Chaperone activity and homo- and hetero-oligomer formation of bacterial small heat shock proteins

Rhizobia are the only bacteria known to induce a multitude of small heat shock proteins (sHsps) upon temperature upshift. The sHsps of Bradyrhizobium japonicum fall into two different classes, class A and class B. Here, we studied the chaperone activity and oligomeric features of two representative members of each class. The purified sHsps were efficient chaperones, as demonstrated by their ability to prevent thermally induced aggregation of citrate synthase in vitro. Homo-oligomer formation of all four sHsps was demonstrated by gel filtration and by two independent co-purification approaches. Mixed oligomers were readily observed between members of the same class, even when these proteins originated from different species such as Escherichia coli and B. japonicum. The chaperone activity of purified hetero-oligomers was indistinguishable from the activity of homo-oligomers. Heteromeric complexes were never obtained between class A and class B sHsps, indicating that hetero-oligomer formation is restricted to sHsps of the same class.     

Rapid kinetic analysis of EF-G-dependent mRNA translocation in the ribosome

Precise and coordinated movement of the tRNA-mRNA complex within the ribosome is a fundamental step during protein biosynthesis. The molecular mechanism for this process is still poorly understood. Here we describe a new sensitive method for monitoring elongation factor G-dependent translocation of the mRNA in the ribosome. In this method, the fluorescent probe pyrene is covalently attached to the 3' end of a short mRNA sequence at position +9. Translocation of the mRNA by one codon results in a significant decrease in the fluorescence emission of pyrene and can be used to directly monitor mRNA movement using rapid kinetic methods. Importantly, this method offers the flexibility of using any tRNA or tRNA analog in order to elucidate the molecular mechanism of translocation. Our results show that the mRNA is translocated at the same rate as the tRNAs, which is consistent with the view that the movement of the tRNAs and the mRNA are coupled in the ribosome. Furthermore, an anticodon stem-loop analog of tRNA is translocated from the ribosomal A site at a rate constant that is 350-fold lower than peptidyl tRNA, indicating that the D stem, T stem and acceptor stem of A site tRNA contribute significantly to the rate of translocation.     

Dopaminergic neuronal differentiation from rat embryonic neural precursors by Nurr1 overexpression

In vitro expanded CNS precursors could provide a renewable source of dopamine (DA) neurons for cell therapy in Parkinson's disease. Functional DA neurons have been derived previously from early midbrain precursors. Here we demonstrate the ability of Nurr1, a nuclear orphan receptor essential for midbrain DA neuron development in vivo, to induce dopaminergic differentiation in naïve CNS precursors in vitro. Independent of gestational age or brain region of origin, Nurr1‐induced precursors expressed dopaminergic markers and exhibited depolarization‐evoked DA release in vitro. However, these cells were less mature and secreted lower levels of DA than those derived from mesencephalic precursors. Transplantation of Nurr1‐induced DA neuron precursors resulted in limited survival and in vivo differentiation. No behavioral improvement in apomorphine‐induced rotation scores was observed. These results demonstrate that Nurr1 induces dopaminergic features in naïve CNS precursors in vitro. However, additional factors will be required to achieve in vivo function and to unravel the full potential of neural precursors for cell therapy in Parkinson's disease.     

Control of simuliid blackflies and mosquitoes withBacillus thuringiensis subsp.israelensis

Summary Bacillus thuringiensis subsp.israelensis produces a -endotoxin which is insecticidal against larvae of mosquitoes and blackflies. The toxin particles introduced into the water at concentrations of a few ng/ml kill rapidly after uptake by target larvae. Breakdown of the function of the gut epithelium is the primary cause for the lethal action of the toxin. The molecular mode of action is not yet understood in its details. However, it appears that the toxin is cytolytic and acts by disruption of membrane permeability systems. Field efficacy ofB. thuringiensis subsp.israelensis has been demonstrated in many countries. Most impressive results were obtained in West Africa where this microbial larvicide has been included in a large field programme to control blackflies, the vector of river blindness.

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Resumen La característica mas importante deBacillus thuringiensis es la producción de un metabolito con propiedades insecticidas durante el proceso de esporulación. Este se deposita normalmente en forma cristalina dentro del esporangio. Dichos cristales al ser ingeridos por larvas de insectos susceptibles se disuelven en el intestino y los polipéptidos liberados son activados mediante proteolisis. Las moléculas tóxicas resultantes matan a los insectos por destrucción del epitelio intestinal. Entre 30 subespecies, la denominadaisraelensis o BT H-14 es activa contra algunas familias de Diptera entre las cuales encontramos especies pertenecientes a los génerosCulex, Aedes, Anopheles ySimulium que son vectores de enfermedades tropicales infecciosas. La subespecieisraelensis, descubierta hace tan solo 10 años, ha sido ya utilizada por varias compañias como la base de potentes insecticidas para el control de los estadios larvales de dichos vectores. La toxina producida por la especieisraelensis esta incluida en una envuelta y tan solo una parte de la inclusión tiene extructura cristalina. La solubilización de la inclusión bajo condiciones reductoras produce una banda prominente en un rango de pesos moleculares entre 25000 y 30000 daltons. Se encuentran ademas bandas adicionales alrededor de 130000 y 65000 daltons. El modo de acción a nivel molecular no esta totalmente dilucidado. Sin embargo, parece ser que la toxina es citolítica y actua alterando los sistemas de permeabilidad de membrana. La eficacia en el campo deBacillus thuringiensis subsp.israelensis ha sido demostrada en muchos paises. Los resultados mas notables se han obtenido en la región oeste de Africa donde la cepa BT H-14 fue empleada en una gran operación de campo para controlar las larvas deSimulium damnosum, vectores de la cegera de rio. Como resultado se redujo la incidencia de la enfermedad. Se conocen distintos procedimientos para incrementar la eficacia de preparaciones comerciales. La modificación de las toxinas mediante ingeniería genética y la mejora de las formulaciones para conseguir una mayor persistencia son ejemplos de dos áreas prometedoras en la cuales se esta investigando en varios laboratorios.

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Résumé La principale caractéristique deBacillus thuringiensis est de produire, au cours de la sporulation, un métabolite insecticide. Celui-ci se dépose habituellement dans le sporange sous forme cristalline. Ces cristaux, lorsqu'ils sont ingérés par des insectes sensibles, se dissolvent dans l'intestin, et les polypeptides ainsi libérés sont activés par protéolyse. Les molécules toxiques qui en résultent tuent les insectes par destruction de l'épithélium intestinal. Parmi les 30 sous-espèces décrites, celle appeléeisraelensis ou BT H-14 est active à l'égard de quelques familles deDiptera parmi lesquelles on trouve des espèces appartenant aux GenresCulex, Aedes, Anopheles etSimulium, qui sont des vecteurs de maladies infectieuses tropicales. La sous-espèceisraelensis, découverte il ya seulement 10 ans, a déjà été utilisée par plusieurs firmes comme base de puissants insecticides pour la lutte contre les stades larvaires de ces vecteurs. La toxine produite par la sous-espèceisraelensis est contenue dans une enveloppe et une partie seulement de l'inclusion présente un arrangement cristallin. La solubilisation de l'inclusion en milieu réducteur donne une bande prédominante, correspondant à un poids moléculaire de 25 à 30.000 daltons. Des bandes accessoires sont situées autour de 130.000 et 65.000 daltons. Le mode d'action au niveau moléculaire n'est pas encore connu en détail. Cependant, il semble que la toxine soit cytolytique et agisse par destruction des systèmes de perméabilité membranaire. L'efficacité sur le terrain deB. thuringiensis subsp.israelensis a été démontrée dans de nombreux pays. Les résultats les plus impressionants ont été obtenus en Afrique Orientale, où BT H-14 a été employé dans une large opération sur le terrain de lutte contre les larves deSimulium damnosum, vectrices de la cécité des rivières. Il existe plusieurs possibilitiés pour accroître l'efficacité des préparations commerciales. La modification de la toxine par manipulation génétique et l'amélioration de la persistance des formules sont deux perspectives prometteuses, actuellement à l'étude dans plusieurs laboratoires.

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Invited paper presented at the VII International Conference on the Global Impacts of Applied Microbiology, Helsinki, 12–16 August 1985. Session 8     

Specific amplification of the ZFY gene to screen sex in man

Summary Using the polymerase chain reaction, a sequence comprising 400 bp of the human ZFY gene was amplified specifically in the male. The method allows detection of the presence of the ZFY gene in the order of 1:10⁴ cells.     

Simultaneous measurement of cholesterol 7 alpha-hydroxylase activity by reverse-phase high-performance liquid chromatography using both endogenous and exogenous [4-14C]cholesterol as substrate

The HPLC-spectrophotometric method (T. Ogishima and K. Okuda (1986) Anal. Biochem. 158, 228-232) for measuring cholesterol 7 alpha-hydroxylase activity was modified by using a C-18 reverse-phase column to separate 7 alpha-hydroxy-4-cholesten-3-one and 4-cholesten-3-one and by adding 7 beta-hydroxycholesterol to each reaction mixture as an internal recovery standard. With this method, we were able to simultaneously measure cholesterol 7 alpha-hydroxylase activity using endogenous cholesterol and exogenous [4-14C]cholesterol as substrate. Rat liver cytosol differentially stimulated (286%) the 7 alpha-hydroxylation of exogenous [4-14C]-cholesterol. In contrast, total cholesterol 7 alpha-hydroxylase activity was stimulated only 35% by cytosol. This method should prove useful for studying mechanisms of cholesterol delivery to cholesterol 7 alpha-hydroxylase.     

Vivid molecular divergence over volcanic remnants: the phylogeography of Megadromus guerinii on Banks Peninsula,New Zealand

Megadromus guerinii, an endemic carabid beetle (Carabidae), is the most common carabid throughout its restricted range on Banks Peninsula, a formation of extinct volcanoes in Canterbury, New Zealand. This study characterises the small-scale phylogeographic patterns of M. guerinii across the formerly volcanically active Banks Peninsula using mitochondrial and ribosomal genes. Between the eastern and western areas of the peninsula, the mitochondrial, but not nuclear, DNA has a well-defined geographic distribution. Specifically, mitochondrial cytochrome c oxidase subunit I (CO1) identifies two distinct groups (> 6% divergence between eastern and western beetles) while ribosomal genes show no discernible pattern. Whether such a pattern represents male-biased dispersal, Wolbachia infection, a recent range expansion of a divergent lineage, or a deeper historic separation is explored. There is potential that male-biased dispersal could have occurred. Wolbachia infection was not detected. We conclude that historical processes have likely separated taxa in the eastern and western peninsula.