α1-Adrenergic Receptor Mediates Arachidonic Acid Release in Spinal Cord Neurons Independent of Inositol Phospholipid Turnover

The alpha 1-adrenergic receptor has been shown to mediate the release of arachidonic acid in FRTL5 thyroid cells and MDCK kidney cells. In primary cultures of spinal cord cells, norepinephrine stimulated release of arachidonic acid (from neurons only) and turnover of inositol phospholipids (from neurons and glia) via alpha 1-adrenergic receptors. These two responses were dissociated by treatment with phorbol ester and pertussis toxin, which inhibited production of inositol phosphates with no appreciable effect on release of arachidonic acid. Extracellular calcium was required for release of arachidonic acid, but not for production of inositol phosphates. The calcium channel blockers nifedipine and verapamil inhibited release of arachidonic acid only. However, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), a compound that blocks intracellular calcium release, diminished production of inositol phosphates, but had little effect on release of arachidonic acid. These results suggest that alpha 1-adrenergic receptors couple to release of arachidonic acid in primary cultures of spinal cord cells by a mechanism independent of activation of phospholipase C, possibly via the activation of phospholipase A2.     

Characterization of inositol 1,4,5-trisphosphate-sensitive (IsCaP) and-insensitive (IisCaP) nonmitochondrial Ca2+ pools in rat pancreatic acinar cells

Summary We have measured Ca²⁺ uptake and Ca²⁺ release in isolated permeabilized pancreatic acinar cells and in isolated membrane vesicles of endoplasmic reticulum prepared from these cells. Ca²⁺ uptake into cells was monitored with a Ca²⁺ electrode, whereas Ca²⁺ uptake into membrane vesicles was measured with⁴⁵Ca²⁺. Using inhibitors of known action, such as the H⁺ ATPase inhibitors NBD-Cl and NEM, the Ca²⁺ ATPase inhibitor vanadate as well as the second messenger inositol 1,4,5-trisphosphate (IP₃) and its analog inositol 1,4,5-trisphosphorothioate (IPS₃), we could functionally differentiate two non-mitochondrial Ca²⁺ pools. Ca²⁺ uptake into the IP₃-sensitive Ca²⁺ pool (IsCaP) occurs by a MgATP-dependent Ca²⁺ uptake mechanism that exchanges Ca²⁺ for H⁺ ions. In the absence of ATP Ca²⁺ uptake can occur to some extent at the expense of an H⁺ gradient that is established by a vacuolar-type MgATP-dependent H⁺ pump present in the same organelle. The other Ca²⁺ pool takes up Ca²⁺ by a vanadate-sensitive Ca²⁺ ATPase and is insensitive to IP₃ (IisCaP). The IsCaP is filled at higher Ca²⁺ concentrations (10^–6 mol/liter) which may occur during stimulation. The low steady-state [Ca²⁺] of 10^–7 mol/liter is adjusted by the IisCaP.It is speculated that both Ca²⁺ pools can communicate with each other, the possible mechanism of which, however, is at present unknown.     

Immunological and chemical characterization of hamster brain polyribosomes-cytomatrix complexes

We have studied the cytoskeletal nature of a brain subcellular fraction previously shown to contain polyribosomes. We have identified the major proteins of this fraction by electrophoretic comparison to a standard cytoskeletal fraction and by immunodetection. These methods have shown the presence of actin, glial fibrillary acidic protein, and neurofilament triplet proteins. We have also studied the effect of various ions and nonionic detergents on the stability of this structure. It was stable in presence of Triton X-100 up to 2% but disrupted by 200 mM K⁺ acetate.Abbreviations CMT cytomatrix - CSK cytoskeleton - DOC sodium deoxycholate - DTT dithiothreitol - EGTA ethylenglycolbis (-Ether)-N,N-N-N-Tetraacetic Acid - GFAP glial fibrillary acidic protein - PR polyribosome - PRCMC polyribosomes-cytomatrix complex     

Morphological and metabolic characterization of a new species of strictly anaerobic rumen fungus: Neocallimastix joyonii

A new strain of strictly anaerobic fungi was isolated from the rumen of sheep. This strain is characterized by a polycentric thallus, an extensive and polynuclear rhizomycelium, polyflagellated zoospores with gamma particle-like bodies. We propose to assign this strain in a new species: Neocallimastix joyonii.     

Androgen synthesis and aromatization by equine corpus luteum microsomes

Whereas mare corpus luteum does not produce androgens or estrogens in vivo, the incubation of mare corpus luteum microsomes with progesterone and NADPH resulted in 17 alpha-hydroxyprogesterone and estrogen production with a small yield of androstenedione. In the presence of an aromatase inhibitor (4-hydroxyandrostenedione), 17 alpha-hydroxyprogesterone and androstenedione were accumulated. Aromatization of testosterone and androstenedione occurred via stereospecific loss of the 1 beta, 2 beta hydrogen atoms and was inhibited by MgCl2, KCl, and EDTA. The Km of estrogen synthetase from equine corpus luteum for testosterone was 18.5 +/- 2.7 nM and for androstenedione was 11.5 +/- 1.5 nM. 19-Norandrogens were aromatized with a slightly higher efficiency than were androgens, but the affinity of the aromatase was lower for 19-norandrogens than for androgens. Our results suggest that aromatases from equine testis and corpus luteum are closely related enzymes. On the other hand, the question arises as to the relationship among the cell origin, the synthetizing abilities, and in vivo production of the corpus luteum in different mammalian species.     

Transfer of amino acids and nitrate from the roots into the xylem of Ricinus communis seedlings

The loading of amino acids and nitrate into the xylem was investigated by collection and analysis of root-pressure exudate from the cut hypocotyl stumps of seedlings of Ricinus communis L. Glutamine was found to be the dominant amino acid in the exudate and also to be the amino acid which is transferred to the xylem most rapidly and accumulated to the greatest extent. The comparison between uptake and xylem loading showed significant differences in specificity between these two transport reactions, indicating a different set of transport systems. Nitrate is transferred to the xylem at a higher relative rate than any amino acid despite the great nitrate-storage capacity of the root system. Thus the supply of nitrate to Ricinus plants leads to enhanced nitrogen allocation to the shoots.     

Sequence-specific assignments of the 1H nuclear magnetic resonance spectra of reduced high-potential ferredoxin (HiPIP) from Chromatium vinosum.

The 1H resonances of the high-potential [4Fe-4S]2+ ferredoxin from Chromatium vinosum have been assigned through conventional sequential methodology applied to 2D NMR spectra. Almost 80% of the residues were identified using standard 2D COSY, HOHAHA, and NOESY pulse sequences. These residues correspond to four segments of the primary structure that do not interact strongly with the iron-sulfur cluster. A minor correction to the amino acid sequence is strongly suggested by these NMR data. Additional protons more sensitive to the proximity of the cluster were assigned by a combination of NOESY experiments with fast repetition rates and short mixing times and of HOHAHA spectra recorded with reduced spin-lock duration aimed at compensating for the short relaxation rates. Hence, the contributions of 79 residues out of 85 were identified in NMR spectra, among which the assignments of 64 residues were completed. Even the fastest relaxing protons, like those of the cysteine ligands, could be correlated, partly because the strong hyperfine shifts isolate them from the crowded diamagnetic region. However, other protons, in particular those involved in NH-S hydrogen bonds with the iron-sulfur cluster, were more difficult to identify, most probably because their relatively broad signals overlapped with those of protons not or less perturbed by the active site. The availability of the major part of the 1H NMR assignments has enabled the detection and identification of many interresidue NOESY cross peaks. These data are in full agreement with the elements of secondary structure previously revealed by X-ray crystallographic analysis of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

d-Ribulokinase from Klebsiella pneumoniae for continuous production of d-(−)-ribulose-5-phosphate

Summary The production of d-ribulose-5-phosphate in an enzyme membrane reactor was examined. Phosphoryl transfer from ATP to d-ribulose was catalysed by d-ribulokinase isolated from Klebsiella pneumoniae. For production of d-ribulose-5-phosphate the phosphoryl donor ATP was used either in stoichiometric or in catalytic amounts. Using catalytic amounts of ATP requires a second enzyme, e.g. pyruvate kinase, to regenerate ATP. The kinetic parameters for d-ribulokinase and pyruvate kinase were determined to calculate the performance of an enzyme membrane reactor for continuous production of d-ribulose-5-phosphate. Both processes operated for more than 200 h. Regardless of whether ATP was used in catalytic or stoichiometric amounts, about the same production parameters were determined. In continuous production space/time yields of 117 g (with ATP regeneration) and 103 g (without ATP regeneration) of d-ribulose-5-phosphate 1^–1 per day were reached.Offprint requests to: D. Gygax     

Continuous microbial production ofl-leucine with cell retention

Summary Continuous production ofl-leucine was carried out withCorynebacterium glutamicum, strain ATCC 13032 starting from-ketoisocaproic acid as the precursor, glucose as the carbon source and ammonium sulphate as the nitrogen source, with biotin in a mineral medium. By means of cross-flow microfiltration or centrifugal separation for cell retention in continuous fermentation an increase in cell density was achieved and the product solution was obtained cell-free. The cells were concentrated to over 70 g cell dry mass/1. In experiments of up to 42 days, conversion rates of 85%–99% andl-leucine yields of 85%–93% were achieved. With a substrate residence time of 3.6 h, 114 mmol/1l-leucine was produced with a space-time yield of 97 g/1 per day. A scale-up of the fermentation volume from 4 to 1001 provided comparable results.     

Analysis of the topological changes induced on cells exposed to adhesive or mechanical stimuli

Fluorescent probes are widely used to study cell structure and function. However, few reports were devoted to a quantitative analysis of the intracellular distribution of fluorescent markers. In the present work, we describe the topographical changes of surface and cytoskeletal markers on individual cells subjected to adhesive or mechanical interaction. Conjugates were prepared with a cytotoxic T-lymphocyte clone and target cells. Specific antigens, membrane phospholipids, surface glycoconjugates, and polymerized actin were labeled with fluorescent antibodies or biochemical probes. The analysis of fluorescence distributions in conjugates demonstrated a selective reorganization of the plasma membrane with a gathering of some molecular species in the intercellular adhesion area. Furthermore, individual phagocytic cells were sucked into glass micropipets, then stained with fluorescent phallacidin to analyze the effect of mechanical efforts on the cytoskeleton organization. The concentration of polymerized actin was found to be similar in mechanicallyinduced protrusions and whole cells. It is concluded that adhesive interactions may result in marked cell polarization and formation of membrane zones with a particular biochemical composition. The submembranar cytoskeleton might play a role in this process.