Carbonic anhydrase inhibition blocks skeletogenesis and echinochrome production in Paracentrotus lividus and Heliocidaris tuberculata embryos and larvae

Carbonic anhydrases (CAs) are a family of widely distributed metalloenzymes, involved in diverse physiological processes. These enzymes catalyse the reversible conversion of carbon dioxide to protons and bicarbonate. At least 19 genes encoding for CAs have been identified in the sea urchin genome, with one of these localized to the skeletogenic mesoderm (primary mesenchyme cells, PMCs). We investigated the effects of a specific inhibitor of CA, acetazolamide (AZ), on development of two sea urchin species with contrasting investment in skeleton production, Paracentrotus lividus and Heliocidaris tuberculata, to determine the role of CA on PMC differentiation, skeletogenesis and on non‐skeletogenic mesodermal (NSM) cells. Embryos were cultured in the presence of AZ from the blastula stage prior to skeleton formation and development to the larval stage was monitored. At the dose of 8 mmol/L AZ, 98% and 90% of P. lividus and H. tuberculata embryos lacked skeleton, respectively. Nevertheless, an almost normal PMC differentiation was indicated by the expression of msp130, a PMC‐specific marker. Strikingly, the AZ‐treated embryos also lacked the echinochrome pigment produced by the pigment cells, a subpopulation of NSM cells with immune activities within the larva. Conversely, all ectoderm and endoderm derivatives and other subpopulations of mesoderm developed normally. The inhibitory effects of AZ were completely reversed after removal of the inhibitor from the medium. Our data, together with new information concerning the involvement of CA on skeleton formation, provide evidence for the first time of a possible role of the CAs in larval immune pigment cells.     

Primary structure and functional properties of the hemoglobin from the free-tailed bat Tadarida brasiliensis (Chiroptera). Small effect of carbon dioxide on oxygen affinity

The hemoglobin of the Free-Tailed Bat Tadarida brasiliensis (Microchiroptera) comprises two components (Hb I and Hb II) in nearly equal amounts. Both hemoglobins have identical beta-chains, whereas the alpha-chains differ in having glycine (Hb I) or aspartic acid (Hb II) in position 115 (GH3). The components could be isolated by DEAE-Sephacel chromatography and separated into the globin chains by chromatography on carboxymethyl-cellulose CM-52. The sequences have been determined by Edman degradation with the film technique or the gas phase method (the alpha I-chains with the latter method only), using the native chains and tryptic peptides, as well as the C-terminal prolyl-peptide obtained by acid hydrolysis of the Asp-Pro bond in the beta-chains. The comparison with human hemoglobin showed 18 substitutions in the alpha-chains and 24 in the beta-chains. In the alpha-chains one amino-acid exchange involves an alpha 1/beta 1-contact. In the beta-chains one heme contact, three alpha 1/beta 1- and one alpha 1/beta 2-contacts are substituted. A comparison with other chiropteran hemoglobin sequences shows similar distances to Micro- and Megachiroptera. The oxygenation characteristics of the composite hemolysate and the two components, measured in relation to pH, Cl-, and 2,3-bis-phosphoglycerate, are described. The effect of carbon dioxide on oxygen affinity is considerably smaller than that observed in human hemoglobin, which might be an adaptation to life under hypercapnic conditions.     

Aggregation of 27 oral bacteria by human whole saliva

Twenty-seven oral strains of the genera Actinomyces (5), Bacteroides (3), and Streptococcus (19) were tested for aggregation by human whole saliva, as well as the effect of culture medium, Ca-ions, and bacteria concentration thereupon. Of the media tested, GF-broth gave rise to less interference by autoaggregation or higher aggregation titers than BHI and TSB, and was used throughout this study. In most cases, Ca-ions (1 mM) only enhanced the rate of induced aggregation, whereas raising the bacteria concentration increased the rate of both induce- and autoaggregation. The final titers, ranging from 1–64, were hardly affected by these parameters, except those of S. rattus HG 59 and S. mutans HG 199, which were respectively increased and decreased by Ca-ions. Saliva-induced aggregation was observed for 21 strains of A. viscosus, A. naeslundii, A. israelii, B. gingivalis, B. intermedius, S. cricetus, S. mutans, S. rattus, S. sanguis, and S. sobrinus, mostly within 15 min to 3 h. Seventeen of these strains also showed autoaggregation, usually well after the onset of induced aggregation. Any potential induced aggregation of B. gingivalis HG 91 was always masked by autoaggregation, as well as that of the S. mutans strains under a particular set of conditions. The aggregation rate and titer varied considerably in a mutually unrelated and strain-dependent way. These microtiterplate data were matched by the 5 spectrophotometric patterns observed for saliva-bacterial interaction, which moreover, gave the better differentiation between induced and autoaggregation. In conclusion, most strains tested can show rapid saliva-induced aggregation in a strain-dependent way, yet strongly affected by the experimental conditions and interference from autoaggregation.     

Control of rat gastric somatostatin release by gamma-aminobutyric acid (GABA)

The influence of gamma-aminobutyric acid (GABA) on gastric somatostatin and gastrin release was studied using an isolated perfused rat stomach preparation. GABA dose-dependently inhibited somatostatin release (maximal inhibition of 44% at 10(-5)M GABA), whereas gastrin secretion was not affected. The GABA agonist muscimol led to a decrease in somatostatin release of similar magnitude. The GABA-induced changes were partially reversed by 10(-5)M atropine. Gastrin secretion was not influenced by either protocol. It is concluded that GABA as a putative neurotransmitter in the enteric nervous system is inhibitory to rat gastric somatostatin release in vitro via cholinergic pathways.     

Nucleotide sequence and evolution of the orangutan ε globin gene region and surrounding Alu repeats

Summary We have mapped and sequenced the globin gene and seven surrounding Alu repeat sequences in the orangutan globin gene cluster and have compared these and other orangutan sequences to orthologously related human sequences. Noncoding flanking and intron sequences, synonymous sites of , , and globin coding regions, and Alu sequences in human and orangutan diverge by 3.2%, 2.7%, and 3.7%, respectively. These values compare to 3.6% from DNA hybridizations and 3.4% from the globin gene region. If as suggested by fossil evidence and molecular clock calculations, human and orangutan lineages diverged about 10–15 MYA, the rate of noncoding DNA evolution in the two species is 1.0–1.5×10^–9 substitutions per site per year. We found no evidence for either the addition or deletion of Alu sequences from the globin gene cluster nor is there any evidence for recent concerted evolution among the Alu sequences examined. Both phylogenetic and phenetic distance analyses suggest that Alu sequences within the and globin gene clusters arose close to the time of simian and prosimian primate divergence (about 50–60 MYA). We conclude that Alu sequences have been evolving at the rate typical of noncoding DNA for the majority of primate history.Presented at the FEBS Symposium on Genome Organization and Evolution, held in Crete, Greece, September 1–5, 1986     

Microculture of single protoplasts of Brassica napus

Protoplasts of Brassica napus L. were cultured individually in a microdroplet system using a synthetic medium with survival rates of more than 70% and division frequencies of up to 65%. Microcallus formation occurred at frequencies of up to 50%. Factors affecting the survival and division of individually cultured protoplasts, such as composition and volume of culture medium, pH, buffering system, osmolarity and genotype, were analyzed.     

On the stoichiometry of the oxidase and monooxygenase reactions catalyzed by liver microsomal cytochrome P-450. Products of oxygen reduction

This laboratory has recently reported that, in a reconstituted enzyme system containing alcohol-induced isozyme 3a of liver microsomal cytochrome P-450, the sum of acetaldehyde generated by the monooxygenation of ethanol and of hydrogen peroxide produced by the NADPH oxidase activity is inadequate to account for the O2 and NADPH consumed. Studies on the stoichiometry have revealed the occurrence of an additional reaction involving an overall 4-electron transfer to molecular oxygen which is presumed to yield water: O2 + 2 NADPH + 2H+----2 H2O + 2 NADP+. The occurrence of a peroxidase reaction in which free H2O2 is reduced to water by NADPH was ruled out. When the 4-electron oxidase activity is taken into account, measurements of NADPH oxidation and O2 consumption are in accord with the amounts of products formed in the presence of various P-450 isozymes, either in the absence or presence of typical substrates, including those which undergo hydroxylation, N- or O-demethylation, or oxidation of hydroxymethyl to aldehyde groups. Of the substrates examined, some had no effect on the oxidase reaction yielding hydrogen peroxide or the 4-electron oxidase reaction, some were inhibitory, and some were stimulatory, but the same substrate did not necessarily have the same effect on the two reactions.     

Vegetative reproduction of trees in some European natural forests

Various means of vegetative reproduction in unexploited forests in western Europe are illustrated with examples. Root suckers are sometimes almost the only method of forest regeneration near the limits of tree growth on the Wadden islands and they can play an important role in forest gaps and riverine forests. Trunk suckers finally replacing their parent trees occur in Alnus, Tilia and Ulmus. Partial uprooting of trees, favoured by special soil conditions, was shown to be an important condition for vegetative reproduction. Temporary survival after uprooting gives opportunity for development of reiterative sprouts, that can replace the vertical axis of a fallen tree. Contact of living branches or even whole stems with soil or mouldering logs favours the growth of adventitious roots. Thus vegetatively reproduced individual trees establish before the uprooted parent tree finally dies. Examples of the clonal spreading of trees are given and a special strategy of layering its branches in pasture woods has been described for beech. Modern forestry rigorously eliminates conditions suitable for the vegetative reproduction of forest trees so their abilities in this respect are often underestimated. Vegetative reproduction seems to be particularly important under circumstances where natural growth is difficult e.g. near the limits of tree growth, on dynamic sites and under heavy shade.