Glucocorticoids Provoke a Shift from α2- to α1-Adrenoreceptor Activities in Cultured Hypothalamic Slices Leading to Opposite Noradrenaline Effect on Corticotropin-Releasing Hormone Release

Abstract: We have shown previously that noradrenaline (NA) stimulated or inhibited the release of corticotropin-releasing hormone (CRH) according to the availability of adrenal steroids. The aim of the present work was to examine whether the changes in the NA modulation of CRH release from hypothalamic neurons result from a steroid-induced plasticity of the adrenergic transduction pathways. From anterior hypothalamic slices cultured in standard medium (i.e., containing adrenal steroids at a final dilution of 61 ± 9 ng/ml), (a) the stimulatory effect of NA on CRH release was reversed in a dose-dependent manner by increasing concentrations of the α₁-adrenoreceptor antagonist prazosin, (b) activation of protein kinase C by acute treatment with phorbol 12-myristate 13-acetate (0.5 µ M , 1 h) mimicked NA stimulation of CRH secretion, and (c) the activation of L-type Ca²⁺ channels by Bay K 8644 also produce an increased CRH secretion. In contrast, the inhibitory effect of NA on CRH secretion from slices cultured in steroid-free medium was markedly reversed by the α₂-adrenoreceptor antagonist yohimbine, by pretreatment with pertussin toxin, or by the addition of 4-aminopyridine, a K⁺-channel blocker. Acute treatment with phorbol 12-myristate 13-acetate did not change the inhibitory NA effect. Moreover, all these effects were reversed by daily corticosterone supplementation, for as long as they were tested. These results are consistent with a steroid-dependent change in the nature of adrenergic receptors and its associated transduction pathways involved in the regulation of CRH secretion in the hypothalamus.     

Expression of Enzymatically Active Rabbit Hemorrhagic Disease Virus RNA-Dependent RNA Polymerase in Escherichia coli

The rabbit hemorrhagic disease virus (RHDV) (isolate AST/89) RNA-dependent RNA-polymerase (3D^pol) coding region was expressed in Escherichia coli by using a glutathione S-transferase-based vector, which allowed milligram purification of a homogeneous enzyme with an expected molecular mass of about 58 kDa. The recombinant polypeptide exhibited rifampin- and actinomycin D-resistant, poly(A)-dependent poly(U) polymerase. The enzyme also showed RNA polymerase activity in in vitro reactions with synthetic RHDV subgenomic RNA in the presence or absence of an oligo(U) primer. Template-size products were synthesized in the oligo(U)-primed reactions, whereas in the absence of added primer, RNA products up to twice the length of the template were made. The double-length RNA products were double stranded and hybridized to both positive- and negative-sense probes.     

FIRST INSIGHTS INTO IMPROVEMENT OF EICOSAPENTAENOIC ACID CONTENT IN PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE) BY INDUCED MUTAGENESIS1

A strain improvement program was initiated based on mutagenesis with the goal of commercial production of eicosapentaenoic acid (EPA)from EPA-overproducing microalgal strains. Two rounds of mutation and selection were conducted using Phaeodactylum tricornutum Bohlin UTEX #640 as the parent strain. After the first round of mutagenesis, a putative mutant (provisionally labeled 114) was obtained. The EPA content (% of dry weight) of this mutant strain was 37% higher than that of the wild type. 114 was further mutated and another putative mutant (provisionally called II242) was isolated, the EPA content of which was 44% higher than that of the wild type. When cultured with aeration in 1-L flasks, EPA content of the wild type and putative mutants 114 and II242 was, 17.3 mg · g^?1, 31.5mg · g^?1, and 38.6 mg · g^?1 dry biomass, respectively. EPA productivity was 3.48 mg · L^?1· d^?1 4.01 mg · L^?1· d^?1, and 4.98 mg · L^?1· d^?1 respectively. These figures compare favorably with many other promising EPA-producing microorganisms and suggest that the use of a single methodology such as mutation and selection is a way to improve the polyunsaturated fatty acid content of microalgae and other microorganisms.     

Pancreatic secretion in rats treated with intra-aortic prostaglandin E1

The effects from one dose of PGE1 on the endocrine pancreatic secretions have been studied in rat. The dose is injected i.a. very near the pancreas in the abdominal aorta at the level of the caeliac artery. Glycemia, insulinemia and glucagonemia are studied after i.v. glucose injection in: a) normal rats; b) rats free from their endogenous rate of PGs by previous treatment with indomethacin i.p. and c) with an excessive rate of PGE1. The treatment with PGE1 produces an inhibitory effect on the insulinic response to glucose, as well as hyperglycemia and hyperglucagonemia. In the cases without the endogenous rat of PGs the insulinic secretion as a response to glucose is greatly improved.     

Depressed lymphoproliferative response in mixed leukocyte reaction after Mis-locus alloimmunization

The proliferation of BALB/C lymphocytes preimmunized with Mis or H-2 incompatible cells in response to alloantigens was studied. The results show that preimmunization with Mis-incompatible spleen cells inhibits the lymphoproliferative response against alloantigens whereas preimmunization with H-2 incompatible spleen cells enhances it. It is suggested that Mis coded determinants activate suppressor mechanisms responsible for the unresponsiveness of these preimmunized lymphocytes against alloantigens.     

Phospholipase C activity-induced fusion of pure lipid model membranes. A freeze fracture study

The structural effects of in situ production of diacylglycerol by phospholipase C in pure lipid model membranes have been examined by freeze fracture electron microscopy. Phospholipase C-activity induces massive aggregation and fusion of large unilamellar lipid vesicles and leads to the formation of a 'sealed' lipid aggregate; the outer membrane of this aggregate appears to be continuous. In some areas lipid arranges into a honeycomb structure; this structure is probably a precursor of a discontinuous inverted (type II) cubic phase. Similarly, enzyme treatment of multilamellar vesicles leads to extensive membrane fusion and vesiculation. Thus morphological evidence is obtained showing the ability of phospholipase C to induce bilayer destabilization and fusion. It is speculated that phospholipase C-induced membrane fusion involves a type II fusion intermediate induced by diacylglycerol produced locally.     

Communicating synapses: types and functional interpretation. Exceptions to Cajal's neuron theory.

The neurons of the dorsal periaqueductal nucleus of the mesencephalon and their synaptic contacts were observed under a transmission electron microscope. We found various types of synapses which constituted an exception to Cajal's neuron theory (law of neuron independence). Some of these synapses had an open communicating or continuity 'passage' between the presynaptic bouton of a neuron (first neuron) and the postsynaptic portion of another neuron (second neuron). The 'communicating' passage (located in the synaptosome) is formed by the continuity of the presynaptic and postsynaptic membrane, and its limits or rims are the reflexion points of the membranes. When only two neurons intervene they could be termed 'simple communicating synapses'. We found three types: I = communicating axosomatic synapses; II = communicating axodendritic synapses, and III = communicating axoaxonic synapses'. When three neurons intervene in the synaptic contact, they could be termed 'complex communicating synapses'. In these, the first and second neurons form a normal synapse, but the lateral portion of the presynaptic bouton of the first neuron also enters into contact with a third neuron, with which it establishes an open communicating or continuity passage. The points of these passages are collateral to the synapse, and may be in the presynaptic or pre-postsynaptic portions simultaneously, communicating collaterally with the third neuron. We found a further three types: IV = complex communicating axosomatic and dendritic synapses; V = complex communicating axoaxonic and somatic synapses, and VI = complex communicating axodendritic and double-somatic synapses. It is suggested that communicating synapses may constitute an exception to Cajal's neuron theory, representing functional states for the acceleration, retardation or modulation of the synaptic function. The neurotransmitters would pass en masse through the communicating passage and the depolarization wave would pass through the rims without being retarded. In the simple communicating synapses, their action would be intensifying. In the complex communicating synapses, their action would be modulating or retarding, since the collateral communicating passage would function as an 'escape valve' through which part of the impulse reaching the presynaptic bouton would escape.     

Characterization and 400-MHz 1H-NMR analysis of urinary fucosyl glycoasparagines in fucosidosis

Fucosyl glycoasparagines accumulating in the urine of a patient with fucosidosis were isolated using reverse-phase HPLC. Structural analysis of 25 glycoasparagines was carried out by combination of methylation and 400-MHz 1H-NMR spectroscopy analyses. The compounds represent different steps in the incomplete catabolism of N-glycosidically linked glycans, as the result of an alpha-L-fucosidase deficiency. All of the glycoasparagines possess a fucose residue alpha-1,6-linked to the GlcNAc 1 residue attached to asparagine. Fucose residues on the peripheral branches were linked either alpha-1,3 to GlcNAc residues (X determinant) or alpha-1,2 to galactose residues (H determinant). The present study allows precise assignments of the NMR parameters for most of the fucosyl linkages occurring in N-glycosidically linked glycans of the N-acetyllactosamine type.     

Glucosidase II from control and ethanol-treated rats. Purification and properties

Liver glucosidase II from control and ethanol-treated rats was purified and its physical and catalytic properties studied. No significant variation was found in the purification and properties of the enzyme from either source (ethanol-treated and control rats), except for activity after storage. Glucosidase II was purified to electrophoretic homogeneity from liver microsomes by solubilization, protamine sulphate precipitation, and anion exchange (DEAE-Sephacel) and affinity (Con A-Sepharose-4B) chromatographies. The native enzyme molecule is a tetramer (Mr 425 +/- 10 kDa) with identical subunits (Mr 106 +/- 3 kDa). Km values, determined at pH 6.8 for the p-nitrophenyl-glucosidase activity of glucosidase II from control and ethanol-treated rats, were 1.20 +/- 0.12 and 1.14 +/- 0.13 mM, respectively. The Arrhenius plot was linear, and the value for the apparent activation energy, calculated from this plot, was 56.64 kJ/mol. The p-nitrophenyl-glucosidase activity of glucosidase II from control and ethanol-treated rats was inhibited to the same extent by NH4+, by the divalent cations Ca2+ and Mg2+, and by methanol, ethanol, 2-propanol, n-propanol, isobutanol and n-butanol.     

Characterization of recF suppressors in Bacillus subtilis

A recF mutation renders Bacillus subtilis cells very sensitive to DNA-damaging agents. Such a recF defect is partially suppressed either by the presence of the recA73 mutation or by the presence of a plasmid-borne, heterologous, single-stranded DNA-binding (ssb) protein gene. Plasmids carrying ssb genes also suppressed the recR and recL defects. Our results suggest that suppression occurs by increasing recombinational repair. The effect of the suppressors may be at the level of induction of the SOS response.