was lower than expected, and secretion was suggested to be a bottleneck. lipBCD plasmids were introduced into S. marcescens recombinants harboring lipA plasmids and the effectiveness of the lipBCD plasmids in elevating LipA productivity was investigated. S. marcescens strains harboring both lipA and lipBCD plasmids showed sevenfold greater extracellular LipA activity than the strain harboring the lipA plasmid alone. A high level of extracellular LipA production (1,300 kU/ml) and high plasmid stability (enough to carry out large-scale cultivation) were observed under non-selective conditions. Addition of L-proline and Tween 80 was effective in increasing cell growth of the recombinant, which led to high LipA production. In batch cultivation using a 30-l jar fermentor, LipA production was achieved at a high level of 5,200 kU/ml. This is the first report describing utilization of ABC exporter for the overproduction of an industrially important extracellular protein.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Proteínas Bacterianas/biosíntesis , Lipasa/biosíntesis , Serratia marcescens/genética , Serratia marcescens/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/genética , Clonación Molecular , Medios de Cultivo , Exopeptidasas/síntesis química , Exopeptidasas/metabolismo , Expresión Génica , Lipasa/genética , Proteínas Recombinantes/metabolismo , Serratia marcescens/crecimiento & desarrollo
2.
Rinsho Shinkeigaku ; 41(2-3): 121-5, 2001.
Artículo en Japonés | MEDLINE | ID: mdl-11481854

RESUMEN

We here reported a 54-year-old female patient with Crow-Fukase syndrome associated with pulmonary plasmacytoma. She was found to have scattered tumor in 1990. Although the tumor had slowly grown for the last 10 years, she showed no clinical symptoms. Numbness and weakness of lower extremities began in June 1999, and she was referred to Kyoto University Hospital on Oct. 21 1999 for evaluation of progressive symptoms. She had skin pigmentation, edema of the lower extremities, lymphadenopathy, muscle weakness and sensory disturbance in a glove-and-stocking distribution. Serological examination showed monoclonal IgG-lambda gammopathy. Serum vascular endothelial growth factor (VEGF) was markedly elevated. Microscopic studies on biopsied sural nerve demonstrated mild decrease of myelinated fibers. Immunohistochemically, the pulmonary tumor was defined as an IgG (lambda type) plasmacytoma. After treatment with melphalan-prednisolone therapy, the neurological symptoms improved along with decrease of serum VEGF levels as well as the size of pulmonary plasmacytoma. This is the first report of a patient with Crow-Fukase syndrome associated with pulmonary plasmacytoma. This case suggests that growth of pulmonary plasmacytoma might have played an important role in the overproduction of VEGF and thus development of Crow-Fukase syndrome.


Asunto(s)
Neoplasias Pulmonares/complicaciones , Síndrome POEMS/etiología , Plasmacitoma/complicaciones , Antineoplásicos Alquilantes/administración & dosificación , Quimioterapia Combinada , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Melfalán/administración & dosificación , Persona de Mediana Edad , Síndrome POEMS/tratamiento farmacológico , Plasmacitoma/tratamiento farmacológico , Prednisolona/administración & dosificación
3.
J Biosci Bioeng ; 91(4): 409-15, 2001.
Artículo en Inglés | MEDLINE | ID: mdl-16233013

RESUMEN

We have isolated a lipase-overproducing mutant, GE14, from Serratia marcescens 8000 after three rounds of N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The mutant GE14 produced 95 kU/ml of extracellular lipase in the lipase medium, which was about threefold higher than that of produced by the original strain 8000. Enzymatic characteristics including specific activity of purified lipases from culture supernatants of GE14 and 8000 were almost same. The lipase gene (lipA) of GE14 contained two base substitutions; one in the promoter region and another in the N-terminal region of the lipA gene without an amino acid substitution. Promoter analysis using lipA-lacZ fusion plasmids revealed that these substitutions were responsible for the increase in the lipA expression level, independently. In contrast, no base substitution was found in the genes encoding the lipase secretion device, the Lip system. In addition, the genes coding for metalloprotease and the cell surface layer protein which are both secreted through the Lip system and associated with extracellular lipase production, also contained no base substitution. The strain GE14 carrying a high-copy-number lipA plasmid produced a larger amount of the extracellular lipase than the recombinant strains of 8000 and other mutants also did, indicating that GE14 was not only a lipase-overproducing strain, but also an advantageous host strain for overproducing the lipase by a recombinant DNA technique. These results suggest that the lipase-overproducing mutant GE14 and its recombinant strains are promising candidates for the industrial production of the S. marcescens lipase.

4.
Diabetologia ; 41(8): 911-7, 1998 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-9726593

RESUMEN

The purpose of this study was to investigate the relationship between complications arising from silent cerebral infarction (SCI) and changes in the levels of serum-soluble adhesion molecules in 82 elderly diabetic patients aged 60 years and older. SCI was found in 43 % of the 82 patients, with incidence increasing in relation to age. The prevalence of SCI was higher in subjects with hypertension, poor metabolic control and increased fibrinolysis. The levels of soluble intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1) and E-selectin (sE-selectin) were higher in diabetic patients than in non-diabetic subjects (p < 0.05, p < 0.001, and p < 0.05, respectively). Also, sICAM-1 and sVCAM-1 were found at increased levels in diabetic patients with SCI compared to those without SCI (p < 0.01 and p < 0.05, respectively). In particular, the level of sICAM-1 was increased in patients with SCI due to perforating arterial occlusion, while the level of sVCAM-1 was increased in patients with SCI due to cortical arterial occlusion. However, no significant difference was found in sE-selectin levels. Overall average of the intima and media thickness (IMT) of the common carotid arteries increased with age. IMT proved to be greater in patients with SCI than in patients without SCI (p < 0.05), and showed a weak but significant positive correlation with sVCAM-1, while no correlation was found with either sICAM-1 or sE-selectin levels. In conclusion, measurement of serum adhesion molecules may be useful for diagnosing the early stages of brain damage and for prophylactic treatment which may prevent the onset or progression of SCI.


Asunto(s)
Moléculas de Adhesión Celular/sangre , Infarto Cerebral/sangre , Diabetes Mellitus Tipo 2/complicaciones , Anciano , Anciano de 80 o más Años , Arterias Carótidas/patología , Infarto Cerebral/complicaciones , Infarto Cerebral/patología , Diabetes Mellitus Tipo 2/sangre , Selectina E/sangre , Femenino , Fibrinólisis , Humanos , Hipertensión/complicaciones , Molécula 1 de Adhesión Intercelular/sangre , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Molécula 1 de Adhesión Celular Vascular/sangre
5.
Diabet Med ; 13(2): 120-4, 1996 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-8641115

RESUMEN

The beneficial effect of long-term treatment with an angiotensin-converting enzyme (ACE) inhibitor on urinary microalbumin excretion (UAE) and renal function was investigated in a 4 year, randomized prospective study in normotensive patients with non-insulin-dependent (Type 2) diabetes mellitus. Sixty-two normotensive patients with Type 2 diabetes mellitus and microalbuminuria but normal renal function were randomized to receive either enalapril 5 mg day-1 or no treatment. In the enalapril-treated patients, UAE was reduced from 115.4 +/- 80.1 to 95.6 +/- 61.7 mg 24 h-1 after 12 months (p < 0.05) and to 75.3 +/- 44.8 mg 24 h-1 after 48 months (p < 0.001). In the untreated group, UAE increased slowly from 93.9 +/- 69.9 to 150.0 +/- 144.5 mg 24 h-1 after 48 months. No changes in creatinine clearance, blood pressure or HbA1C were seen in either group during the 4-year period. In normotensive Type 2 diabetic patients with early stage of diabetic microalbuminuria. This effect is long-lasting and probably independent of the antihypertensive action of the drug.


Asunto(s)
Albuminuria/tratamiento farmacológico , Inhibidores de la Enzima Convertidora de Angiotensina/administración & dosificación , Diabetes Mellitus Tipo 2/complicaciones , Nefropatías Diabéticas/tratamiento farmacológico , Enalapril/administración & dosificación , Anciano , Albuminuria/etiología , Análisis de Varianza , Presión Sanguínea/efectos de los fármacos , Diabetes Mellitus Tipo 2/fisiopatología , Nefropatías Diabéticas/orina , Esquema de Medicación , Estudios de Seguimiento , Humanos , Persona de Mediana Edad , Estudios Prospectivos
6.
Diabet Med ; 12(5): 392-6, 1995 May.
Artículo en Inglés | MEDLINE | ID: mdl-7648800

RESUMEN

An in vitro incubation study was conducted to investigate whether increased activity of the polyol pathway in human neutrophils under diabetic conditions resulted in a decrease of superoxide anion produced by NADPH oxidase on the membrane of neutrophils. Lucigenin-enhanced chemiluminescence (CL) to phorbol myristate acetate as respiratory burst and sorbitol levels in neutrophils after incubation with glucose and an aldose reductase (AR) inhibitor, SNK-860 (SNK) were measured. Sorbitol levels increased from 0.210 +/- 0.029 nmol 10(7) cells-1 in 5 mmol l-1 glucose to 0.446 +/- 0.036 nmol 10(7) cells-1 in 40 mmol l-1 glucose, while CL decreased from 0.542 +/- 0.034 cpm cell-1 in 5 mmol l-1 glucose to 0.430 +/- 0.018 cpm cell-1 in 40 mmol l-1 glucose. The addition of 10 mumol l-1 SNK normalized the increased sorbitol levels in neutrophils exposed to 40 mmol l-1 glucose and improved, but did not normalize, the decrease in CL induced by 40 mmol l-1 glucose (p < 0.001). Galactose (40 mmol l-1) also reduced CL, which was improved by the addition of SNK (p < 0.01). These results suggest that impaired respiratory burst induced by high-glucose concentrations is caused by competition for NADPH resulting from increased polyol pathway activity and/or glycation and that an AR inhibitor may be capable in part of preventing increased susceptibility to infection in diabetic patients.


Asunto(s)
Aldehído Reductasa/antagonistas & inhibidores , Glucosa/farmacología , Imidazoles/farmacología , Imidazolidinas , Neutrófilos/efectos de los fármacos , Sorbitol/sangre , Adulto , Glucemia/metabolismo , Estudios de Casos y Controles , Galactosa/farmacología , Humanos , Mediciones Luminiscentes , Manitol/farmacología , Valores de Referencia , Acetato de Tetradecanoilforbol/farmacología
7.
Clin Ther ; 16(6): 1007-15, 1994.
Artículo en Inglés | MEDLINE | ID: mdl-7697680

RESUMEN

We investigated the usefulness of the C64 quantitative tuning fork in assessing vibratory sensation in patients with diabetic neuropathy. The vibratory sensation scores determined by using the C64 quantitative tuning fork were significantly correlated with severity of subjective symptoms of numbness, cold, desensitization, spontaneous pain in the upper and lower extremities, and vertigo, as well as measurements of neurologic function (threshold of vibratory sensation, the coefficients of variation of the R-R intervals of the electrocardiogram at rest, and the motor nerve conduction velocity [MNCV] of the lower extremities; P < 0.05 to 0.01). Subsequently, we evaluated the effects of niceritrol on neurologic function in the same patients by using the C64 quantitative tuning fork, other neurologic function tests, and the change in severity of subjective symptoms before and after therapy. Niceritrol significantly improved measures of neurologic function (vibratory sensation scores, MNCV of the upper extremities, and sensory nerve conduction velocity of the lower extremities; P < 0.05 to 0.001), and subjective symptoms (numbness, cold, desensitization, and spontaneous pain in the lower extremities; P < 0.01 to 0.001). Our results suggest that the C64 quantitative tuning fork is a convenient and objective tool for assessing the severity of diabetic neuropathy and that niceritrol is useful for the treatment of this disease.


Asunto(s)
Neuropatías Diabéticas/tratamiento farmacológico , Niceritrol/uso terapéutico , Glucemia , Neuropatías Diabéticas/fisiopatología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Umbral Sensorial , Índice de Severidad de la Enfermedad , Vibración
8.
Diabetes Care ; 17(5): 420-4, 1994 May.
Artículo en Inglés | MEDLINE | ID: mdl-8062609

RESUMEN

OBJECTIVE: To determine whether long-term treatment with an angiotensin-converting enzyme (ACE) inhibitor has a beneficial effect on the urinary microalbumin excretion and renal function in non-insulin-dependent diabetes mellitus (NIDDM) patients, enalapril (5 mg/day) was administered for 48 months. RESEARCH DESIGN AND METHODS: -Fifty-two patients with NIDDM who had persistent microalbuminuria in the range of 20-300 mg/24 h, serum creatinine < 106.1 microM (1.2 mg/dl), supine systolic blood pressure (BP) < 150 mmHg, supine diastolic BP < 90 mmHg, and HbA1c < 10% were divided into four groups. Twenty-six patients with normotension were divided at random into two groups; one group received enalapril (5 mg/day) (NE group), the other did not receive enalapril (NC group). In the same way, 26 other patients who were already well-controlled with nifedipine (30 mg/day) over a long-term period (4-6 years) were divided at random into two groups; one received enalapril (5 mg/day) (HE group), the other did not receive enalapril (HC group). RESULTS: After 48 months, urinary albumin excretion (UAE) was markedly reduced in group NE from 102.4 x/divided by 1.3 to 55.5 x/divided by 1.3 mg/24 h (P < 0.005), whereas no significant change occurred in group NC. In the well-controlled hypertensive groups, a significant reduction in UAE occurred in group HE (P < 0.05), whereas no significant change occurred in group HC. No changes in creatinine clearance, BP, or blood glucose control were seen during the study. CONCLUSIONS: Treatment with enalapril for 48 months may have a beneficial effect on the decline of microalbumin excretion in NIDDM patients.


Asunto(s)
Albuminuria/tratamiento farmacológico , Diabetes Mellitus Tipo 1/fisiopatología , Enalapril/uso terapéutico , Hipertensión/fisiopatología , Acetilglucosaminidasa/orina , Presión Sanguínea , Peso Corporal , Colesterol/sangre , Creatinina/metabolismo , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/orina , Nefropatías Diabéticas/prevención & control , Hemoglobina Glucada/análisis , Humanos , Hipertensión/complicaciones , Hipertensión/tratamiento farmacológico , Persona de Mediana Edad , Nifedipino/uso terapéutico , Triglicéridos/sangre , Microglobulina beta-2/orina
9.
Diabet Med ; 10(2): 134-8, 1993 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-8458189

RESUMEN

The efficacy and safety of acarbose therapy (100 mg tds for 24 weeks) was investigated in a placebo-controlled double-blind study in patients with non-insulin dependent diabetes mellitus who could not achieve satisfactory glycaemic control by diet alone. In the acarbose group, the 2 h postprandial blood glucose and haemoglobin A1 levels decreased significantly from 14.0 mmol l-1 to 11.3 mmol l-1 and from 11.1% to 9.7%, respectively. In the placebo group, the 2 h postprandial blood glucose (14.4 mmol l-1 to 14.2 mmol l-1) and the hemoglobin A1 level (10.3% to 9.9%) showed no significant changes. A 75 g oral glucose tolerance test was performed before and after the study, the difference not being significant in either the acarbose group or the placebo group. The incidence of side-effects (mainly gastrointestinal symptoms such as flatulence and abdominal distension) was high at 78.9% in the acarbose group and 61.1% in the placebo group. However, there was no significant difference between the groups, and side-effects in the acarbose group tapered during the trial, suggesting that some at least were not related to the drug. From these findings, it was concluded that acarbose is an effective new treatment for diet treated non-insulin-dependent diabetic patients.


Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Trisacáridos/uso terapéutico , Acarbosa , Adulto , Anciano , Glucemia/metabolismo , Colesterol/sangre , HDL-Colesterol/sangre , Diabetes Mellitus Tipo 2/sangre , Método Doble Ciego , Femenino , Enfermedades Gastrointestinales/inducido químicamente , Prueba de Tolerancia a la Glucosa , Hemoglobina Glucada/análisis , Humanos , Insulina/sangre , Masculino , Persona de Mediana Edad , Placebos , Factores de Tiempo , Triglicéridos/sangre , Trisacáridos/efectos adversos
10.
Biotechnol Appl Biochem ; 12(2): 129-40, 1990 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-2184837

RESUMEN

A method for reducing endotoxin contamination in various solutions by immobilized histidine is described. Immobilized histidine is a porous adsorbent suitable for the adsorption of endotoxin with a high affinity over a wide range of pH and temperature and at low ionic strength (gamma/2 less than or equal to 0.1). When a purified endotoxin originating from Escherichia coli UKT-B was studied, the apparent dissociation constant between endotoxin and the adsorbent was 7.3 X 10(-13) M. The adsorbent was able to remove various kinds of endotoxin originating from gram-negative bacteria; the concentration of endotoxin was reduced from 1000 to less than 0.01 ng/ml in water. It is shown that the adsorbent specifically adsorbs endotoxin provided that the adsorption conditions are properly selected. Some examples of the specific removal of endotoxin from high-molecular-weight physiologically active substances such as tumor necrosis factor and lysozyme are shown.


Asunto(s)
Contaminación de Medicamentos , Endotoxinas/aislamiento & purificación , Histidina , Proteínas/aislamiento & purificación , Adsorción , Escherichia coli , Bacterias Gramnegativas , Concentración de Iones de Hidrógeno , Peso Molecular , Muramidasa/aislamiento & purificación , Concentración Osmolar , Soluciones , Temperatura , Factor de Necrosis Tumoral alfa/aislamiento & purificación
11.
Horm Metab Res ; 20(7): 387-90, 1988 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-3139540

RESUMEN

Myo-inositol uptake by erythrocytes from humans, rabbits and rats was studied with an isotope technique. In human erythrocytes, the inhibitory effect on myo-inositol uptake was stronger with glucose than with ouabain. However, an aldose reductase inhibitor (ONO-2235, 100 microM) or insulin (200 microU/ml) failed to correct the decrease in myo-inositol uptake in packed RBC, produced by either 10 mM glucose or 2mM ouabain. Ten mM ouabain had an inhibitory effect on myo-inositol uptake in all species, but an inhibitory effect was not observed with 20 mM glucose in rabbit erythrocytes. The results suggest that myo-inositol uptake by erythrocytes may be dependent on the active transport system via sodium-ATPase and that erythrocytes may not be a suitable model to monitor the possible effect of an aldose reductase inhibitor on myo-inositol concentrations in other tissues concerned with diabetic complications.


Asunto(s)
Aldehído Reductasa/antagonistas & inhibidores , Eritrocitos/metabolismo , Glucosa/farmacología , Inositol/metabolismo , Ouabaína/farmacología , Deshidrogenasas del Alcohol de Azúcar/antagonistas & inhibidores , Animales , Humanos , Técnicas In Vitro , Conejos , Ratas , Ratas Endogámicas , Especificidad de la Especie
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