RESUMEN
Methods that visualize subsets as well as the entire enteric neuron population are not readily available or have proved to be unreliable. Therefore, we attempted to combine NADPH-d histochemistry, AChE histochemistry, and CGRP immunohistochemistry, techniques that mark subsets of enteric neurons, with a technique that appeared to visualize the entire enteric neuron population, the cuprolinic blue staining method. To guarantee representative staining results, the individual staining methods were modified by using microwaves. In addition, this preserved the characteristics of each of the individual techniques. The distribution of NADPH-d, AChE, and CGRP corresponded well with previous morphological and physiological reports. Consequently, the different combinations gave rise to rapid, useful, and ready-to-use double labeling techniques. Their main advantage is that they simultaneously visualize the total population as well as subsets of enteric neurons.
Asunto(s)
Sistema Nervioso Entérico/citología , Histocitoquímica/métodos , Técnicas para Inmunoenzimas , Indoles , Neuronas/química , Compuestos Organometálicos , Acetilcolinesterasa/metabolismo , Animales , Animales Recién Nacidos , Péptido Relacionado con Gen de Calcitonina/análisis , Colorantes , Sistema Nervioso Entérico/enzimología , Yeyuno/inervación , Microondas , Neuronas/enzimología , PorcinosRESUMEN
Pseudomonas oleovorans can grow on linear alkanes and alkenes in the hexane to dodecane range by virtue of enzymes encoded by the alk genes. By introducing selected alk genes into Pseudomonas strains and by supplying alkanes in the growth medium as a bulk liquid phase, specific alkane oxidation products can be accumulated in the alkane phase. We review the genetics and enzymology of the alk system and the potential of bioconversions in two-liquid-phase bioreactors, and suggest that such systems might eventually allow the biotechnological production of intermediate value compounds.
Asunto(s)
Alcanos/metabolismo , Alquenos/metabolismo , Pseudomonas/enzimología , Biotecnología , Fermentación , Microbiología Industrial , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Oxidación-Reducción , Plásmidos , Pseudomonas/genética , Pseudomonas/crecimiento & desarrollo , Especificidad por SustratoRESUMEN
Two Pseudomonas strains (PpG777 and PaG158) were derived from the parent isolate Pseudomonas incognita (putida). Strain PpG777 resembles the parental culture in growth on linalool as a source of carbon and slight growth on p-cymene, whereas PaG158 grows well on p-cymene, but not on linalool or other terpenes tested, and has a P. aeruginosa phenotype. Curing studies indicate that linalool metabolism is controlled by an extrachromosomal element whose loss forms a stable strain PaG158 with the p-cymene growth and P. aeruginosa phenotype characters. The plasmid can be transferred by PpG777 to both P. putida and P. aeruginosa strains. Surprisingly, the latter assume the P. putida phenotype. We conclude that the genetic potential to oxidize p-cymene is inherent in PpG777 but expression is repressed. Similarly, this observation implies that support of linalool oxidation effectively conceals the P. aeruginosa character.
Asunto(s)
Monoterpenos , Plásmidos , Pseudomonas aeruginosa/genética , Pseudomonas/genética , Terpenos/metabolismo , Monoterpenos Acíclicos , Conjugación Genética , Cimenos , Genotipo , Mutación , Oxidación-Reducción , Fenotipo , Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Especificidad de la EspecieRESUMEN
The growth of Pseudomonas oleovorans on n-octane was characterized by the formation of intracellular structures. These inclusions were isolated and characterized. Morphologically, they resembled the poly-beta-hydroxybutyrate granules found in Bacillus cereus, as shown by freeze-fracture electron microscopy. The elemental analysis of isolated granules showed, however, that they do not contain poly-beta-hydroxybutyric acid. Instead, the analysis was consistent with a C8 polyester, which interpretation was supported by the fatty acid analysis of hydrolyzed granules. From the evidence presented here, we conclude that P. oleovorans forms poly-beta-hydroxyoctanoate granules when grown on n-octane.
Asunto(s)
Gránulos Citoplasmáticos/ultraestructura , Octanos/metabolismo , Pseudomonas/ultraestructura , Caprilatos/análisis , Fraccionamiento Celular , Gránulos Citoplasmáticos/análisis , Técnica de Fractura por Congelación , Microscopía Electrónica , Pseudomonas/metabolismoRESUMEN
We have optimized and compared the synthesis of 1,2-epoxyoctane from 1-octene by resting and by growing cells of Pseudomonas oleovorans. The net production of 1,2-epoxyoctane by resting cells never exceeded 0.6 mg/ml of suspension. In contrast, P. oleovorans produced much more epoxide when it was grown on high levels of 1-octene. To raise the total production of epoxide, the octene layer was repeatedly transferred to fresh, growing cultures of P. oleovorans. By using this approach, a maximum of 28 mg of epoxide was synthesized per ml of total culture, resulting in the accumulation of ca. 75 mg of epoxide per ml in the octene phase.
RESUMEN
ADP and ATP with a spin-label linked to the terminal phosphate are activators of glutamate dehydrogenase and bind to the same site as the activator ADP. There is hardly any interaction with the coenzyme site. Glutamate dehydrogenase can be modified with a ketone spin-label at a site in the active centre[Andree and Zantema, (1978) Biochemistry, 17, 778--783]. The spin-labelled activators interact with ketone spin-labelled glutamate dehydrogenase in the same way as with native glutamate dehydrogenase relative to the activator site, but show a stronger binding to the coenzyme site. Upon binding to the coenzyme site a spin-spin interaction between the ketone spin-label and the spin-labelled activators is observed. Nuclear magnetic resonance studies of the linewidth of 2-oxoglutarate and NADP+ bound to their functional sites on glutamate dehydrogenase without and with spin-labels result in distances between the ligand nuclei and the spin-labels. The results show that NADP+ binds in an open conformation consistent with the conformation in other dehydrogenases. The activator ADP binds in the neighbourhood of the active centre, but with very little or no overlap with the coenzyme site.
Asunto(s)
Glutamato Deshidrogenasa , Hígado/enzimología , Adenosina Difosfato , Adenosina Trifosfato , Animales , Sitios de Unión , Bovinos , Espectroscopía de Resonancia por Spin del Electrón , Activación Enzimática , Radicales Libres , Glutamato Deshidrogenasa/metabolismo , Técnicas In Vitro , Ácidos Cetoglutáricos/metabolismo , Cetonas , Cinética , Espectroscopía de Resonancia Magnética , Conformación Molecular , NADP/metabolismo , Conformación Proteica , Marcadores de SpinRESUMEN
The effect of toluene on Escherichia coli has been examined. In the presence of Mg2+, toluene removes very little protein, phospholipid, or lipopolysacharide from E. coli. In the absence of Mg2+, or in the presence of EDTA, toluene removes considerably more cell material, including several specific cytoplasmic proteins such as malate dehydrogenase (EC 1.1.1.37). In contrast, glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and glutamate dehydrogenase (EC 1.4.1.4) are not released at all under the same conditions. Cells treated with toluene in the presence of Mg2+ remain relatively impermeable to pyridne nucleotides, while cells treated with toluene in the presence of EDTA become permeable to these compounds. Freeze-fracture electron microscopy shows that toluene causes considerable damage to the cytoplasmic membrane, while the outer membrane remains relatively intact. These results indicate that the permeability characteristics of toluene-treated cells depend at least partly on the state of the outer membrane after the toluene treatment.