Comparison of surface proteomes of enterotoxigenic (ETEC) and commensal Escherichia coli strains

Pathogenesis of enterotoxigenic Escherichia coli (ETEC) infections involves colonization of the small intestine mediated by cell-surface fimbriae (CS) or colonization fimbriae antigens (CFA). However, protection against reinfection of ETEC is also conferred by somatic antigens rather than by virulence factors. To discover ETEC specific somatic antigens, the surface proteome of the ETEC H10406 strain was compared with that of non-pathogenic E. coli K12 strains. In this study, we were using stable isotope labelling with amino acids in cell culture (SILAC) technology for the labelling and relative quantification of surface proteins in order to identify polypeptides that are specifically present on ETEC strains. Outer membrane proteins were isolated, separated by gel electrophoresis, and identified by mass spectrometry. Twenty-three differentially expressed cell-surface polypeptides of ETEC were identified and evaluated by bioinformatics for protein vaccine candidates. The combination of being surface-exposed and present differentially makes these polypeptides highly suitable as targets for antibodies and thus for use in passive or active immunisation/vaccination.     

Transfer and fate of male secretions deposited in the spermatophore of females of Acanthoscelides obtectus Say (Coleoptera Bruchidae)

During mating, males of Acanthoscelides obtectus deposit a spermatophore in the female genital tract. Spermatophore structure subsequently undergoes considerable modification, especially the central portion, which becomes vacuolated. Two methods were used to show that certain male secretions could thus pass into female haemolymph. When young males were injected with [¹⁴C]-arginine or [¹⁴C]-histidine, the accessory glands actively incorporated the isotope and the resulting spermatophores were radioactive. After mating, spermatophore radioactivity declined and then appeared in the haemolymph of females and in the oöcytes after a delay. Immunoelectrophoresis also showed that antigens appeared in the haemolymph of females after mating which reacted against male-gland antiserum. This technique, however, did not enable us to detect the presence of male antigens in the oöcytes formed after mating. The fate of some male secretions in the female and their physiological importance in the control of the female reproductive function were analysed in the present work.     

Identification of four classes of cell surface antigens appearing at gastrulation in sea urchin embryos

An antiserum to isolated membranes of gastrula-stage embryos of the sea urchin Lytechinus variegatus was characterized by absorption and cell agglutination specificities. The antiserum was found to recognize four distinct classes of antigens on the embryonic cell surface: (1) an early embryonic class or “maternal” class present from the earliest stages of development, (2) an embryonic class of antigens which appeared on all cells beginning at gastrulation, (3) a class of antigens present on ectoderm cells, and (4) a class of antigens present on endoderm cells. All four classes of antigens were shown indirectly to be synthesized on embryonic mRNA since a hybrid embryo of the cross Tripneustes ♀ × Lytechinus ♂ expressed all four classes of Lytechinus-specific antigens beginning at gastrulation. Each class was Lytechinus specific in that hybrid cells were agglutinated if beyond the beginning of gastrulation, while normal Tripneustes ♀ × Tripneustes ♂ cells were not agglutinated.     

Somatic and gametic resources in male rock shrimp, Rhynchocinetes typus: effect of mating potential and ontogenetic male stage

Investment in somatic and gametic resources may vary during the ontogeny of an organism. In the rock shrimp, Rhynchocinetes typus, males pass through several stages during ontogeny, and a dominance order reflects this developmental order (typus<intermedius<robustus). During mating in a competitive environment, subordinate males use a sneaking tactic characterized by rapid placement of many spermatophores, but dominant males transfer few spermatophores during matings. We therefore hypothesized that males in the different ontogenetic stages (1) invest differently in somatic and gametic growth and (2) differ in their ability to engage in multiple matings. The relative weight of the hepatopancreas (adjusted for total body weight), an organ related to somatic growth, was significantly lower in robustus males than in all other ontogenetic stages examined. The relative weight of the vas deferens (Vd), a measure of sperm reserves, was significantly higher in robustus males than in males in the other stages. In typus males that had mated once, the total weight of Vd was significantly lower than in unmated typus males, but no such difference was found in robustus males. All robustus males mated successfully with five females during 5 consecutive days, but many typus males failed to mate after the second or third day. Typus males that mated successfully with females placed significantly more spermatophores than did robustus males during the first mating but not in subsequent matings. The results suggest that robustus males, in contrast to typus males, can invest more in sperm production and due to their ability to defend a female, can use spermatic resources economically allowing them to mate with subsequent females. We conclude that, during ontogeny, R. typus males invest simultaneously in somatic and gametic growth in accordance with their mating behaviour and chance to mate. In this and other crustacean species, male resource investment during ontogeny thus may depend on their probabilities at different ontogenetic stages for obtaining mating opportunities. Copyright 2003 Published by Elsevier Ltd on behalf of The Association for the Study of Animal Behaviour.      

Frequent diploidisation of haploid Armillaria ostoyae strains in an outdoor inoculation experiment

Very little is known about the biology and ecology of haploid Armillaria strains in nature. In this outdoor inoculation experiment, we assessed the virulence of six haploid Armillariaostoyae strains along with their diploid parent towards 2-year-old seedlings and 4-year-old saplings of Norway spruce (Picea abies), and determined their ability to colonise freshly cut stumps. As inoculum source an Armillaria-colonised hazelnut (Corylus avellana) stem segment was inserted into the soil substrate. Re-isolations from mycelial fans at the root collar of infected trees or stumps were made. Surprisingly, not a single haploid re-isolate could be recovered. Microsatellite genotyping of 133 re-isolates suggests that the inoculated haploid strains were diploidised either by mating propagules (basidiospores or haploid mycelia) already present in the soil substrate or naturally disseminated in the course of the experiment from nearby forests. Consequently, no conclusion about the infectious ability of haploid Armillaria mycelia under natural conditions can be drawn. Nonetheless, the diploid half-sib families resulting from the diploidisation showed varying degrees of virulence, with a high correlation between the experiment with 2-year-old seedlings and 4-year-old saplings. Despite extensive genotyping of re-isolates, no evidence for somatic recombination between haploid mating propagules and diploidised mycelia was detected, suggesting that this is an uncommon phenomenon in A. ostoyae.     

Rvs161p Interacts with Fus2p to Promote Cell Fusion in Saccharomyces cerevisiae

FUS7 was previously identified by a mutation that causes a defect in cell fusion in a screen for bilateral mating defects. Here we show that FUS7 is allelic to RVS161/END6, a gene implicated in a variety of processes including viability after starvation, endocytosis, and actin cytoskeletal organization. Two lines of evidence indicate that RVS161/END6's endocytic function is not required for cell fusion. First, several other endocytic mutants showed no cell fusion defects. Second, we isolated five function-specific alleles of RVS161/FUS7 that were defective for endocytosis, but not mating, and three alleles that were defective for cell fusion but not endocytosis. The organization of the actin cytoskeleton was normal in the cell fusion mutants, indicating that Rvs161p's function in cell fusion is independent of actin organization. The three to fourfold induction of RVS161 by mating pheromone and the localization of Rvs161p-GFP to the cell fusion zone suggested that Rvs161p plays a direct role in cell fusion. The phenotypes of double mutants, the coprecipitation of Rvs161p and Fus2p, and the fact that the stability of Fus2p was strongly dependent on Rvs161p's mating function lead to the conclusion that Rvs161p is required to interact with Fus2p for efficient cell fusion.     

Deletion of the Bombyx mori odorant receptor co-receptor (BmOrco) impairs olfactory sensitivity in silkworms

Olfaction plays an essential role in many important insect behaviors such as feeding and reproduction. To detect olfactory stimuli, an odorant receptor co-receptor (Orco) is required. In this study, we deleted the Orco gene in the Lepidopteran model insect, Bombyx mori, using a binary transgene-based clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 system. We initially generated somatic mutations in two targeted sites, from which we obtained homozygous mutants with deletion of a 866 base pair sequence. Because of the flight inability of B. mori, we developed a novel method to examine the adult mating behavior. Considering the specialization in larval feeding, we examined food selection behavior in Orco somatic mutants by the walking trail analysis of silkworm position over time. Single sensillum recordings indicated that the antenna of the homozygous mutant was unable to respond to either of the two sex pheromones, bombykol or bombykal. An adult mating behavior assay revealed that the Orco mutant displayed a significantly impaired mating selection behavior in response to natural pheromone released by a wild-type female moth as well as an 11:1 mixture of bombykol/bombykal. The mutants also exhibited a decreased response to bombykol and, similar to wild-type moths, they displayed no response to bombykal. A larval feeding behavior assay revealed that the Orco mutant displayed defective selection for mulberry leaves and different concentrations of the volatile compound cis-jasmone found in mulberry leaves. Deletion of BmOrco severely disrupts the olfactory system, suggesting that BmOrco is indispensable in the olfactory pathway. The approach used for generating somatic and homozygous mutations also highlights a novel method for mutagenesis. This study on BmOrco function provides insights into the insect olfactory system and also provides a paradigm for agroforestry pest control.     

Synchronization of somatic embryogenesis in a carrot cell suspension culture

Synchronization of somatic embryogenesis was achieved in a carrot (Daucus carota L. cv. “Kurodagosun”) suspension culture by sieving the initial heterogeneous cell population, by density gradient centrifugation in Ficoll solutions, and by subsequent repeated centrifugations at a low speed (50g) for a short time (5 seconds), followed by transferring the cell clusters obtained, which were composed of 3 to 10 cells, to a medium containing zeatin (0.1 micromolar) but no auxin. The frequency of embryo formation reached more than 90%, and synchrony of the embryogenetic process was observed at least in the early stages of the process. The system established in the present work provides a useful system for biochemical research into the mechanisms of somatic embryogenesis.     

Metagonimus yokogawai: a 100-kDa Somatic Antigen Commonly Reacting with Other Trematodes

This study was undertaken to characterize the properties of a 100 kDa somatic antigen from Metagonimus yokogawai. Monoclonal antibodies (mAbs) were produced against this 100 kDa antigen, and their immunoreactivity was assessed by western blot analysis with patients'' sera. The mAbs against the 100 kDa antigen commonly reacted with various kinds of trematode antigens, including intestinal (Gymnophalloides seoi), lung (Paragonimus westermani), and liver flukes (Clonorchis sinensis and Fasciola hepatica). However, this mAb showed no cross-reactions with other helminth parasites, including nematodes and cestodes. To determine the topographic distribution of the 100 kDa antigen in worm sections, indirect immunoperoxidase staining was performed. A strong positive reaction was observed in the tegumental and subtegumental layers of adult M. yokogawai and C. sinensis. The results showed that the 100 kDa somatic protein of M. yokogawai is a common antigen which recognizes a target epitope present over the tegumental layer of different trematode species.     

Serological analysis of eleven strains ofRhizobium japonicum

The present communication reports a serological analysis of eleven strains ofRhizobium japonicum. The slow-diffusing thermostable antigens were found to be suitable for the basic differentiation of the somatic serogroups inRhizobium japonicum. One to three precipitation bands of the slow-diffusing thermostable antigens, one to two bands of the fast-diffusing thermostable antigens and one to three bands of the thermolabile antigens were detectable in the whole cell cultures ofR. japonicum by means of the immunodiffusion technique. Two basic somatic serogroups were differentiated on the basis of the slow-diffusing thermostable antigens. The thermolabile antigens were identical in most of the strains.The author is greatly indebted to Mrs. M. Kabelovà for technical assistance.This investigation forms part of a contribution prepared by the Czechoslovak National Committee for the International Biological Programme (Section PP: Production Processes).     

Transposable DNA elements and life history traits: II. Transposition of P DNA elements in somatic cells reduces fitness, mating activity, and locomotion of Drosophila melanogaster

Some transposable DNA elements in higher organisms are active in somatic cells, as well as in germinal cells. What effect does the movement of DNA elements in somatic cells have on life history traits? It has previously been reported that somatically active P and mariner elements in Drosophila induce genetic damage and significantly reduce lifespan. In this study, we report that the movement of P elements in somatic cells also significantly reduces fitness, mating activity, and locomotion of Drosophila melanogaster. If other elements cause similar changes in life history traits, it is doubtful if transposable DNA elements remain active for long in somatic cells in natural populations.     

Effect of the California red scale Aonidiella aurantii sex pheromone on the natural parasitism by Aphytis spp. in Mediterranean citrus

Mating disruption has proved successful against California red scale (CRS), Aonidiella aurantii Maskell (Hemiptera: Diaspididae) in Mediterranean citrus. Although mating disruption does not affect negatively the parasitism by Aphytis melinus DeBach (Hymenoptera: Aphelinidae), a CRS parasitoid introduced to the Mediterranean, there is no information regarding its potential effect on the native Aphytis species. In the present study, the effect of CRS mating disruption on the field parasitism inflicted by Aphytis spp. has been assessed and compared to a mineral oil and a control treatment. In order to confirm the effectiveness of the mating disruption we also evaluated its effect on the captures of the CRS males and on fruit infestation. Moreover, the potential role of the CRS sex pheromone as kairomone for the Aphytis species was also evaluated by comparing captures of parasitoids on sticky traps with or without pheromone. Significantly lower CRS male captures and fruit damage were registered in the mating disruption respect to the control or oil treatments indicating that mating disruption was effective. In September, when compared to the control, parasitism by Aphytis spp. was significantly lower in the mating disruption and mineral oil treatments and crucially no Aphytis chrysomphali Mercet were registered in the mating disruption treatment. Finally, while the captures of both A. melinus and Aphytis lepidosaphes (Mercet) were not significantly different between traps with or without pheromone, A. chrysomphali captures were significantly higher in traps baited with CRS pheromone. These results suggest a possible kairomonal effect of the CRS pheromone on A. chrysomphali.     

Telomere length status of somatic cell sheep clones and their offspring

This study was carried out to determine the telomere length status of sheep clones and their offspring, and to examine telomere dynamics and chromosomal abnormalities in culture propagated donor cells. Skin samples were collected from somatic cell nuclear transfer-derived sheep clones, and three of their progeny generated by natural mating. Samples were collected from control animals (n = 35), spanning in age from 1 month to 36 months of age. Genomic DNA was extracted from cell/tissue samples and their telomere lengths were assessed by terminal restriction fragment (TRF) analysis. Results revealed: that (a) sheep clones derived from cultured somatic cells have shortened telomere lengths compared to age-matched controls; (b) the offspring derived from natural mating between clones had normal telomere lengths compared to their age-matched counterparts; and donor cell cultures beyond 20 population doublings had significantly (P < 0.05) shortened telomeres and exhibited a higher numerical and structural chromosomal abnormalities.     

Characterization of antisera against mouse teratocarcinoma OTT6050: Molecular species recognized on embryoid bodies,preimplantation embryos,and sperm

Antisera raised in rabbits by hyperimmunization with small embryoid bodies of the transplantable teratocarcinoma OTT6050 recognize several distinct antigenic protein species on the surfaces of cells of the immunogen. Some of these antigens were found on the cells of preimplantation mouse embryos, on cells from parietal yolk sac carcinoma, and on mouse sperm. These antigens have been distinguished by polyacrylamide electrophoresis of the immune precipitates from detergent extracts of lactoperoxidase-iodinated cells. The intact embryoid bodies from the ascitic form of the OTT6050 teratocarcinoma exhibited five major protein bands (approximate MW 150K, 115K, 82K, 48K, and 12K), one band that ran at the dye front of the gels (R_f ≥1) and one minor band (approximate MW 22K). Two different rabbit antisera recognized an essentially identical pattern of antigens which, however, varied on the different cell types tested. Antisera were also elicited in syngeneic male mice using glutaraldehyde-fixed or irradiated OTT6050 embryoid bodies. The isoantisera had very poor titers in comparison to the absorbed xenoantisera, as assessed by complement-mediated cytotoxic activity against the immunizing cell types. Complement-mediated cytotoxicity could also be demonstrated using parietal yolk sac carcinoma cells, preimplantation mouse embryos from all cleavage stages, blastocysts, and immunosurgically isolated inner cell masses, as targets. The complexity of the antisera generated by intact embryoid bodies described here indicates that these structures bear multiple antigenic specificities not present on adult somatic cells, some of which are stage-specific embryonic polypeptides.     

Study of Developmental Stages of Methylosinus trichosporium with the Aid of Fluorescent-Antibody Staining Techniques

When stained by using an indirect fluorescent-antibody technique, Methylosinus trichosporium displayed an uneven fluorescence. Exospores and the polar tips of some vegetative cells displayed a more intense fluorescence than the other cells. Cross-absorption of the specific anti-M. trichosporium immunoglobulin G with exospores resulted in no fluorescence of exospores or exospore regions of sporulating vegetative cells. This demonstrated that antigens were present on exospores and exospore regions of vegetative cells that are different from vegetative cell antigens. Taking advantage of this phenomenon, three fluorescentantibody staining techniques were developed which were used to study the life cycle of M. trichosporium.     

The RAG cell line defines a new complementation group of MHC class II deficiency

We previously described RAG, a mouse adenocarcinoma cell line, as deficient for the induction of major histocompatibility (MHC) class II antigens by IFN-, but responding normally for MHC class I antigen stimulation and anti-viral protection. We had established that the fusion of RAG with various human cell lines restored the induction of MHC class II antigens, whenever the human chromosome 16 was present in somatic cell hybrids. Here we show that the RAG cell line does not exhibit any induction by IFN- ofDMA, DMB, and theinvariant chain (Ii) mRNAs, and that the induction is restored in somatic cell hybrids containing human chromosome 16. In order to define the gene (designatedF16) affected in the RAG cells, we performed a complementation analysis by fusing RAG with previously described human cell lines defective for MHC class II antigen expression (e.g., BLS cell lines), and which belong to five different complementation groups. Our data show that the resulting somatic cell hybrids present an inducible expression of mouse MHC class II antigens, Ii, DMA, and DMB. Therefore, the RAG cell line represents a yet undescribed cellular mutant affected in the expression of MHC class II antigens. In addition, we demonstrate that MHC class II antigens can be constitutively expressed in the RAG cell line when transfected with the cDNA encoding humanCIITA driven by the RSV LTR promoter. Since the complementation analysis assessed that F16 and CIITA are distinct, our data suggest that F16 is required for the expression of CIITA.     

Serological identification of Bacillus thuringiensis and related bacteria isolated in Japan

Distribution of Bacillus thuringiensis and related sporeforming bacteria in Japan was investigated and it was found that most of the crystalliferous isolates belonged to B. thuringiensis serotypes 3a, 4a:4b, 7, and 8. Serotypes 1, 3a:3b, 4a:4c, and 11 were rarely isolated. H antigens of 189 isolates of acrystalliferous sporeformers were analyzed and 26 isolates were agglutinated by B. thuringiensis H antisera against serotypes 3a, 4a:4b, 5a:5c, 6, 7, 8, 10, 11, and 12 at high serum dilutions. Heat-stable somatic antigens of these isolates differed significantly from that of reference B. thuringiensis.