Long-Term Effect of Forskolin on the Activation of Adenylyl Cyclase in Astrocytes

Abstract: Long-term (48-h) forskolin treatment of rat astroglial cells led to a slight decrease (30–40%) in the response to isoproterenol, vasoactive-intestinal peptide, guanyl 5'-(βγ-imido)diphosphate, guanosine 5'- O -(3-thiotriphosphate) [GTP(S)], and AIF₄⁻ in crude membrane fractions. In contrast, the acute stimulatory effect of forskolin was increased by 1.25–1.5-fold. These two opposite effects of forskolin were mediated by a cyclic AMP-dependent mechanism. No changes in G_sα, G_iα, or Gβ protein levels could be determined by immunoblotting using specific antisera. No significant differences were observed in the ability of G proteins extracted from control and forskolin-treated cells to reconstitute a full adenylyl cyclase activity in membranes from S49 cyc⁻ cells, lacking G_sα protein. G_sα proteins were detected in two pools of membranes, one in the heavy sucrose fractions and the other in light sucrose fractions. Forskolin treatment of the cells shifted G_sα protein toward the light-density membranes. We did not find any significant change in the distribution of adenylyl cyclase. In contrast to the decreased stimulation of adenylyl cyclase activity by agonists acting via G_sα, observed in the crude membrane fraction, the responses of adenylyl cyclase to forskolin as well as to GTP(S) were increased in the purified plasma membrane fractions. These results may indicate that sensitization of the catalyst appears to be the dominant component in the astroglial cell response to long-term treatment by forskolin.     

Membrane receptors for estrogen,progesterone, and testosterone in the rat brain: Fantasy or reality

Summary 1. There are numerous circumstantial evidence supporting the concept that steroid hormones control cellular function by means other than the nuclear receptor steroid binding mechanism. It is the intent of this report to present evidence indicating that steroids bind to specific sites in neuronal membranes.2. Some of the criteria to define steroid membrane receptors using steroid-BSA conjugates that can be radioiodinated to desired specific activity have been fulfilled for each of the three sex steroids using crude synaptosomal membrane preparations (P2 fractions) from the CNS of female and male rats. Ligand binding for each of the three steroids indicate high-affinity and high-capacity sites with distinct brain selectivity and stereospecificity. For example, 17-E-6-[¹²⁵I]BSA binds hypothalamic P2 fractions (HYP-P2) with an estimatedK _d of about 3±0.7 nM (X ± SE;n=3), whereas the cerebellum P2 (CB-P2) fractions bind the ligand with aK _d of 34±7 nM and, aB _max of 3 and 42 pmol/mg protein, respectively. Estrogen and testosterone binding fit best a one-single site, while progesterone binding sites can be best represented by a two-binding site, one high-affinity (K _d=1–2 nM) and one low affinity (K _d=62 nM), in CB-P2 fractions from intact adult female rat brain. Kinetics studies for T-3-[¹²⁵I]BSA indicate that the estimatedK _d of 30±2 nM for the olfactory bulb P2 fractions (OB-P2) from male rats is in good agreement withK _d values computed from Scatchard-derived data using the LIGAND algorithm.3. 17-E-6-[¹²⁵I]BSA binding sites are stereospecific and appears to be present as early as 5 days of age in both the OB- and the CB-P2 fractions without changes during development. In contrast, P-6-[¹²⁵I]BSA binding sites are practically absent during days 5 and 12 and appear by day 22.4. Finally, membrane receptor molecules for estrogen and progesterone have been isolated and purified by affinity chromatography and characterized by PAGE and Western blot. Microsequencing of one of the membrane estrogen binding proteins indicates that the high-affinity site corresponds to the OSCP subunit of the proton ATP synthase.5. It remains to be determined if P and T also bind to this complex enzyme or if they bind to other subunits of the family of proton ATPases. Overall the data indicate that steroid hormones conjugated to BSA are important tools to study the reality of membrane steroid receptors.     

Ichnology and sedimentology of the early Permian deep-water deposits from the Lercara-Roccapalumba area (Western Sicily,Italy)

Summary Diverse and abundant trace fossils of the deep-waterNereites ichnofacies have been found in well-dated Early Permian deep-water turbidites (Lercara Formation) of western Sicily (Italy). Conodonts indicate a latest Artinskian to Cathedralian (late Early Permian) age. Microfossils (pelagic conodonts, albaillellid Radiolaria, paleopsychrospheric ostracods, foraminiferal associations dominated byBathysiphon), trace fossils (deep-bathyal to abyssalNereites ichnofacies) and sedimentologic data collectively indicate a deep-water environment for the Early Permian turbidites of the Lercara Formation. The dominance ofAgrichnium and of thePaleodictyon subichnogeneraSquamodictyon andMegadictyon suggests that this icnofauna is closely related in ichnotaxonomic composition to other late Paleozoic deep-water ichnofaunas. The occurrence ofAcanthorhaphe. Dendrotichnium andHelicoraphe, to date only reported from Cretaceous or Tertiary flysch deposits, suggests that the entire ichnofauna corresponds well to previously documented Silurian-Tertiary flysch ichnofaunas. Eight new ichnospecies and a new ichnosubgenus,Megadictyon, are described.     

Early ordovician reefs from Argentina: Stromatoporoid vs stromatolite origin

Summary Late Arenigian biohermal reef mounds and biostromes within the shallow-marine platform facies of the upper San Juan Formation of the Precordillera (Western Argentina) represent a new Early Ordovician reef type. The meter-sized reefs are dominated byZondarella communis n.g. n. sp. The new taxon is characterized by domical, bulbous and laminar morphotypes exhibiting growth layers and thin horizontal and vertical as well as intermingled skeletal elements included within different sets. The fossil maybe compared with stromatolites and stromatoporoids but an interpretation as primitive stromatoporoids is favoured.     

Comparison of ELISA and immunoblotting techniques for the detection of cherry mottle leaf virus

Formaldehyde treated cherry mottle leaf virus (ChMLV) and the isolated coat protein were used successfully for the production of polyclonal and monoclonal antibodies. The monoclonal antibodies had a titre of 1:51 200 and consisted of IgG1 and IgG2. The antibodies reacted with all 11 isolates of ChMLV, from five locations in Canada and the USA, included in this study. Several serological procedures were assessed to compare their sensitivity for detecting ChMLV. Plate-trapped antigen ELISA (PTA-ELISA) and dot-blot immunobinding assay (DBIA), using virus specific MAbs, were the most sensitive tests in this study. Triple antibody sandwich ELISA (TAS-ELISA) and Western blot were found to be less sensitive. Dilution of the samples appeared to increase the sensitivity of both PTA-ELISA and Western blot detection. Young leaves and flowers of Prunus avium were the best tissue for detecting the virus which could also be detected in the fruit and leaves of P. tomentosa. April and May were optimal for detection of the virus in the field, whereas both April to May and August to September were optimal for screenhouse-grown plants.     

Keratella distribution in North Patagonian lakes (Argentina)

The distribution of Keratella species from 15 different lakes in North Patagonia (Argentina) was analysed. The genus was not present at altitudes above 1000 m. K. tropica was restricted to Patagonian Plateau lakes with a comparatively high conductivity. A morphometric analysis of the widely distributed K. cochlearis was performed. Results showed three groups of K. cochlearis corresponding to Andean lakes, Patagonian Plateau lakes and a Patagonian Reservoir.     

Electro-immunoblotting characterization ofPseudo-nitzschia multiseries andP. pungens antigens recognized by antibodies directed against whole cells

Separate polyclonal antibodies have previously been developed against the domoic-acid-producingPseudonitzschia multiseries (=Pseudo-nitzschia pungens f.multiseries) and the non-toxicP. pungens (=P. pungens f.pungens). These antibodies bind to the surface of the diatoms as shown by immunofluorescence studies. Here we examine the molecular nature of the antigens by Western blotting (electro-immunoblotting) analysis. The major antigens for both polyclonal antibodies migrated as high molecular-weight diffuse bands, mostly remaining in the stacking gel, using an SDS-PAGE system. The antibodies prepared againstP. multiseries strongly labelled the high molecular-weight antigens of allP. multiseries strains tested and showed little reactivity towardsP. pungens extracts on Western blots.P. pungens antibodies strongly labelled high molecular-weightP. pungens antigens and faintly labelled a fewP. multiseries antigens. The selectivity of the antibodies for their respective species correlates with the results of the immunofluorescence experiments, suggesting that the antigens examined in this study are responsible for the selective labelling in immunofluorescence studies. The electrophoretic mobility and the antibody labelling of antigens were not altered by proteolytic digestion of cell pellets. However, disruption of carbohydrates in the pellets by treatment with periodic acid resulted in loss of the antigen. These data suggest that the major antigens of toxicP. multiseries and non-toxicP. pungens are high molecular-weight (°>100kDa) polysaccharides located on the surface of these diatoms.Author for correspondence     

人白介素6受体功能区片段在E．coli中的表达

通过ＤＮＡ体外重组技术,以ｐＥＴ－３ｂ为表达载体,构建了重组表达质粒ｐＥＴ－６Ｒ（Ｂ）和ＰＥＴ－６Ｒ（Ｂ）４,分别编码２８ｋＤ的ｈＩＬ－６Ｒ配基结合区片段及其５３ｋＤ的二联体蛋白,并为酶切分析和ＤＮＡ序列分析所证实。ＳＤＳ－ＰＡＧＥ分析表明,含有重组表达质粒的菌株可分别表达出２８ｋＤ的蛋白ｒＩＬ６Ｒ－２８和５３ｋＤ的ｒＩＬ６Ｒ－５３。重组蛋白分别占菌体总蛋白的４５％和２９％左右。重组蛋白主要以包涵体形式存在,Ｗｅｓｔｅｒｎ印迹表明重组蛋白具有ＩＬ－６Ｒ的抗原性。     

Arable acronyms analysed – a review of integrated arable farming systems research in Western Europe

Arable production has come under increasing economic and environmental pressures, especially in the last decade. These have derived from over-production, decreased farm incomes and a concern with the possible environmental effects of intensive pesticide use associated with such intensive cultivation. A number of long-term research programmes on integrated farming systems and their sustainability have recently been completed or are currently under way. In the UK, these include the ‘Boxworth’ project, ‘SCARAB’, TALISMAN’, RISC, ‘LINK Integrated Farming Systems’, ‘LIFE’ and the demonstration-only programme ‘LEAF’. These projects are reviewed in terms of their objectives, designs and results to date, and are compared with some parallel programmes in Germany, the Netherlands, Switzerland and France.