Reconstitution of the 9 S estrogen receptor with heat shock protein 90

As a first step in the investigation of the reconstitution of steroid hormone receptor systems, we studied the reconstitution of 9 S estrogen receptor (ER) from purified vero ER, which is the estradiol binding subunit, and heat shock protein 90 (hsp 90). By using a phosphate buffer containing molybdate, thiocyanate, dimethylformamide, glycerol, etc., vero ER could be converted to 9 S ER with hsp 90, but not with the control protein, ovalbumin. Inactivation of ER during the reconstitution was suppressed partially by hsp 90, but not by ovalbumin. Like native 8 S ER, the reconstituted ER was sedimented at about 8.9 S and 4.6 S on glycerol gradient centrifugation in low and high salt buffers, respectively.     

NADPH-cytochrome P-450 oxidoreductase gene organization correlates with structural domains of the protein

cDNA clones to rat liver NADPH-cytochrome P-450 oxidoreductase were used to isolate genomic clones from a Wistar-Furth inbred rat genomic DNA library. Fifteen exons containing the coding region and 3'-nontranslated segment of the P-450 reductase gene were identified, spanning 20 kilobases of DNA contained in 3 lambda-Charon 35 clones. The organization of this single copy gene reveals a general correspondence between exons and structural domains of the protein, with the segment responsible for anchoring the reductase to the microsomal membrane and several segments involved in FMN, FAD, and NADPH binding encoded by discrete exons.     

Biosynthesis of Prostacyclin in Rat Cerebral Microvessels and the Choroid Plexus

Abstract: Microvessels, predominantly capillaries, were isolated from rat cerebrum by a modification of published procedures. The morphology and purity of the preparations were monitored by light and electron microscopy and by enrichment in alkaline phosphatase, γ-glutamyl transpeptidase, and prostacyclin synthetase. A reversed-phase high-pressure liquid chromatographic method was used in the purification of prostaglandins after extraction from aqueous incubation solutions. Prostacyclin synthesis in brain is localized in cerebral blood vessels and capillaries. The endogenous biosynthetic capacity of the isolated cerebral capillary fractions for prostacyclin, measured as its chemically stable breakdown product, 6-keto-prostaglandin F_1α, was 11 ng/mg protein/10 min. Choroid plexus and intact surface vessels synthesized 6-keto-prostaglandin F_1α at 37 and 35 ng/mg protein/10 min, respectively. The prostacyclin-synthesizing enzyme of the cerebral capillaries also converted the exogenously added prostaglandin endoperoxides to 6-keto-prostaglandin F_1α. Comparison of the synthesis of prostaglandins 6-keto-F_1α, E₂, and F_2α showed that 6-keto-prostaglandin F_1α was the major prostaglandin formed in the microvessels, in the larger surface vessels, and in the choroid plexus. Prostaglandin D₂ was not detected. Prostacyclin synthesis by the cerebral vasculature is similar to that in other blood vessels and cultured human endothelial cells. Possible physiological roles of prostacyclin in the cerebral microvasculature are discussed with special regard to the autoregulation of cerebral blood flow.     

Immunological and biochemical characterization of nuclear envelope reduced nicotinamide adenine dinucleotide phosphate-cytochrome c oxidoreductase.

NADPH-cytochrome c oxidoreductase (EC 1.6.99.2) activity innate to rat liver nuclear envelope displays antigenic identity with the corresponding microsomal enzyme in a standard Ouchterlony double immunodiffusion test. As with the microsomal enzyme, the nuclear envelope enzyme is selectively released by restricted proteolysis and may be quantitatively isolated from the supernatant phase of the digest by immunoprecipitation. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the immunoprecipitates reveals that the oxidoreductase has a molecular weight of 72,000 regardless of its membrane of origin. Radial immunodiffusion titration demonstrates that nuclear envelope contains about one-third the level of NADPH-cytochrome c oxidoreductase (0.21%) as compared to microsomal membrane (0.71%) on a weight basis. By comparison, the specific activity of the nuclear envelope enzyme was half that of the microsomal enzyme. Turnover studies employing NaH¹⁴CO₃ indicate that the half-lives for the nuclear envelope and microsomal enzymes are indistinguishable, each being approximately 55 h.     

Tritosol: A new scintillation cocktail based on Triton X-100

A scintillation cocktail consisting of 3.0 g PPO, 257 ml Triton X-100, 106 ml ethanol, 37 ml ethylene glycol, and 600 ml xylene is described. A linear relationship between counting efficiency and the external standard ratio could be demonstrated over a wide range of quenching. The counting efficiencies (unquenched) for³H are about 47%, for¹⁴C about 87%, and for⁴⁵Ca about 80%.     

PERTICA,A NEW GENUS OF DEVONIAN PLANTS FROM NORTHERN MAINE

A new genus of Devonian age fossil plants is described from the Trout Valley Formation of northern Maine. Abundant compression material permits a rather complete understanding of its morphology. Pertica quadrifaria Kasper and Andrews, gen. et sp. nov., was an erect plant, perhaps a meter tall, with a pseudomonopodial main axis and dichotomous side branches. The side branches were arranged in a clockwise spiral (from base to apex) and were tetrastichous. They dichotomized numerous times, with the intervals between dichotomies decreasing distally. The ultimate branchlets bore numerous sporangia in dense clusters. Other side branches were completely sterile. Pertica quadrifaria is classified in the Subdivision Trimerophytina of Banks. Its evolutionary significance rests in the fact that it is a link in the chain of increasingly complex early vascular land plants.     

Zur Spezifität des histochemischen Carboanhydratase-Nachweises im inselorgan der Bauchspeicheldrüse

Zusammenfassung Die enzymatische Aufspaltung von Kobalthydrogenkarbonaten durch das Ferment CAH führt beim Häuslerschen Fermentnachweis über eine sekundäre Visualisation der Kobaltkarbonatniederschläge durch Umwandlung in Kobaltsulfid zur Markierung der Fermentaktivität am Schnitt. Untersuchungen am Pankreas zeigen, daß die Aussagekraft fermentmarkierender Niederschläge durch eine unspezifische Fällung zweiwertiger, nichtfermentgebundener Zinkionen beeinträchtigt wird. Das inkretorische Parenchym des Pankreas enthält keine CAH, wird aber intensiv imprägniert durch seinen Reichtum an nichtfermentgebundenen Zinkionen. Nach Abfangen dieser Metallionen durch Metallchelatbildner (Dithizon, NDDC) fehlt eine Kobaltsulfidschwärzung der Inselzellen.Im exkretorischen Parenchym führt dagegen die Bebrütung von Kryostatschnitten zu spezifischer Fermentmarkierung. Aus sterischen Gründen (tertiäre Bindung des zweiwertigen Metalls im Enzymmolekül) ist das CAH-Zink einer Chelatbindung mit Dithizon oder NDDC nicht zugänglich, die Aktivität der CAH in den Gangepithelien der Bauchspeicheldrüse wird durch Chelatbildner nicht beeinflußt.Die Spezifität des Häuslerschen Fermentnachweises ist unbestritten. Zu fordern ist aufgrund der vorliegenden Ergebnisse aber eine Kontrolle der Fermentreaktion durch Chelatbildner, um eine gleichzeitige unspezifische Imprägnation zweiwertiger Metallionen (insbesondere des Zinks) im Kryostatschnitt auszuschließen.

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Summary In the Häusler incubation method the demonstration of the catalytic activity of carbonic anhydrase in tissue sections involves an enzymatic splitting of cobalt hydrogencarbonates. The resulting carbonate precipitates are secondarily visualized by being transformed into cobalt sulfide. Examination of the pancreas by the modified Häusler reaction indicates that the specificity of the precipitates marking the enzyme is masked by an unspecific precipitation of bivalent zinc ions that are not bound to the enzyme. The endocrine parenchyma of the pancreas does not contain any carbonic anhydrase, but is intensely impregnated because of its abundance of zinc ions bound to insulin or glucagon. If these metallic ions are sequestered by the use of metal chelating agents (dithiozone, sodium diethyldithiocarbamate), the unspecific precipitates in the islets of Langerhans are eliminated.In the exocrine parenchyma, however, the incubation of cryostat sections results in a specific demonstration of the enzyme. The steric arrangement of zinc ions within the enzymic molecules (trivalent complexes of bivalent metal ions) prevents a complex linkage of dithiozone or sodium diethyldithiocarbamate to carbonic anhydrase. The activity of carbonic anhydrase in the epithelium of the ducts is not at all inhibited by chelating agents.The specificity of the Häusler incubation method for demonstrating carbonic anhydrase is uncontested. The results show, however, that in histochemical studies of the enzyme the use of chelating agents is necessary as a control to exclude a simultaneous unspecific precipitation of bivalent metal ions (especially zinc) in cryostat sections.

     

Non-condensation of one segment of a chromosome No. 2 in a male with an otherwise normal karyotype (and severe hypospadias)

Summary A child with severe hypospadias is presented, whose karyotype showed in about 11% of mitoses of peripheral blood one member of chromosome pair No. 2 with a non-condensed region near the centromere. The non-condensed segment does not show late replication, however, it is situated very close to the late replicating segment of the long arms of chromosome No. 2. The nature and possible implications of this kind of aberration are discussed. It is held that non-condensation can produce localized chromosome breaks by a mechanism possibly different from any of the classical breakage mechanisms.