Gene regulation of shell banding in a land snail from Israel

The Israeli land snail, Xeropicta vestalis, offers a particularly clear example of gene regulation in relation to natural selection, in that within each population the appropriate phenotype is generated only at the correct part of the animals life cycle, and a contrasting phenotype develops when the forces of natural selection change. In the mountains of Jerusalem, where the winter is cold, the shells are dark. Westwards, towards the coastal plain where the winter is warmer, the shells gradually become paler. As dark shells absorb more radiation than pale ones, this clinal variation in morph frequencies can be explained in thermal terms. (Banded shells are also more cryptic than non-banded shells, so that in the mountains visual selection by predators may be an additional force which favours dark shells.) Xeropicta vestalis is an annual, semelparous species: the snails hatch in winter, become mature within one year, reproduce, and then die. In the coastal plain the snails are active throughout most of the year, and they have a long period in which to grow to reach adult size. In the mountains and hills, on the other hand, the snails are active for only a very short period. They spend most of their lives as small snails, in a state of aestivation. Xeropicta vestalis must be dark in mountains because when it finally awakens, it must very rapidly and hastily reach reproduction size. A dark shell, by speeding up temperature-dependent processes in this critical stage, assists the snail to mature rapidly. Shell darkness varies with age: in the mountains and hills the shells are moderately dark when they hatch, but become darker whilst growing in early winter. In the coastal plain also, the snails are moderately dark when they hatch; but here they become paler, whilst growing in winter and spring. In both cases, each snail is darker in the colder months and paler in the hot ones. A strategy of gene regulation of shell colour is thus favoured when the subsequent forces of the environment are very contrasting in their direction, very severe—yet also very predictable.     

Cellular effects of N(4-ethoxyphenyl)p-benzoquinone imine, a p-phenetidine metabolite formed during peroxidase reactions

The interaction of N-(4-ethoxyphenyl)p-benzoquinone imine (NEPBQI), a metabolite formed during peroxidase catalyzed metabolism of p-phenetidine, with GSH and its effects in isolated rat hepatocytes were investigated.

When reacted with GSH NEPBQI formed both a mono- and a diglutathione conjugate as well as GSSG. Formation of glutathione conjugates and GSSG also occurred when NEPBQI was added to isolated hepatocytes. The formation of GSSG was, however, only detectable if the hepatocytes had been pretreated with the GSSG reductase inhibitor BCNU (1,3-bis-(2-chloroethyl-1-nitrosourea).

Similarly, NEPBQI caused a rapid decrease in cellular free protein thiols when added to hepatocytes, however this was expressed at higher concentrations than for effects on GSH. The protein thiol decrease was correlated with protein binding of NEPBQI.

NEPBQI was also shown to be toxic to isolated hepatocytes. At a concentration of 400 μM extensive bleb formation was followed by loss of cell membrane integrity and cell death.

To assess further the subcellular metabolism of NEPBQI microsomes and cytosol was used. NEPBQI was found to be preferentially reduced by cytochrome P-450 reductase in the microsomes whereas DT-diaphorase catalyzed its reduction in cytosol. NEPBQI did not undergo significant redox cycling since no formation of O was observed. Thus, the cytotoxic effect of NEPBQI appears to be due to protein arylation rather than redox cycling.     

The historv of introductions of Bufo mariuk (Amphibia: Anura); a natural experiment in evolution

The Central American toad, Bufo marinus , has been extensively introduced throughout the Caribbean and Pacific regions and is now one of the most widespread of terrestrial vertebrates. Details, such as the sources and dates of introductions, the number of individuals introduced and the fates of the introduced populations have been documented and are described. The availability of this historical information makes the introductions of great potential value as a series of evolutionary experiments. They can be used to study the way in which allopatric populations diverge genetically and the effects of population bottlenecks of known size on the genetic characteristics of populations, and to examine theories of rapid speciation caused by genetic revolutions associated with founder events.     

Characterization of p80, a novel nuclear and cytoplasmic protein in dinoflagellates.

The presence of myosin in dinoflagellates was tested using an anti-Acanthamoeba castellanii myosin II polyclonal antibody on the heterotrophic dinoflagellate Crypthecodinium cohnii Seligo. Western blots revealed the presence of a unique band of 80 kDa in total protein extracts and after immunoprecipitation. Expression of this 80 kDa protein appeared constant during the different phases of the cell cycle. In protein extracts from various other dinoflagellates, this 80 kDa protein was detected only in the autotrophic species Prorocentrum micans Ehr. Screening of a C. cohnii cDNA expression library with this antibody revealed a cDNA coding for an amino acid sequence without homology in the databases. However, particular regions were detected: - a polyglutamine repeat domain in the N-terminal part of the protein, - four peptide sequences associated with GTP-binding sites, - a sequence with slight homology to the rod tail of Caenorhabditis elegans myosin II, -a sequence with homology to a human kinesin motor domain. Immunocytolocalization performed on C. cohnii thin sections with a polyclonal antibody raised against the recombinant protein showed p80 to be present both within the nucleus and in the cytoplasm. Labelling was widespread in the nucleoplasm and more concentrated at the periphery of the permanently condensed chromosomes. In the cytoplasm, labelling appeared in a punctate region close to the nucleus and in the flagellum. Potential functions of this novel protein are discussed.     

Morphological studies of Asian rice and its related wild species and the recognition of a new Australian taxon

In order to clarify the taxonomy and the interrelationships among Asiatic cultivated rice, Oryza sativa , and its related wild species ( O. rufipogon, O. nivara and O . barthii ), 41 morphological characters were examined. Numerical taxonomic methods were used to analyse the data and to illustrate species relationships.
Distinctive differences among the materials studied suggest the retention of O. rufipogoon, O. nivara and O . sativa as three distinct species. The origin of O. sativa from O. nivara through domestication is discussed. An annual wild taxon from Australia, which had been classified as a form of O. nivara , is shown to be distinct from typical O. nivnra and is raised to specific rank. This species has been named O. meridionalis Ng.     

Differentiation in morphology and habitat partitioning of genetically characterized Gammarus fossarum forms (Amphipoda) across a contact zone

Evolutionary processes do not affect all character types equally. Depending on environmental effects, morphological and ecological evolution may be uncoupled from molecular evolution. A natural contact zone between two forms of the amphipod Gammarus fossarum was used to assess the levels of differentiation in genetic, morphological and habitat characters. About 50 individuals per population were analysed for six enzyme loci in 72 populations and four size-standardized morphometric traits in a subset of 32 populations. Nine environmental parameters per population were used to characterize the habitat. All three character sets revealed significant divergences between G. fossarum forms. The eastern form A of G. fossarum differs from the western form B by longer setae at the second antennae (males) and longer exopodits at die third uropods (males and females). The variance partitioning within populations, among populations and between forms, was compared within the three character sets. The relative variance component between forms in genetic characters appeared to be 10-fold greater in comparison to morphological traits, indicating morphological stasis during speciation. The inter-form environmental differentiation was shown to be low, but consistent between pure populations. The eastern form A seems to prefer higher altitudes, a substrate type of gravel, stones and leaves, shading, and water with higher conductivity and pH man the western form B.     

A plant transformation vector with a minimal T-DNA II. Irregular integration patterns of the T-DNA in the plant genome

A minimal T-DNA binary vector was used for Agrobacterium-mediated transfer of a chimeric T4 lysozyme gene located next to the left border, and transgenic potato plants which expressed T4 lysozyme protein were identified and further analysed. Frequent rearrangements of T4 lysozyme transgenes were detected. A vector derivative containing two matrix associated regions (MARs) flanking its multiple cloning site was constructed. In transgenic potato plants, reduced variability in gene expression due to position effects was detected. When either the donor vector contained MAR sequences, or when vector pPCV701 which contains a pBR322 fragment next to the left border were used, only relatively few rearrangements were observed. However, when the T4 lysozyme gene was driven by a CaMV 35S promoter modified by multiplied enhancer region carrying either 2 or 4 elements, frequent rearrangements were again obtained.     

Polymorphic segregation in Arctic charr Salvelinus alpinus (L.) from Vatnshlidarvatn, a shallow Icelandic lake

We studied the salmonid fish Arctic charr, Salvelinus alpinus , in a small and shallow landlocked lake in NW Iceland. The lake is productive but die only fish present is Arctic charr. Despite the apparent absence of discrete benthic and limnetic habitats for fish, two forms of Arctic charr are found in the lake. They show subde differences in morphology related to swimming performance and manoeuvrability, but differences in life history such as growth, and age and size at sexual maturation are more pronounced. Both forms have benthic feeding habits with one form consuming greater number of species than the other. We suggest that the segregation of these forms is based on the evolution of a specialist from a local generalist and that this has been made possible by the absence of a common fish competitor in similar lakes, the threespined stickleback Gasterosteous aculeatus.     

Isolation and identification of a fucose-containing ganglioside from bovine thyroid gland

Bovine thyroid glands are known to contain a complex array of gangliosides. One of the predominant gangliosides was isolated and analyzed by gas-liquid chromatography and mass spectrometry. The carbohydrate composition was fucose, N-acetylneuraminic acid, galactose, N-acetylgalactosamine, and glucose in molar ratios of 1:1:2:1:1. The structure of the ganglioside was identified as:

     

Evidence for massive clonal growth in the invasive weed Fallopia japonic a (Japanese Knotweed)

Clonal growth in introduced populations of Japanese Knotweed ( Fallopia juponica ) in Britain was assessed using RAPDs (Randomly Amplified Polymorphic DNA). A total of 150 British samples was analysed for genetic variation using ten arbitrary decamer primers, and compared with data from 16 samples of other introduced populations from Europe and the U.S.A. All samples produced an identical multi-primer RAPD profile. Accepting that RAPD profile identity need not equate to genet identity, based on the sensitivity of these markers for detecting genetic diversity in related taxa and on the absence of male fertile individuals of this species in Britain, we interpret this result as consistent with the presence of a single, exceptionally widespread clone. This clone must represent one of the world's largest vascular plants.     

Involvement of a 40-kDa glycoprotein in food recognition, prey capture, and induction of phagocytosis in the protozoon Actinophrys sol

A 40-kDa glycoprotein (gp40) was identified as a Con A-binding adhesive substance of the heliozoon Actinophrys sol for immobilizing and ingesting prey flagellates. Isolation and partial characterization of gp40 showed that: 1) gp40 is a major Con A-binding protein of Actinophrys with a molecular weight of 40 kDa, and is stored in secretory granules called extrusomes; 2) gp40 was purified by Con A-affinity chromatography, and the N-terminal amino acid sequence was determined as H2N-KVLK-FEDDFDTFDLQ; 3) prey flagellates became adhered to gp40-immobilized agarose beads; 4) phagocytosis of Actinophrys was induced against gp40-immobilized agarose beads; and 5) solubilized gp40 induced exocytosis of extrusomes and cell fusion of heliozoons. These results indicate that gp40 is a multi-functional secretory protein of Actinophrys, which is required in correct targeting of the heliozoon to food organisms as well as in self-recognition.     

Neomycin Is an Agonist at a Polyamine Site on the N-Methyl-D-Aspartate Receptor

Neomycin appears as a full agonist and spermidine as a partial agonist at the site where polyamines enhance 1-[1-(2-thienyl)cyclohexyl][3H]piperidine ([3H]TCP) binding on the N-methyl-D-aspartate (NMDA) receptor. Other aminoglycosides also enhance [3H]TCP binding with efficacies roughly proportional to the number of primary amine groups. The polyamine antagonists ifenprodil and arcaine inhibit enhancement of [3H]TCP binding by spermidine or neomycin. The inhibition of [3H]TCP binding by arcaine is apparently competitively reduced by neomycin and spermidine, supporting a common site. Diethylenetriamine (previously described as a polyamine antagonist) may be a partial agonist. Enhancement by neomycin or spermidine is not additive to that of Mg2+, consistent with competition of Mg2+ and spermidine or neomycin at the site where these compounds enhance [3H]TCP binding. Polyamines also enhance the binding of the competitive antagonist 2-(2-carboxypiperazin-4-yl)[3H]propyl-1-phosphonic acid ([3H]CPP). Neomycin, which does not enhance [3H]CPP binding, inhibits the enhancement by spermidine. That this site is distinct from the site where spermidine and neomycin increase [3H]TCP binding is supported by different pharmacology. Arcaine and diethylenetriamine do not inhibit spermidine enhancement of [3H]CPP binding. Mg2+ also does not compete with the spermidine enhancement of [3H]CPP binding. Ifenprodil inhibits the spermidine enhancement of [3H]CPP binding. The data suggest two or more polyamine sites, with arcaine selective for the site that enhances [3H]TCP binding. Neomycin is an agonist at one polyamine site and antagonist to the second.     

伪狂犬病病毒TK-/gG-缺失株的构建及其生物学特性研究

将伪狂犬病病毒TK^-／gG^-／LacZ^ 突变株的基因组DNA与含有缺失的gG基因的转移质粒pUSKBB共转染猪肾传代细胞PK-15,待完全病变后收获病毒进行空斑试验,用PCR筛选gG缺失的重组病毒。空斑纯化3次后,随机挑取空斑进行PCR扩增,证实所获得的病毒为均一的TK^-／gG^-缺失株。遗传稳定性试验表明该重组病毒能在PK-15细胞上稳定遗传,动物试验表明该缺失株对Balb／c小鼠极为安全且能保护Balb／c小鼠抵抗致死量PRV强毒的攻击。该突变株的获得为我国伪狂犬病的控制和根除奠定了基础。     

Participation of peroxisomal beta-oxidation system in the chain-shortening of a xenobiotic acyl compound

A drug, (E)-3-[4-(1-imidazolylmethyl)phenyl]-2-propenoic acid, was metabolized to 4-(1-imidazolylmethyl)benzoic acid in isolated hepatocytes of rats, which was enhanced markedly by the pretreatment of rats with clofibrate. With liver homogenates, the formation of the CoA-ester of this drug and its subsequent chain-shortening were demonstrated. In the series of these reactions, acyl-CoA synthetase, CoA, ATP and NAD were required, whereas cyanide did not inhibit the reaction. These results indicate that peroxisomes are capable of shortening the acyl side-chains of drugs by the beta-oxidation, giving an additional suggestion on the functions of peroxisomes.     

Secondary productivity and energy flow in a tropical pond

Monthly changes in density and biomass of a Pila globosa population were estimated in the littoral area of the pond Idumban. Mean density of active snail was 10.4, equivalent to 76 g dry weight/m² during 1973 and 6.5, equivalent to 45 g/m² during 1974. Total population size of the snail decreased from 9.2 × 10⁶ individuals, equivalent to 6.5 ton during 1973 to 6.3 × 10⁶ snails, equivalent to 4.4 ton during 1974. The period from December to May represented the time of abundance and active growth. Mortality assessed from marking and recapture as well as from monthly changes in population density, averaged to 2.7 snails/m²/month or 20% of the density. Growth estimated by marking and recapture suggested that the snail required a period of over 4 years to attain a body (wet) weight of 35 g. Laboratory experiments revealed that young (<4 g), intermediate (4 g><24 g) and old (>24 g) P. globosa grew at the rate of 4.0, 1.5 and 0.3 mg dry weight/g live weight/day. Using these values and the size-wise population density data, net productivity of the snail was estimated as 74 and 40 g/m²/year in 1973 and 74, respectively. The snail exhibited an efficiency of 70% for absorption and 10% for conversion. Using these values, it was further possible to estimate rates of feeding and absorption for the population. Consumption amounted to 1039 g/m² in 1973 and 560 g/m² in 1974. The efficiencies of exploitation, gross and net productions were 21, 7 and 10%, respectively; ecological efficiency amounted to 1.4% only.     

Isolation of a fast decay in submillisecond Chlorella luminescence

Using a simple He-Ne (632.8-nm) laser phosphoroscope steady-state luminescence from Chlorella pyrenoidosa was studied from 50 μs to 1.1 ms between 1 ms long exciting flashes. The following results were obtained: (1) prior freezing or ultraviolet irradiation changed the time course of the luminescence to a rapid decay with a half-time of about 110 μs; (2) 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) suppressed the 110-μs luminescence; (3) spectrally, all observed luminescence was, within possible error, identical to fluorescence; (4) no effect on the luminescence intensity from pulsed magnetic fields up to 30 kgauss was observed; (5) the relative fluorescence yield, measured simultaneously with luminescence, was found to be constant.Our principal conclusions, supported mainly by experiments with DCMU, are: (1) the 110-μs decay is a distinct component of the total steady-state luminescence; (2) prior freezing or ultraviolet irradiation isolates this component of the luminescence by suppressing all other components; (3) the half-time and intensity of this component are temperature independent in the interval 0–22 °C.     

Development of a Protein-free Medium with Iron Salts Replacing Transferrin for a Human-Human Hybridoma

Many protein-free media have been deveoped, because protein-free media are usually more economical than serum-free or serumcontaining media and facilitate the purification ofbioactive materials. We evaluated various iron salts and chelating agents replacing transferrin to develop a protein-free medium for a human-human hybridoma, HB4C5, and found out that ferric citrate was favorable for the production and the productivity of monoclonal antibodies.     

Phenotypic plasticity in reproductive traits: importance in the life history of Helix aspersa (Mollusca: Helicidae) in a recently colonized habitat

Reproductive traits of the land snail Helix aspersa Mtiller were investigated under artificial conditions from two samples, one collected from a population exposed to unpredictable human pressures in its natural environment, i.e. a recently created polders area with intensive agriculture, and the other from a snail farm in which animals were reared under constant conditions denned as 'optimal' for growth and reproduction. Results were compared with data collected from natural populations of the same region (Brittany) and from habitats spanning the environmental heterogeneity of the range of the species. A large part of the variation among populations could be explained by different phenotypic covariances between shell size, clutch size and egg size, but not by the number of clutches per snail. Thus, the higher egg production of snails from the polders was related to (i) a strong correlation between clutch size and shell size, shell size being in the upper limit of the overall range for the region concerned; (ii) an uncommonly low egg weight in comparison with the 'norm' of Helix aspersa , this trait seeming to be involved in a trade-off with clutch size. Second clutches were smaller than the first ones, but their eggs were significantly heavier. This difference may be linked to a size-dependent mortality of juveniles during winter which arises in all populations in which hibernation occurs as an adaptation to low temperatures. In addition to the selective regime usually involved for populations of helicid snails from Western Europe, several unpredictable mortality factors occurred in the polders area: herbicide and pesticide treatments (lethal for young snails), human predation (lethal for adults) and burning (letiial for all snails). Life-history patterns of Helix aspersa are discussed in relation to its ability to successfully colonize a large range of habitats modified by humans, to such an extent that it can become a pest.