Glycoproteins from Teratocarcinomas and Organs of Adult Mice: Analysis by Affinity Chromatography on Lectin-conjugated Agarose1

Glycoproteins were isolated from particulate fraction of four teratccarcinomas and several organs of adult mice by affinity chromatography on lectins conjugated with agarose [concanavalin A (Con A), wheat germ agglutinin (WGA), Ricinus communis agglutinin (RCA), peanut agglutinin (PNA) and Dolichos biflorus agglutinin (DBA)] and were compared by SDS gel electrophoresis. Glycoprotein components were found to be very similar in three lines of solid teratocarcinoma, namely F9, STT-2 and OTT-10A. Teratocarcinoma OTT6050, which is an ascitic form called embryoid bodies, also gave glycoprotein profiles somewhat similar to those of other teratocarcinomas. However, glycoprotein profiles of most adult organs were significantly different from those of teratocarcinomas. The following points were of special interest. 1) RCA receptors from the four teratocarcinomas gave a strong band with an apparent molecular weight 145,000 daltons, which was either weak or absent in the receptors from adult organs. 2) The WGA receptors of all adult organs except muscle and small intestine gave more intense bands than those of the teratocarcinomas. 3) Glycoproteins with molecular weights of more than 240,000 daltons were present in WGA receptors, RCA receptors and PNA receptors of teratocarcinoma OTT 6050, but not in the receptors of other teratocarcinomas.     

Southern elephant seal (Mirounga leonina) II. Studies of milk protein fractions by gel electrophoresis

 Milk protein fractions during various stages of lactation in the southern elephant seal Mirounga leonina were analysed. Twelve milk samples were taken from ten females throughout the lactation period during 1990 and 1991 at Stranger Point, King George Island, South Shetland Islands. Milk samples were subjected to polyacrylamide gel electrophoresis (PAGE). Samples from different days of lactation gave similar qualitative electrophoretic patterns. True protein content was significantly higher (P<0.05) at the beginning of lactation, and then remained constant until weaning. Caseins and whey proteins each consisted of several protein entities (four and five distinct bands respectively). Casein constituted only about 30% of the protein nitrogen, the remaining 70% being derived from whey proteins. There was some variation in concentration of casein and whey proteins as a function of time (P<0.0.5). Received: 28 July 1993/Accepted: 25 July 1995     

Deep sympatric mtDNA divergence in the autumnal moth (Epirrita autumnata)

Deep sympatric intraspecific divergence in mtDNA may reflect cryptic species or formerly distinct lineages in the process of remerging. Preliminary results from DNA barcoding of Scandinavian butterflies and moths showed high intraspecific sequence variation in the autumnal moth, Epirrita autumnata. In this study, specimens from different localities in Norway and some samples from Finland and Scotland, with two congeneric species as outgroups, were sequenced with mitochondrial and nuclear markers to resolve the discrepancy found between mtDNA divergence and present species‐level taxonomy. We found five COI sub‐clades within the E. autumnata complex, most of which were sympatric and with little geographic structure. Nuclear markers (ITS2 and Wingless) showed little variation and gave no indications that E. autumnata comprises more than one species. The samples were screened with primers for Wolbachia outer surface gene (wsp) and 12% of the samples tested positive. Two Wolbachia strains were associated with different mtDNA sub‐clades within E. autumnata, which may indicate indirect selection/selective sweeps on haplotypes. Our results demonstrate that deep mtDNA divergences are not synonymous with cryptic speciation and this has important implications for the use of mtDNA in species delimitation, like in DNA barcoding.     

Identification,characterization, and developmental profile of a high molecular weight,juvenile hormone-binding protein in the hemolymph of the migratory grasshopper,Melanoplus sanguinipes

In the hemolymph of Melanoplus sanguinipes, a high molecular weight juvenile hormone binding protein (JHBP) was identified by photoaffinity labelling and found to have a M_r of 480,000. The JHBP, purified using native gel electrophoresis followed by electroelution, has an equilibrium dissociation constant for JH III of 2.1 nM and preferentially binds JH III over JH I. Antibody raised against JHBP recognized only the 480,000 band. Under denaturing conditions the native JHBP gave a single band with a M_r 78,000. The antibody against native JHBP recognized only the 78,000 protein in SDS-treated hemolymph samples, indicating that JHBP is a hexamer in this species. The concentration of JHBP fluctuates in both the sexes during nymphal and adult development in parallel with total protein content of hemolymph. © 1995 Wiley-Liss, Inc.     

Isolation and characterization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains from different grain habitats in Turkey

Bacillus thuringiensis (Bt) is a gram-positive, spore-forming bacterium and it produces insecticidal crystal (cry) proteins during sporulation. Because the genetic diversity and toxic potential of Bt strains differ from region to region, strains have been collected and characterized all over the world. The aim of this study is to isolate Bt strains in grain-related habitats in Turkey and to characterize them on the basis of crystal morphology, cry gene content, and chromosomal and plasmid DNA profiles. Four approaches were taken analysis with phase contrast (PC) microscopy, polymerase chain reaction (PCR), pulsed field gel electrophoresis (PFGE) and plasmid isolation. Ninety-six samples were collected from Central Anatolia and the Aegean region. Bt was isolated from 61 of 96 samples (63.5) and 500 Bt-like colonies were obtained. One hundred and sixty three of the colonies were identified as Bt based on cry protein formation using PC microscopy. Among the examined colonies, the overall proportion identified (as Bt index) was 0.33. We found that 103 isolates were positive for the five different cry genes (cry1, cry2, cry3, cry4 and cry9) examined with PCR. In addition, plasmid profiling of 37 cry gene-positive isolates indicated that the 15 kb plasmid band was present in all isolates; however, 11 of 37 isolates had more than one plasmid band at different sizes. Finally, chromosomal DNA profiling by PFGE gave rise to different DNA patterns for isolates containing the same cry gene which suggests a high level of diversity among the Bt strains isolated.     

Interannual Variation in Nut Abundance Is Related to Agonistic Interactions of Foraging Female Japanese Macaques (Macaca fuscata)

The importance of dominance status to foraging and ultimately survival or reproductive success in wild primates is known; however, few studies have addressed these variables simultaneously. We investigated foraging and social behavior among 17 adult female Japanese macaques (Macaca fuscata) on Kinkazan Island, northern Japan, from September to November in 2 consecutive years (2004 and 2005) to determine whether interannual variation in food availability was related to variation in agonistic interactions over food resources and the feeding behavior of individuals of different dominance rank. We compared energy obtained with daily energy requirements and also examined the effect of variation in feeding behavior on female survival and reproductive success. Fruiting conditions differed considerably between the 2 yr: of four nut-producing species, the nuts of only Torreya nucifera fruited in 2004, whereas all four species, particularly Fagus crenata, produced nuts in abundance in 2005. The abundance and average crown size of trees of Torreya nucifera were smaller than those of Fagus crenata, and there was a higher frequency of agonistic interactions during 2004, when dominant, but not subordinate, individuals were able to satisfy daily energy requirements from nut feeding alone through longer nut feeding bouts. In contrast, all macaques, regardless of their dominance rank, were able to satisfy their energy requirements by feeding on nuts in 2005. Subordinate macaques appeared to counter their disadvantage in 2004 by moving and searching for food more and maintaining larger interindividual distances. Several lower-ranking females died during the food-scarce season of 2004, and only one dominant female gave birth the following birth season. In contrast, none of the adult females died during the food-scarce season of 2005, and 12 females gave birth the following birth season. These findings suggest that an interaction between dominance rank and interannual variation in food availability are related to macaque behavior, survival, and reproduction.     

Induced resistance to Mexican bean beetles in soybean: variation among genotypes and lack of correlation with constitutive resistance

Fourteen genotypes (varieties) of soybean (Glycine max) were screened for levels of induced resistance to Mexican bean beetle (Epilachna varivestis) damage, and a subset of 6 of those varieties was screened for levels of constitutive resistance to Mexican bean beetles. Experiments were carried out in the greenhouse, with damage imposed by Mexican bean beetle larvae, and levels of resistance measured by a choice test bioassay with adult beetles. We found significant variation among soybean genotypes in levels of both induced and constitutive resistance. We found no significant correlation between levels of induced and constitutive resistance measured in the same genotypes. We compare these results to past work on resistance in the soybean-Mexican bean beetle system, consider the implications of variation in both types of resistance for plant-herbivore interactions in agricultural and natural systems, and discuss the relationship between induced and constitutive resistance. Received: 30 November 1998 / Accepted: 25 June 1999     

Microsatellite Variation Among Red Snapper (Lutjanus campechanus) from the Gulf of Mexico

Allelic variation at a total of 20 nuclear-encoded microsatellites was examined among adult red snapper (Lutjanus campechanus) sampled from 4 offshore localities in the Gulf of Mexico. The number of alleles at the 20 microsatellites ranged from 5 to 20; average (± SE) direct count heterozygosity values ranged from 0.148 ± 0.025 to 0.902 ± 0.008. No significant departures from expectations of Hardy-Weinberg equilibrium were found for any locus within samples, and genotypes at pairs of microsatellites appeared to be randomly associated, i.e., in genotypic equilibrium. Tests of homogeneity in allele distributions among the 4 localities were nonsignificant for 19 of the microsatellites. Allele distribution at microsatellite Lca 43 was heterogeneous among localities before (but not after) Bonferroni corrections for multiple tests executed simultaneously. Tests of homogeneity in the distribution of individual alleles at Lca 43 gave similar results: one low frequency allele was distributed heterogeneously among samples before, but not after, Bonferroni correction. Molecular analysis of variance indicated that more than 99% of variation at each microsatellite was distributed within sample localities. These results generally are consistent with the hypothesis of a single population (stock) of red snapper in the northern Gulf of Mexico. Received September 25, 2000; accepted January 16, 2001     

Development of 22 new microsatellite loci for fishers (Martes pennanti) with variability results from across their range

We developed 22 new microsatellite loci for the fisher (Martes pennanti), a North American mesocarnivore. The loci were developed with samples from the southern Sierra Nevada Mountains in California, and were screened with samples from this population and four other populations. We observed a range of six to 21 polymorphic loci per population, with the Sierra Nevada population exhibiting markedly lower levels of variation compared to the other four.     

Development of Vitellogenin-ELISA,an In Vivo Bioassay,and Identification of Two Vitellogenesis-Inhibiting Hormones of the Tiger Shrimp <Emphasis Type="BoldItalic">Penaeus monodon</Emphasis>

A sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of vitellogenin in the hemolymph of Penaeus monodon is reported. Lipovitellin from the mature ovary was purified using hydroxylapatite column chromatography and used as the standard protein, which was serially diluted from 3336 to 6.51 μg and gave a linear plot. Sensitivity results showed ELISA was insensitive to samples that did not contain vitellogenin or lipovitellin. Specificity results showed the degree of the discrimination of the assay between positive samples (having vitellogenin or lipovitellin) and negative samples (devoid of vitellogenin or lipovitellin). Reproducibility studies showed that the intraassay coefficient of variation was 5.1% (n= 11) and the interassay coefficient of variation was 5.15% (n= 13). Separation of X-organ sinus gland peptides using a reversed phase column gave a total of 37 fractions. Two fractions were found to reduce the hemolymph vitellogenin concentration in a time-dependent manner and could be identified as vitellogenesis-inhibiting hormones I and II. Received February 2, 2001; accepted June 24, 2001     

Deconstructing a Complex Molecular Phenotype: Population-Level Variation in Individual Venom Proteins in Eastern Massasauga Rattlesnakes (<Emphasis Type="Italic">Sistrurus c. catenatus</Emphasis>)

Identifying the molecular basis for complex adaptations such as the toxic proteins used by venomous snakes to subdue and digest prey is an important step in understanding the evolutionary and functional basis for such traits. Recent proteomics-based analyses have made possible the identification of all constituent proteins in whole venom samples. Here we exploit this advance to study patterns of population-level variation in venom proteins from 254 adult eastern massasauga rattlesnakes (Sistrurus c. catenatus) collected from 10 populations. Analysis of presence–absence variation in specific proteins from 1D PAGE gels shows that: (1) The frequency spectra for individual protein bands is U-shaped with a large number of specific proteins either being consistently “common” or “rare” across populations possibly reflecting functional differences. (2) Multivariate axes which summarize whole venom variation consist of bands from all major types of proteins implying the integration of functionally distinct components within the overall venom phenotype. (3) There is significant differentiation in venom proteins across populations and the specific classes of proteins contributing to this differentiation have been identified. (4) Levels of population differentiation in venom proteins are not correlated with levels of neutral genetic differentiation, or genetically effective population sizes which argues that patterns of venom variation are not simply a consequence of population structure but leaves open the role of selection in generating population differences in venom. Our results identify particular classes of venom proteins and their associated genes as being fruitful targets for future studies of the molecular and functional basis for this complex adaptive phenotype.     

Resolution of microbial mixtures by free flow electrophoresis

Summary The resolution of bacterial mixtures by free flow electrophoresis (FFE) was not affected by the position of the microbes on the growth curve and approximately 70% of the individual cells applied were recovered as viable cells. The dependence of bacterial electrophoretic mobility on the pH, salt concentration, and viscosity of the electrolyte was determined. Suspending media and running electrolyte were developed which allowed collection of samples of>99% purity within two minutes of introduction of a mixture of Escherichia coli and Staphylococcus aureus. Most bacterial strains migrated in a single band, although some migrated in more than one band. Escherichia coli was resolved from each of 10 different species. The considerable variation in mobility found in 21 different E. coli strains, however, appears to preclude use of FFE as a method of species identification.     

Acrylamide gel electrophoresis of seed proteins from someSolanum (sectionSolanum) species

Polyacrylamide gel electrophoresis was used to investigate the seed proteins of 36 accessions belonging toSolanum sect.Solanum (Solanaceae). These accessions represented 20 species, of four differing ploidy levels, and included infraspecific morphological variants. The resultant band patterns tended to reflect the morphological differences and genetical isolation displayed by many of the species. The most variable band patterns were encountered in the taxa with the greatest infraspecific variation, while many of the more morpho-genetically distinct taxa seemed to have species-specific band patterns.Good matches were found between the band patterns of artificial hybrids, those of their known parents, and mixtures of the parental protein extracts. This illustrates the potential use of such a technique for pinpointing possible genome donors of natural hybrids, and especially of polyploids. These comparative band patterns confirmed experimental work on the origin of the hexaploidS. nigrum from the diploidS. americanum and the tetraploidS. villosum, and also supported the suggestion thatS. nigrum contains two genomes from the diploidS. sarrachoides, but not four genomes of the diploidS. americanum.     

Development of a chromatographic method for the isolation and detection of hygromycin B in biological fluids

An affinity chromatography method was developed for the purification of hygromycin B from biological fluids. Lysozyme and α-lactalbumin were immobilized on an N-hydroxysuccinimide activated agarose support. Hygromycin B solubilized in water was bound by the proteins and subsequently eluted using 10 mM sodium citrate buffer, pH 4.0. Hygromycin B was purified from swine plasma, bovine serum and bovine milk samples using a combination of ion-exchange chromatography for initial clean-up of spiked biological samples followed by affinity chromatography. Thin layer chromatographic analysis of the isolated hygromycin B revealed one band with the same R_F value as the hygromycin B standard.     

Variability in the calcification rate of Acropora acuminata measured with radioisotopes

Summary Under standard experimental conditions, sample groups of Acropora acuminata gave calcification results having a coefficient of variation approaching 50%. Because of this, the resolution of radioisotope measurements approaches the magnitude of the effect of light on calcification rate. Differences in calcification rate between samples appear to be caused by short-term variations in calcification rate of samples, rather than size differences between samples. The coefficient of variation associated with results can be decreased by relatively straightforward modifications to the standard technique.     

Microbial Hydrogenation of Nitroolefins

Microorganisms which hydrogenate 2-nitro-1-phenyl-1-propene were screened in type cultures and soil samples. Some actinomycetes belonging to Rhodococcus, Nocardia and Mycobacterium asymmetrically reduced the substrate and gave optically active 2-nitro-1-phenylpropane. Among them, Rhodococcus rhodochrous IFO 3338 gave the best results. The saturated compound was obtained quantitatively, when cultivation was carried out for 3 days at 30°C with a substrate concentration of 0.4%. The optical purity of the product was seriously affected by the pH of the medium. The more acidic the medium, the higher the enantiomeric excess. The results suggested that non-enzymatic racemization of the product takes place even under neutral conditions. Other substrates, such as 2-nitro-1-propene were also converted to optically active 2-nitro-1-substituted propane.     

Identification and characterization of a novel postlarval hemolymph protein from Manduca sexta

The identification, purification and characterization of a new postlarval specific hemolymph protein from Manduca sexta is described. Incorporation of [³⁵S]methionine into Manduca sexta hemolymph proteins in vivo was investigated as a function of development. A major protein band of M_r ≈ 50,000 was highly labeled during the prepupal and adult stage but not in feeding larvae. This postlarval protein (PLP) was isolated from adult male hemolymph and its chemical and immunological properties determined. PLP is a basic protein (pI ～8.6). Electrophoresis under denaturing conditions reveals a subunit M_r ≈ 50,000 while the native protein has an apparent M_r ～ 85,000 by gel permeation chromatography. Anti-PLP serum recognized PLP but not other hemolymph proteins on immunoblots. In vitro translation of fat body mRNA followed by immunoprecipitation revealed that fat body is the site of PLP synthesis. Quantitation of PLP levels in hemolymph throughout development was performed and suggests PLP may play a role in adult development of M. sexta.     

The life expectancy of phlebotomine sandflies: first field estimates from southern France

Abstract. 1. A field study of Phlebotomus ariasi Tonnoir, the vector of Leishmania infantum Nicolle in southern France, addressed the following questions: Is it possible to estimate reliably the life expectancy of this sandfly; can spatial or temporal variation in the life expectancy be detected, and is such variation significant for disease transmission? 2. Life expectancy was estimated by examining follicular relics in the ovaries of more than ten thousand females caught in light traps at seven sites in the Cévennes and the Garrigues, throughout their active period in 1985 and 1986. Whilst the distinction between nulliparous and parous flies was easily made, assessments of the number of times a parous fly had laid eggs were unreliable. Best estimates of life expectancy were therefore calculated from the parous rate. 3. Large samples collected from one site in the Cevennes in both years gave very similar estimates of life expectancy. 4. There was also no significant difference between estimates obtained from the Cévennes and the Garrigues, despite their distinct vegetation and climates. Therefore, large regional differences in sandfly population size and the prevalence of canine leishmaniasis cannot be explained by a difference in adult survival rate. 5. With no systematic annual or regional variation, a useful mean life expectancy can be calculated from the data collected at all sites in both years. It is 1.54 (SE 0.04) ovarian cycles. However, this estimate is sensitive to the assumption that survival rate is a discrete rather than a continuous variable. 6. Local variation in the parous rate may be associated with the proximity of traps to P.ariasi emergence sites.     

Cloning, expression, purification, and activity assay of proteins related to D-lactic acid formation in Lactobacillus rhamnosus

Two proteins that might be responsible for D-lactic acid (D-LA) formation were screened from the genome database of Lactobacillus rhamnosus GG. The coding genes of the two proteins in L. rhamnosus CASL, ldhD1 and ldhD2, were cloned and expressed in Escherichia coli Rosetta with an inducible expression vector pETDuet™-1 (Novagen, Darmstadt, Germany), respectively. The two purified proteins, LdhD-1 and LdhD-2, migrated as a single protein band separately, both corresponding to an apparent molecular mass between 35 kDa and 45 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activities of LdhD-1 and LdhD-2 catalyzing pyruvate to LA were 0.02 U/mg and 0.21 U/mg, respectively. The configuration of LA converted from pyruvate was determined using high-performance liquid chromatography equipped with a chiral column. Only D-LA was detected when LdhD-1 and LdhD-2 were tested. In summary, the two proteins cloned and expressed in this study were most probably responsible for D-LA formation during fermentation of L. rhamnosus CASL.