Lipoproteins of the egg perivitelline fluid of Pomacea canaliculata snails (Mollusca: Gastropoda)

The lipid and protein composition of the perivitelline fluid of the eggs of Pomacea canaliculata was investigated. Two lipoproteins (PV 1 and PV 2) and one lipoprotein fraction (PV 3) were detected for the first time in gastropods. They represent 57.0, 7.5, and 35.5% of the egg total proteins, respectively. PV 1 is a glyco-carotene-protein complex with characteristics of a very high-density lipoprotein (VHDL). It has 0.33% lipids, mainly free sterols and phospholipids. The particle has a MW of 300 Kd and is composed of three subunits of 35, 32, and 28 Kd, respectively. PV 2 particle is a VHDL of 400 Kd and 3.75% lipids. The major lipid classes are free sterols and phospholipids and also have significant quantities of energy-providing triacylglycerides and free fatty acids. It is composed of two apoproteins of 67 and 31 Kd. PV 3 density corresponds to a high-density lipoprotein (HDL). It was fractionated into two subfractions "h" and "p". Fraction "h" contains 5.16% lipids, mainly free sterols, phospholipids, and free fatty acids, and two particles of 100 and 64 Kd. Dissociating electrophoresis showed two subunits of 34 and 29 Kd. Fraction "p" is composed of a single particle of 26 Kd that contains 9.5% lipids, which represents 30% of the total egg lipids. It has high levels of a carotenoid pigment. Besides it contains free fatty acids, hydrocarbons, sterified sterols, and triacylglycerides. These three fractions are probably the major supply of lipids and amino acids for the developing embryo.     

Cell lineage of the prototroch of Patella vulgata (Gastropoda, Mollusca)

The trochophore larva of the archaeogastropod mollusc Patella vulgata has a well-developed locomotory organ, the ciliated prototroch. This structure is formed from specific founder cells, the trochoblasts. Two methods were employed to determine the composition and cell lineage of the prototroch. Fluorescent cell-lineage tracer injection in trochoblasts and trochoblast founder cells was used to show how the various trochoblasts became incorporated into the prototroch. Scanning electron microscopy was used to study both differentiation, more specifically ciliation, of trochoblasts and localization of trochoblasts in the prototroch. The results obtained with both methods are in accordance with each other. During early development all trochoblasts involved in prototroch formation become cell cycle-arrested and develop cilia. Subsequently, the trochoblasts shift in position to form a circular prototroch and a number of trochoblasts deciliate. As a result of these processes the mature prototroch consists of a number of heavily ciliated cells as well as a number of deciliated cells. Comparison of prototrochs from a number of spiralian species shows that this structure is very conserved during evolution. The significance of this is discussed.     

Histochemical estimation of lipid amounts in cells of the hepato-pancreatic gland of Lymnaea stagnalis (L.) (Gastropoda, Pulmonata) naturally infected with digenetic larvae

OBJECTIVE: We wished to determine the abnormality responsible for Generalized Resistance to Thyroid Hormone in a family with this syndrome. DESIGN: Molecular biological studies were performed on a mutant human thyroid hormone receptor beta (hTR beta) cloned from fibroblasts of the patient. PATIENTS: The patient is from a previously reported family with typical features of Generalized Resistance to Thyroid Hormone, demonstrating goitre, elevated thyroid hormone levels, slightly elevated TSH, and retarded bone age. MEASUREMENTS: A cDNA for hTR beta 1 was cloned using specific oligonucleotide primers from fibroblast DNA. A mutant hTR beta 1 expression vector was constructed, and an in-vitro expressed mutant receptor was tested for T3 binding. Receptor binding to DNA was studied in a DNA cellulose assay and gel mobility shift assay. RESULTS: Two mutations were found in the cloned hTR beta. One was silent but the second changed arginine 438 to histidine. The mutation was present in RNA and genomic DNA, as shown by allele-specific amplification. The mutated receptor had reduced T3 binding affinity but demonstrated normal binding in a DNA cellulose assay and in a gel mobility shift assay. The receptor did not have altered heat sensitivity. CONCLUSIONS: In the T sibship with Generalized Resistance to Thyroid Hormone, resistance to thyroid hormone is apparently produced by a substitution of a histidine for arginine at amino acid 438, which causes reduced binding of receptor to T3, although the receptor remains able to bind to DNA and, for this reason, functions as a dominant negative in affected subjects who are heterozygous with one normal and one mutated allele.     

On the ultrastructure of the abdominal sense organ of the giant scallop, Placopecten magellanicus (gmelin)

The long tentacles of the Giant scallop Placopecten magellanicus (Gmelin) have been examined with light, scanning, and transmission electron microscopy. Three types of ciliated cells have been observed, one of which is located in specialised papillae born on the distal third of the tentacle. There are two separate cell types within the papillae. Type I cells are non-ciliated supporting cells, which form a capsule within which are found the Type II cells. These cells bear up to five cilia at their apices, and it is suggested that these are the receptor cells of the organ. No function has yet been determined for the receptors, but is suggested that they might be mechanoreceptors. A third cell type, Type III cells, occur at the base of the papillae. These cells bear many cilia and also macrocilia. Another ciliated cell type occurs on the proximal two thirds of the tentacle. These cells bear many cilia that are thought to be motile and not sensory.     

Morphology and histochemistry of the mandibular gland of the Australian brush-tail possum Trichosurus vulpecula (Marsupialia)

A histological, ultrastructural and histochemical study of the mandibular gland of the Australian possum Trichosurus vulpecula revealed essentially similar features as those described earlier for the mandibular gland of the taxonomically relatively unrelated American opossum Didelphis. The secretory endpieces consist of a branched tubular part, composed of serous cells whose secretion granules contain neutral glycoproteins, and terminal acini, consisting of seromucous cells containing small amounts of sialomucins. Relatively short intercalated ducts lead to striated ducts of variable ultrastructural appearance. The striated ducts run in bundles in the center of each sublobule of the gland. The possible functional significance of the abundance and variability in ultrastructure of the striated ducts is discussed.     

Double fluorescence analysis of human cytomegalovirus (HCMV) infected human fibroblast cultures by flow cytometry: increase of class I MHC expression on uninfected cells and decrease on infected cells

Cultured human foreskin fibroblasts (HFF) were infected with different multiplicities of infection (moi 0.001-0.1) of human cytomegalovirus (HCMV) strain AD 169 or a clinical isolate. Percentage of infected cells was determined by analysis of immediate early (IEA), early (EA), and late (LA) virus antigen expression with flow cytometry or by immunoperoxidase staining. Changes in the expression of class I MHC surface molecules were demonstrated by comparing the mean fluorescence intensities of infected HFF cultures with those of mock infected cell cultures by flow cytometry. At day three post infection single fluorescence analysis showed that infected HFF cultures split into low and high density class I MHC bearing cells. The addition of anti-interferon beta reduced the expression of class I MHC, distinctly. The assumption that infected cells down-regulate and uninfected cells up-regulate their expression of class I MHC molecules was demonstrated by double fluorescence analysis both with flow cytometry and fluorescence microscopy. Analysis of class I MHC-antigen expression versus immediate (IEA, mab E13), early (EA, mab 9221), or late (LA, mab BM219) virus antigen expression yielded three cell populations of HCMV infected HFF cultures three days post infection: 1. uninfected cells with an increase of class I MHC, 2. high density class I MHC, IEA and/or EA expressing cells, and 3. low class I MHC, IEA, EA and LA expressing cells.     

Histogenesis of perineal gland cells from the pangolin (Manis javanica Demarest, 1821, Pholidota, Mammalia) during organ culture in diffusion chambers]

OBJECTIVES: To examine the association between intraoperative cerebral microembolic signals (MES) and postoperative neuropsychological deficit in patients with valve replacement (VR) and patients with coronary artery bypass grafting (CABG). METHODS: Neuropsychological examination (10 tests) was performed 1-2 days before and 2 months after surgery (VR (n=26) and CABG (n=14)). The intraoperative number of cerebral MES were monitored from the right middle cerebral artery using transcranial Doppler. RESULTS: A higher number of cerebral MES were detected in VR patients with neurological deficit (6/26) compared with those without deficit (20/26) (MES median number 2083 v 645, p=0.04). No such difference was found in patients with CABG (2/14 v 12/14) (MES median number 50 v 112, p=0.2). CONCLUSION: A high number of MES were detected in patients with VR with neuropsychological deficit. In patients with CABG with such a deficit, a low number of MES were detected. This difference in number may be explained by relatively more gaseous emboli in the first and more solid in the second. However, the results in the patients with CABG should be interpreted with caution due to the few patients in this subgroup.     

Transient and sustained expression of FMRFamide-like immunoreactivity in the developing nervous system of Lymnaea stagnalis (Mollusca, Pulmonata)

1. In the present study we have investigated the ontogeny of FMRFamide expression in the snail, Lymnaea stagnalis, from its first appearance to its distribution in young adults. 2. The first FMRFamide-like immunoreactive (FaLI) cells within CNS appear by E45 embryonic stage (premetamorphic veliger). The number of FaLI neurons increases throughout both pre- and post-hatching development. 3. Both transient and sustained expression of FMRFamide-like immunoreactivity by specific sets of neurons occurs. Two cells which transiently express immunoreactivity appear outside the future CNS by the stage E45. Other population of transient FaLI neurons includes bilaterally symmetric groups of cells in the cerebral and pedal ganglia during posthatching stages P1 (hatchlings) to P5 (juveniles). All other immunostained cells which appear during development maintain their transmitter phenotype into adulthood. 4. The possible role of FMRFamide-related peptides in the processes of morpho- and neurogenesis is discussed.     

Immunochemical investigations of cytochrome P450 forms/epitopes (CYP1A, 2B, 2E, 3A and 4A) in digestive gland of Mytilus sp

Western blot analysis of microsomes and partially purified cytochrome P450 (CYP) from digestive gland of Mytilus edulis was carried out using polyclonal antibodies to hepatic Perca fluviatilis CYP1A, Oncorhynchus mykiss CYP3A and rat CYP2B, CYP2E and CYP4A isoforms. Multiple CYP bands were detected in partially purified CYP compared to single bands for microsomes for anti-CYP1A, anti-CYP2B, anti-CYP2E and anti-CYP3A. In contrast, anti-CYP4A showed two distinct bands for both. The apparent molecular weights in kD (mean +/- range or S.D.; n = 2-4) for partially purified CYP were 42.5 +/- 0.5 and 48.1 +/- 0.3 (2 bands, anti-CYP1A); 67.4 +/- 0.7, 52.8 +/- 0.6, 44.5 +/- 2.5 (3 bands, anti-CYP3A); 52.8 +/- 0.7, 48.1 +/- 1.1 and 43.9 +/- 1.1 (3 bands, anti-CYP2B); 52.7 +/- 0.8 and 47.2 +/- 0.2 (2 bands, anti-CYP2E); 50.9 +/- 0.3 and 44.1 +/- 0.2 kD (2 bands, anti-CYP4A). Digestive gland microsomes of Mytilus galloprovincialis from a polluted compared to a clean field site showed higher levels of bands recognised by anti-CYP1A, anti-CYP2E and anti-CYP4A, but not anti-CYP2B and anti-CYP3A (P < 0.05), indicative of independent regulation of different CYP forms. Overall, the apparent molecular weight and field studies indicate at least five different digestive gland CYP forms.     

The effect of age on the ultrastructure of the mid-gut cells of the hymenopteran Nasonia vitripennis (Walk.) (Hymenoptera, Pteromalidae)

The ultrastructure of the mid-gut cells of aged female Nasonia vitripennis is described. The mid-gut is a shrunken and distorted organ in the aged animal. The individual cells are highly disorganised and the organelle components are altered. The small lipid droplets formed in the apical cell region do not coalesce to form the large central lipid inclusions characteristic of the young animal. The rough endoplasmic reticulum is reduced and some of the mitochondria enlarge. The mid- and apical cell regions also contain large numbers of cytolysosomes. The basal cell region is essentially unchanged, but the channels formed by the infolded basal plasma membranes are dilated. The changes observed are discussed in relation to previous observations on other insect species.     

Interactions of gonadal steroids and pesticides (DDT, DDE) on gonaduct growth in larval tiger salamanders, Ambystoma tigrinum

The nervous system is dependent upon thyroid hormones for normal development, and we previously reported that developmental Aroclor 1254 (A1254) exposure caused hypothyroxinemia, hearing loss and other behavioral changes in rats. (Goldey et al., 1995a; Herr et al., 1996). The hypothesis that A1254-induced hypothyroxinemia may have contributed to the observed functional changes was tested in primiparous Long-Evans rats given daily oral doses of corn oil (control) or 8 mg/kg of Aroclor 1254 from gestation day (GD) 6 through postnatal day (PND) 21. In addition, from PND 4 to PND 21, all pups in one-half of the litters received daily, subcutaneous injections of saline or 100 micrograms/kg thyroxine (T4), to yield four groups of litters: corn oil plus saline (CO-S),. corn oil plus T4 (CO-T4), Aroclor 1254 plus saline (PCB-S), and Aroclor 1254 plus T4 (PCB-T4). We measured thyroid hormone concentrations (T4 and T3) in serum collected from 7-, 14-, and 21-day-old pups. The kinetics of the injected T4 were also monitored in the CO-T4 and PCB-T4 groups on PND 7 and 21 by measuring T4 and T3 at 1, 3, 5, 8, and 24 h after injection. Circulating T4 concentrations were dramatically depleted in the PCB-S group relative to CO-S. The kinetics study indicated that T4 therapy raised circulating T4 concentrations following in the PCB-T4 pups to near CO-S concentrations, but only for approximately 6 h postinjection, and T4 concentrations fell precipitously thereafter to near PCB-S concentrations. In accord with previous studies, PCB-S pups showed early eye opening, an effect which was exacerbated by T4 injection (in both the CO-T4 and the PCB-T4 groups). Motor activity (figure-eight maze) testing also replicated our finding of an age-dependent, transient reduction in motor activity on PND 15 that was significantly attenuated in the PCB-T4 group. Similarly, we again found reduced acoustic startle amplitudes on PND 23 and low-frequency (1 kHz) hearing loss in animals tested as adults (the latter determined by reflex modification audiometry). Importantly, the hearing loss at 1 kHz in PCB-exposed animals was significantly attenuated by T4 replacement therapy. These data suggest the hypothesis that hypothyroxinemia is involved in PCB-induced alterations in motor and auditory function, while other effects (e.g., eye opening) appear to have a different mechanism of action.     

The effects of colony-stimulating factor-1 on the number and ultrastructure of osteoclasts in toothless (tl) rats and osteopetrotic (op) mice

The role of colony-stimulating factor-1 (CSF-1 or M-CSF) in osteoclast development is illustrated by observations that administration of exogenous CSF-1 increases osteoclast number and improves the skeletal sclerosis of two osteopetrotic mutations, toothless (tl) in the rat and osteopetrotic (op) in the mouse. We examined the effects of CSF-1 treatment on the number and ultrastructure of osteoclasts in the tibial metaphysis of normal and mutant animals of both stocks to understand the similarities and differences between these two mutations. Osteoclasts from normal animals of both stocks were abundant and possessed the ultrastructural features of active cells. These included apical areas in contact with mineralized surfaces with tightly apposed clear zones, extensive ruffled borders, and a vacuolated cytoplasm with numerous mitochondria. In toothless rats osteoclasts were difficult to locate and those present had poorly defined ruffled borders, fewer cytoplasmic vacuoles, and a basal membrane with both smooth and ruffled areas. Large lipid accumulations were often found near tl osteoclasts. Osteoclasts in op mice were difficult to find, but more numerous than in tl rats. Unlike tl osteoclasts, those of op mice possessed very well developed ruffled borders, small clear zones, and large electron-dense cytoplasmic inclusions. These cells also had unusual basal membranes with both smooth and ruffled regions. CSF-1 treatment increased the number of osteoclasts in both mutant stocks, normalizing the numbers in op mice, but not tl rats. CSF-1 injections caused dramatic changes in the morphology of tl osteoclasts, including increased incidence and size of ruffled borders and cytoplasmic vacuolization. The growth factor had little effect on ruffled borders or clear zones in op mice. Interestingly, mutant osteoclasts of both stocks exhibited a ruffled basal membrane in response to CSF-1 treatment. This increase in membrane ruffling may reflect the ability of CSF-1 to promote rapid formation of osteoclasts from mononuclear precursors in a more permissive microenvironment. Our data indicate that CSF-1 is not required for the development of at least some osteoclasts. The differences in response to CSF-1 treatment which we report lead us to speculate that additional factors may be involved in osteoclastogenesis.     

Phylogenetic relationships inferred from the sequence and secondary structure of ITS1 rRNA in Albinaria and putative Isabellaria species (Gastropoda, Pulmonata, Clausiliidae)

It has been suggested that liking for the taste, flavour and aroma of, for example, coffee and tea is acquired through the process of classical conditioning, involving association of these orosensory cues with the psychopharmacological consequences of caffeine ingestion. Accordingly, this study investigated caffeine reinforcement by assessing changes in preference for a novel drink consumed with or without caffeine. Particular care was taken to use "ecologically valid" procedures; that is, overnight caffeine abstinence followed by a cup-of-coffee equivalent dose of caffeine (70 mg) at breakfast. Caffeine had no significant effects on drink preference or mood in subjects with habitually low intakes of caffeine. In contrast, moderate users of caffeine developed a relative dislike for the drink lacking caffeine and showed somewhat lowered mood following overnight caffeine abstinence (e.g., less lively, clearheaded and cheerful), which was significantly improved by caffeine. These together with other recent results strongly suggest that, in everyday life, caffeine reinforcement can occur as the result of the alleviation by caffeine of the adverse effects of overnight caffeine abstinence (negative reinforcement). They also demonstrate the utility of this flavour-conditioning procedure, which could be applied in the wider investigation of the reinforcing properties of drugs.     

Observations on ultrastructure of the anterior adhesive areas and other anterior glands in the monogenean, Monocotyle spiremae (Monocotylidae), from the gills of Himantura fai (Dasyatididae)

Observations with the light microscope and the scanning and transmission electron microscopes have shown that the anterior end of Monocotyle spiremae has 8 slit-like apertures on the ventrolateral margins at each side of the mouth. Gland cells located next to the pharynx produce rod-shaped secretory bodies that are conveyed in ducts that open on to the surfaces of rounded lobes inside "reservoirs" behind each ventral, slit-like aperture. Rod-shaped secretory bodies are extruded into the "reservoirs" and appear to combine and form a homogeneous secretion which may bond the ventrolateral regions of the head of the parasite to a substrate. At no stage, however, were intact rods observed outside the duct endings. Gland cells that produce an ovoid secretory body also supply the head of M. spiremae, but ducts from these open dorsal and anterior to the mouth in a region where the parasite is not known to attach. There appears to be little or no chance for the ventral rods and the dorsal ovoid secretion to mix. This is the first record of a monogenean parasite with a single type of secretion supplying the ventral surfaces of the anterior end. The rods in M. spiremae differ in some respects from the rod-shaped bodies recorded previously among gyrodactylid, dactylogyrid, capsalid and acanthocotylid monogeneans.     

Studies on the mechanism of action of a novel anorectic agent, (--)-threo-chlorocitric acid

(--)-threo-Chlorocitric acid, a novel and potent anorectic agent in rats and dogs, decreased food intake to a comparable extent in rats fed chow, low fat, or high fat diets. This inhibition of food intake was not the result of conditioned aversion. Unlike (--)-threo-hydroxycitric acid, a structurally related compound which inhibits fatty acid synthesis, (--)-threo-chlorocitric acid did not suppress fatty acid synthesis in an isolated hepatocyte system or in vivo. (--)-threo-Chlorocitric acid significantly delayed the rate of gastric emptying. Of the four stereoisomers of chlorocitric acid, (--)-threo-chlorocitric acid was the most active both in delaying gastric emptying and producing anorexia in rats. It is suggested that the mechanism by which (--)-threo-chlorocitric acid may suppress food intake is through a reduction of gastric emptying.