A Go-like protein in Drosophila melanogaster and its expression in memory mutants.

G proteins couple receptors for extracellular signals to several intracellular effector systems and play a key role in signalling transduction mechanisms. In particulate preparations of Drosophila melanogaster heads, only one substrate for pertussis toxin at 39-40 kd was detected. This substrate, which showed only one isoform when analysed by isoelectric focusing, was recognized by immunoblotting and immunoprecipitation techniques using a polyclonal antibody against the alpha subunit of the Go protein purified from bovine brain and can be thus considered as a Go-like protein. Antibodies obtained against a carboxy-terminal sequence of the alpha subunit of Go (but not of Gi1 or Gi2) and against an internal sequence shared by all the alpha subunits, were also able to cross-react with the alpha subunit of this protein in insects. We have also studied the Go-like protein in several D.melanogaster mutants, primarily in memory and learning mutants. In these mutants there was a sex-dependent enhancement in pertussis toxin-catalysed ADP-ribosylation with respect to the wild-type. This increase could be attributed in part to an increase in the alpha subunit of the Go-like protein, as revealed by immunoblotting with anti-Go alpha polyclonal antibody. This report constitutes the first evidence for the participation of a Go protein in learning and memory.     

Chemical mechanism of lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung. pH-dependence of kinetic parameters.

Lysophosphatidylcholine: lysophosphatidylcholine acyltransferase is an enzyme that catalyses two reactions: hydrolysis of lysophosphatidylcholine and transacylation between two molecules of lysophosphatidylcholine to give disaturated phosphatidylcholine. Following the kinetic model previously proposed for this enzyme [Martín, Pérez-Gil, Acebal & Arche (1990) Biochem. J. 266, 47-53], the values of essential pK values in free enzyme and substrate-enzyme complexes have now been determined. The chemical mechanism of catalysis was dependent on the deprotonation of a histidine residue with pK about 5.7. This result was supported by the perturbation of pK values by addition of organic solvent. Very high and exothermic enthalpy of ionization was measured, indicating that a conformational re-arrangement in the enzyme accompanies the ionization of the essential histidine residue. These results, as well as the results from previous studies, enabled the proposal of a chemical mechanism for the enzymic reactions catalysed by lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung.     

Penetration of intact bovine ova with ram sperm in vitro

In culture, mature bovine ovarian oocytes were fertilized in vitro with freshly ejaculated ram spermatozoa treated with heparin. The zona pellucida does not prevent penetration of ram spermatozoa. The penetration rate varied between 10 and 84%, and in most instances, after 24 hr of culture, two normal-looking pronuclei and sperm tail were present in the cytoplasm. These results suggest that the zona pellucida of bovine oocytes does not represent a barrier for the penetration of ram spermatozoa.     

Ultrastructural localization of silver-staining nuclear proteins at the onset of transcription in early bovine embryos.

Argyrophilic nuclear proteins, known to be functionally associated with ribosomal genes, were localized, in four-, eight-, and 16-cell bovine embryo blastomere nuclei using two different silver-staining procedures. Within the eight-cell cleavage stage by the process of embryonal nucleologenesis in the cow embryo the full-capacity ribosome-producing machinery is established. In the four-cell embryo, many patches and islands of argyrophilic (Ag+) material were detected in the nucleoplasm. The nucleolus-precursor bodies (NPBs), composed uniformly of a homogeneous compact mass, were completely devoid of any silver staining. On the other hand, clear-cut localization of argyrophilic proteins was detected during the eight-cell stage either inside the transforming NPBs or in the close vicinity, or in the already differentiated nucleolus. In compact, nonvacuolated NPB, an intensive Ag+ area was detected, in the form of a lenticle, at the periphery of the NPB. During and following vacuolation of the NPB, no Ag+ was detected inside these vacuoles. It was seen, however, in the dense fibrillar nucleolar component surrounding the smaller vacuoles formed at the time of the establishment of nucleolar structure. Ag+ areas were seen repeatedly in the vicinity of NPBs, probably a part of the nucleolus-associated chromatin or, alternatively, representing the extranucleolar bodies. In blastomere nuclei of 16-cell embryos, already possessing reticulated nucleoli known from intensively synthesizing somatic cells, the silver-staining pattern corresponded to the usual situation in differentiated cells: slight staining of fibrillar centers, heavy labelling in the dense fibrillar component, and absence of silver deposits in the granular component.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid colorimetric detection of in vitro amplified DNA sequences

A colorimetric assay to detect immobilized amplified nucleic acids has been designed. This approach provides a rapid assay, suitable for clinical diagnosis, to analyze DNA sequences amplified by the polymerase chain reaction. The specific DNA sequences are captured on a solid support by the use of a recombinant fusion protein consisting of the Escherichia coli lac repressor and staphylococcal protein A. The biotin streptavidin system is used to detect the immobilized material. Positive samples can be analyzed by direct solid-phase sequencing. Here, we show that this nonradioactive concept can be used for analysis of Staphylococci and Streptococci and for specific detection of the protozoa Plasmodium falciparum in clinical samples.     

Phytoecological importance,mutual redundancy and phytological threshold values of certain climatic factors

A combination of methods (intensity of indication, floristic and mesological redundancy analysis, beta-diversity analysis, principal components analysis and Wildi's interactive ranking procedure) were used to evaluate redundancy and relative phytoecological importance among 80 climatic variables in Galicia (N.W. Spain). The information they contained was found to be adequately summarized by just 3 factors thought to play a major role in regulating the distribution of the species considered in the study area and similar areas: Baudiere's QE index, mean minimum temperature in the coldest month and mean temperature range in the coldest month. For these three factors, phytoclimatic thresholds were determined by examining beta-diversity and were used to define phytoclimatic zone types.

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Resumen Se valora la redundancia e importancia fitoecológica relative de 80 variables climáticas en Galicia (N.O. de España) empleando una combinacion de diferentes metodos (intensidad indicadora, análisis de la redundancia florística y mesológica, análisis de beta-diversidad, análisis de componentes principales y el método de ordenación interactiva de Wildi). La información contenida en esta variables es adecuadamente resumida por 3 factores que juegan un papel predominante en regular la distribución de las especies consideradas en el area de estudio y areas similares: el índice QE de Baudiere, la temperatura media de las mínimas del mes más frío y la oscilación térmica del mes más frío. Para estos factores el análisis de la beta-diversidad permitió determinar los umbrales fitoclimáticos utilizados en la definición y cartografía de las zonas fitoclimáticas.