Syrticola mediterraneus n. sp., a harpacticoid copepod from the Bay of Calvi,Corsica

Syrticola mediterraneus n. sp. belonging to the family Cylindropsyllidae Sars (sensu Lang, 1948) is described from the Bay of Calvi (Corsica). The species is closely related to (?) Notopontia galapagoensis Mielke, 1982 and Syrticola flandricus Willems & Claeys, 1982. The former species is now transferred to the genus Syrticola as Syrticola galapagoensis n. comb.     

Effect of postnatal development on calcium currents and slow charge movement in mammalian skeletal muscle

Single- (whole-cell patch) and two-electrode voltage-clamp techniques were used to measure transient (Ifast) and sustained (Islow) calcium currents, linear capacitance, and slow, voltage-dependent charge movements in freshly dissociated fibers of the flexor digitorum brevis (FDB) muscle of rats of various postnatal ages. Peak Ifast was largest in FDB fibers of neonatal (1-5 d) rats, having a magnitude in 10 mM external Ca of 1.4 +/- 0.9 pA/pF (mean +/- SD; current normalized by linear fiber capacitance). Peak Ifast was smaller in FDB fibers of older animals, and by approximately 3 wk postnatal, it was so small as to be unmeasurable. By contrast, the magnitudes of Islow and charge movement increased substantially during postnatal development. Peak Islow was 3.6 +/- 2.5 pA/pF in FDB fibers of 1-5-d rats and increased to 16.4 +/- 6.5 pA/pF in 45-50-d-old rats; for these same two age groups, Qmax, the total mobile charge measurable as charge movement, was 6.0 +/- 1.7 and 23.8 +/- 4.0 nC/microF, respectively. As both Islow and charge movement are thought to arise in the transverse-tubular system, linear capacitance normalized by the area of fiber surface was determined as an indirect measure of the membrane area of the t-system relative to that of the fiber surface. This parameter increased from 1.5 +/- 0.2 microF/cm2 in 2-d fibers to 2.9 +/- 0.4 microF/cm2 in 44-d fibers. The increases in peak Islow, Qmax, and normalized linear capacitance all had similar time courses. Although the function of Islow is unknown, the substantial postnatal increase in its magnitude suggests that it plays an important role in the physiology of skeletal muscle.     

Immobilization of acetylcoenzyme A synthetase and the preparation of an enzyme reactor for the synthesis of [11C]acetylcoenzyme A

The enzyme acetylcoenzyme A synthetase (acetate-CoA ligase (AMP forming), EC 6.2.1.1) from Saccharomyces cerevisiae (baker's yeast) is used for the synthesis of 1 mumol [11C]acetylcoenzyme A. (CoA-[11C]Ac). A screening of the immobilization of the enzyme on differently derivatized controlled pore glass beads (50 nm pore size and 125-180 micron particle size) was performed. Several silanes, spacer arms and terminal reactive groups were tested. The immobilized enzyme was subjected to storage stability tests. From these experiments, the method of choice was selected: immobilization on CNBr-activated controlled pore glass. The immobilized parameters were optimized further to improve the activity of the enzyme-loaded glass beads. The latter were packed in a glass column. The kinetic properties of the column were investigated and optimized to obtain an almost complete conversion of 1 mumol acetate into acetylcoenzyme A (CoA-Ac) within a few minutes. This is realized with an enzyme reactor (13.0 x 0.5 cm) containing 6.12 U active acetylcoenzyme A synthetase immobilized onto 1 g controlled pore glass.     

Direct assay for phosphotransacetylase and acetyl-coenzyme A carboxylase by high-performance liquid chromatography

A simple and specific assay to measure the activity of two coenzyme A derivative-processing enzymes, i.e., phosphotransacetylase (EC 2.3.1.8) and acetyl-coenzyme A carboxylase (EC 6.4.1.2), is described. The assay is based on the HPLC analysis of the short-chain coenzyme A derivatives formed by the enzymatic reaction, viz., acetyl-CoA and malonyl-CoA. For this purpose, ion-pair reversed-phase HPLC conditions are optimized. Furthermore, the influence of several variables on the enzyme reaction is studied in order to get maximum activity. Due to its short analysis time, good selectivity, and chromatogram information, HPLC proves to be an excellent method for the assay of these enzymes.     

Isolation and identification of two isomeric trihydroxy octadecenoic acids with prostaglandin E-like activity from onion bulbs (Allium cepa)

Two fractions with prostaglandin E-like activity were isolated from onion (Allium cepa) by using XAD-2 adsorption, silicic acid column chromatography and thin layer chromatography. The fractions were analyzed by gas chromatography/mass spectrometry and were characterized as isomeric mixtures of 9,10,13-trihydroxy-11-octadecenoic and 9,12,13-trihydroxy-10-octadecenoic acid, which are lipoxygenase metabolites of linoleic acid. Bio-assay, for which cascade superfusion was used and the rabbit coeliac and mesenteric arteries and the rat fundus strip were employed as assay organs, was utilized to monitor the bio-active profile throughout the isolation procedures. The activity of 1 microgram of the pharmacologically active fractions T1 and T2 was found to be equivalent to that of respectively 1.33 and 0.63 ng of prostaglandin E2.     

Mechanism of complement-induced stimulation of prostacyclin production by isolated rabbit peritoneum

The interaction between the complement system and prostaglandin synthesis has not thoroughly been explored, although both mediators are known to be involved in inflammatory reactions and endotoxic shock. When rabbit peritoneum, a rich source of prostacyclin forming activity was incubated in serum in which the complement system was activated (CVF, LPS, zymosan), the tissue produced significantly more PGI2, when compared with appropriate controls, indicating that by activation of the complement, factors were generated that stimulated PGI2 biosynthesis. Further results indicated that tryptic cleavage products of complement factor C3 and C5 also led to the appearance of PGI2 releasing principles with a molecular weight of about 7000-11000. The stimulation of PGI2 biosynthesis was explained by enhanced release of AA, and not due to increased activity of cyclo-oxygenase or PGI2 synthetase. Our results suggest that complement-derived products may promote the supply of prostaglandins at the site of inflammation.     

Molecular evolution of voltage-sensitive ion channel genes: on the origins of electrical excitability

We have analyzed nucleic acid and amino acid sequence alignments of a variety of voltage-sensitive ion channels, using several methods for phylogenetic tree reconstruction. Ancient duplications within this family gave rise to three distantly related groups, one consisting of the Na+ and Ca++ channels, another the K+ channels, and a third including the cyclic nucleotide-binding channels. A series of gene duplications produced at least seven mammalian homologues of the Drosophila Shaker K+ channel; clones of only three of these genes are available from all three mammalian species examined (mouse, rat, and human), pointing to specific genes that have yet to be recovered in one or another of these species. The Shaw-related K+ channels and the Na+ channel family have also undergone considerable expansion in mammals, relative to flies. These expansions presumably reflect the needs of the high degree of physiological and neuronal complexity of mammals. Analysis of the separate domains of the four-domain channels (Ca++ and Na+) supports their having evolved by two sequential gene duplications and implies the historical existence of a functional two-domain channel.      

Fully automated measurements by light microscopy of tissue sections using a cellular array computer

Software was developed for the acquisition, segmentation and analysis of microscopic OD-images on a VICOM digital image processor, extended with a VISIOMORPH morphoprocessor board. The delineation algorithms for peroxisomes, lysosomes, and nuclei in liver, kidney, and adrenal gland sections start by thresholding the difference between the original image and a low pass filtered version. The resulting binary mask is then processed by morphological operations in order to produce an object overlay. The efficiency of the programs is evaluated by comparing delineated objects at different OD-levels, created by varying the stain or by multiplying the original pixel values with constant factors. Manual delineation on some images is also used as a reference. More complex algorithms are used for the delineation of muscle fibres in ATP-ase-stained sections and immunocytochemically labelled cells in monolayer preparations. Muscle images from parallel sections with different stainings are matched with a coordinate transform, enabling the transfer of the object mask from a single delineated image to the unprocessed images and thus obtain all necessary information for fibre classification. After segmentation, the OD-images and their object overlays are fed into a data extraction program, measuring for each delineated object user-selected features. Data are sent to a VAX for statistical interpretation.     

Mass spectral characterization of two onion constituents with prostaglandin E-like activity as lipoxygenase metabolites of linoleic acid

Onions ($\text{Allium cepa}$ have been used in folk medicine for the treatment of hypertension and gastrointestinal ulcers, which are cases where the administration of prostaglandins is considered. Using bioassay guided fractionation and gas chromatography/mass spectrometry (GC/MS) we have isolated two biologically active fractions with PGE-like properties and have characterized these products as lipoxygenase metabolites of linoleic acid.For the isolation of the active principles, onion bulbs were homogenized in phosphate buffer and the PGE-like products were isolated using Amberlite XAD-2 absorption, silicic acid column chromatography and silicagel thin layer chromatography. The PGE-like activity was estimated in a cascade superfusion system in which the isolated rabbit coeliac artery, the rabbit mesenteric artery and the rat fundus were used as assay organs.For the GC/MS characterization, various types of volatile derivatives were prepared in order to facilitate the structure elucidation. Derivatization included hydrogenation, methyl ester formation, n-butyl boronate formation and trimethylsilylation. The active fractions yielded identical electron-impact mass spectra, indicating that they are stereoisomeric, and each fraction was identified as a mixture of two positional isomers, i.e. of 9,12,13-trihydroxy-10-octadecenoic and of 9,10,13-trihydroxy-11-octadecenoic acid. With regard to the structure elucidation of the latter isomers, the mixed hydrogenated, n-butyl boronate, methyl ester, TMS-ether derivatives were shown to be of particular value for the determination of the vicinal diol function.The isomeric trihydroxylated octadecenoic acids have been described for the first time as metabolites of linoleic acid in wheat flour incubates. In this system, the trihydroxylated octadecenoic acids were shown to be formed through a sequence of three reactions, including an initial 9- or 13-lipoxygenation yielding hydroperoxy octadecadienoic acids, followed by rearrangement into unstable allylic epoxy hydroxy octadecenoic acids, which in turn are hydrolyzed to trihydroxy octadecenoic acids. Furthermore, structurally related trihydroxy octadecadienoic acids have also recently been isolated from a plant source, more specifically from the roots of $\text{Bryonia Alba}$, which are used for the same medicinal purposes as onion bulbs.     

An improved breeding method for solitarious locusts

In order to unravel the physiological, endocrine, and behavioral differences between gregarious and solitarious forms of the desert locust, Schistocerca gregaria (Forsk.) (Orthoptera, Acrididae), a constant supply of rather large numbers of solitary individuals has to be guaranteed. This represents a bottleneck, mainly because of the intensity of the labor involved and limited laboratory accommodation. The method we describe here substantially reduces the space and manpower needed. The survival rate we obtained in the solitarised population was relatively high, reaching about 55%. The optimal rearing temperature proved to be 32–36 °C. Cabbage leaves and oat flakes sufficed for feeding all year round. Special racks have been designed that enable high density stacking and easy handling. The solitarisation process was monitored over ten consecutive generations. Changes in morphometrics, eye stripes, color, and behavior were recorded, of which only morphometrics, temperature related development, and mortality are discussed. A shift towards the solitarious phase was recorded, with clear differences between gregarious, 1^st generation and 7^th to 10^th generation solitarious locusts.