Substance P in the suprachiasmatic nucleus of the rat: an immunohistochemical and in situ hybridization study

Using a biotin-streptavidin-horseradish peroxidase (HRP) immunohistochemical technique the distribution of substance P-immunoreactive neuronal elements was investigated in the rat suprachiasmatic nucleus (SCN). Substance P-immunoreactive nerve fibres and varicosities were distributed throughout the suprachiasmatic nucleus, with the largest accumulation in its ventral part. Because this location overlaps with the innervation of retinal afferents, the distribution and density of substance P-immunoreactive fibres in bilaterally enucleated rats were compared to normal rats. The density of substance P-immunoreactive fibres and nerve terminals in the ventral part of the suprachiasmatic nuclei was reduced in the rats with bilateral destruction of the optic nerves, whereas the density of fibres and nerve terminals in the dorsal part as well as other retinal target areas in the thalamus and mesencephalon was unaffected. In rats pretreated with an intraventricular injection of colchicine several substance P-immunoreactive perikarya were identified in the suprachiasmatic nucleus. The immunoreactive neurons, measuring 9.7 m±1.1 m in diameter, were frequently observed in the central core of the nucleus and to a lesser extent in the dorsomedial and ventrolateral subparts. Using in situ hybridization histochemistry pre-protachykinin-A mRNA was found in the same part of the SCN indicating that synthesis of substance P takes place in SCN neurons. Using a double immunohistochemical approach applying diaminobenzidine and benzidinedihydrochloride as chromagens substance P-, vasoactive intestinal peptide (VIP)-, and vasopressin/neurophysin-immunoreactivities were identified in the same brain section. The substance P-immunoreactive perikarya constituted a separate population of SCN neurons, which were not vasopressin-, neurophysin- or VIP-immunoreactive. Taken together, these observations show that substance P is contained in the retinohypothalamic pathway and within a group of SCN cell bodies, indiating that substance P may play a role in the generation and entrainment of circadian rhythmicity.     

The low temperature,rapid dissolution of gellan away from root cultures

Summary A buffer system consisting of 50 mM Tris-HCl-TRIZMA base plus 10 mM EDTA was used to rapidly dissolve gellan gels used for maintaining transformed carrot root cultures. The optimum conditions of pH 7.5 in the presence of 10 mM EDTA for dissolving gellan were first worked out on a model test system containing 0.4% gellan, 0.025% MgSO₄·7H₂O, and blue dye. The conditions were then tested on gellan gels (0.2% gellan plus nutrients) containing carrot roots. This gel dissolution system was rapid (18 to 20 min), did not require heating, and could also be efficiently performed at 4 °C. Furthermore, the buffer system used for gel dissolution is a standard one used for plant cell fractionation studies.     

Diseases and Injuries Associated with Mortality of Hatchery Reared Baltic Cod (Gadus morhua L.) Larvae

A cod hatching plant was established in 1992 on the island of Bornholm in the Baltic Sea in order to elucidate the possibilites for restocking of cod fry in this brackishwater system. The disease prevalence in 3 batches of hatchery-reared yolksac larvae from the Baltic cod (Gadus morhua L.) was monitored during the posthatch period. High prevalences of bacteriosis/mycosis, lordosis/scoliosis, injuries and protozoan endoparasitism were recorded. Vibrio sp. and Vibrio anguillarum serovar 04, 06, 08 in addition to nontypable strains and saprolegniaceous fungi were isolated from the larvae. The dinoflagellate-like endoparasites were located in the yolksac of the cod larvae.     

Effect of Dietary Magnesium on Post Mortem Phosphocreatine Utilization in Skeletal Muscle of Swine: A Non-Invasive Study Using 31P-NMR Spectroscopy

The effect of dietary magnesium on the post mortem PCr (phosphocreatine) decay in muscle of heterozygote malignant hyperthermia pigs was studied after in vivo exposure to a combination of halothane and succinylcholine. The pigs were anaesthetized with halothane and succinylcholine was injected in the ear vein. Immediately after initiation of the depolarizing neuromuscular blocking effect of succinylcholine the animals were captive-bolt stunned. The PCr decay, reflecting ATP turnover, was followed in situ by 31P-NMR spectroscopy in the biceps femoris muscle for the subsequent 40-70 min post mortem. In 3 of the 4 experiments, the Mg-fed pig had a significantly reduced rate of PCr hydrolysis compared to the control animal. The mechanism of this magnesium effect is unknown.     

Atypical Aeromonas salmonicida Isolated from Diseased Turbot (Scophtalmus maximus L.)

Atypical Aeromonas salmonicida were isolated from 3 outbreaks of disease among farmed turbot (Scophthalmus maximus L.) in 3 different farms, 1 from Norway (Nl) and 2 from Denmark (DK1 and DK2). In all 3 cases, the incidence of disease and mortality was high and the main characteristic pathological finding was skin ulcers and septicaemia. The isolated bacteria were subjected to a thorough phenotypic and genotypic examination and comparison in the laboratory. All 3 isolates belonged to A. salmonicida but dis-played some very different biochemical properties. However, the 2 Danish strains, DK1 and DK2 had identical ribotypes but different from that of Nl, whereas the plasmid pro-files of DK1 and Nl were identical but different from that of DK2. These observations emphasize the need for an improvement of our understanding of the taxonomy and epi-demiology of atypical A. salmonicida.     

Acute Post-Treatment Enterocolitis in 13 Horses Treated in a Norwegian Surgical Ward

Case histories, results of laboratory analyses, treatment regimens and outcome are de-scribed for 13 horses that developed acute enterocolitis after various surgical treatments. Twelve of the 13 horses had been cast in lateral recumbency, and 10 of those 12 had re-ceived general anesthesia. Eleven horses had been treated with antimicrobial drugs. Most horses (11) developed diarrhea 2 days after initiation of treatment, with a variation from 1 to 5 days. The most apparent symptoms were fever, up to 40.5°C, elevated heart rate, discoloration of mucous membranes, and diarrhea. Dehydration, acidosis and leu-kopenia were marked at the time clinical signs occurred. Packed cell volume and total serum protein values were elevated. Therapy was directed toward replacement of fluid losses, restoration of acid-base balance, and counteraction of inflammation. Seven horses (54%) died or were euthanized. At necropsy, pathological changes showed large variations between individuals. Salmonella spp. were not isolated from any of the horses, and no common causative agent was identified. Preventive efforts to avoid post-treatment enterocolitis have been taken with regard to preoperative routines, premedic-ation and anesthesia, with apparent success.     

Purification and characterization of dihydroorotate dehydrogenase A from Lactococcus lactis, crystallization and preliminary X-ray diffraction studies of the enzyme.

Lactococcus lactis is the only organism known to contain two dihydroorotate dehydrogenases, i.e., the A- and B-forms. In this paper, we report the overproduction, purification, and crystallization of dihydroorotate dehydrogenase A. In solution, the enzyme is bright yellow. It is a dimer of subunits (34 kDa) that contain one molecule of flavin mononucleotide each. The enzyme shows optimal function in the pH range 7.5-9.0. It is specific for L-dihydroorotate as substrate and can use dichlorophenolindophenol, potassium hexacyanoferrate (III), and, to a lower extent, also molecular oxygen as acceptors of the reducing equivalents, whereas the pyridine nucleotide coenzymes (NAD+, NADP+) and the respiratory quinones (i.e., vitamins Q6, Q10 and K2) were inactive. The enzyme has been crystallized from solutions of 30% polyethylene glycol, 0.2 M sodium acetate, and 0.1 M Tris-HCl, pH 8.5. The resulting yellow crystals diffracted well and showed little sign of radiation damage during diffraction experiments. The crystals are monoclinic, space group P21 with unit cell dimensions a = 54.19 A, b = 109.23 A, c = 67.17 A, and beta = 104.5 degrees. A native data set has been collected with a completeness of 99.3% to 2.0 A and an Rsym value of 5.2%. Analysis of the solvent content and the self-rotation function indicates that the two subunits in the asymmetric unit are related by a noncrystallographic twofold axis perpendicular to the crystallographic b and c axes.     

Cloning and expression in Escherichia coli of the obtusifoliol 14α-demethylase of Sorghum bicolor (L.) Moench, a cytochrome P450 orthologous to the sterol 14α-demethylases (CYP51) from fungi and mammals

Obtusifoliol 14β-demethylase from Sorghum bicolor (L.) Moench has been cloned using a gene-specific probe generated using PCR primers designed from an internal 14 amino acid sequence. The sequence identifies sorghum obtusifoliol 14α-demethylase as a cytochrome P450 and it is assigned to the CYP51 family together with the sterol 14α-demethylases from fungi and mammals. The presence of highly conserved regions in the amino acid sequences, analogous substrates and the same metabolic role demonstrate that the sterol 14α-demethylases are orthologous enzymes. The sterol 14α-demethylases catalyse an essential step in sterol biosynthesis as evidenced by the absence of a 14α-methyl group in all known functional sterols. A functional sorghum obtusifoliol 14α-demethylase was expressed at high levels in Escherichia coli and purified using an efficient method based on temperature-induced Triton X-114 phase partitioning. The recombinant purified enzyme produced a type I spectrum with obtusifoliol as substrate. Reconstitution of purified recombinant enzyme with sorghum NADPH—cytochrome P450 reductase in dilaurylphosphatidylcholine micelles confirms that obtusifoliol 14α-demethylase catalyses the 14α-demethylation of obtusifoliol to 4α-methyl-5α-ergosta-8,14,24(28)-trien-3β-ol as evidenced by GC—MS. The isolation of a cDNA clone encoding the plant sterol 14α-demethylase, combined with the previously isolated cDNA clones for fungal and mammalian sterol 14α-demethylases, provides an important tool in the rational design of specific inhibitors towards the individual sterol 14α-demethylases.