Selection and gene flow between microenvironments: the case of Drosophila at Lower Nahal Oren,Mount Carmel,Israel

Drosophila melanogaster and D. simulans from contrasted microenvironments (south- and north-facing slopes of Lower Nahal Oren Canyon in Israel) were tested for genetic differentiation at microsatellite loci, which might be linked to differential adaptation to local ecological factors. No overall genetic differentiation was observed in either species. This indicates that the contrasted selective pressures on the two sides of the valley are not strong enough to cause population subdivision in highly mobile species such as Drosophila. Significant differences in allele frequencies were observed at two microsatellite loci, but on the whole the level of divergence we observed is far lower than has been reported previously.     

Genomic distribution of copia-like elements in laboratory stocks of Drosophila melanogaster

The genomic location of five copia-like transposable elements has been compared by the Southern technique in laboratory lines of Oregon R and Canton S strains of Drosophila melanogaster. The results show that extensive rearrangements have taken place in the few decades of separation between the stocks and suggest that transposition occurs at a sizable rate.     

Expression of Veratrum alkaloid teratogenicity in the mouse

Jervine, a steroidal alkaloid found as a minor constituent in the teratogenic range plant Veratrum californicum, has produced similar terata in sheep, rabbit, hamster, and chick, although the sensitivity to the alkaloid varies in the different species. Sprague Dawley rats and Swiss Webster mice are relatively insensitive. The aim of this study was to determine the teratogenic potential of jervine in three strains of mice and to ascertain if the response is strain dependent. One strain, Swiss N:GP(S), was retested since a Swiss Webster strain had been found previously to be jervine-resistant. In addition, we tested C57BL/6J and A/J, which are known to differ in their response to the teratogenic action of steroids and vitamin A. Mice were treated by gavage with single doses of jervine (70, 150, or 300 mg/kg body weight) on either day 8, 9, or 10 of gestation. Jervine was teratogenic to C57BL/6J and A/J mice but not to N:GP(S). The induced terata included cleft lip with or without cleft palate, isolated cleft palate, mandibular micrognathia or agnathia, and limb malformations. Fetal teratogenicity and maternal and fetal toxicity were highly correlated. The prevalence of each defect and fetal death was a function of strain, dose, and time of treatment. Maternal death was higher in C57BL/6J than in A/J mice. Although some of the terata were similar, the response pattern between strains was different from corticosteroids and vitamin A for both sensitive period and the strain dose response. An effect on differentiation of chondrocyte precursors may account for many of the defects, but an earlier lethal effect on differentiation of neural crest cells or precordal mesenchyme may also occur.     

Interferon Studies with Japanese and U.S. Rubella Virus Strains

Japanese strains of rubella virus have been claimed not to be teratogenic, and tests on three Japanese strains showed that they induced high levels of interferon in human placental cell cultures obtained from conceptuses ranging from 13 to 24 weeks'' gestational age, whereas two strains derived from the U.S.A. induced low levels. Both Japanese and U.S. strains induced similar but low levels in fetal lung cell cultures and leucocyte preparations. A representative Japanese strain and a U.S. strain were both interferon-sensitive. If indeed a strain can be shown to be non-teratogenic it could lead to an alternative, safer rubella vaccine.     

Differential expression of heat shock proteins in Drosophila embryonic cells following metal ion exposure

Drosophila embryonic cells were exposed to a number of metal ions that have been previously reported to act as teratogens in mammalian systems, including some known to induce heat shock (stress) proteins in a variety of model systems. This study examined the effects of these ions both on differentiation of muscles and neurons and on the induction of heat shock proteins. Metals such as arsenate, cadmium, and mercury all inhibited neuron and/or muscle differentiation in Drosophila embryonic cultures, while they also induced the entire set of heat shock proteins. Two metal ions, nickel and zinc, were shown to induce only the 22- and 23-K proteins, a pattern similar to that seen in "classical" teratogens reported previously. None of the metals tested induced only the 26- and 27-K proteins. These results suggest that there exist different regulatory mechanisms responsible for the heat shock response.     

The effects of inbreeding and low temperature on the pattern of chromosome synapsis in the ovarian nurse nuclei of Drosophila melanogaster strains

The effects of inbreeding and low temperature on the pattern of homologous chromosome synapsis in ovarian nurse cell nuclei of Drosophila melanogaster strains were studied. Exposure to decreased temperature (16 degrees C) caused a noticeable increase in the rate of asynapses of homologous chromosomes, whereas this effect was insignificant for F30 inbreeding generation. Long-term inbreeding has a substantial effect on the relative positions of chromosomes in the nurse cell nuclei. This is visually evident only in the interstrain hybrids between highly inbred strains LA (F923) and HA (F923) or between either strain and laboratory strain Canton S or the flies from the natural population, where abnormalities in homologous chromosome synapsis are observed in virtually all nuclei.     

Classical reward conditioning in Drosophila melanogaster

Negatively reinforced olfactory conditioning has been widely employed to identify learning and memory genes, signal transduction pathways and neural circuitry in Drosophila. To delineate the molecular and cellular processes underlying reward-mediated learning and memory, we developed a novel assay system for positively reinforced olfactory conditioning. In this assay, flies were involuntarily exposed to the appetitive unconditioned stimulus sucrose along with a conditioned stimulus odour during training and their preference for the odour previously associated with sucrose was measured to assess learning and memory capacities. After one training session, wild-type Canton S flies displayed reliable performance, which was enhanced after two training cycles with 1-min or 15-min inter-training intervals. Higher performance scores were also obtained with increasing sucrose concentration. Memory in Canton S flies decayed slowly when measured at 30 min, 1 h and 3 h after training; whereas, it had declined significantly at 6 h and 12 h post-training. When learning mutant t beta h flies, which are deficient in octopamine, were challenged, they exhibited poor performance, validating the utility of this assay. As the Drosophila model offers vast genetic and transgenic resources, the new appetitive conditioning described here provides a useful tool with which to elucidate the molecular and cellular underpinnings of reward learning and memory. Similar to negatively reinforced conditioning, this reward conditioning represents classical olfactory conditioning. Thus, comparative analyses of learning and memory mutants in two assays may help identify the molecular and cellular components that are specific to the unconditioned stimulus information used in conditioning.     

Proteolytic activity of Staphylococcus xylosus strains on pork myofibrillar and sarcoplasmic proteins and use of selected strains in the production of "Naples type" salami

AIMS: The aim of this study was to determine the proteolytic activities of Staphylococcus xylosus strains on sarcoplasmic and myofibrillar proteins in order to evaluate the suitability of selected strains as starter cultures in the processing of a dry fermented pork sausage. METHODS AND RESULTS: The proteolytic activity of 27 strains of Staphylococcus xylosus on sarcoplasmic and myofibrillar proteins was determined by agar plate method, o-phtaldialdehyde (OPA) spectrophotometric assay and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Four strains were selected for the formulation of six starter cultures to use in the production of "Naples type" salami. The proteolytic contribution of starters was determined by SDS-PAGE, comparing the protein profile of inoculated sausages with that of uninoculated sausages after 0, 15 and 33 days of ripening. The results showed that the proteolytic activity of some strains, determined by the agar plate method, were not confirmed by electrophoretic and spectrophotometric assays. In fact, of 24 strains of Staphylococcus xylosus able to hydrolyse muscle protein extracts on agar plate, only 12 strains were shown to change SDS-PAGE profile of pork proteins. The SDS-PAGE profile of sarcoplasmic proteins extracted from all sausages showed that the major changes were produced with starters S3, S4 and S5 after 15 days of ripening. Also myofibrillar proteins undergo major changes after 15 days of ripening and the protein profiles showed the same pattern in all samples, except for the sausages produced with starter S4. CONCLUSIONS: The results of this work showed that the muscle protein extracts hydrolysis test is suitable for preliminary screening of Staphylococcus xylosus strains on the basis of their proteolytic activity. However, evaluation of muscle protein hydrolysis in a food model system could then be more appropriate for selecting micro-organisms for use as starter cultures for fermented sausages. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of the findings is discussed with reference to the formulation of starter cultures for the dry fermented sausages production.     

Lipid Patterns of Selected Microorganisms as Determined by Gas-Liquid Chromatography

The cellular lipid patterns of seven strains of microorganisms were examined by gas-liquid chromatography in this preliminary study. The chloroform methanol-soluble lipids were extracted by the Soxhlet method from dried cultures which had been grown at 25 +/- 2 C for 18 hr with mechanical shaking. The cellular extract was methylated by use of a low temperature sulfuric acid method, and the resulting methyl esters were chromatographed. Considerable differences in the lipid patterns among the seven microorganisms tested indicated that this method might be useful for the identification of closely related microbial genera, and possibly for species differentiation.     

Male age,host effects and the weak expression or non-expression of cytoplasmic incompatibility in Drosophila strains infected by maternally transmitted Wolbachia

In Drosophila melanogaster, the maternally inherited endocellular microbe Wolbachia causes cytoplasmic incompatibility (CI) in crosses between infected males and uninfected females. CI results in a reduction in the number of eggs that hatch. The level of CI expression in this species has been reported as varying from partial (a few eggs fail to hatch) to nonexistent (all eggs hatch). We show that male age in this host species has a large impact on the level of CI exhibited and explains much of this variability. Strong CI is apparent when young males are used in crosses. CI declines rapidly with male age, particularly when males are repeatedly mated. Wolbachia from a Canton S line that was previously reported as not causing CI does in fact induce CI when young males are used in crosses, albeit at a weaker level than in other D. melanogaster strains. The strain differences in CI expression are due to host background effects rather than differences in Wolbachia strains. These results highlight the importance of undertaking crosses with a range of male ages and nuclear backgrounds before ascribing particular host phenotypes to Wolbachia strains.     

Strain-specific differentiation of lactococci in mixed starter culture populations using randomly amplified polymorphic DNA-derived probes.

A hydrophobic grid membrane filtration (HGMF) colony hybridization assay was developed that allows strain-specific differentiation of defined bacterial populations. The randomly amplified polymorphic DNA (RAPD) fingerprinting technique was used to identify potential signature nucleic acid sequences unique to each member of a commercial cheese starter culture blend. The blend consisted of two closely related Lactococcus lactis subsp. cremoris strains, 160 and 331, and one L. lactis subsp. lactis strain, 210. Three RAPD primers (OPX 1, OPX 12, and OPX 15) generated a total of 32 products from these isolates, 20 of which were potential strain-specific markers. Southern hybridization analyses revealed, that the RAPD-generated signature sequences OPX15-0.95 and a 0.36-kb HaeIII fragment of OPX1-1.0b were specific for strains 331 and 210, respectively, within the context of the test starter culture blend. These strain-specific probes were used in a HGMF colony hybridization assay. Colony lysis, hybridization, and nonradioactive detection parameters were optimized to allow specific differentiation and quantitation of the target strains in the mixed starter culture population. When the 210 and 331 probes were tested at their optimal hybridization temperatures against single cultures, they detected 100% of the target strain CFUs, without cross-reactivity to the other strains. The probes for strains 210 and 331 also successfully detected their targets in blended cultures even with a high background of the other two strains.     

The ecdysoneless1 gene regulates metabolism of the juvenile hormone and dopamine in Drosophila melanogaster

The dopamine (DA) content and the level of juvenile hormone (JH) degradation were studied in females of the wild-type Canton S strain and the ecdysoneless1 (ecd1) mutant, which does not produce ecdysone at a restrictive temperature (29 degrees C). Exposure at the restrictive temperature considerably increased the JH-hydrolyzing activity and the DA content in five-day ecd1 females compared with flies of both strains growing at 19 degrees C and Canton S females exposed at 29 degrees C. In one-day ecd1 females, the level of JH degradation also increased at the restrictive temperature, but the DA content was low. The effect of ecdysone deficiency on the stress response in Drosophila melanogaster females was studied using changes in DA content and JH degradation were used as indices. The ecd1 mutation did not prevent the initiation of the stress response in females exposed at the restrictive temperature, but changed its intensity (stress reactivity). The interaction of 20-hydroxyecdysone with JH and DA in regulating Drosophila reproduction under normal conditions and in stress is discussed.     

In vitro long-term development of cultured inner ear stem cells of newborn rat

The adult mammalian auditory receptor lacks any ability to repair and/or regenerate after injury. However, the late developing cochlea still contains some stem-cell-like elements that might be used to regenerate damaged neurons and/or cells of the organ of Corti. Before their use in any application, stem cell numbers need to be amplified because they are usually rare in late developing and adult tissues. The numerous re-explant cultures required for the progressive amplification process can result in a spontaneous differentiation process. This aspect has been implicated in the tumorigenicity of stem cells when transplanted into a tissue. The aim of this study has been to determine whether cochlear stem cells can proliferate and differentiate spontaneously in long-term cultures without the addition of any factor that might influence these processes. Cochlear stem cells, which express nestin protein, were cultured in monolayers and fed with DMEM containing 5% FBS. They quickly organized themselves into typical spheres exhibiting a high proliferation rate, self-renewal property, and differentiation ability. Secondary cultures of these stem cell spheres spontaneously differentiated into neuroectodermal-like cells. The expression of nestin, glial-fibrillary-acidic protein, vimentin, and neurofilaments was evaluated to identify early differentiation. Nestin expression appeared in primary and secondary cultures. Other markers were also identified in differentiating cells. Further research might demonstrate the spontaneous differentiation of cochlear stem cells and their teratogenic probability when they are used for transplantation.     

The effects of inbreeding and low temperature on the pattern of chromosome synapsis in the ovarian nurse cell nuclei of Drosophila melanogaster strains

The effects of inbreeding and low temperature on the pattern of homologous chromosome synapsis in ovarian nurse cell nuclei of Drosophila melanogaster strains were studied. Exposure to decreased temperature (16°C) caused a noticeable increase in the rate of asynapses of homologous chromosomes, whereas this effect was insignificant for F₃₀ inbreeding generation. Long-term inbreeding has a substantial effect on the relative positions of chromosomes in the nurse cell nuclei. This is visually evident only in the interstrain hybrids between highly inbred strains LA (F₉₂₈) and HA (F₉₂₈) or between either strain and laboratory strain Canton S or the flies from the natural population, where abnormalities in homologous chromosome synapsis are observed in virtually all nuclei.     

Induction of chromosome aberrations by chemical mutagens in neural ganglia of Drosophila melanogaster

Chromatid aberrations induced by various concentrations of bleomycin, cyclophosphamide and mitomycin C were analyzed in neural ganglia of third-instar larvae of Drosophila melanogaster. A clear dose response was observed with increasing dose after treatment with bleomycin and mitomycin C, whereas no effect was observed after treatment with cyclophosphamide. A comparison with published data for the induction of sex-linked recessive lethals showed that, at least for the 3 drugs tested, the use of both tests eliminates false negatives and might comprise a useful procedure for testing mutagenicity in Drosophila.     

An evaluation of a novel chick cardiomyocyte micromass culture assay with two teratogens/embryotoxins associated with heart defects

This study was aimed at determining whether the chick cardiomyocyte micromass (MM) system could be employed to predict the teratogenicity/embryotoxicity of exogenous chemicals. Two documented teratogens/embryotoxins, sodium valproate (the sodium salt of valproic acid; VPA) and all-trans retinoic acid (tRA), were used in the initial phase of the study. White Leghorn 5-day-old embryo hearts were dissociated to produce a cardiomyocyte suspension in Dulbecco's Modified Eagle's Medium. Cultures were incubated at 37 degrees C in 5% CO(2) in air, and observations were made every 24 hours over 5 days, for the detection of beating. Culture viability was assessed by using the resazurin reduction assay for determining culture activity and the kenacid blue assay for determining cell number. It was found that tRA significantly reduced cell activity and beating, whilst not affecting total cell number. VPA up to 500 microM induced no cytotoxicity in the MM cardiomyocyte cultures, whilst all the VPA concentrations tested reduced beating. The results demonstrate the potential of the chick cardiomyocyte MM culture assay to identify teratogens/embryotoxins that alter functionality, which may result in a teratogenic outcome, whilst not causing cytotoxicity (direct embryotoxicity). This could form part of a screen for developmental toxicity related to cardiac function, whilst limb cultures and brain cultures based on the same system could be relevant to teratogenic effects on those tissues.     

Amylase gene expression in intraspecific and interspecific somatic transformants of Drosophila.

The Amylase locus in Drosophila melanogaster normally contains two copies of the structural gene for alpha-amylase, a centromere-proximal copy, Amy-p, and a distal copy, Amy-d. Products of the two genes may display discrete electrophoretic mobilities, but many strains known to carry the Amy duplication are characterized by a single amylase electromorph, e.g., Oregon-R, which produces the mobility variant AMY-1. A transient expression assay was used in somatic transformation experiments to test the functional status of the Amy genes from an Oregon-R strain. Plasmid constructs containing either the proximal or distal copy were tested in amylase-null hosts. Both genes produced a functional AMY-1 isozyme. Constructs were tested against an AMY-3 reference activity produced by a coinjected plasmid that contains the Amy-d3 allele from a Canton-S strain. With reference to the internal control, the Amy-p and Amy-d genes from Oregon-R expressed different relative activity levels for AMY-1 in transient assays. The transient expression assay was successfully used to test the functional status of Amy-homologous sequences from strains of other species of Drosophila characterized by a single amylase elctromorph, namely, Drosophila pseudoobscura ST and Drosophila miranda S 204. The amylase-null strain of D. melanogaster provided the hosts for these interspecific somatic transformation experiments.     

Electrical resistance of bacterial culture suspension as indicator of epidemic and sporadic variants of Shigella

For the first time method of measurement of electric resistance of bacterial suspension was used for differentiation of "epidemic" and "sporadic" (further used without quotes) variants of Shigella species. Study of 40 strains of S. flexneri and S. sonnei revealed significant differences in electric resistance of cultures isolated during epidemic outbreaks after the growth during 1 or 6 hours - 18.1+/-8.3 kOhm and 84.6+/-7.8 kOhm respectively (increase in 4.9-fold; p<0.01), while non significant differences were observed in cultures isolated from sporadic cases after the growth during 1 or 6 hours - 21.1+/-9.3 and 57.1+/-11.3 kOhm respectively (increase in 2.5-fold; p>0.05). Express-method for differentiation of epidemic and sporadic variants of Shigella can be useful for timely detection of outbreaks beginning (preoutbreak).     

Distribution of hobo transposable elements in natural populations of Drosophila melanogaster.

Forty-six strains derived from American and French natural populations of Drosophila melanogaster were tested for the presence and activity of hobo elements by using Southern blotting and a gonadal dysgenesis assay. The oldest available strains exhibited weak detectable hybridization to the hobo-element probe and revealed neither hobo-activity potential nor hobo-repression potential. In contrast, all recently collected strains harbored hobo sequences and revealed a strong hobo-repression potential but no strong hobo-activity potential. On the basis of restriction-enzyme analysis, old strains appear to have numerous fragments hybridizable to hobo sequences, several probably conserved at the same locations in the genome of the tested strain and others dispersed. In recently isolated strains, and unlike the situation in the published sequence of the cloned hobo108 element, a PvuII site is present in the great majority of full-sized hobo elements and their deletion derivatives. When the genetic and molecular characteristics are considered together, the available evidence is consistent with the hypothesis of a worldwide hobo-element invasion of D. melanogaster during the past 50 years. Comparison of data from the I-R and P-M systems suggests that the putative invasion followed the introduction of the I element but preceded that of the P element. This hypothesis poses the problem of the plausibility of three virtually simultaneous element invasions in this species. Such a possibility might be due to a modification of the genetic structure of American populations of D. melanogaster during the first part of the 20th century.