Multiple sources of esterase enzymes in the crop juice ofCepaea (Mollusca: Helicidae)

Summary Previous workers have (a) compared pulmonate crop juice and digestive gland extracts and found a close similarity in the enzymic complements from these two sources, and (b) located specific enzymes within the various cell types of the digestive gland. The digestive gland seems to be the major source of extracellular enzymes but what is not clear is which of the enzymes associated with particular intracellular structures are actively secreted into the crop juice. The present study has used polyacrylamide disc gel electrophoresis to investigate the digestive gland and crop juice esterases ofCepaea nemoralis andC. hortensis. It appears that only some of the digestive gland esterases are specifically secreted. The variation shown in crop juice esterases suggests three independent sources in the digestive gland. Less detailed studies ofHelix aspersa andArianta arbustorum also indicate multiple sources of extracellular esterases.     

Agar quality of commercial agarophytes from different geographical origins: 1. Physical and rheological properties

Six economically important species ofGracilaria, from a number of commercial sources around the world, andGracilariopsis lemaneiformis, collected from two Japanese localities, were used as the sources of raw material for the evaluation of agar quality. Agar-agar was extracted by pretreatment with various concentratrions of NaOH (0%, 3%, 5%, 7%, 10%) incubated at 80 °C for 2 h. Agar yield, viscosity, dynamic gelling and melting temperature and gel texture were determined for 1.5% agar gels. The highest agar yield was obtained fromG. gracilis from Argentina (39.5%), while the lowest was from BrazilianG. gracilis (13.37%). Dynamic gelling temperature was highest in the agar fromG. gracilis from Turkey (59 °C) and lowest in the non-alkali treated agar isolated fromG. edulis from Indonesia (46 °C). Melting temperature ranged from 96 °C in the agars from the JapaneseGracilariopsis andG. chilensis from Chile to 69 °C in the non-alkali treated agar fromG. edulis from Indonesia. In general, all species produced an agar with high gel strength after treatment with 5% NaOH, except forG. chilensis and the twoGracilariopsis species, which produced an agar with high gel strength after treatment with 3, 7 and 10% NaOH. The highest gel strength (2056 ± 13.6 cm^–2) and hardest gel (261 ± 19.89 g mm^–2) were obtained fromG. lemaneiformis from Japan (Oita Prefecture) after treatment with 7 and 10% NaOH respectively. The lowest gel strength (351 ± 93 cm^–2) was obtained fromG. gracilis from Brazil after treatment with 3% NaOH. The softest gel (66.31 ± 9.63 g mm^–2) was isolated fromG. tenuistipitata from China, after treatment with 3% NaOH. The most flexible gel (11.62 ± 0.31 g mm^–2 × 10²) was obtained fromG. chilensis from Chile after treatment with 3% NaOH.Author for correspondence     

Purification and biochemical characterization of a cadmium metallothionein from the digestive gland of the Antarctic scallop Adamussium colbecki (Smith, 1902)

A cadmium-binding protein was purified from the digestive gland of the Antarctic scallop, Adamussium colbecki, and biochemically characterized. Purification procedures included gel permeation and anion exchange chromatography, followed by preparative polyacrylamide gel electrophoresis. Our results demonstrate that the A. colbecki cadmium-binding protein has the general properties of metallothioneins: low molecular weight of about 10 kDa, spectroscopic features typical of cadmium thiolate clusters and high metal (cadmium) content. Analysis of amino acid composition reveals the absence of aromatic amino acids, histidine, methionine and arginine. Asparagine and glutamine are also absent. The A. colbecki metallothionein shows high levels of glycine (14%), aspartic acid (14%), glutamic acid (11%) and a low lysine content (4%); the A. colbecki metallothionein shows a lower cysteine content (12%) compared to other metallothioneins (17–30%) purified from both vertebrate and invertebrate organisms. The presence of a metallothionein in the digestive gland of A. colbecki suggests that in cold-ocean-adapted molluscs the heavy metal homeostasis mechanisms may have evolved similarly to those of organisms living in temperate marine environments, although the A. colbecki cadmium-binding protein shows a typical amino acidic composition that might reflect a peculiar physiological role. Accepted: 7 August 2000     

Metallothionein research in terrestrial invertebrates: Synopsis and perspectives

While most of metallothionein research during the past years has been carried out on mammals or vertebrates, only relatively few studies have been directed towards invertebrates. Even fewer investigations have focussed on terrestrial invertebrates. The best studied metallothioneins and/or metallothionein genes among terrestrial invertebrates are those from an insect species (Drosophila melanogaster), a nematode (Caenorhabditis elegans) and some terrestrial gastropods (Helix pomatia, Arianta arbustorum). From these few examples it already appears that terrestrial invertebrate metallothioneins provide intriguing models to better understand the multiplicity of functions of these proteins and their evolution within the animal kingdom. Like in mammals, metallothioneins in terrestrial invertebrates seem to perform different functions simultaneously. This is exemplified by terrestrial gastropods, which are able to accumulate different metals in different tissues, in which metal-specific metallothionein isoforms or conformation forms are expressed, allowing these organisms to detoxify more efficiently nonessential trace elements such as cadmium, and at the same time to maintain the homeostasis of essential trace elements such as copper. A major proportion of metallothionein research in terrestrial invertebrates addresses the ecophysiological and ecotoxicological significance of these proteins with regard to the increasing risk due to chemical pollution. One promising aspect in this concern is the potential utilization of metallothioneins as biomarkers for risk assessment in terrestrial environments.     

Comparison of growth rate in culture,dry matter content,agar content and agar quality of two New Zealand red seaweeds,Gracilaria chilensis Bird,McLachlanet Oliveira andGracilaria truncata Kraft

Plant growth rates and agar characteristics were compared for two agarophytes,Gracilaria chilensis (Gracilariales, Rhodophyta) andG. truncata, so that the suitability ofG. truncata for mariculture could be assessed.G. chilensis plants grew steadily in the laboratory culture system at rates of 5–8% day^-1 (mean Relative Growth Rate) throughout the 6 week experimental period, with no decline in health.G. truncata plants grew at rates of 2–4% day^-1 for 5 weeks, but during the sixth week their apical tips became necrotic and growth rates fell to zero. There was no significant difference in the dry matter content (as a percentage of their wet weight) between the two species ofGracilaria, with values falling in the range of 16–19%. Slightly higher agar yields were obtained from alkali-treatedG. chilensis (17–20% dry matter) than from untreatedG. truncata (16–18%) although the agar fromG. truncata formed softer gels from which it was more difficult to recover. The gel strength of untreated agar extracted fromG. chilensis was very low (ca. 100 g cm^-2 for a 1% gel) but when an alkali treatment step was included in the extraction process, it increased to 520 g cm^-2 for a 1% gel. Contrary to an earlier report, untreatedG. truncata agar also had a very low gel strength (ca. 100 g cm^-2 for a 1.5% gel), but it rose to only 167 g cm^-2 after alkaline treatment.     

Accumulation of cadmium and copper by the terrestrial snail Arianta arbustorum L.: kinetics and budgets

Summary Specimens of the terrestrial gastropod Arianta arbustorum were fed on cadmium- or copper-enriched agar plates with the aim of performing an input/output analysis and of studying the distribution of these metals in several organs of the snails. After a feeding period of 20 days about 45% of cadmium were lost. 36% accumulated in the hepatopancreas, where a cadmium concentration of more than 500 g/g was measured. The efficiency of cadmium assimilation decreased from about 90% at the beginning to about 55% after 20 days. Copper was distributed more evenly than cadmium, but the main site of copper storage seemed to be the foot/mantle tissues, where 49% of the ingested copper were found. The efficiency of copper assimilation always exceeded 95%. The patterns of distribution and assimilation of copper and cadmium are discussed in relation to differences in the cytological and biochemical detoxification mechanisms which exist for these metals in molluscs.     

Nerve cells and synaptic connections in the intestinal nerve of the snail,Helix pomatia L.

Summary The ultrastructure of nerve cells and the finestructural organization of synaptic contacts have been investigated in the intestinal nerve in the snail Helix pomatia. Three types of nerve cells, occurring singly or in groups, can be distinguished on the basis of the ultrastructure of their perikaryon and content of granules. The peripheral output of these nerve cells has been verified by retrograde CoCl₂ and NiCl₂ staining. Both axosomatic and axo-axonic specialized synaptic contacts occur in the intestinal nerve. Presynaptic elements of these synaptic contacts contain 100–120 nm granular vesicles or 120–200 nm neurosecretory-like granules. Following intracellular horseradish peroxidase (HRP) labelling of identified central neurons responsible for peripheral regulatory processes, several labelled axons running toward the periphery can be followed throughout the branches of the intestinal nerve. These labelled axon processes (either primary axon or small collaterals) form specialized synaptic contacts, inside the intestinal nerve, and are always in a postsynaptic position. The occurrence of peripheral axo-somatic and axo-axonic synapses provides a morphological basis for integrative processes taking place in the intestinal nerve (peripheral nervous system) of Helix pomatia.     

Analysis of chimeric UmuC proteins: identification of regions inSalmonella typhimurium UmuC important for mutagenic activity

UnlikeEscherichia coli, the closely related bacteriumSalmonella typhimurium is relatively unresponsive to the mutagenic effects of DNA-damaging agents. Previous experiments have suggested that these phenotypic differences might result from reduced activity of theS. typhimurium UmuC protein. To investigate this possibility, we have taken advantage of the high degree of homology between the UmuC proteins ofE. coli andS. typhimurium and have constructed a series of plasmid-encoded chimeric proteins. The possibility that the phenotypic differences might be due to differential expression of the respective UmuC proteins was eliminated by constructing chimeric proteins that retained the first 25 N-terminal amino acids of either of the UmuC proteins (and presumably the same translational signals), but substituting the remaining 397 C-terminal amino acids with the corresponding segments from the reciprocal operon. Constructs expressing mostlyE. coli UmuC were moderately proficient for mutagenesis whereas those expressing mostlyS. typhimurium UmuC exhibited much lower frequencies of mutation, indicating that the activity of the UmuC protein ofS. typhimurium is indeed curtailed. The regions responsible for this phenotype were more precisely localized by introducing smaller segments of theS. typhimurium UmuC protein into the UmuC protein ofE. coli. While some regions could be interchanged with few or no phenotypic effects, substitution of residues 212–395 and 396–422 ofE. coli UmuC with those fromS. typhimurium resulted in reduced mutability, while substitution of residues 26–59 caused a dramatic loss of activity. We suggest, therefore, that the primary cause for the poor mutability ofS. typhimurium can be attributed to mutations located within residues 26–59 of theS. typhimurium UmuC protein.     

Isolation and identification of proteolipid proteins injimpy mouse brain

Proteolipids were isolated from 20 day old normal andjimpy mouse brain by extraction into chloroform-methanol (21, w/v), delipidated by size-exclusion HPLC, and analyzed by SDS-PAGE, Western blots, amino acid analyses, and N-terminal sequencing. SDS-PAGE showed that a major proteolipid fromjimpy mouse brain had an apparent molecular weight of 23 kDa, intermediate to that of PLP and DM-20 from normal mouse brain. Western blots with 3 different antibodies which recognize residues 200–224, 116–150, and 270–276 respectively recognized immunoreactive material in normal andjimpy PLP. Since antibody reactive with 270–276 did not recognizejimpy PLP, an altered C-terminus of thejimpy protein is suggested. These results demonstrated that a PLP can be partially purified fromjimpy mouse brain. Amino acid analyses failed to show the predicted increase in cysteinyl residues (predicted from cDNA) injimpy PLP. However, whenjimpy brain proteolipids were subjected to N-terminal sequencing, Gly, Leu, Leu, Gly the first four amino acids of PLP were detected. Thus, the partial purification of a proteolipid fromjimpy mouse brain, whose characteristics (apparent molecular weight, immunoreactivity, N-terminal sequence and relative net charge) strongly suggested that PLP of altered size is present injimpy mouse brain.Abbreviations BCIP 5-bromo-4-chloro-3-indolyl phosphate toluidine salt - MBP myelin basic protein - NBT -nitro blue tetrazolium chloride - PITC phenylisothiocyanate - PLP myelin proteolipid protein - PVDF polyvinylidene difluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis Special issue dedicated to Dr. Marjorie B. Lees.     

Common antigenic determinants in the glycoproteins of plants,molluscs and insects

An antiserum raised against -fructosidase isolated from the cell walls of suspension-cultured carrot cells cross-reacts with many plant proteins and hemocyanin ofHelix pomatia. The shared epitope appears to be a small complex glycan with a (1–2)-linked xylose residue attached to the -linked mannose residue of the core of an asparagine-linked oligosaccharide. There is strong cross-reactivity with the proteins of many seed plants, molluscs and insects, and no cross-reactivity with the proteins of fungi, algae, mosses, ferns, or any of the vertebrates tested. Xylose-containing glycans appear to increase the immunogenicity of the proteins to which they are attached, and we suggest that they may be responsible for some allergic responses of people that are repeatedly exposed to plant or insect proteins.     

Cultivation and carrageenan yield and quality ofKappaphycus alvarezii in the waters of Vietnam

The carrageenan-producing red algaKappaphycus alvarezii (Doty) Doty was brought to Vietnam from Japan in 1993. Branch fragments of this species were cultivated in a pond, lagoon, inlet and offshore in Vietnam for the first time. The best daily growth rate (DGR) of plants grown in the lagoon area attained 9–11 % day^–1 in May to June (cold season). The water temperature and salinity in this area ranged from 27.2–32.4 °C and 31.4–33.7 °C, respectively. DGR of plants grown in the inlet ranged from 7 to 9% day^–1 in June. Grazing by fish has been observed to occur in this area. The DGR of plants grown in the pond ranged from 5–6% in January–July, but decreased to less than 4% day^–1 in August (hot season). K. alvarezii in Vietnam showed a carrageenan yield of 18.8–24.6% and gel strength of 1566–1712 g cm^–2. These values are similar ones obtained fromK. alvarezii cultivated in the Philippines and Indonesia.     

Chemical and cytological changes during the autolysis of yeasts

Summary Cell suspensions ofSacharomyces cerevisiae, Kloeckera apiculata andCandida stellata were autolyzed in phosphate buffer, pH 4.5, for up to 10 days. Cell dry weights decreased by 25–35% after 10 days. Based on initial cell dry weight, the soluble autolysate consisted of: carbohydrate (principally polysaccharide) 3–7%; organic acids 3–6%; protein 12–13%; free amino acids 8–12%; nucleic acid products 3–5%; and lipids 1–12%. The main organic acids in autolysates were propionic, succinic and acetic and the main amino acids were phenylalanine, glutamic acid, leucine, alanine and arginine. Approximately 85–90% of cellular RNA and 25–40% of cellular DNA were degraded during autolysis. Both neutral lipid and phospholipid components were degraded, with neutral lipids but not phospholipids being found in autolysates. Scanning and transmission electron micrographs showed retention of cell wall structure and shape during autolysis, but there was extensive intracellular disorganization withinS. cerevisiae andC. stellata. There were differences in the autolytic behavior ofK. apiculata compared withS. cerevisiae andC. stellata.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Extracellular mannanases and galactanases from selected fungi

Summary Eight thermophilic fungi were tested for production of mannanases and galactanases. Highest mannanase activities were produced byTalaromyces byssochlamydoides andTalaromyces emersonii. Mannanases from all strains tested were induced by locust bean gum except in the case ofThermoascus aurantiacus, where mannose had a greater inducing effect. Locust bean gum was also the best inducer of -mannosidase and galactanase except in the case ofT. emersonii where galactose was a better inducer of both these enzymes. Highest mannanase activity was produced byTalaromyces species when peptone was used as nitrogen source whereas sodium nitrate promoted maximum production of this enzyme byThielavia terrestris andT. aurantiacus. The pH optima of mannanases from the thermophilic fungi were in the range 5.0–6.6 and contrasted with the low pH optimum (3.2) of the enzyme fromAspergillus niger. Galactanases had pH optima in the range 4.3–5.8. The mannanase fromT. emersonii and the galactanase fromT. terrestris were most thermostable, each retaining 100% activity for 3 h at 60°C.     

Modulation of synaptic events by heavy metals in the central nervous system of mollusks

Summary 1. The effects of heavy metals (Pb²⁺, Hg²⁺, and Zn²⁺) on synaptic transmission in the identified neural network ofHelix pomatia L. andLymnaea stagnalis L. (Gastropoda, Mollusca) were studied, with investigation of effects on inputs and outputs as wells as on interneuronal connections.2. The sensory input running from the cardiorenal system to the central nervous system and the synaptic connections between central neurons were affected by heavy metals.3. Lead and mercury (10^–5–10^–3 M) eliminated first the inhibitory, then the excitatory inputs running from the heart to central neurons. At the onset of action lead increased the amplitude of the excitatory postsynaptic potentials, but blockade of sensory information transfer occurred after 10–20 min of treatment.4. The monosynaptic connections between identified interneurons were inhibited by lead and mercury but not by zinc. Motoneurons were found to be less sensitive to heavy metal treatment than interneurons or sensory pathways.5. The treatment with Pb²⁺ and Hg²⁺ often elicited pacemaker and bursting-type firing in central neurons, accompanied by disconnection of synaptic pathways, manifested by insensitivity to sensory synaptic influences.6. Zn²⁺ treatment also sometimes induced pacemaker activity and burst firing but did not cause disconnection of the synaptic transmission between interneurons.7. A network analysis of heavy metal effects can be a useful tool in understanding the connection between their cellular and their behavioral modulatory influences.     

Cyclodextrin production and genetic characterization of cyclodextrin glucanotranferase of <Emphasis Type="Italic">Paenibacillus graminis</Emphasis>

Paenibacillus graminis strains were described recently as cyclodextrin (CD) producers. Cyclodextrins are produced by cyclodextrin glucanotransferase (CGTase) which has not been characterized in P. graminis. Similar amounts of α- and β-CDs were produced by P. graminis (MC22.13) and P. macerans (LMD24.10^T). Primers were designed to sequence the gene encoding CGTase from P. graminis. A phylogenetic tree was constructed and P. graminis CGTase protein showed to be closer (79.4% protein identity) to P. macerans |P31835|. Hybridization studies suggested that the gene encoding CGTase is located in different positions in the genomes of P. macerans and P. graminis.     

Comparative analysis of agglutinins from hemolymph and albumin gland ofHelix pomatia

Summary The hemolymph ofHelix pomatia contains a weak agglutinating activity. This lectin concentration was calculated to be about 1.8 g·ml^–1. Among the different red blood cells tested, pronase-treated sheep erythrocytes were found to be the most suitable indicator cells. Their agglutination could be inhibited by GalNAc and GlcNAc. The serum agglutinin was isolated by affinity chromatography using Sephadex G-200 as the matrix. It exhibited a single band in discontinuous PAGE. In the presence of SDS, subunits of 27000 daltons were obtained which, after addition of 2-mercaptoethanol, partly dissociated into 13000-dalton subunits. The biochemical properties observed were compared with those of the well-known blood group A-specific lectin from the albumin gland ofH. pomatia.Abbreviations GalNac N-acetyl-galactosamine - GlcNAc N-acetyl-glucosamine - SDS sodium dodecylsulfate     

Identification of a cadmium-binding protein from a cadmium-resistant variant of human lymphoblastoid cells (WI-L2)

Cadmium-binding protein synthesis and induction by cadmium chloride were studied in the human lymphoblastoid cell line WI-L2. Lymphoblasts were adapted to growth in 5 μM cadmium chloride (Cd^r) and these cells were 2.5-fold more resistant to cadmium than the parental line. There was no difference in the cellular protein profile between the parental line and lymphoblasts grown for a short period, less than 10 days, in cadmium chloride as measured by [³⁵S]cysteine labelling and SDS-polyacrylamide gel electrophoresis. A basal level of cadmium binding protein was apparent, however, by gel filtration. The Cd^r lymphoblasts were found to synthesize a substantial amount of cadmium-binding protein, approximately 25-fold more than the parental line. The cadmium-binding protein has the following properties which are consistent with its being a metallothionein: (1) [³⁵S]Cysteine-labelled protein eluted at a on a Sephadex G-75 column; (2) the molecular weight was estimated as 11 kDa on 7–17% SDS polyacrylamide gels; (3) the protein was heat-stable; (4) the unlabelled protein bound ¹⁰⁹Cd²⁺.     

Differences in the envelope proteins ofChlamydia pneumoniae,Chlamydia trachomatis,andChlamydia psittaci shown by two-dimensional gel electrophoresis

Analysis by two-dimensional gel electrophoresis of theN-laurylsarkosinate(Sarkosyl)-insoluble envelope complexes ofl-^[35]S-cysteine-iabeled elementary bodies ofChlamydia pneumoniae strain IOL-207,Chlamydia trachomatis serovar LGV2, D, and F, andChlamydia psittaci strain 6BC showed differences in the molecular charges of chlamydial outer membrane proteins. The apparent isoelectric point (pI) of the major outer membrane protein ofC. pneumoniae strain IOL-207 was 6.4, whereas the pI of the major outer membrane protein of theC. trachomatis andC. psittaci strains differed little from one another, ranging from 5.3 to 5.5. The 60-kDa cysteinerich protein ofC. pneumoniae was the only 60-kDa chlamydial protein with a pI value (5.9) more acidic than that of the corresponding major outer membrane protein. As a general rule, the charges of both the 60-kDa and the lowmolecular-mass (12–15 kDa) cysteine-rich proteins were widely variable, depending on the strain. However, in cach individual strain, the variation of the charge of the 60-kDa protein had a compensatory change in the lowmolecular-mass cysteine-rich protein.     

The monomeric and dimeric mannose-binding proteins from the Orchidaceae speciesListera ovata andEpipactis helleborine: sequence homologies and differences in biological activities

The Orchidaceae speciesListera ovata andEpipactis helleborine contain two types of mannose-binding proteins. Using a combination of affinity chromatography on mannose-Sepharose-4B and ion exchange chromatography on a Mono-S column eight different mannose-binding proteins were isolated from the leaves ofListera ovata. Whereas seven of these mannose-binding proteins have agglutination activity and occur as dimers composed of lectin subunits of 11–13 kDa, the eighth mannose-binding protein is a monomer of 14 kDa devoid of agglutination activity. Moreover, the monomeric mannose-binding protein does not react with an antiserum raised against the dimeric lectin and, in contrast to the lectins, is completely inactive when tested for antiretroviral activity against human immunodeficiency virus type 1 and type 2. Mannose-binding proteins with similar properties were also found in the leaves ofEpipactis helleborine. However, in contrast toListera only one lectin was found inEpipactis. Despite the obvious differences in molecular structure and biological activities molecular cloning of different mannose-binding proteins fromListera andEpipactis has shown that these proteins are related and some parts of the sequences show a high degree of sequence homology indicating that they have been conserved through evolution.Abbreviations EHMBP Epipactis helleborine mannose-binding protein - LOMBP Listera ovata mannose-binding protein Note: The nucleotide sequences reported in this paper will appear in the Genbank/EMBL Data library with the accession numbers L18894, L18895 and U07787.