RESUMEN

UV-induced melanogenesis is a well known physiological response of human skin exposed to solar radiation; however, the signaling molecules involved in the stimulation of melanogenesis in melanocytes following UV exposure remain unclear. In this study we induced melanogenesis in vitro in normal human epidermal melanocytes using a single irradiation with UVA at 1 kJ/m2 and examined the potential involvement of mitogen-activated protein kinases (MAPK) as UVA-responsive signaling molecules in those cells. UVA irradiation did not affect the proliferation of melanocytes, but it did increase tyrosinase mRNA expression, which reached a maximum level 4 hr after UVA irradiation. The amount of tyrosinase protein, as quantitated by immunoblotting, was also increased at 24 hr following UVA irradiation. Among the MAPK examined, extracellular signal-related kinase (ERK) 1/2 was phosphorylated within 15 min of UVA irradiation, but no such phosphorylation was observed for c-Jun N-terminal kinases (JNK) or p38. Accordingly, the activity of ERK1/2 was also increased shortly after UVA irradiation. These responses of ERK1/2 to UVA irradiation were markedly inhibited when cells were pre-treated with N-acetyl-L-cysteine, an antioxidant, or with suramin, a tyrosine kinase receptor inhibitor. The formation of (6-4)photoproducts or cyclobutane pyrimidine dimers was not detected in cellular DNA after UVA irradiation. These findings suggest that a single UVA irradiation-induced melanogenesis is associated with the activation of ERK1/2 by upstream signals that originate from reactive oxygen species or from activated tyrosine kinase receptors, but not from damaged DNA.

Asunto(s)

Células Epidérmicas , Melanocitos/metabolismo , Melanocitos/efectos de la radiación , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Rayos Ultravioleta , Acetilcisteína/farmacología , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Daño del ADN/efectos de la radiación , Activación Enzimática/efectos de la radiación , Epidermis/metabolismo , Epidermis/efectos de la radiación , Depuradores de Radicales Libres/farmacología , Humanos , Melaninas/metabolismo , Melaninas/efectos de la radiación , Melanocitos/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/efectos de la radiación , Proteína Quinasa 3 Activada por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/efectos de la radiación , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/efectos de la radiación , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/metabolismo , Monofenol Monooxigenasa/efectos de la radiación , Fosforilación/efectos de la radiación , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

The diazepam binding inhibitor (DBI), initially isolated as an endogenous 10-kDa polypeptide from the brain, has the ability to displace ligands from benzodiazepine binding sites on gamma-aminobutyric acid (GABA) receptors. However, DBI is widely distributed outside the brain, with the highest expression in the intestine. The present in situ hybridization study revealed the cellular expression of DBI mRNA throughout the gastrointestinal tract of mice, showing it to be intensely expressed in the spinous layer in the stratified squamous epithelium of the oral cavity, esophagus and forestomach, in surface mucous cells in the glandular stomach, and in columnar (absorptive) cells of the intestinal villi. A precise identification of DBI-expressing cell types was confirmed immunohistochemically, although the expressing cells detectable by the two histochemical methods differed slightly in their extension. Noteworthily, DBI always coexisted with the fatty acid binding protein (FABP), which participates in the uptake and metabolic processing of long chain fatty acids. In addition to the biochemical finding that DBI is identical with the acyl-CoA binding protein (ACBP), the distributional patterns of DBI and its colocalization with FABPs suggests its involvement in the absorption and metabolism of lipid in the epithelia of the digestive tract.

Asunto(s)

Proteínas Portadoras/análisis , Inhibidor de la Unión a Diazepam/análisis , Mucosa Intestinal/química , Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Proteínas Supresoras de Tumor , Animales , Células CACO-2 , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Inhibidor de la Unión a Diazepam/inmunología , Esófago/química , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Humanos , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones , Mucosa Bucal/química , ARN Mensajero/análisis , Conejos , Ratas , Ratas Sprague-Dawley , Estómago/química

RESUMEN

An osteochondroma is a common developmental tumor of bone characterized by abnormal peri-physeal ectopic enchondral ossification. This results in a cartilage-capped subperiosteal bony projection, which may be either sessile or pedunculated. These lesions are said to grow until skeletal maturity. The cartilage cap is thought to become thinner as the patient ages beyond skeletal maturity. Apparent growth beyond skeletal maturity may be a sign of malignant conversion, usually to a chondroma. Osteochondromas are usually appreciated in the first decades of life, and are most commonly located in the extremities, usually in the knees, ankles, or wrists. Clinical complaints generally relate to the mass effect of the lesion. Solitary osteochondromas of the axial skeleton are less common and may present with a neurological deficit. We report on such a case, in a woman significantly older than other cases described in the literature.

Asunto(s)

Calcinosis/etiología , Vértebras Cervicales/patología , Osteocondroma/complicaciones , Compresión de la Médula Espinal/etiología , Neoplasias de la Columna Vertebral/complicaciones , Anciano , Calcinosis/patología , Calcinosis/cirugía , Descompresión Quirúrgica , Femenino , Humanos , Imagen por Resonancia Magnética , Compresión de la Médula Espinal/patología , Compresión de la Médula Espinal/cirugía

RESUMEN

We examined the protective effect of gamma-glutamylethylamide (theanine) on ischemic delayed neuronal death in field CA1 of the gerbil hippocampus. One microliter of theanine from each three concentrations (50, 125 and 500 microM) was administered through the lateral ventricle 30 min before ischemia. Transient forebrain ischemia was induced by bilateral occlusion of the common carotid arteries for 3 min under careful control of brain temperature at approximately 37 degrees C. Seven days after ischemia, the number of intact CA1 neurons in the hippocampus was assessed. Ischemia-induced neuronal death in hippocampal CA1 region was significantly prevented in a dose-dependent manner in the theanine-pretreated groups. These findings indicate that theanine might be useful clinically for preventing ischemic neuronal damage.

Asunto(s)

Isquemia Encefálica/tratamiento farmacológico , Antagonistas de Aminoácidos Excitadores/farmacología , Glutamatos/farmacología , Hipocampo/efectos de los fármacos , Degeneración Nerviosa/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Daño por Reperfusión/tratamiento farmacológico , Animales , Isquemia Encefálica/patología , Isquemia Encefálica/fisiopatología , Recuento de Células/estadística & datos numéricos , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Gerbillinae , Ácido Glutámico/análogos & derivados , Ácido Glutámico/metabolismo , Hipocampo/patología , Hipocampo/fisiopatología , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Daño por Reperfusión/patología , Daño por Reperfusión/fisiopatología

RESUMEN

Various kinds of acute pathological events in the central nervous system, such as ischemia, hemorrhage, and trauma, often cause brain edema. The edema may advance for days or weeks while inducing extensive damage in neural function, regardless of the extent of the original damage, and often results in death. Delayed edema is thought to be vasogenic; however, the mechanism underlying edema induction remains unknown. We found delayed vascular cell proliferation with a blood-brain barrier breakdown in and around the gerbil CA1 hippocampus, a region known to be involved in delayed apoptotic neuronal death 2-6 days after transient ischemia. Vascular cell proliferation, assessed by (3)H-thymidine incorporation, was most prominent 4-6 days after ischemia, and extravasation of exogenously applied dye or endogenous serum albumin from blood vessels was observed concomitantly. We propose neovascularization in delayed neuronal death as a cause of brain edema advancing days after neurological events.

Asunto(s)

Barrera Hematoencefálica , Neovascularización Patológica , Neuronas/patología , Animales , Vasos Sanguíneos/patología , Edema Encefálico/etiología , Edema Encefálico/patología , Edema Encefálico/fisiopatología , Isquemia Encefálica/complicaciones , Isquemia Encefálica/patología , Isquemia Encefálica/fisiopatología , Muerte Celular , División Celular , Gerbillinae , Masculino , Células Piramidales/patología

RESUMEN

A fungus with the ability to utilize a metalcyano compound, tetracyanonickelate (II) ¿K2[Ni (CN)4]; TCN¿, as its sole source of nitrogen was isolated from soil and identified as Fusarium oxysporum N-10. Both intact mycelia and cell-free extract of the strain catalyzed hydrolysis of TCN to formate and ammonia and produced formamide as an intermediate, thereby indicating that a hydratase and an amidase sequentially participated in the degradation of TCN. The enzyme catalyzing the hydration of TCN was purified approximately ten-fold from the cell-free extract of strain N-10 with a yield of 29%. The molecular mass of the active enzyme was estimated to be 160 kDa. The enzyme appears to exist as a homotetramer, each subunit having a molecular mass of 40 kDa. The enzyme also catalyzed the hydration of KCN, with a cyanide-hydrating activity 2 x 10(4) times greater than for TCN. The kinetic parameters for TCN and KCN indicated that hydratase isolated from F. oxysporum was a cyanide hydratase able to utilize a broad range of cyano compounds and nitriles as substrates.

Asunto(s)

Cianuros/metabolismo , Fusarium/aislamiento & purificación , Fusarium/metabolismo , Níquel/metabolismo , Microbiología del Suelo , Secuencia de Aminoácidos , Biodegradación Ambiental , Medios de Cultivo , Fusarium/clasificación , Fusarium/crecimiento & desarrollo , Hidroliasas/química , Hidroliasas/metabolismo , Cinética , Datos de Secuencia Molecular

RESUMEN

The joint capsule exhibits a unique cellular lining in the luminal surface of the synovial membrane. The synovial intimal cells, termed synoviocytes, are believed to be responsible for the production of synovial fluid components, for absorption from the joint cavity, and for blood/synovial fluid exchanges, but their detailed structure and function as well as pathological changes remain unclear. Two types of synoviocytes, macrophagic cells (type A cells) and fibroblast-like cells (type B cells) have been identified. Type A synoviocytes are non-fixed cells that can phagocytose actively cell debris and wastes in the joint cavity, and possess an antigen-presenting ability. These type A cells, derived from blood-borne mononuclear cells, can be considered resident macrophages (tissue macrophages) like hepatic Kupffer cells. Type B synoviocytes are characterized by the rich existence of rough endoplasmic reticulum, and dendritic processes which form a regular network in the luminal surface of the synovial membrane. Their complex three-dimensional architecture was first revealed by our recent scanning electron microscopy of macerated samples. The type B cells, which are proper synoviocytes, are involved in production of specialized matrix constituents including hyaluronan, collagens and fibronectin for the intimal interstitium and synovial fluid. The proliferative potentials of type B cells in loco are much higher than type A cells, although the transformation of subintimal fibroblasts into type B cells can not be excluded. In some mammals, type B cells show features suggesting endocrine and sensory functions, but these are not recognized in other species. The synoviocytes, which form a discontinuous cell layer, develop both fragmented basement membranes around the cells and junctional apparatus such as desmosomes and gap junctions. For an exact understanding of the mechanism of arthritis, we need to establish the morphological background of synoviocytes as well as their functions under normal conditions.

Asunto(s)

Articulaciones/fisiología , Membrana Sinovial/citología , Membrana Sinovial/fisiología , Animales , Caballos , Inmunohistoquímica , Articulaciones/ultraestructura , Laminina/fisiología , Modelos Biológicos , Ratas , Membrana Sinovial/ultraestructura , Articulación Temporomandibular/ultraestructura

Asunto(s)

Sistema Nervioso Autónomo/fisiología , Cromograninas/metabolismo , Glándulas Salivales/metabolismo , Acetilcolina/farmacología , Animales , Sistema Nervioso Autónomo/efectos de los fármacos , Cromogranina A , Humanos , Inmunoensayo , Inmunohistoquímica , Ratones , Norepinefrina/farmacología , Péptidos/metabolismo , Ratas , Glándulas Salivales/efectos de los fármacos , Estrés Fisiológico/fisiopatología

RESUMEN

We cloned and sequenced the gene encoding an NADPH-dependent aldehyde reductase (ARII) in Sporobolomyces salmonicolor AKU4429, which reduces ethyl 4-chloro-3-oxobutanoate (4-COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate. The ARII gene is 1,032 bp long, is interrupted by four introns, and encodes a 37,315-Da polypeptide. The deduced amino acid sequence exhibited significant levels of similarity to the amino acid sequences of members of the mammalian 3beta-hydroxysteroid dehydrogenase-plant dihydroflavonol 4-reductase superfamily but not to the amino acid sequences of members of the aldo-keto reductase superfamily or to the amino acid sequence of an aldehyde reductase previously isolated from the same organism (K. Kita, K. Matsuzaki, T. Hashimoto, H. Yanase, N. Kato, M. C.-M. Chung, M. Kataoka, and S. Shimizu, Appl. Environ. Microbiol. 62:2303-2310, 1996). The ARII protein was overproduced in Escherichia coli about 2, 000-fold compared to the production in the original yeast cells. The enzyme expressed in E. coli was purified to homogeneity and had the same catalytic properties as ARII purified from S. salmonicolor. To examine the contribution of the dinucleotide-binding motif G(19)-X-X-G(22)-X-X-A(25), which is located in the N-terminal region, during ARII catalysis, we replaced three amino acid residues in the motif and purified the resulting mutant enzymes. Substrate inhibition of the G(19)-->A and G(22)-->A mutant enzymes by 4-COBE did not occur. The A(25)-->G mutant enzyme could reduce 4-COBE when NADPH was replaced by an equimolar concentration of NADH.

Asunto(s)

Acetoacetatos/metabolismo , Aldehído Reductasa/genética , Aldehído Reductasa/metabolismo , Butiratos/metabolismo , Hongos Mitospóricos/enzimología , Hongos Mitospóricos/genética , Aldehído Reductasa/química , Secuencia de Aminoácidos , Animales , Clonación Molecular , Intrones , Cinética , Mamíferos , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Sitio-Dirigida , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato

RESUMEN

A DNA fragment carrying the genes coding for EcoO109I endonuclease and EcoO109I methylase, which recognize the nucleotide sequence 5'-(A/G)GGNCC(C/T)-3', was cloned from the chromosomal DNA of Escherichia coli H709c. The EcoO109I restriction-modification (R-M) system was found to be inserted between the int and psu genes from satellite bacteriophage P4, which were lysogenized in the chromosome at the P4 phage attachment site of the corresponding leuX gene observed in E. coli K-12 chromosomal DNA. The sid gene of the prophage was inactivated by insertion of one copy of IS21. These findings may shed light on the horizontal transfer and stable maintenance of the R-M system.

Asunto(s)

Enzimas de Restricción-Modificación del ADN/genética , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Escherichia coli/genética , Genes Bacterianos , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Bacterianos , Clonación Molecular , Enzimas de Restricción-Modificación del ADN/química , Desoxirribonucleasas de Localización Especificada Tipo II/química , Escherichia coli/enzimología , Datos de Secuencia Molecular , Análisis de Secuencia de ADN

RESUMEN

Chromogranin A (CgA) is a member of a family of highly acidic proteins, chromogranins, which are co-stored in the adrenergic neurons and paraneurons and co-released with adrenaline and noradrenaline (NAd) in response to adequate stimulation. The present study provides novel evidence that CgA-like immunoreactivity (IR) is stored in the exocrine cells in the granular convoluted tubule, and is secreted into saliva by stimulation with NAd and acetylcholine (ACh) in the isolated and perfused rat submandibular gland. NAd at 1 microM produced maximum secretion of CgA-like IR (<< 0.9 mM) and a marked increase in salivary flow. Further increases in NAd concentration (10 or 100 microM) yielded concentration-dependent decreases in both responses. ACh at 1 microM produced maximum salivary flow and a slight elevation of CgA-like IR secretion (6 microM); 100 microM ACh decreased the salivary flow but increased the CgA-like IR secretion (0.6 mM). Electron microscopic examination showed vigorous compound exocytosis of secretory granules in the cells of the granular convoluted tubule when the submandibular gland was stimulated with 1 microM NAd. These results provide an experimental basis for the view that the salivary CgA-like IR secretion may be a sensitive and quantitative index of the activity of the sympathetic nervous system innervating the gland.

Asunto(s)

Acetilcolina/farmacología , Cromograninas/análisis , Norepinefrina/farmacología , Saliva/metabolismo , Glándula Submandibular/efectos de los fármacos , Animales , Cromogranina A , Masculino , Microscopía Electrónica , Perfusión , Radioinmunoensayo , Ratas , Ratas Wistar , Saliva/química , Estrés Psicológico , Glándula Submandibular/metabolismo , Glándula Submandibular/ultraestructura

RESUMEN

We found a novel subtype of prostaglandin (PG) I(2) receptor (IP(2)) expressed in the central nervous system. Recently we have demonstrated that (15R)-16-m-tolyl-17,18,19, 20-tetranorisocarbacyclin (15R-TIC) and 15-deoxy-16-m-tolyl-17,18,19, 20-tetranorisocarbacyclin (15-deoxy-TIC), IP(2)-specific ligands, significantly prevented high (50%) oxygen-induced apoptotic neuronal death in cultured hippocampal neurons. We report here a potent neuroprotective effect of such analogs on delayed neuronal death of hippocampal CA1 neurons following transient ischemia for 3 min in gerbils. (15S)-16-m-tolyl-17,18,19,20-tetranorisocarbacyclin (15S-TIC), which nonselectively acts both on the PGI(2) receptor expressed in the peripheral tissue (IP(1)) and on IP(2), also showed a neuroprotective effect on such an ischemic model at higher doses than those for 15R-TIC and 15-deoxy-TIC. These PGI(2) analogs did not affect brain temperature, indicating that the agents showed the neuroprotective effect not by a hypothermic effect, but rather by the direct action on neurons.

Asunto(s)

Isquemia Encefálica/tratamiento farmacológico , Epoprostenol/análogos & derivados , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Animales , Temperatura Corporal/efectos de los fármacos , Isquemia Encefálica/patología , Isquemia Encefálica/fisiopatología , Muerte Celular/efectos de los fármacos , Epoprostenol/química , Epoprostenol/farmacología , Gerbillinae , Neuronas/patología , Fármacos Neuroprotectores/química , Células Piramidales/efectos de los fármacos , Células Piramidales/patología , Estereoisomerismo

RESUMEN

Prostacyclin (PGI2) is a critical regulator of the cardiovascular system, via dilatation of vascular smooth muscle and inhibition of platelet aggregation (Moncada, S. 1982, Br. J. Pharmacol., 76, 3). Our previous studies demonstrated that a novel subtype of PGI2 receptor, which is clearly distinct from a peripheral subtype in terms of ligand specificity, is expressed in the rostral region of the brain, e.g. cerebral cortex, hippocampus, thalamus and striatum, and that (15R)-16-m-17,18,19,20-tetranorisocarbacyclin (15R-TIC) and 15-deoxy-16-m-17,18,19,20-tetranorisocarbacyclin (15-deoxy-TIC) specifically bind to the central nervous system (CNS)-specific PGI2 receptor. Here, we report that these CNS-specific PGI2 receptor ligands, including PGI2 itself, prevented the neuronal death. They prevented apoptotic cell death of hippocampal neurons induced by high (50%) oxygen atmosphere, xanthine + xanthine oxidase, and serum deprivation. IC50s for neuronal death were approximately 30 and 300 nM for 15-deoxy-TIC and 15R-TIC, respectively, which well correlated with the binding potency for the CNS-specific PGI2 receptor. 6-Keto-PGF1alpha (a stable metabolite of PGI2), peripheral nervous system-specific PGI2 ligands and other prostaglandins (PGs) than PGI2 did not show such neuroprotective effects. In vivo, 15R-TIC protected CA1 pyramidal neurons against ischaemic damage in gerbils. These results indicate that CNS-specific PGI2 ligands have neuronal survival-promoting activity both in vitro and in vivo, and may represent a new type of therapeutic drug for neurodegeneration.

Asunto(s)

Supervivencia Celular/fisiología , Sistema Nervioso Central/fisiología , Epoprostenol/fisiología , Neuronas/fisiología , Animales , Autorradiografía , Isquemia Encefálica/patología , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Sistema Nervioso Central/citología , Epoprostenol/análogos & derivados , Epoprostenol/farmacología , Femenino , Gerbillinae , Hipocampo/metabolismo , Hipocampo/patología , Hiperoxia/patología , Ligandos , Masculino , Neuronas/efectos de los fármacos , Células Piramidales/efectos de los fármacos , Ratas , Ratas Wistar , Receptores de Epoprostenol , Receptores de Prostaglandina/biosíntesis , Xantina/toxicidad , Xantina Oxidasa/toxicidad

RESUMEN

The levels of extracellular glutamate were measured in the dentate gyrus by using an in vivo brain microdialysis method to determine whether the ischemia-induced glutamate release might be correlated with the neuronal vulnerability to ischemia. A microdialysis membrane was placed in CA4 (vulnerable to ischemia) and the molecular and granule cell layers of the dentate gyrus (resistant to ischemia) of gerbils. A significant increase in glutamate levels was induced in the normal dentate gyrus during 10-min ischemia. The increase was completely suppressed during the first 5 min of ischemia when CA4 neurons were eliminated. Thus, it was indicated that during the first 5 min of ischemia glutamate was released mostly from CA4 neurons but not from granule cells of the dentate gyrus. During the second half of 10-min ischemia, a significant increase in glutamate release was induced even in the dentate gyrus where CA4 neurons were eliminated; this increase was significantly suppressed by inhibiting proliferation of astrocytes. A large part of glutamate that was released during the second half of 10-min ischemia was considered to be attributable to glutamate release from astrocytes.

Asunto(s)

Giro Dentado/irrigación sanguínea , Giro Dentado/metabolismo , Ácido Glutámico/metabolismo , Ataque Isquémico Transitorio/metabolismo , Animales , Astrocitos/química , Astrocitos/metabolismo , Giro Dentado/citología , Gerbillinae , Proteína Ácida Fibrilar de la Glía/análisis , Masculino , Microdiálisis , Neuronas/metabolismo

RESUMEN

The synovial membrane displays a superficial cellular lining composed of two types of synoviocytes: "absorptive" macrophages (type A cells) and "secretory" fibroblast-like cells (type B cells). The types are intermingled and extend a variety of processes, rendering the cellular architecture of the synovial membrane difficult to visualize. Previous electron microscopic and histochemical studies failed to demonstrate the entire shape of synoviocytes, except our immunohistochemical study for protein gene product 9.5 in the horse joint. The present SEM study is the first to demonstrate the three-dimensional ultrastructure of synoviocytes as well as their distribution in the synovial membrane, using macerated samples from the horse carpal joints. The equine synovial membrane was largely covered by conspicuously developed synovial villi. Type A synoviocytes were closely similar to macrophages in regard to surface structure, and showed uneven distribution with the densest occurrence around the tips of the synovial villi. In the basal half of villi, type B synoviocytes, which were situated in close proximity to the synovial cavity, projected thick processes horizontally and intertwined to form a regular network of processes on the synovial surface. Those in the upper half of the villi were located in the abluminal layers and protruded an antenna-like process into the joint cavity with tips covered with long microvilli, in addition to forming the superficial plexus of processes. Type B cells were also provided with fine, membranous extensions that tended to cover the surface of synovial intima. The meshwork of horizontal processes, the antenna-like processes, and the membranous processes imply advantages in not only secretion but also sensation and regulation of the barrier function in the synovial membrane.

Asunto(s)

Caballos/anatomía & histología , Articulaciones/ultraestructura , Membrana Sinovial/citología , Animales , Cartílago Articular/citología , Cartílago Articular/fisiología , Cartílago Articular/ultraestructura , Femenino , Fibroblastos/citología , Fibroblastos/fisiología , Fibroblastos/ultraestructura , Macrófagos/citología , Macrófagos/fisiología , Macrófagos/ultraestructura , Masculino , Microscopía Electrónica de Transmisión de Rastreo/veterinaria , Membrana Sinovial/fisiología , Membrana Sinovial/ultraestructura

RESUMEN

The 4.4-kb PstI fragment harboring the gene encoding 3-hexulose-6-phosphate synthase, rmpA, which was previously cloned from the chromosome of an obligate methylotroph, Methylomonas aminofaciens 77a, was investigated in detail. In addition to the rmpA gene, the fragment contained three open reading frames encoding transaldolase (rmpD), IS10-R (rmpI), and 6-phospho-3-hexuloisomerase (PHI) (rmpB). The rmpB gene product was overproduced in Escherichia coli cells, purified to homogeneity, and then enzymatically identified as PHI. The gene organization of the ribulose monophosphate pathway enzymes together with a transposon, IS10-R, is discussed from both evolutionary and regulatory aspects.

Asunto(s)

Aldehído-Liasas/genética , Genes Bacterianos , Methylococcaceae/genética , Ribulosafosfatos/genética , Transaldolasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Elementos Transponibles de ADN/genética , Methylococcaceae/enzimología , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Ribulosafosfatos/metabolismo , Eliminación de Secuencia

RESUMEN

A carboxylesterase that is responsible for conversion of 1,4-butanediol diacrylate (BDA) to 4-hydroxybutyl acrylate (4HBA) was found in Brevibacterium lines IFO 12171, and purified to homogeneity. The purified enzyme was active toward a variety of diesters of ethylene glycol, 1,4-butanediol, and 1,6-hexanediol. The K(m) and kcat of the enzyme for BDA were 3.04 mM and 203,000 s-1, respectively. The reaction with the purified enzyme gave 98 mM 4HBA from 100 mM BDA for 60 min. The enzyme gene was cloned from the chromosomal DNA of the bacterium. The open reading frame encoding the enzyme was 1176 bp long, corresponding to a protein of 393 amino acid residues (molecular mass = 42,569 Da). The deduced amino acid sequence contained the tetra peptide motif sequence, STTK, and the serine residue was confirmed to be the catalytic center of BDA esterase by site-directed mutagenesis for several amino acid residues. The gene was expressed in Escherichia coli under the control of the lac promoter, and the gene product (a fusion protein with 6 amino acid residues from beta-galactosidase) showed the same catalytic properties as the enzyme from the parent strain.

Asunto(s)

Acrilatos/metabolismo , Brevibacterium/enzimología , Butileno Glicoles/metabolismo , Hidrolasas de Éster Carboxílico/genética , Escherichia coli/genética , Secuencia de Aminoácidos , Secuencia de Bases , Hidrolasas de Éster Carboxílico/aislamiento & purificación , Hidrolasas de Éster Carboxílico/metabolismo , Cromatografía Líquida de Alta Presión , Clonación Molecular , Escherichia coli/enzimología , Regulación Bacteriana de la Expresión Génica/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad por Sustrato

RESUMEN

The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate [(R)-CHBE] using Escherichia coli cells, which coexpress both the aldehyde reductase gene from Sporobolomyces salmonicolor and the glucose dehydrogenase (GDH) gene from Bacillus megaterium as a catalyst was investigated. In an organic solvent-water two-phase system, (R)-CHBE formed in the organic phase amounted to 1610 mM (268 mg/ml), with a molar yield of 94.1% and an optical purity of 91.7% enantiomeric excess. The calculated turnover number of NADP+ to CHBE formed was 13,500 mol/mol. Since the use of E. coli JM109 cells harboring pKAR and pACGD as a catalyst is simple, and does not require the addition of GDH or the isolation of the enzymes, it is highly advantageous for the practical synthesis of (R)-CHBE.

Asunto(s)

Acetoacetatos/metabolismo , Aldehído Reductasa/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Glucosa Deshidrogenasas/metabolismo , Aldehído Reductasa/genética , Bacillus/enzimología , Bacillus/genética , Escherichia coli/crecimiento & desarrollo , Glucosa 1-Deshidrogenasa , Glucosa Deshidrogenasas/genética , Hongos Mitospóricos/enzimología , Hongos Mitospóricos/genética , Oxidación-Reducción , Plásmidos/genética , Estereoisomerismo , Transformación Bacteriana

RESUMEN

Fibroblast-like (Type B) synoviocytes are cells in the synovial membrane that are responsible for production of both synovial fluid and the extracellular matrix in the synovial intima. Immunostaining of the horse synovial membrane for protein gene product (PGP) 9.5, which is a neuron-specific ubiquitin C-terminal hydrolase, demonstrated selective localization of the immunoreactivity in a synoviocyte population different from acid phosphatase-positive Type A synoviocytes. The immunoreactive cells were lined up in the synovial intima and extended dendritic processes towards the joint cavity to form a dense plexus on the surface. Electron microscopic examination clearly identified the PGP 9.5-immunoreactive cells as Type B synoviocytes characterized by developed rough endoplasmic reticulum and free ribosomes. Immunoreactivity for PGP 9.5 was diffusely distributed throughout the cytoplasm, including the tips of fine processes. Western and Northern blot analyses could not distinguish the corresponding protein and mRNA obtained from the brain and synovial membrane. The existence of the neuron-specific PGP 9.5 in Type B synoviocytes suggests a common mechanism regulating the protein metabolism between neurons and synoviocytes, and also provides a new cytochemical marker for identification of the cells.

Asunto(s)

Caballos/metabolismo , Membrana Sinovial/metabolismo , Tioléster Hidrolasas/metabolismo , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Encéfalo/metabolismo , Clonación Molecular , Humanos , Técnicas para Inmunoenzimas , Masculino , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Neuronas/metabolismo , Ratas , Alineación de Secuencia , Tioléster Hidrolasas/genética , Ubiquitina Tiolesterasa

RESUMEN

The cytotoxicity test of neutral red (NR) uptake in normal rabbit corneal epithelial cells (CornePack((R))) was validated as an alternative method to the Draize rabbit eye irritation test (Draize test). We tested 38 cosmetic ingredients as well as isotonic sodium chloride solution in three phases of the validation study. The test procedures were controlled among participating laboratories under a common standard operating procedure (SOP). The concentration of test substances that showed a 50% reduction in NR uptake relative compared with controls (median NR uptake concentration: NR(50), mug/ml) was determined and compared with in vivo Draize scores. Six laboratories participated in the first phase of the validation study, seven in the second, and five in the third. The average interlaboratory coefficient of variation (CV) was 32.9%. The correlation and rank correlation coefficients between the maximal average Draize total scores (MAS) and NR(50) were -0.583 and 0.587, respectively. When the anionic detergents were excluded from analysis, the correlation coefficient increased to -0.738. When the cut-off point for positive and negative irritation was set at MAS of 15 and the predictability of this method was assessed by liner regression line, six substances (two acids, two alkanolamines and two alcohols) were false negative. Through this project, it appeared that CornePack, supplied in kit form with frozen secondary cultured cell in serum-free medium, could provide an effective, highly sensitive and simple preliminary screen for determining the cytotoxicity of substances. These results suggested that CornePack might have the potential to predict the MAS if definite criteria can be established for the compounds to be applicable. However, it is important to understand the nature of CornePack responses since its NR(50) profile was quite different from other cytotoxicity assays.