Glycosylation of high-affinity thrombin receptors appears necessary for thrombin binding.

Monosaccharide binding competition, lectin affinity chromatography, and glycosylation inhibitors have been used to determine if glycosylation plays a role in thrombin-receptor interactions. Mannose appeared to specifically inhibit thrombin binding to mouse embryo (ME) and hamster fibroblasts. Concanavalin A bound to antibody-purified receptor fractions, and was used as an affinity ligand to purify receptor fractions that retained thrombin binding activity. Cells treated with tunicamycin (6.25 ng/ml) for 24 h lost approximately 35% of their high-affinity thrombin binding sites, yet binding of receptor monoclonal antibody TR-9 was not affected, indicating that the receptor was present in the membrane, but unable to bind thrombin. Thus thrombin receptor glycosylation may be directly involved in thrombin binding.     

Zeitaufwand für das Ernten und Verstecken von Kiefernsamen bei Dünnschn?bligen Tannenh?hern (Nucifraga caryocatactes macrorhynchos) im Fernen Osten Russlands

Zusammenfassung In einem Vorkommen des Dünnschnäbligen Tannenhähers an der Küste des Ochotskischen Meeres im Fernen Osten Sibiriens wurde das Ernten und Verstecken von Samen aus den Zapfen der Zwergzirbelkiefer (Pinus pumila) untersucht. Der Inhalt von durchschnittlich 2,8 Zapfen, das sind etwa 80 Samen, wurde in der gefüllten Kehltasche transportiert und auf eine Anzahl unter niedriger Zwergstrauchvegetation gelegener Bodenverstecke verteilt. Die Verstecke wurden in annähernd linearer Anordnung ohne Bevorzugung einer bestimmten Himmelsrichtung angelegt. Die Versteckserien enthielten im Median 79, maximal mehr als 120 Samen, das Einzelversteck durchschnittlich 19,6 Samen. Das Ernten und Leeren eines Zapfens geschah im Schnitt innerhalb von 47 s. Für das Verstecken einer Füllung des Kehlsacks benötigten die Vögel ca. 170 s. Für das gesamte Beschaffen und Verstecken eines einzelnen Kiefernsamens errechnet sich ein durchschnittlicher Zeitbedarf von 3,26 s. Nach 20 Tagen war der Zapfenvorrat in der lokalen Kiefernpopulation erschöpft. Jeder Häher hat nach den Hochrechnungen bis zu 100.000 Samen vergraben.

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Harvesting and caching capacities of Thin-billed Nutcrackers in the Russian Far East

Summary At the Ochotskian sea coast Thin-billed Eurasian Nutcrackers (Nucifraga caryocatactes macrorhynchos) harvested seeds of ripe cones of the brush pinePinus pumila in late summer. The mean number of seeds carried in their sublingual pouch was 80, which respresents the harvestable contents of 2.8 cones. These were distributed in an average of 5 caches, exclusively in the soil under low tundra vegetation. Caches were organized in nearly straight lines. Series contained a mean of 82.7 seeds, single caches a mean of 19.6 seeds. Plucking one cone and harvesting its seeds took 47 seconds on average. The caching of a complete pouchful took on average 123.4 seconds. The time invested for harvesting and caching one single seed was calculated at 3.26 seconds. Within three weeks in July, an average individual bird was calculated to have cached a total of up to 100,000 seeds.

     

Ein Vogelruf, dessen Form sich im Jahresablauf ?ndert: der Verbeugungstriller bei der Brandente (Tadorna tadorna)

Zusammenfassung Der Verbeugungstriller männlicher Brandenten, eine epigame Lautäußerung, weist sowohl in syntaktischen wie auch phonetischen Parametern zyklische jahreszeitliche Veränderungen auf. Die Gesamtdauer der Lautäußerung vergrößert sich allmählich im Laufe des Frühjahrs. Dafür sind eine Verlängerung des tonalen Eingangselements und eine Vermehrung der Elemente der abschließenden Phrase verantwortlich. Dagegen verkürzt sich das 2. Element gleichzeitig. In allen untersuchten Elementen erhöht sich die Tonhöhe parallel zur Verlängerung des Rufes. Diese Veränderungen des Ausdrucksverhaltens geben vermutlich Änderungen innerer Zustandsgrößen wieder.

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Seasonally changing bird call: the trill call of male Shelducks (Tadorna tadorna)

Both syntactic and phonetic features of the trill call accompanying the whistle-shake in adult male Shelducks are subject to cyclic annual changes. In the course of spring, the duration of the whole call increases gradually. This is caused by a prolongation of the first call element and insertion of additive elements in the final phrase. In contrast the second element is shortened by about 10 ms. At the same time, the pitch of all measured elements is raised. These findings are discussed in the framework of hormonal regulation and communicative function.

     

Enhanced susceptibility to cetyl fluoride (CF) and cetyl fluoroacetate (CFA) in DDT-resistant insects

Two compounds showing negative correlation to DDT-resistance in larvae of Musca domestica were found, namely cetyl fluoride (CF) and cetyl fluoroacetate (CFA). Larvae of the DDT-resistant housefly strains K₁ and TP were affected more strongly by CFA incorporated in the breeding medium than larvae of the normal reference strains Sv and S-Rome. The same held true for CF, except that the effect on strain TP was doubtful. The chlordane-resistant, strain R-Sard. had a normal response to CF and CFA. In larvae of several DDT-resistant, laboratory-developed strains of Anopbeles atroparvus, negative correlation to resistance was noted for CF, but not for CFA and cetyl cyanide. The effect of CF applied again only to DDT-resistance, not to dieldrin-resistance.No negative correlation to resistance was found in adults of houseflies and anophelines with regard to CF, CFA, cetyl chloroacetate, cetyl cyanide, cetyl thiocyanate and cetyl bromide.

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Zusammenfassung Cetylfluorid (CF) war gegen Hausfliegenlarven des Schweizer DDT-resistenten Stammes K₁ etwas giftiger als gegen die normalsensiblen Vergleichsstämme Sv und S-Rome; für den DDT-resistenten italienischen Stamm TP war diese Erscheinung jedoch fraglich. Cetylfluorazetat (CFA) wirkte etwas besser auf Larven der beiden DDT-resistenten Fliegenstämme als auf die der Vergleichsstämme. Gegen Anopheleslarven verschiedener DDT-resistenter Stämme, die alle von dem gleichen normalen Ausgangsstamm selektioniert wurden, war CF etwas besser wirksam, als gegen den normalsensiblen Ausgangsstamm. Dagegen konnte weder für CFA noch für Cetylzyanid erhöhte Empfindlichkeit bei resistenten Anopbeleslarven nachgewiesen werden. Die oben geschilderten Preferentialtoxizitäten für resistente Stämme bezogen sich nur auf DDT-Resistenz und waren für Chlordan-oder Dieldrin-Resistenz nicht zutreffend.Ausser dem in früheren Arbeiten geschilderten Fall (Cetylbromazetat bei adulten Hausfliegen) konnten keine neuen Beispiele für durch Resistenz induzierte, erhöhte Empfindlichkeit (Ascher 1958b, 1960) bei Adulttieren der Hausfliege und der Anopbelesmücke gefunden werden; es wurden zu diesem Zweck CF, CFA, Cetylchlorazetat, Cetylzyanid, Cetylrhodanid und Cetylbromid getestet, jedoch waren die Etgebnisse negativ. Verschiedene Cetyl-phosphorsäureester, Cetyl-polyäthylenaether und Alkylbrommalonate waren entweder überhaupt unwirksam oder wiesen bei vorhandener schwacher Wirksamkeit weder gegen Larven noch gegen Adulttiere resistenter Stämme eine preferentielle Toxizität auf.

     

Oxidation of methyl derivatives of pteridin-4-one, lumazine and related pteridines by bovine milk xanthine oxidase.

1. Pteridin-4-ones, methylated at nitrogen or carbon, N-methylated lumazines and related oxopteridines were studied as substrates of a highly purified bovine milk xanthine oxidase (xanthine : oxygen oxidoreductase, EC 1.2.3.2). 2. The enzyme can oxidise at high rates both uncharged and anionic substrates. Variation of enzymic activity with pH is mainly due to pH-dependent changes in the active enzymic center. 3. Milk xanthine oxidases at different stages of purification convert pteridin-4-one into the 4,7-dione (compound 13 in this article). 4. Methylation at C-6 in the pyrazine moiety enhances enzymic attack at C-2 in the pyrimidine ring. N-Methylation may increase or reduce rates of oxidation. 5. For oxidation at C-2, the most favorable form of the substrate bears a double bond at C(2) = N(3). Attack at C-7 is enhanced strongly in structures bearing a double bond at C(6) = C(7). 6. In general, pteridines react with xanthine oxidase as non-hydrated molecules. However, oxidation of 8-methyllumazine at C-7 may take place by dehydrogenation of the 7-CHOH group of the covalently hydrated molecule.     

Quantitative and time-resolved monitoring of organelle and protein delivery to the lysosome with a tandem fluorescent Halo-GFP reporter

Lysosomal degradative compartments hydrolyze macromolecules to generate basic building blocks that fuel metabolic pathways in our cells. They also remove misfolded proteins and control size, function, and number of cytoplasmic organelles via constitutive and regulated autophagy. These catabolic processes attract interest because their defective functioning is linked to human disease and their molecular components are promising pharmacologic targets. The capacity to quantitatively assess them is highly sought-after. Here we present a tandem-fluorescent reporter consisting of a HaloTag-GFP chimera appended at the C- or at the N-terminus of select polypeptides to monitor protein and organelle delivery to the lysosomal compartment. The Halo-GFP changes color on fluorescent pulse with cell-permeable HaloTag ligands and, again, on delivery to acidic, degradative lysosomal compartments, where the fluorescent ligand-associated HaloTag is relatively stable, whereas the GFP portion is not, as testified by loss of the green fluorescence and generation of a protease-resistant, fluorescent HaloTag fragment. The Halo-GFP tandem fluorescent reporter presented in our study allows quantitative and, crucially, time-resolved analyses of protein and organelle transport to the lysosomal compartment by high resolution confocal laser scanning microscopy, antibody-free electrophoretic techniques and flow cytometry.     

Expression, secretion and processing of hirudin in E. coli using the alkaline phosphatase signal sequence

A DNA fragment coding for the E. coli phoA signal peptide was synthesized and inserted into the expression vector pKK223-3. A single HindIII restriction site is located just at the end of the signal sequence. A gene coding for the proteinase inhibitor hirudin, which has previously been synthesized, was inserted into this HindIII site. The hybrid protein was expressed under control of the tac-promoter and secreted into the periplasm of E. coli. From the periplasmic fraction two processed proteins were isolated. One of these was identical with desulfatohirudin and also had similar biological properties.     

Biliary lipid secretion in patients with heterozygous familial hypercholesterolemia and combined hyperlipidemia. Influence of bezafibrate and fenofibrate

Serum and biliary lipid metabolism were examined in 13 patients with different types of hyperlipoproteinemia before and after 4 weeks of treatment with either bezafibrate or fenofibrate. In patients with heterozygous familial hypercholesterolemia (FH), bezafibrate (n = 5) and fenofibrate (n = 7) produced a similar significant reduction of total cholesterol, LDL-cholesterol, and triglycerides by 21, 23, and 32%, respectively. In patients with familial combined hyperlipidemia (CHL), only triglycerides decreased markedly. Biliary lipid secretion rates in patients with heterozygous FH were not different from those of young male volunteers, indicating that a reduction of hepatic LDL receptors did not affect hepatic elimination of cholesterol or bile acids. Biliary cholesterol secretion increased significantly from 57 to 75 mg/hr during bezafibrate therapy (n = 8) and from 62 to 71 mg/hr during fenofibrate therapy (n = 9). No consistent change in bile acid or phospholipid secretion was observed. The elevated output of biliary cholesterol increased cholesterol saturation significantly from 147 to 185% and from 152 to 173% during administration of bezafibrate and fenofibrate, respectively. The present study indicates that treatment with bezafibrate or fenofibrate is effective in lowering LDL cholesterol in patients with heterozygous FH, but both drugs increase cholesterol saturation of bile, which might enhance the risk of cholesterol gallstone formation.     

Altered differentiation and clustering of Sertoli cells in transgenic mice showing a Sertoli cell specific knockout of the connexin 43 gene

Histological analysis revealed that Sertoli cell specific knockout of the predominant testicular gap junction protein connexin 43 results in a spermatogenic arrest at the level of spermatogonia or Sertoli cell-only syndrome, intratubular cell clusters and still proliferating adult Sertoli cells, implying an important role for connexin 43 in the Sertoli and germ cell development. This study aimed to determine the (1) Sertoli cell maturation state, (2) time of occurrence and (3) composition, differentiation and fate of clustered cells in knockout mice. Using immunohistochemistry connexin 43 deficient Sertoli cells showed an accurate start of the mature markers androgen receptor and GATA-1 during puberty and a vimentin expression from neonatal to adult. Expression of anti-Muellerian hormone, as a marker of Sertoli cell immaturity, was finally down-regulated during puberty, but its disappearance was delayed. This observed extended anti-Müllerian hormone synthesis during puberty was confirmed by western blot and Real-Time PCR and suggests a partial alteration in the Sertoli cell differentiation program. Additionally, Sertoli cells of adult knockouts showed a permanent and uniform expression of GATA-1 at protein and mRNA level, maybe caused by the lack of maturing germ cells and missing negative feedback signals. At ultrastructural level, basally located adult Sertoli cells obtained their mature appearance, demonstrated by the tripartite nucleolus as a typical feature of differentiated Sertoli cells. Intratubular clustered cells were mainly formed by abnormal Sertoli cells and single attached apoptotic germ cells, verified by immunohistochemistry, TUNEL staining and transmission electron microscopy. Clusters first appeared during puberty and became more numerous in adulthood with increasing cell numbers per cluster suggesting an age-related process. In conclusion, adult connexin 43 deficient Sertoli cells seem to proliferate while maintaining expression of mature markers and their adult morphology, indicating a unique and abnormal intermediate phenotype with characteristics common to both undifferentiated and differentiated Sertoli cells.     

A novel surface protein of Trichomonas vaginalis is regulated independently by low iron and contact with vaginal epithelial cells

Background  

Trichomonosis caused by Trichomonas vaginalis is the number one, non-viral sexually transmitted disease (STD) that affects more than 250 million people worldwide. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections by pathogens. No reports are available of IgA-reactive proteins and the role, if any, of this class of antibody in the control of this STD. The availability of an IgA monoclonal antibody (mAb) immunoreactive to trichomonads by whole cell (WC)-ELISA prompted us to characterize the IgA-reactive protein of T. vaginalis.