The possible involvement of protein kinase C and phospholipase A2 inHydra tentacle regeneration

The participation of protein kinase C (PKC) in the regeneration of tentacles ofHydra vulgaris was studied. Regeneration was induced by 1,2-sn-dioctanoyl-glycerol (diC₈) and the novel diterpenoidic diacylglycerol verrucosin B (VB), a potent PKC activator extracted from marine sources. VB substantially increasedHydra average tentacle number (ATN) at concentrations 10,000 times lower than those needed for diC₈ to exert an analogous effect. When both synthetic and natural VB analogues were tested, the structure/activity relationship found inHydra tentacle regeneration was identical to that known for DAG-induced activation of PKC in vitro. VB-induced increase of ATN was strongly counteracted by the PKC inhibitors sphingosine and A3, but was not synergic with a tenfold increase of extracellular Ca²⁺ concentration or with an increase of intracellular Ca²⁺ concentration obtained either with the ionophore A23187 or with thapsigargin. This suggested the involvement of a non-Ca²⁺-dependent PKC in VB-triggeredHydra tentacle regeneration. The involvement of phospholipase A₂ (PLA₂) activation inHydra regenerative processes was studied using the novel site-specific inhibitor of the enzyme, oleyloxyethylphosphorylcholine (OOPC), which brought about a striking inhibition of ATN in the low molar range. This effect was reversed by arachidonic acid (AA), while an enhancement of ATN was also observed with an inhibitor of AA uptake from membrane phospholipids, thus suggesting that PLA₂-catalysed liberation of AA is involved inHydra tentacle regeneration. OOPC also blocked verrucosin B-induced PKC-mediated enhancement of ATN, thus suggesting that this effect is also mediated by PLA₂ activation. ATN was increased also by compound 48/80, a direct activator of pertussis toxin-sensitive GTP-binding proteins, and this effect was counteracted by pertussis toxin pretreatment. None of the known AA cascade inhibitors exhibited an effect on ATN comparable to that exerted by OOPC, but, surprisingly, the cycloxygenase inhibitor indomethacin strongly enhanced ATN, thus suggesting that prostanoids might effect a negative control onHydra regenerative processes. This represents the first attempt so far reported to study the implication of more than one biochemical pathway as a signalling event in the hydroid regenerative processes.     

Allozyme polymorphism and linkage disequilibrium ofAdh and α-Gpdh loci in wine cellar and field populations ofDrosophila melanogaster

Summary Over three years, theAdh and -Gpdh loci have been studied in two cellar populations ofDrosophila melanogaster and in two field populations which were each near to one of the cellars. Analyses of gene frequencies indicate that the divergence among subpopulations is greater in theAdh locus than in the -Gpdh locus. Selection for or againstAdh ^S allele acting on theIn(2L)t inversion influences of the -Gpdh alleles. This phenomenon may contribute to explain the maintenance of theAdh and -Gpdh polymorphism and of theIn(2L)t inversion.     

Dominance for enzyme activity inDrosophila melanogaster

Summary A regulatory element tightly linked to theGpdh locus inDrosophila melanogaster has been isolated from a natural population. Flies homozygous for second chromosomes bearing the element,H31, have half the GPDH activity of normal homozygotes. Heterozygotes betweenH31 andF orS alleles exhibit dominance in GPDH activity. Heterozygotes betweenH31, F orS andDf(2L) GdhA have half the diploid level. The contribution of theS allele to the activity inS/H31 heterozygotes is more than four times that ofH31. The regulatory element distinguishingH31 is tightly linked to theGpdh ⁺ locus.     

Development of DNA probes for cytotoxin and enterotoxin genes in enteric bacteria

Summary DNA probes to identify the genes encoding toxins in enteric bacteria have been developed. Use of these probes reduces the number of animals required for toxicity testing, as suspect bacteria can be directly tested for the presence of toxin. We have augmented the gene probes available by developing probes against theEscherichia coli enterotoxin LTII and shiga toxin fromShigella dysenteriae 1.The LTII gene fromE. coli 357900 was identified and characterised and a suitable internal probe was obtained. The LTII gene was found not to be common among enterobacteriae from various geographical locations. Isolates predominately of animal origin from Nigeria and Thailand hybridized with the probe.The shiga toxin gene was isolated fromS. dysenteriae 1 by a combination of in vivo and in vitro methods. An internal probe was identified and used against different serogroups ofShigella andE. coli isolated. The probe was found to hybridize withS. dysenteriae 1 isolates and also someS. flexneri andS. sonnei strains. Representatives were tested for toxin production and found to produce toxin at low levels.     

Diasteroisomeric ichthyotoxic acylglycerols from the dorsum of two geographically distinct populations ofArchidoris nudibranchs

Two marine opisthobranchs,Archidoris tuberculata from Asturias (N. Spain) andArchidoris carvi from Patagonia (S. Argentina), contain in their dorsum diasteroisomeric ichthyotoxic acylglycerols esterified in position 1-sn by antipodal diterpenoid acids.     

Properties and modes of action of specific and non-specific phospholipid transfer proteins

Summary We have described the mode of action of the phosphatidylcholine transfer protein (PC-TP), the phosphatidylinositol transfer protein (PI-TP) and the non-specific lipid transfer protein (nsL-TP) isolated from bovine and rat tissues. PC-TP and PI-TP specifically bind one phospholipid molecule to be carried between membranes. PC-TP, and most likely PI-TP as well, have independent binding sites for thesn-1- andsn-2-fatty acyl chains. These sites have different properties, which may explain the ability of PC-TP and PI-TP to discriminate between positional phospholipid isomers. nsL-TP, which is identical to sterol carrier protein 2, transfers all common phospholipids, cholesterol and oxysterol derivatives between membranes. This protein is very efficient in mediating a net mass transfer of lipids to lipid-deficient membranes. Models for its mode of action, which is clearly different from that of PC-TP and PI-TP, are presented.     

Hydra vulgaris ω10-lipoxygenase is used in vivo to synthesize new α-linolenic acid metabolites

Previous studies conducted in cytosolic extracts of the freshwater hydrozoanHydra vulgaris led to the finding of an abundant 11(R)-lipoxygenase catalyzing the peroxidation of polyunsaturated fatty acid (PUFAs) on the tenth carbon atom from the aliphatic end (10 peroxidation). Here we describe experiments aimed at identifying the actual metabolites generated in vivo by such enzymic activity. Homogenates ofH. vulgaris polyps were analyzed by HPLC. This showed the presence of three major components chromatographically identical to three metabolites obtained when incubating the homogenates with exogenous -linolenic acid (-LA). The presence, in extracts of polyps prelabelled with [¹⁴C]--linolenic acid, of radioactive metabolites displaying the same chromatographic properties, substantiated the hypothesis that the natural products isolated in vivo are derived from -LA. Gas chromatographic analyses revealed that this was the most abundant PUFA in both free and phosphoglyceride-bound fatty acid pools. [¹H]-NMR analysis of the endogenous substances, carried out in comparison with products obtained from exogenously incubated -LA, indicated that their structures were those of 9-hydroxy-, 9-hydroperoxy- and 9-keto-octadeca-10E-12Z-15Z-trienoic acids (9--HOTrE,-HPOTrE and-KOTrE).Hydra homogenates transformed 9--HPOTrE partly into 9--HOTrE and partly into 9--KOTrE. Chiral phase HPLC conducted on 9--HOTrE established that this metabolite was composed mostly of theR anantiomer. These observations, and the finding that the presence of exogenous arachidonic acid in incubated homogenates significantly reduces the production of -LA metabolites, provide strong evidence that these compounds are produced by an enzymic activity identical to the previously-describedH. vulgaris (R)-10-lipoxygenase. Further experiments suggested that -LA, acting as the native substrate for this enzyme, is mainly esterified on the 2 position ofHydra phosphoglycerides, and that the production of the -LA metabolites described here for the first time from natural sources, can be potentially enhanced in vivo by stimuli activating phospholipase A₂.     

Aragusterol C: A novel halogenated marine steroid from an Okinawan sponge,Xestospongia sp., possessing potent antitumor activity

A novel chlorinated steroid, aragusterol C, was isolated from an Okinawan marine sponge of the genusXestospongia. The compound strongly inhibited the proliferation of KB cells in vitro, and also showed potent in vivo antitumor activity against L1210 cells in mice. The complete structure of aragusterol C was determined by spectroscopic analysis and X-ray crystallographic analysis.     

In vitro inhibition by dopamine of 5-hydroxytryptamine-stimulated ovarian maturation in the red swamp crayfish,Procambarus clarkii

Dopamine inhibits 5-hydroxytryptamine-stimulated maturation of the ovaries of the red swamp crayfish,Procambarus clarkii, in vitro just as it does in vivo. This in vitro inhibition appears to be due to inhibition of release of the gonad-stimulating hormone from the brain and thoracic ganglia. However, it is possible that in vivo dopamine also triggers release of the gonad-inhibiting hormone.     

Optical monitoring of activity of many neurons in invertebrate ganglia during behaviors

Summary Optical methods for monitoring neuron activity were developed because these methods lend themselves to simultaneous multiple-site measurements. With the use of new voltage-sensitive dyes, the dye-related pharmacology and photodynamic damage appear to be relatively unimportant. Using multiple-site measurements made with a 124-element photodiode array, we estimated that approximately 30 of the 200 neurons present in theNavanax buccal ganglion make action potentials during feeding and that approximately 300 of the 1100 neurons present in theNavanax buccal ganglion make are active during the gill-withdrawal reflex. The fact that a light mechanical touch to the siphon skin activated such a large number of neurons in the abdominal ganglion suggests that understanding the neuronal basis of the gill-withdrawal reflex and its behavioral plasticity may be forbiddingly difficult.     

La posizione occupata dalla fosforilcolina,citidindifosfatocolina, α-glicerilfosforilcolina e da altri corpi analoghi nel ciclo metabolico di alcuni fosfatidi encefalici

Summary The rate of turnover of individual P-compounds, related to the metabolic cycle of some phosphatides, was followedin vivo in the brain of rats a few days old after³²P administration. The phosphorylcholine is the precursor of the cytidinediphosphatecholine; this in turn may be the precursor of lecithin. Thel--glyceryl-phosphorylcholine represents the degredation product of lecithin. The specific activity curves of the phosphorylethanolamine and phosphorylse rine are similar to that of the phosphorylcholine; likewise thel--glycerylphos-phorylethanolamine is similar to the activity curve of thel--glycerylphosphorylcholine.     

A specific protein in the genusRhynchosciara (Diptera,Sciaridae)

Summary Proteins immunologically related toRhynchosciara americana larval protein 10 occur in the hemolymph and ovaries of five different fly species of the genusRhynchosciara. Electrophoretic analyses showed these proteins to have a mol.wt similar to that of theR. americana protein 10 (43,000), e.g. theR. hollanderi protein 44,300, theR. milleri protein 45,500.     

Research Articles¶Naturally occurring 2′-O-methylpurine nucleosides with hypotensive properties

2′-O-Methylinosine (1) has been isolated for the first time and shown to be an intrinsic hypotensive principle. Its probable in vivo precursor, 2′-O-methyladenosine (3), showed stronger and even orally potent hypotensive activity. Resistance of the methyladenosine (3) against adenosine deaminase is thought to contribute to its long-lasting activity. The effect of both nucleosides (1 and 3) was not accompanied with any significant change in heart rate, which is often observed with adenosine. Received 2 October 1997; accepted 28 October 1997     

Effect of intercalating mutagens on crossing-over inDrosophila melanogaster females

Summary In order to evaluate the effect of several intercalating compounds on crossing-over inDrosophila melanogaster females, acridine orange, acriflavine, chloroquine, ethidium bromide and quinacrine were fed separately to larvae ofy ct f/+++ genotype. Our results show that acridine orange, acriflavine and ethidium bromide increase significantly the recombination frequency at thect-f region and support the view that, for intercalating agents, there is a relationship between clastogenic activity and female recombination induction.     

A new 9,11-secosterol,stellettasterol from a marine spongeStelletta sp

A new 9,11-secosteroid, stellettasterol (1) was isolated from a Japanese marine sponge,Stelletta sp.; its structure was determined by spectroscopic analysis. All NMR signals of1 were unambiguously assigned by application of various 2D NMR techniques. Stellettasterol exhibited antifungal activity againstMortieralla ramannianus.Part 62 of the Bioactive Marine Metabolites series. Part 61: Fusetani, N., Takahashi, M., and Matsunaga, S., Tetrahedron, in press.     

Ein chemisch und pharmakologisch neuartiger Antagonist von pathophysiologisch bedeutsamen Aminen,Kininen und kininbildenden Faktoren

Summary CIBA 21 401-Ba, a glucofuranoside derivative (ethyl-3,5,6-O-benzyl-d-glucofuranoside), antagonizes in vitro the smooth-muscle action of a large number of biogenic amines and polypeptides, the accelerated migration of leucocytes induced by endotoxin, and the Schultz-Dale phenomenon. In vivo, the compound shows anti-inflammatory and anti-allergic effects and improves the survival rate of infected mice treated with suboptimal doses of a sulphonamide.     

Presence of benzodiazepine binding sites (receptors) and amplification thereof by imprinting inTetrahymena

Summary LiveTetrahymena cells bound³H-diazepam specifically, as demonstrated by autoradiographic evidence of displacement of about 25% of labeled diazepam in the presence of a 1000-fold amount of cold diazepam. The³H-diazepam bound to membrane preparations isolated from untreated (control) cells was not displaced by cold diazepam, whereas cells involved in primary interaction (imprinting) with diazepam showed amplification and specificity of diazepam binding in both in vivo (cell suspension) and in vitro (pellicle) systems, as well as displacement of bound label in the presence of 1000-fold cold diazepam. It appears that diazepam induced imprinting and, consequently, also the formation of specific receptors inTetrahymena.     

Effect of xanthurenic acid on P-450-dependent biotransformation by molting glands in vitro

Incubation of molting glands from the crayfishProcambarus clarkii (Y-organ) and the silkwormBombyx mori (prothoracic gland) with 23,24-[²H₄]-2-deoxyecdysone resulted in the production of deutero-ecdysone; this biotransformation was inhibited in the presence of xanthurenic acid. When the experiments were performed under an¹⁸O₂ atmosphere, the¹⁸O atom was introduced into ecdysone, as confirmed by mass spectrometry. We therefore suggest that xanthurenic acid inhibits P-450-dependent hydroxylation of 2-deoxyecdysone. However, deutero-2-deoxyecdysone was not converted to 3-dehydroecdysone when using Y-organs in vitro, although it is a major product. We therefore conclude that the biosynthetic pathway of ecdysteroids inP. clarkii branches at an early step.     

The toxicity of twoBacillus thuringiensis δ-endotoxins to gypsy moth larvae is inversely related to the affinity of binding sites on midgut brush border membranes for the toxins

Summary The-endotoxin fromBacillus thuringiensis subspecieskurstaki strain HD1-9 is almost 400 times more potent than the-endotoxin from strain HD-73 as a gypsy moth larvicide. The two-endotoxins compete for a high-affinity binding site on the brush border membrane of larval gypsy moth midguts. The affinity for the-endotoxin from strain HD-73 is much greater than the affinity for the-endotoxin from strain HD1-9.