Narthecium ossifragum Associated Photosensitization in Sheep in The Faroe Islands

Hepatogenous photosensitization in sheep is an important problem in various parts of the world. Most photosensitization diseases are associated with ingestion of plant or fungal toxins. The lily, Narthecium ossifragum, has long been associated with photosensitization in lambs in western Norway (Ender 1955, Flåøyen 1993) and in the northern regions of the British Isles (Ford 1964).     

The gap junctional intercellular communication is no prerequisite for the stabilization of xenobiotic metabolizing enzyme activities in primary rat liver parenchymal cells in vitro

Summary In primary monocultures of adult rat liver parenchymal cells (PC), the activities of the xenobiotic metabolizing enzymes microsomal epoxide hydrolase (mEH_b), soluble epoxide hydrolase (sEH), glutathione S-transferases (GST), and phenolsulfotransferase (ST) were reduced after 7 d to values below 33% of the initial activities. Furthermore, the gap junctional intercellular communication (GJIC), measured after microinjection by dye transfer, decreased from 90% on Day 1 to undetectable values after 5 d in monoculture. Co-culture of PC with nonparenchymal rat liver epithelial cells (NEC) increased (98% on Day 1) and stabilized (82% on Day 7) the homotypic GJIC of PC. Additionally, most of the measured xenobiotic metabolizing enzyme activities were well stabilized over 1 wk in co-culture. Because GJIC is one of several mechanisms playing an important role in cell differentiation, the importance of GJIC for the stabilization of xenobiotic metabolizing enzymes in PC was investigated. PC in monoculture were, therefore, treated with 2% dimethyl sulfoxide (DMSO), a differentiation promoting factor, and 1,1,1-trichloro-2,2,-bis (p-chlorophenyl) ethane (DDT) (10 μg/ml), a liver tumor promotor and inhibitor of GJIC, was given to co-cultures of PC with NEC. DMSO significantly stabilized (68% on Day 7), while DDT significantly inhibited (8% on Day 7) homotypic GJIC of PC in the respective culture systems. In contrast, the activities of mEH_b, sEH, GST, and ST were not affected in the presence of DMSO or DDT. These results lead to the assumption that the differentiation parameters measured in this study (i.e., homotypic GJIC and the activities of xenobiotic metabolizing enzymes) are independently regulated in adult rat liver PC.     

Comparison of voice stress reactivity under psychological stress test and simulated Mir docking manoeuvre

The relation between the results of laboratory tests and results of the interesting professional work is a very difficult one. The same is true for the relation between training and real performance. The pilot controlled MIR-docking manoeuvre is in reality a very seldom but very important one. The training of this manoeuvre is a very special professional work and only for cosmonauts available. But the objective estimation of the performance and the used objective and subjective needs is nevertheless necessary. Even for the subjective "stress"-feelings during task solving it is often impossible to get reliable data without any physiological measurements. The use of electrodes, cables and equipments is hardly limited during real work. Sometimes only the voice is available over more or less quality communication channels. So it is the case during the training and the real pilot controlled MIR-docking manoeuvre. Based on findings in the literature and own experiments analyzing the relation between fundamental voice frequency (F0) and subjective stress feelings we tested the hypothesis that a on-line measurement of F0 can be used for stress evaluation during standardised training sessions of MIR-docking manoeuvres.     

Synthetic substrate analogues for UDP-GlcNAc: Manα1-6R β(1-2)-N-acetylglucosaminyltransferase II. Substrate specificity and inhibitors for the enzyme

UDP-GlcNAc: Man1-6R (1-2)-N-acetylglucosaminyltransferase II (GlcNAc-T II; EC 2.4.1.143) is a key enzyme in the synthesis of complexN-glycans. We have tested a series of synthetic analogues of the substrate Man1-6(GlcNAc1-2Man1-3)Man-O-octyl as substrates and inhibitors for rat liver GlcNAc-T II. The enzyme attachesN-acetylglucosamine in 1-2 linkage to the 2-OH of the Man1-6 residue. The 2-deoxy analogue is a competitive inhibitor (K _i=0.13mm). The 2-O-methyl compound does not bind to the enzyme presumably due to steric hindrance. The 3-, 4- and 6-OH groups are not essential for binding or catalysis since the 3-, 4- and 6-deoxy and -O-methyl derivatives are all good substrates. Increasing the size of the substituent at the 3-position to pentyl and substituted pentyl groups causes competitive inhibition (K _i=1.0–2.5mm). We have taken advantage of this effect to synthesize two potentially irreversible GlcNAc-T II inhibitors containing a photolabile 3-O-(4,4-azo)pentyl group and a 3-O-(5-iodoacetamido)pentyl group respectively. The data indicate that none of the hydroxyls of the Man1-6 residue are essential for binding although the 2- and 3-OH face the catalytic site of the enzyme. The 4-OH group of the Man-O-octyl residue is not essential for binding or catalysis since the 4-deoxy derivative is a good substrate; the 4-O-methyl derivative does not bind. This contrasts with GlcNAc-T I which cannot bind to the 4-deoxy-Man- substrate analogue. The data are compatible with our previous observations that a bisectingN-acetylglucosamine at the 4-OH position prevents both GlcNAc-T I and GlcNAc-T II catalysis. However, in the case of GlcNAc-T II, the bisectingN-acetylglucosamine prevents binding due to steric hindrance rather than to removal of an essential OH group. The 3-OH of the Man1-3 is an essential group for GlcNAc-T II since the 3-deoxy derivative does not bind to the enzyme. The trisaccharide GlcNAc1-2Man1-3Man-O-octyl is a good inhibitor (K _i=0.9mm). The above data together with previous studies indicate that binding of the GlcNAc1-2Man1-3Man- arm of the branched substrate to the enzyme is essential for catalysis. Abbreviations: GlcNAc-T I, UDP-GlcNAc:Man1-3R (1-2)-N-acetylglucosaminyltransferase I (EC 2.4.1.101); GlcNAc-T II, UDP-GlcNAc:Man1-6R (1-2)-N-acetylglucosaminyltransferase II (EC 2.4.1.143); MES, 2-(N-morpholino)ethane sulfonic acid monohydrate.     

Structure determination of the disialylated poly-(N-acetyllactosamine)-containingO-linked carbohydrate chains of equine chorionic gonadotropin

The disialylated poly-(N-acetyllactosamine)-containingO-linked oligosaccharide alditols, released by alkaline borohydride treatment of the enzymicallyN-deglycosylated β-subunit of equine chorionic chonadotropin, were purified by fast protein liquid chromatography (FPLC) on Mono Q and analysed by fast ion bombardment mass spectrometry (FAB-MS) and¹H-NMR spectroscopy. The identified oligosaccharide alditols have the following structure: $$\begin{gathered} Neu5Ac\alpha 2 - 3\left[ {Gal\beta 1 - 4GlcNAc\beta 1 - 3} \right]_{0 - 4} Gal\beta 1 - 4GlcNAc\beta 1 - 6 \hfill \\ \begin{array}{*{20}c} { \backslash } \\ { GalNAc - ol} \\ { /} \\ {Neu5Ac\alpha 2 - 3Gal\beta 1 - 3} \\ \end{array} \hfill \\ \end{gathered}$$      

Occurrence of osteoblast necroses during ossification of long bone cortices in mouse fetuses

Previous investigations concerned with in vitro osteogenesis and mineralization have revealed some indication of a participation of cell necroses in the course of calcification. These observations were confirmed by in vivo investigations on desmoid ossification in fetal mouse calvariae, where abundant necrotic osteoblasts were found at the mineralization border and in the osteoid. In the present study, ossification of long bone cortices from fetal mice was investigated by use of electron microscopy. Specimens obtained from the collection of the Institute of Anatomy, Free University of Berlin (mouse fetuses, forearm; rat fetuses, forearm) were reinvestigated for control purposes. In all cases, mineralization of osteoid was accompanied by cell necroses. Cell degeneration was characterized by swelling of the endoplasmic reticulum and loss of the plasma membrane resulting in freely distributed vesicular structures. Cell debris was incorporated within the mineral. Initially, cell necroses in the perichondrium occurred in the region surrounding the hypertrophic cartilage and the matrix of which showed spots of endochondral mineralization. Necrotic osteoblasts occurred simultaneously with mineralization of the osteoid. During further ossification of the long bone cortices, the number of necrotic cells increased markedly. In addition to necrotic cells, healthy osteoblasts, osteocytes and perichondral tissue were present, indicating that an artifact can be excluded. The importance of cell necroses in the process of mineralization is as yet unclear. Possibly, the cells act as calcium and/or phosphate stores, which are liberated by cell death to increase the amount of mineral constituents at sites of mineralization.     

Immunocytochemical localization of Na+/K+-ATPase and V-H+-ATPase in the salivary glands of the cockroach,Periplaneta americana

The acinous salivary glands of the cockroach (Periplaneta americana) consist of four morphologically different cell types with different functions: the peripheral cells are thought to produce the fluid component of the primary saliva, the central cells secrete the proteinaceous components, the inner acinar duct cells stabilize the acini and secrete a cuticular, intima, whereas the distal duct cells modify the primary saliva via the transport of water and electrolytes. Because there is no direct information available on the distribution of ion transporting enzymes in the salivary glands, we have mapped the distribution of two key transport enzymes, the Na⁺/K⁺-ATPase (sodium pump) and a vacuolar-type H⁺-ATPase, by immunocytochemical techniques. In the peripheral cells, the Na⁺/K⁺-ATPase is localized to the highly infolded apical membrane surface. The distal duct cells show large numbers of sodium pumps localized to the basolateral part of their plasma membrane, whereas their highly folded apical membranes have a vacuolar-type H⁺-ATPase. Our immunocytochemical data are supported by conventional electron microscopy, which shows electrondense 10-nm particles (portasomes) on the cytoplasmic surface of the infoldings of the apical membranes of the distal duct cells. The apically localized Na⁺/K⁺-ATPase in the peripheral cells is probably directly involved in the formation of the Na⁺-rich primary saliva. The latter is modified by the distal duct cells by transport mechanisms energized by the proton motive force of the apically localized V-H⁺-ATPase.     

Nitric oxide synthase in the rat carotid body and carotid sinus

The participation of nitric oxide synthase (NOS) in the innervation of the rat carotid body and carotid sinus was investigated by means of NADPH-diaphorase histochemistry and NOS immunohistochemistry using antisera raised against purified neuronal NOS and a synthetic tridecapeptide. NOS was detected in 23% of neurons at the periphery of the carotid bodies. Some negative neurons were surrounded by NOS-positive terminals. NOS-containing varicose nerve fibres innervated the arterial vascular bed and, to a lesser extent, the islands of glomus cells. These fibres persisted after transection of the carotid sinus nerve and are probably derived from intrinsic neurons. Large NOS-positive axonal swellings in the wall of the carotid sinus were absent after transection of the sinus nerve, indicating their sensory origin. The results suggest a neuronal nitrergic control of blood flow, neuronal activity and chemoreception in the carotid body, and an intrinsic role of NO in the process of arterial baroreception.     

Role of sodium ions for sulfate transport and energy metabolism in Desulfovibrio salexigens

The sodium ion gradient and the membrane potential were found to be the driving forces of sulfate accumulation in the marine sulfate reducer Desulfovibrio salexigens. The protonmotive force of –158 mV, determined by means of radiolabelled membrane-permeant probes, consisted of a membrane potential of –140 mV and a pH gradient (inside alkaline) of 0.3 at neutral pH_out. The sodium ion gradient, as measured with silicone oil centrifugation and atomic absorption spectroscopy, was eightfold ([Na⁺]_out/[Na⁺]_in) at an external Na⁺ concentration of 320 mM. The resulting sodium ionmotive force was –194 mV and enabled D. salexigens to accumulate sulfate 20000-fold at low external sulfate concentrations (<0.1 M). Under these conditions high sulfate accumulation occurred electrogenically in symport with three sodium ions (assuming equilibrium with the sodium ion-motive force). With increasing external sulfate concentrations sulfate accumulation decreased sharply, and a second, low-accumulating system symported sulfate electroneutrally with two sodium ions. The sodium-ion gradient was built up by electrogenic Na⁺/H⁺ antiport. This was demonstrated by (i) measuring proton translocation upon sodium ion pulses, (ii) studying uptake of sodium salts in the presence or absence of the electrical membrane potential, and (iii) the inhibitory effect of the Na⁺/H⁺ antiport inhibitor propylbenzilylcholin-mustard HCl (PrBCM). With resting cells ATP synthesis was found after proton pulses (changing the pH by three units), but neither after pulses of 500 mM sodium ions, nor in the presence of the uncoupler tetrachorosalicylanilide (TCS). It is concluded that the energy metabolism of the marine strain D. salexigens is based primarily on the protonmotive force and a protontranslocating ATPase.Abbreviations MOPS morpholinopropanesulfonic acid - TCS tetrachlorosalicylanilide - PrBCM propylbenzilylcholin-mustard HCl - Tris tris(hydroxymethyl)aminomethane - TPP⁺ bromide tetraphenylphosphonium bromide