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.     

Kinetics and thermodynamics of lactate dehydrogenases from beef heart, beef muscle, and flounder muscle.

The eight rate constants for a four-step ordered ternary-complex mechanism have been compared for lactate dehydrogenases (EC1.1.1.27) from three sources, beef heart, beef muscle, and flounder muscle. The rate constants were determined at temperatures ranging from 5 degrees C to 50 degrees C, and the corresponding activation parameters deltaG not equal to, deltaH not equal to, and deltaS not equal to were calculated. Significant differences are noted for the values for the three types of enzyme. The relative heights of the activation barriers are much the same in all three cases, differences in kinetic behavior resulting mainly from differences in the stable binary and ternary enzyme-substrate complexes. These complexes are, in general, at lower free-energy and enthalpy levels of the beef-heart and beef-muscle enzymes than for the flounder-muscle enzyme. A high degree of compensation is found between the enthalpies and entropies of activation, resulting in relatively small differences between the free energies (and rates) for homologous steps with different enzymes. Analysis of the results, on the assumption that the compensation effect is due to weak-bonding effects, suggests that there are fewer weak bonds in the stable complexes of the muscle enzymes.     

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%.     

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.     

How to Report Record Open‐Circuit Voltages in Lead‐Halide Perovskite Solar Cells

Open‐circuit voltages of lead‐halide perovskite solar cells are improving rapidly and are approaching the thermodynamic limit. Since many different perovskite compositions with different bandgap energies are actively being investigated, it is not straightforward to compare the open‐circuit voltages between these devices as long as a consistent method of referencing is missing. For the purpose of comparing open‐circuit voltages and identifying outstanding values, it is imperative to use a unique, generally accepted way of calculating the thermodynamic limit, which is currently not the case. Here a meta‐analysis of methods to determine the bandgap and a radiative limit for open‐circuit voltage is presented. The differences between the methods are analyzed and an easily applicable approach based on the solar cell quantum efficiency as a general reference is proposed.     

Desert breath—How fog promotes a novel type of soil biocenosis,forming the coastal Atacama Desert’s living skin

The Atacama Desert is the driest non‐polar desert on Earth, presenting precarious conditions for biological activity. In the arid coastal belt, life is restricted to areas with fog events that cause almost daily wet–dry cycles. In such an area, we discovered a hitherto unknown and unique ground covering biocenosis dominated by lichens, fungi, and algae attached to grit‐sized (~6 mm) quartz and granitoid stones. Comparable biocenosis forming a kind of a layer on top of soil and rock surfaces in general is summarized as cryptogamic ground covers (CGC) in literature. In contrast to known CGC from arid environments to which frequent cyclic wetting events are lethal, in the Atacama Desert every fog event is answered by photosynthetic activity of the soil community and thus considered as the desert's breath. Photosynthesis of the new CGC type is activated by the lowest amount of water known for such a community worldwide thus enabling the unique biocenosis to fulfill a variety of ecosystem services. In a considerable portion of the coastal Atacama Desert, it protects the soil from sporadically occurring splash erosion and contributes to the accumulation of soil carbon and nitrogen as well as soil formation through bio‐weathering. The structure and function of the new CGC type are discussed, and we suggest the name grit–crust. We conclude that this type of CGC can be expected in all non‐polar fog deserts of the world and may resemble the cryptogam communities that shaped ancient Earth. It may thus represent a relevant player in current and ancient biogeochemical cycling.     

The enigmatic Crimean green lizard (Lacerta viridis magnifica) is extinct but not valid: Mitogenomics of a 120-year-old museum specimen reveals historical introduction

It has been proposed that Lacerta viridis magnifica Sobolevssky, 1930 represents an extinct species or subspecies of green lizard endemic to the southern Crimea. Using NGS protocols optimized for heavily degraded DNA, we sequenced the complete mitogenome of one of the originally formalin-preserved specimens collected in the late 19th century. A comparison with sequence data of other green lizards revealed that L. v. magnifica is a junior synonym of the northern subspecies of the western green lizard (L. b. bilineata Daudin, 1802), which occurs at least 1,500 km away, beyond the distribution ranges of other green lizards. In medieval times, a Genoese colony existed in the Crimean region where the extinct green lizards occurred. Until the early 20th century, close ties to Italy persisted, and locals of Genoese descent sent their children for education to Italy, where L. b. bilineata occurs. This suggests that the extinct Crimean green lizards have been introduced accidentally or intentionally from Italy. Our study exemplifies the value of historical formalin-preserved museum specimens for clarifying the status of questionable rare or extinct taxa.     

SANS (USH1G) regulates pre-mRNA splicing by mediating the intra-nuclear transfer of tri-snRNP complexes

Splicing is catalyzed by the spliceosome, a compositionally dynamic complex assembled stepwise on pre-mRNA. We reveal links between splicing machinery components and the intrinsically disordered ciliopathy protein SANS. Pathogenic mutations in SANS/USH1G lead to Usher syndrome—the most common cause of deaf-blindness. Previously, SANS was shown to function only in the cytosol and primary cilia. Here, we have uncovered molecular links between SANS and pre-mRNA splicing catalyzed by the spliceosome in the nucleus. We show that SANS is found in Cajal bodies and nuclear speckles, where it interacts with components of spliceosomal sub-complexes such as SF3B1 and the large splicing cofactor SON but also with PRPFs and snRNAs related to the tri-snRNP complex. SANS is required for the transfer of tri-snRNPs between Cajal bodies and nuclear speckles for spliceosome assembly and may also participate in snRNP recycling back to Cajal bodies. SANS depletion alters the kinetics of spliceosome assembly, leading to accumulation of complex A. SANS deficiency and USH1G pathogenic mutations affects splicing of genes related to cell proliferation and human Usher syndrome. Thus, we provide the first evidence that splicing dysregulation may participate in the pathophysiology of Usher syndrome.