Molecular cut-off values of dialysis membranes for alginate and kappa-carrageenan oligosaccharides

The effective molecular weight cut-off values of dialysis membranes for carrageenan and alginate oligosaccharides were evaluated by gel permeation chromatography and nuclear magnetic resonance spectroscopy. For the different membranes tested, i.e. Medicell, Spectra Por 1000D and 3500D, the porous sizes are analogous to tri- and tetrasaccharides. A simple dialysis can be used to recover the majority of the oligosaccharides produced by a carrageenase or an alginate lyase digestion.     

Mannuronan C-5 epimerase activity in protoplasts of Laminaria digitata

Biosynthesis of alginate in algae may be studied by following the cell wall regeneration of brown seaweed protoplasts in culture. The enzyme mannuronan C-5 epimerase will control the composition of the alginate being synthetized.Freshly isolated protoplasts from the thallus of young Laminaria digitata plants showed only low expression of this enzyme. However, after prolonged periods in culture, this activity increased 15-fold. The synthesis of C-5 epimerase by the protoplasts is probably essential for the formation of a new cell wall.After cellular disruption by osmotic shock and centrifugation, most of the epimerase activity resided in the pellet fraction. This may indicate that the enzyme is membrane associated.     

Improved mating efficiency inAcetabularia acetabulum: recovery of 48–100% of the expected zygotes

Summary We have improved zygote recovery 11–1,000 fold by optimizing the physiology of gamete release and mating inAcetabularia acetabulum. Gamete release was affected by agar purity, concentration, and volume/gametangial pair. Cold pre-treatment of gametangia (14–30 d at 10°C in the dark) synchronized subsequent gamete release at 21°C in the light. Cold pre-treatment was nearly twice as effective in synchronizing subsequent gamete release when intact, gametangia-bearing caps rather than isolated gametangia were pretreated. Synchronizing gamete release doubled mating efficiency. In a wild-type laboratory strain ofA. acetabulum, there were 1,561±207 gametes/gametangium which had half-lives of 14.5 d in 0.1% seawater-agar. We recovered 48–93% of the expected numbers of zygotes from a mass mating of 8 to 1,226 gametangia and 11–128% of the expected numbers of zygotes from mating single gametangial pairs: the large range in the calculated mating efficiency may be attributable to the variation in the numbers of gametes made per gametangium. Zygote recovery from single gametangial pairs was highly dependent on the volume of mating matrix. In addition, most zygotes recovered were unattached to any other zygotes in the subsequent generation (> 95% single cells from matings of 1–500 gametangial pairs). Our improvements in mating conditions and zygote recovery (1) have facilitated cell manipulation and culture ofA. acetabulum in the laboratory; and (2) have made controlled crosses for selection and genetic analysis of mutants feasible. These advances have removed a major barrier to genetic analysis of development inAcetabularia.Abbreviations LB Luria-Bertani bacteriological broth - SE standard error of the mean - T_g agar gelling temperatures - DAPI 4,6-diamidino-2-phenylindole     

Microtubule-associated distribution of specific granules and secretion of atrial natriuretic factor in primary cultures of rat cardiomyocytes

A close spatial relationship between specific granules containing atrial natriuretic factor (ANF) and microtubules was demonstrated in primary cultures of neonatal rat cardiac myocytes. For the detection of specific granules and microtubules, the myocytes were double immunolabelled with antibodies against -ANF and -tubulin and examined by conventional fluorescence or laser scanning confocal microscopy. In addition, the ultrastructural distribution of specific granules was demonstrated by electron microscopy. In the atrial myocytes, ANF was stored in numerous specific granules that were mainly localized in the perinuclear sarcoplasm. In the ventricular myocytes, however, a minority of the cells (10%) exhibited limited ANF immunoreactivity after 4 days in culture. Microtubules were present throughout the sarcoplasm of the myocytes. They were most densely packed in the perinuclear regions. Depolymerization of the microtubules with nocodazole was followed by dispersal of ANF immunostaining both in the atrial myocytes and in the ventricular myocytes exhibiting ANF immunoreactivity. When the microtubules were allowed to recover, the perinuclear distribution of specific granules, as seen in non-treated myocytes, reappeared. Measurements of secreted immunoreactive ANF by radioimmunoassay revealed that the secretion of ANF from atrial myocytes into the medium was significantly reduced following nocodazole treatment, whereas a similar decrease in secretion from ventricular myocytes was not observed. These findings indicate that ANF-containing specific granules are closely associated with microtubules within the myocytes. It is suggested that secretion of ANF from the atrial myocytes, in contrast to the ventricular myocytes, is microtubule-dependent.     

The signal recognition particle database (SRPDB).

The SRPDB (signal recognition particle database) provides annotated SRP RNA sequences from Eucaryotes and Archaea, phylogenetically ordered and aligned with their bacterial equivalents. We also make available representative RNA secondary structure diagrams, where each base pair is proven by comparative sequence analysis. New to this release are 17 SRP RNA sequences (a total of 64 sequences) and alignments of proteins SRP19 and SRP54 with their RNA binding sites.     

Visual performance of the toad (Bufo bufo) at low light levels: retinal ganglion cell responses and prey-catching accuracy

The accuracy of toad snapping towards moving worm dummies under various levels of dim illumination (from absolute threshold to moonlight) was videorecorded and related to spike responses of retinal ganglion cells exposed to equivalent stimuli. Some toads (at ca. 16 °C) successfully snapped at dummies that produced only one photoisomerization per 50 rods per second in the retina, in good agreement with thresholds of sensitive retinal ganglion cells. One factor underlying such high sensitivity is extensive temporal summation by the ganglion cells. This, however, is inevitably accompanied by very long response latencies (around 3 s near threshold), whereby the information reaching the brain shows the dummy in a position where it was several seconds earlier. Indeed, as the light was dimmed, snaps were displaced successively further to the rear of the dummy, finally missing it. The results in weak but clearly supra-threshold illumination indicate that snaps were aimed at the advancing head as seen by the brain, but landed further backwards in proportion to the retinal latency. Near absolute threshold, however, accuracy was too good, suggesting that the animal had recourse to a neural representation of the regularly moving dummies to correct for the slowness of vision.     

Egg attendance and brooding by males of the giant water bugLethocerus medius (Guerin) in the field (Heteroptera: Belostomatidae)

Males of the giant water bug Lethocerus medius(Guerin) typify their monobasic subfamily, the Lethocerinae, in that they do not brood eggs attached to their backs as do males of all members of the subfamily Belostomatinae. Exclusive male parental investment as expressed in the Belostomatinae is extremely rare behavior among animals, and evolution of the trait is obscure. Lethocerus mediusmales apparently remain with their mates through oviposition and are consistently found in attendance of eggs after the female has departed. This behavior may enhance paternity assurance at no cost in opportunity for polygyny. Two double clutches of eggs were found, from which we infer the potential for polygynous matings and shared parental investment. Male L. mediusbrood attended egg clutches above the surface of the water, where they may moisten them, shade them, and defend them against predation. Egg attendance/brooding by L. mediusand other Lethocerusspecies may represent a plesiomorphic state from which paternal back- brooding evolved in the Belostomatinae.     

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.     

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.