Immunocytochemical colocalization of adhesive proteins with clathrin in human blood platelets: further evidence for coated vesicle-mediated transport of von Willebrand factor,fibrinogen and fibronectin

Coated membranes and vesicles play an important role in receptor-mediated endocytosis and intracellular trafficking in various cell types, and are also present in blood platelets. Platelets take up certain proteins from the blood plasma, such as von Willebrand factor and fibrinogen, and these substances are transferred to storage granules. The receptors for these plasma proteins on the platelet plasma membrane have been well characterized, but morphological evidence for their transport to the storage granules is not yet available. In an attempt to clarify this aspect, we employed postembedding immunocytochemistry on platelets embedded in the acrylic resin LR White. Clathrin as the major coat component of coated vesicles was localized in the cytoplasm, on the plasmic faces of -granules and the open canalicular system, and on the plasmic face of the plasma membrane. Colocalizations of the adhesive proteins, von Willebrand factor, fibrinogen and fibronectin, with clathrin could be observed at the same typical locations as coated vesicles were seen in Araldite-embedded material. These colocalizations have not been reported to date and furnish further evidence for a coated vesicle-mediated transport of blood plasma-derived adhesive proteins from their receptors on the outer plasma membrane to the -granules.     

Die postembryonale Entwicklung und die funktionelle Anatomie des Gnathosoma in der Milbenfamilie Erythraeidae (Acarina,Prostigmata)

Zusammenfassung Das Gnathosoma von sieben Arten der Milbenfamilie Erythraeidae wurde untersucht. Die postembryonale Entwicklung der Cuticula-Strukturen, der Muskulatur und der Drüsen wird beschrieben.Das Gnathosoma der Larven weist Cheliceren auf, die aus Grundglied und klauenförmigem Digitus mobilis bestehen. Sie liegen dem Infracapitulum dorsal auf und inserieren an einem Paar Capitular-Apophysen. Bei den Protound Tritonymphen unterliegt das Gnathosoma der Histolyse und wird anschließend jeweils neu ausgebildet. Bei der Deutonymphe und beim Adultus ist es gegenüber der Larve stark abgewandelt: Die Tracheenstämme sind aus den Capitular-Apophysen der Larve abzuleiten. Die Stech-Cheliceren entsprechen dem Grundglied der larvalen Cheliceren, und ihre medialen Protraktoren gehen aus den Levator-Muskeln der Cheliceren der Larve hervor.Der Weg der Sekrete der podocephalischen und infracapitulären Drüsen über die Dorsalfläche des Infracapitulum erfährt mit dem Erwerb schmaler Stech-Cheliceren eine radikale Umstellung des Schutzes gegen Austrocknung. Während bei der Larve die breiten Cheliceren-Grundglieder den Sekretweg überdecken, und der Raum zwischen den Grundgliedern und dem Infracapi-tulum zusätzlich durch das ölartige Sekret der Intercheliceraldrüse ausgefüllt wird, schließen sich bei der Deutonymphe und beim Adultus die Lateralkiele der Genae über den Stech-Cheliceren zusammen, und der hintere Abschnitt des Cervix wird in das Idiosoma eingesenkt. Dadurch wird der Cervix zu einem internen Cervicalkanal umgebildet.Der Mundraum wird bei den aktiven Stadien durch die lipidartigen Sekrete der Buccal- und Labialdrüsen geschützt.Die phylogenetischen Beziehungen der Parasitengona innerhalb der Prostigmata und der Erythraeidae innerhalb der Parasitengona werden diskutiert.

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Postembryonic development and functional anatomy of the gnathosoma in the family Erythraeidae (Acarina, Prostigmata)

Summary The gnathosoma of seven species of the Erythraeidae was investigated. The postembryonic development of the cuticular structures, the musculature and the glands are described.In the larval gnathosoma, the chelicerae consist of a basal segment and a claw. They rest upon the dorsal surface of the infracapitulum. The basal segments are attached to a pair of capitular apophyses (sigmoid pieces). During the two molts larva to protonymph and deutonymph to tritonymph, the gnathosoma is histolysed. Directly afterwards it is rebuilt. Compared to the larva, in the deutonymph and in the adult it undergoes profound changes: The capitular apophyses are transformed to parts of the tracheae, the basal segment of each chelicera to a styliform chelicera, and the levator muscles of the chelicerae to medial protractors of the styliform chelicerae.The secretions of the podocephalic and infracapitular glands proceed along the dorsal surface of the infracapitulum to the buccal cavity. In the larva, the way of the secretions is protected against desiccation by the broad basal segments of the chelicerae, that cover the infracapitulum. In addition the oily secretion of the intercheliceral gland seals the space between the infracapitulum and the chelicerae. By obtaining styliform chelicerae the protection against desiccation undergoes a radical change: The lateral ridges of the infracapitulum join above the chelicerae, and the posterior part of the cervix is transferred back into the idiosoma. Thus the cervix is transformed into an internal canal.In the active instars, the buccal cavity is protected by the lipid-like secretions of the buccal glands and labial glands.The phylogenetic relationships of the Parasitengona within the Prostigmata, and of the Erythraeidae within the Parasitengona are discussed.

     

Improved analysis of hemagglutination assays for quantitation of lectin activity

A method to quantitate lectin activity based on hemagglutination assay in microtiter plates is described. In addition to the normal method of visual titer evaluation an electronic particle counter is used for counting of nonagglutinated cells in the microtiter wells; this allows a rapid, quantitative determination of the amount of lectin required to agglutinate 50% of the countable single cells. It is also recommended that counting results should be related to a standard curve of concanavalin A to improve the reproducibility of the assay.     

Cynoglossopsis somaliensis,sp. nov., und die GattungCynoglossopsis (Boraginaceae)

Cynoglossopsis somaliensis H. Riedl, sp. nov., is described as a new species of the hitherto monotypic genusCynoglossopsis Brand which is closest related toCynoglossum L. but has to be included inBoraginoideae-Eritrichieae from the way the nutlets are attached to the gynobasis.

     

Calcium and magnesium levels in isolated cardiac mitochondria from mice injected with isoproterenol

Summary Calcium (Ca) and Magnesium (Mg) are determined by atomic absorption flame spectrometry in isolated cardiac mitochondria from mice receiving subcutaneous injections of DL-isoproterenol HC1 (ISO), and in mitochondria of untreated controls. In the controls, mitochondria were isolated in the presence or absence of ruthenium red. On the absence of ruthenium red in the isolation medium, mitochondrial Ca levels increase by about 300%, while levels of Mg remain unchanged. Focal myocardial necrosis following a single ISO-injection is shown by electron microscopy. Ca and Mg levels are largely unaffected by a single dose of ISO until 24 h after the injection. A slight increase in Ca occurs in the 48 h samples. When multiple injections of ISO are given every 12th hour for 48 h, 72 h and 96 h, respectively, endogenous Ca and Mg increase significantly. It is suggested that this increase might be associated with ISO-induced cardiac hypertrophy rather than with the pharmacological effects of ISO per se.This work was supported by grants from The Norwegian Council on Cardiovascular Disease and from The Norwegian Research Council for Science and the Humanities     

1H NMR studies at 360 Mhz of the methyl groups in native and chemically modified basic pancreatic trypsin inhibitor (BPTI)

In the ¹H NMR spectra obtained at 360 MHz after digital resolution enhancement, the multiplet resonances of the methyl groups in the basic pancreatic trypsin inhibitor (BPTI) were resolved. With suitable double irradiation techniques the individual methyl resonances were assigned to the different types of aliphatic amino acid residues. Furthermore, from pH titration and comparison of the native protein with chemically modified BPTI, the resonance lines of Ala 16 in the active site and Ala 58 at the C-terminus were identified. Potential applications of the resolved methyl resonances as natural NMR probes for studies of the molecular conformations are discussed.     

Verwertung von Trimethylammoniumverbindungen durch Acinetobacter calcoaceticus

Zusammenfassung Die Verwertung von Carnitin und Carnitinderivaten (O-Acylcarnitine, Carnitincarboxyl-derivate) und strukturverwandten Trimethylammoniumverbindungen (Betaine und Stickstoffbasen) durch Acinetobacter calcoaceticus wurde anhand des Wachstums und des quantitativen Nachweises der Metabolite untersucht. Der Stamm wuchs auf l-Carnitin, l-O-Acylcarnitinen und -Butyrobetain als jeweils einziger C-Quelle. Der Verbrauch dieser Verbindungen und das Wachstum korrelierten mit der Spaltung der C-N-Bindung und mit dem gebildeten Trimethylamin. d-Carnitin wurde metabolisiert, wenn als zusätzliche C-Quelle l-Carnitin im Nährmedium vorhanden war, oder wenn die Bakterien mit l-oder dl-Carnitin vorinkubiert worden waren. Mit d-Carnitin als einziger C-Quelle wuchsen die Bakterien jedoch nicht. Die Bakterien oxidierten Cholin zu Glycinbetain in Gegenwart einer zusätzlichen C-Quelle, Glycinbetain selbst wurde nicht assimiliert. In Hinsicht auf den Abbau quaternärer Stickstoffverbindungen besitzt Acinetobacter calcoaceticus im Vergleich zu anderen Carnitin-verwertenden Bakterienarten einen für ihn charakteristischen Stoffwechselweg.

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Utilization of trimethylammonium-compounds by Acinetobacter calcoaceticus

The utilization of carnitine and carnitine derivatives (O-acylcarnitines, carnitine carboxylderivatives) and structure-related trimethylammonium-compounds (betaines and nitrogen-bases) by Acinetobacter calcoaceticus was studied by means of the control of growth and the quantitative detection of metabolites. The strain grew only on l-carnitine, l-O-acylcarnitines, and -butyrobetaine as the sole carbon sources. The utilization of these compounds and the growth correlated with the cleavage of the C-N bond and thereby with the formation of trimethylamine. d-Carnitine was metabolized, if an additional carbon source, like l-carnitine, was present in the incubation mixture, or if the bacteria were preincubated with l-or dl-carnitine, but no growth was observed on d-carnitine as the sole carbon source. The bacteria oxidized choline to glycinebetaine in the presence of additional carbon sources, glycinebetaine itself was not assimilated. With regard to the catabolism of quaternary nitrogen compounds Acinetobacter calcoaceticus shows a different pathway in comparison with other bacterial species metabolizing carnitine.

     

Progesterone in the uterus. VIII. Uptake of corticosterone and incorporation of progesterone under concurrent injection of corticosterone into rat uterus

A study of the subcellular distribution of radioactivity in rat uterus after injection of labelled corticosterone showed that the radioactivity was observed in all fractions from 5 min. to 120 min. A maximum uptake was observed 10 min. after application of the labelled steroid. Competitive uptake of radioactive progesterone and unlabelled corticosterone was assayed 10 min. after injection of the hormone mixture. The ratio between radioactive progesterone and unlabelled corticosterone was 1 : 1 and 1 : 2 (moles:moles), respectively. Compared with control experiments with rats which had received radioactive progesterone alone, the results gave evidence that progesterone found in all subcellular fractions and in the total homogenate was not depressed by unlabelled corticosterone. However, unlabelled progesterone reduced the tritiated progesterone in uterine tissue. This observation demonstrates that the uptake of progesterone by rat uterus is specific.     

The time course of glucagon action on the utilization of [U-14C]palmitate by isolated hepatocytes

The time course of glucagon action on the utilization of [U-¹⁴C]palmitate by isolated hepatocytes was studied. Ten minutes incubation of the cells after hormone addition was required in order to observe increased oxidation and decreased esterification of the labeled palmitate. The acid-soluble, labeled oxidation products could be separated into two main fractions, glucose and ketone bodies. Initially, glucagon directed the flux of radioactivity toward glucose and CO₂. After prolonged incubation in the presence of glucagon, labeled ketone bodies, as well as labeled glucose and ¹⁴CO₂, were increased. This effect was most marked as regards glucose. The results indicate that glucagon induces a rapidly onset stimulation of the rates of Krebs cycle and gluconeogenesis, while increased oxidation and decreased esterification of palmitate are time-delayed corresponding to the establishment of a lower level of glycerophosphate. About 10% of the glucose carbon formed by gluconeogenesis originated from the fatty acid when cells from fasted rats were incubated in the presence of alanine and [U-¹⁴C]palmitate.