Spingophosphonolipids in microsomes of Diplodon delodontus. Distribution and effect on the membrane microviscosity

• 1.1. The distribution of ceramide aminoethylphosphonate (CAEP) in microsomal membranes obtained from different tissues of the bivalve mollusc Diplodon delodontus was determined.
• 2.2. The concentration of CAEP reached from 9 to 19% of the total microsomal polar lipids, depending on the kind of tissue.
• 3.3. Palmitic acid was the main fatty acid in the ceramide moiety, followed by stearic and eicosamonoenoic acids.
• 4.4. Artificial membranes were prepared with microsomal phospholipids or phospholipids plus sterols, with and without the addition of CAEP.
• 5.5. It was shown that the phosphonate confers minor mobility to the membranes. This effect is more effective when the membrane contains the natural sterols and the phospholipids are unsaturated.

     

Transport of hydrocarbons by the lipophorin of insect hemolymph

When [I-¹⁴C]acetate was injected into the American cockroach, the labeled acetate was incorporated preferentially into the hydrocarbon fraction and, subsequently, the labeled hydrocarbon was released into the hemolymph where it was associated with the lipophorin (formerly called diacylglycerol-carrying lipoprotein). The label was traced to the three hydrocarbons, n-pentacosane, 3-methylpentacosane and 6,9-heptacosadiene that had been shown previously to be associated with the lipophorin. The specific capacity of lipophorin to accept hydrocarbons from oenocytes, which are believed to be the site of hydrocarbon synthesis, was demonstrated in vitro, and the uptake of hydrocarbon by lipophorin was retarded by respiratory poisoning. When lipophorin containing ¹⁴C-labeled hydrocarbon was injected into the hemocoele of cockroach, the labeled hydrocarbon soon appeared at the cuticular surface where it was deposited specifically. The above observations and our previous data support the postulate that insect lipophorin serves as the true carrier molecule for the transport of hydrocarbons from the site of synthesis (oenocyte) to the site of deposition (cuticle), in addition to its function of transporting diacylglycerol and cholesterol from the fat body and intestine.     

Transport of cholesterol from the endoplasmic reticulum to the plasma membrane

We have studied the transport of newly synthesized cholesterol from the endoplasmic reticulum to the plasma membrane in Chinese hamster ovary cells using a cell fractionation assay. We found that transport is dependent on metabolic energy, but that the maintenance of the high differential concentration of cholesterol in the plasma membrane is not an energy-requiring process. We have tested a variety of inhibitors for their effect on cholesterol transport and found that cytochalasin B, colchicine, monensin, cycloheximide, and NH4Cl did not have any effect. The cholesterol transport process shows a sharp temperature dependence; it ceases at 15 degrees C, whereas cholesterol synthesis continues. When synthesis occurs at 15 degrees C, the newly synthesized cholesterol accumulates in the endoplasmic reticulum and in a low density, lipid-rich vesicle fraction. These results suggest that cholesterol is transported via a vesicular system.     

Transport of maternal cholesterol to the fetus is affected by maternal plasma cholesterol concentrations in the Golden Syrian hamster

The fetus has a high requirement for cholesterol and synthesizes cholesterol at elevated rates. Recent studies suggest that fetal cholesterol also can be obtained from exogenous sources. The purpose of the current study was to examine the transport of maternal cholesterol to the fetus and determine the mechanism responsible for any cholesterol-driven changes in transport. Studies were completed in pregnant hamsters with normal and elevated plasma cholesterol concentrations. Cholesterol feeding resulted in a 3.1-fold increase in the amount of LDL-cholesterol taken up by the fetus and a 2.4-fold increase in the amount of HDL-cholesterol taken up. LDL-cholesterol was transported to the fetus primarily by the placenta, and HDL-cholesterol was transported by the yolk sac and placenta. Several proteins associated with sterol transport and efflux, including those induced by activated liver X receptor, were expressed in hamster and human placentas: NPC1, NPC1L1, ABCA2, SCP-x, and ABCG1, but not ABCG8. NPC1L1 was the only protein increased in hypercholesterolemic placentas. Thus, increasing maternal lipoprotein-cholesterol concentrations can enhance transport of maternal cholesterol to the fetus, leading to 1) increased movement of cholesterol down a concentration gradient in the placenta, 2) increased lipoprotein secretion from the yolk sac (shown previously), and possibly 3) increased placental NPC1L1 expression.     

Uptake of cholesterol and cholestanol by the intestine,hemolymph, and fat body of Heliothis zea

The effect of the concentration and structure of dietary sterol on its uptake and distribution in the intestine, hemolymph and fat body was studied in sixth-instar larvae of Heliothis zea. When cholesterol (cholest-5-en-3β-ol) was inoculated per os into the foregut of larvae, it was rapidly taken up by the intestine. Some of the dietary sterol then passed into the hemolymph, primarily via the midgut, during at least the first 9 h after inoculation, while at least 7% of the dose remained associated with the intestine. The amount of dietary sterol per 0.10 g of hemolymph increased until it reached 3–6% of the dose after 9 h. The amount of sterol per 0.10 g of the fat body increased to as much as 5% of the dose after 10 h. As the concentration of sterol in the dose increased from 0.3 to 15 μg/4 μl, the amount of sterol associated with the intestine, hemolymph, and fat body also increased. When cholesterol was inoculated intrahemocoelically, instead of per os, the amount of sterol in the hemolymph decreased, for at least the first 8 h after inoculation, and may have been absorbed, at least in part, by the intestine. The absence of a double bond in cholestanol (5α-cholestan-3β-ol) had no significant effect, at least 5 h after inoculation, on the uptake and distribution of this sterol in the intestine, hemolymph, and fat body of the larva. The results of this study indicate that although larvae of H. zea fed cholestanol have a slower rate of growth than those fed cholesterol, this may be due to differences in the utilization of the two sterols rather than to differences in their uptake by the tissues.     

Transport of cortisol,progesterone and cholesterol across isolated mesentery

Summary Isolated rat mesentery, mounted in a diffusion cell, is used as a model for the study of vascular endothelium permeability characteristics. The passage of tracer molecules is measured in the absence of osmotic or hydrostatic pressure gradients across the mesentery. The permeability coefficient of the membrane for cortisol and progesterone is similar. When bound to transcortin, cortisol crosses mesentery at a significantly slower rate. Metyrapone diatartrate increases by 30% the passage of free and of transcortinbound cortisol, but is without effect on the passage of progesterone or glucose in the same conditions. When the transfer of cholesterol across mesentery is studied, a high percentage of the tracer is trapped by the membrane.     

Differential Pavlovian conditioning in the mollusc Pleurobranchaea

The present differential Pavlovian conditioning experiments on the sea slug Pleurobranchaea californica extend conditioning described in a preceding paper and provide the conditioning foundation for studies reported in another accompanying paper comparing learned behavior in whole animals with the behavior and motor patterns of electrophysiological preparations. All animals received two appetitive-conditioned stimuli (CSs), one derived from beer (Sbr) and the other derived from squid muscle (Ssq), in different temporal relationships to an electric shock unconditioned stimulus (UCS). Two groups of animals were run concurrently. One group (n = 19) received Sbr as the CS+ in close temporal pairing with the UCS, and Ssq as the CS- explicitly unpaired with the UCS (Sbr +/Ssq-). The second group (n = 20) received the opposite contingencies (Sbr-/Ssq+). All animals received only one day of conditioning involving 5 trials with an intertrial interval of 2 h. There were two replicate experiments, each involving about half of the total n, and each yielding similar results as the sum we report here. Before conditioning, animals exhibited feeding behavior (extension of the proboscis and bite-strike responses) to both stimuli at similar low thresholds. Conditioning produced long-term behavioral changes in all animals throughout the 4.5-day postconditioning observation period. However, only the Sbr+/Ssq- animals consistently exhibited the appropriate differentially conditioned food-aversion behavior which consisted of strong withdrawal and high-threshold feeding responses to Sbr, and low-threshold feeding responses to Ssq. We discuss the possibility that such differences between Sbr+/Ssq- and Sbr-/Ssq+ conditioning may arise either from inherent differences in the responses of the animals to Sbr and Ssq, or, as seems more likely to us, from training and testing effects produced by differences in the compositions of the two stimuli.     

Feeding neural networks in the mollusc Aplysia

Aplysia feeding is striking in that it is executed with a great deal of plasticity. At least in part, this flexibility is a result of the organization of the feeding neural network. To illustrate this, we primarily discuss motor programs triggered via stimulation of the command-like cerebral-buccal interneuron 2 (CBI-2). CBI-2 is interesting in that it can generate motor programs that serve opposing functions, i.e., programs can be ingestive or egestive. When programs are egestive, radula-closing motor neurons are activated during the protraction phase of the motor program. When programs are ingestive, radula-closing motor neurons are activated during retraction. When motor programs change in nature, activity in the radula-closing circuitry is altered. Thus, CBI-2 stimulation stereotypically activates the protraction and retraction circuitry, with protraction being generated first, and retraction immediately thereafter. In contrast, radula-closing motor neurons can be activated during either protraction or retraction. Which will occur is determined by whether other cerebral and buccal neurons are recruited, e.g. radula-closing motor neurons tend to be activated during retraction if a second CBI, CBI-3, is recruited. Fundamentally different motor programs are, therefore, generated because CBI-2 activates some interneurons in a stereotypic manner and other interneurons in a variable manner.     

Tyrosinase localization in mollusc shells

In molluscan shellfish, pigmentation is frequently observed in the calcified shell, but the molecular basis of this process is not understood. Here, we report two tyrosinase proteins (Pfty1 and Pfty2) found in the prismatic shell layer of the pearl oyster Pinctada fucata; this layer is recognized as the pigmented region in P. fucata. The protein sequences were deduced from the corresponding cDNAs and confirmed by MALDI-TOF/TOF analysis. The sequences suggest that both tyrosinases have two copper-binding sites in similar N-terminal domains that are homologous to tyrosinases of cephalopods and hemocyanins of gastropods. In turn, this suggests that bivalve tyrosinases are evolved from a common ancestral copper-binding protein in the mollusc. Pfty1 and Pfty2 were specifically expressed in the mantle, and their expression in the mantle is different from each other, suggesting that these tyrosinases have distinctive roles in melanogenesis in shells.     

Transport of a hydrophobic biosynthetic precursor by lipophorin in the hemolymph of a geometrid female moth which secretes an epoxyalkenyl sex pheromone

Previous experiments with a geometrid species, Ascotis selenaria cretacea, have suggested that a pheromonal C19 3,4-epoxy-6,9-diene is biosynthesized from the corresponding 3,6,9-triene produced outside a pheromone gland and transported to it via hemolymph after association with lipophorin. In order to clarify this transport, high-density lipophorin (HDLp) in the female moths showing two bands (apoLp I with ca. 250 kDa and apoLp II with ca. 80 kDa) on an SDS-PAGE was purified by KBr equilibrium density-gradient ultracentrifugation, and the association of the triene was confirmed by GC-MS analysis of a solvent extract from the isolated protein. Next, the role of HDLp was revealed by a topical application of the deuterated trienyl precursor to the abdomens of the females. The trienyl precursor was associated with HDLp. In their pheromone glands, the triene and the deuterated epoxy pheromone were detected, indicating movement of the triene via the hemolymph. Experiments with male moths of A. s. cretacea and female moths of Bombyx mori showed the same association of HDLp with the triene topically applied. This result suggested that the adult females of A. s. cretacea did not develop HDLp specialized in the triene transport. Furthermore, the topical application of a mixture including the trienyl precursor and two other related hydrocarbons showed equal amounts of association by HDLp but selective delivery of the precursor to pheromone glands in the A. s. cretacea females.     

Spectral and acid-base properties of hemolymph plasma and its fractions in the gastropod pulmonate mollusc <Emphasis Type="Italic">Achatina fulica</Emphasis>

A set of normal biochemical indices of the hemolymph plasma in the gastropod pulmonate mollusc Achatina fulica was described in this study. A comparative analysis of the whole plasma and its subfractions enriched with and depleted of the oxygen-carrying protein hemocyanin was carried out using spectrophotometry and spectrofluorimetry. Individual features of the absorption spectra were analyzed using fourth derivatives. An optimal method to estimate the protein concentration was identified. The buffer capacity, equivalence points and pK values of the dominant buffer groups were calculated to characterize the acid-base properties of hemolymph plasma and its subfractions. A major role in maintaining the hemolymph buffer capacity was shown to belong to the bicarbonate buffering system. The results of this study are compared with the data for Helix pomatia available in literature. In the future, the tested indices will be used to devise ecotoxicological criteria for environmental assessment.     

Pleural-buccal interneurons in the pteropod mollusc Clione limacina

Paired, Phe-Met-Arg-Phe-NH₂-ergic pleural-to-buccal projecting neurons of the pleural ganglia were suggested to be responsible for feeding arrest associated with defensive withdrawal in freshwater and terrestrial pulmonate molluscs. In the present study, the pleural-to-buccal projecting cells were, for the first time, identified in a representative opisthobranch, the carnivorous marine pteropod Clione limacina. Two symmetric neurons of its pleural ganglia were found to be similar to the pulmonate pleural-to-buccal projecting neurons in the number of neurons, positions of their cell bodies in the central nervous system, a unique, indirect route of their axon, electrotonic coupling of the left and right cells, and expression of Phe-Met-Arg-Phe-NH₂-like immunoreactivity and inhibitory action on neurons participating in the motor program for feeding. In their turn, pleural-to-buccal projecting neurons receive excitatory inputs from the protractor interneurons involved in the feeding rhythm generation. Also, it was demonstrated that the pleural-to-buccal projecting cells activity positively correlates with spontaneous and induced acceleration of the locomotor rhythm. Accordingly, stimulation of the cerebral command neuron for locomotion, cell CPA1, excited pleural-to-buccal projecting neurons. We conclude that the neuronal network underlying feeding behavior in both pulmonate and opisthobranch molluscs is similarly linked to defensive behavior by pleural Phe-Met-Arg-Phe-NH₂-ergic neurons, thus indicating evolutionary conservation of these pleural-buccal projections. Accepted: 22 June 1999     

Oxidative stress and histological alterations produced by dietary copper in the fresh water bivalve Diplodon chilensis

The aim of this work was to study the oxidative stress effects and histological alterations caused by dietary copper on the filter-feeding freshwater mussel Diplodon chilensis. Bivalves were fed during 6 weeks with the green algae Scenedesmus vacuolatus previously exposed to copper. Metal concentration in algae cultures and bivalve digestive gland was measured by TXRF. A maximum accumulation of 0.49 μg Cu/mg protein was detected at week 6. Also at this week, the hepatosomatic index (HSI) showed the highest decrease (50%) in response to Cu exposure. SOD and GST activities were significantly increased at weeks 4, 5 and 6, reaching an activity on average 50% higher than in controls for GST. CAT activity and GSH increased significantly at weeks 5 and 6. Despite this response, oxidative damage measured as TBARS and carbonyl groups contents increased significantly at weeks 4, 5 and 6, respectively. Digestive tubule and duct atrophy and cell-type replacement in treated mussels were observed by histological studies. The presence of intracellular rhodanine-positive granules, suggests copper accumulation in intracellular vacuoles of digestive cells.     

Early development of the aplacophoran mollusc Chaetoderma

The early development of the trochophore larva of the aplacophoran Chaetoderma nitidulum (Mollusca: Caudofoveata = Chaetodermomorpha) is described using scanning and transmission electron microscopy and using fluorescence staining and confocal laser scanning microscopy of the muscle system. The most important features include the presence of a prototroch and a telotroch, serially arranged glandular cells (spiculoblasts) at the dorsal mantle, the occurrence of a ventral suture as a possible remnant of a former foot sole, and the presence of a pair of protonephridia. The development of the early anlagen of the circular body wall muscles does not show the anterior–posterior mode of formation that is typical for annelids, thus strengthening the hypothesis of a non‐segmented ancestry of Mollusca.     

Plasticity of feeding behavior in the opisthobranch mollusc Navanax

Several forms of plasticity of feeding behavior were investigated in Navanax. Navanax is a gastropod mollusc which lacks a radula, and ingests prey with suction caused by rapid pharyngeal expansion. Feeding is little affected by handling or posture and is resistant to the noxious stimulus of cutting through the body wall. Feeding is affected by arousal, as shown by a decrease in latency following initial exposure to food. The feeding response also habituates, as shown by an increase in latency when feeding responses are elicited without allowing animals to engulf food. The latency increase is not likely to be due to motor fatigue, since it can be partially reversed by dishabituation with an alternate prey species. Continued feeding causes satiation, as shown by increased feeding latencies and eventual cessation of feeding after a mean weight gain of 42.0% of initial animal weight (N = 12). Another form of plasticity occurs when Navanax are presented with food too large to swallow whole. Navanax either cease to respond after a few presentations, perhaps due to habituation, or maintain suction on the partially swallowed prey for an extended period of time. During extended sucks, the more deeply ingested portions are digested.     

Innervation of the intestine in the bivalve mollusc Chione stutchburyi

Summary The innervation of the gut of the venerid bivalve mollusc, Chione stutchburyi, has been examined by fluorescence histochemistry, electron microscopy and autoradiography. Specific green and yellow varicose fluorescent fibres indicate the presence of dopaminergic and serotonergic axons, respectively. Three different types of axons can be distinguished by the morphological characteristics of their vesicles. Type I axons contain predominantly small granular vesicles (average diameter 65 nm), Type II axons possess large granular vesicles (average diameter 100 nm) and Type III axons contain large opaque vesicles (average diameter 150 nm). The granular vesicles in both Types I and II axons react positively to dichromate, and their granularity is reduced by reserpine indicating that they are monoaminergic. Only Type I axons accumulate tritiated dopamine and are selectively damaged by 6-hydroxydopamine. It is concluded that Type I axons are dopaminergic. Type II axons are serotonergic: they alone take up tritiated 5-hydroxytryptamine, and 5,7-dihydroxytryptamine selectively causes degenerative changes in these axons. Type III axons contain an unidentified neurotransmitter substance. The large opaque vesicles of these axons do not react to dichromate and are unaffected by reserpine, 6-hydroxydopamine or 5,7-dihydroxytryptamine.     

Anti-tumor factor in insects' hemolymph

We have purified a lectin from the hemolymph of Sarcophaga peregrina larvae, obtained after injury of their body wall. This lectin recognizes galactose residues and suggested to be involved in the defense mechanism of this insect. We also demonstrated that this lectin induced cytotoxic effects on tumor cells in the presence of murine macrophages. It was found that the murine macrophages had Sarcophaga lectin binding proteins. Using pupal hemocytes and fatbody of this insect, we established an in vitro system that mimics dissociation of the fatbody in vivo. New membrane protein, induced on the surface of the hemocytes at pupation, suggested to participate in the recognition of the fatbody.