Prolactin receptor expression by splenocytes from rats in various hormonal states

Prolactin (PRL) is mitogenic for lymphocytes in vitro , but the responsiveness of lymphocytes depends on the in vivo hormonal status of the rats from which the cells were obtained. Lymphocytes from ovariectomized (OVX) rats, but not from rats in oestrus or from male rats, respond to prolactin; administration of oestradiol to OVX rats diminishes the response. In order to determine if a correlation exists between lymphocyte responsiveness to prolactin and levels of cell surface prolactin receptors (PRL-R) expression, the percentage of splenocytes and each splenocyte subpopulation expressing surface PRL-R from rats of various hormonal states (OVX, oestradiol-injected OVX, oestrus and male) was analysed by single-colour and dual-colour flow cytometric analysis. We found that approximately 20% of splenocytes expressed surface PRL-R regardless of hormonal states ( n =16). The majority (85%) of PRL-R positive splenocytes were B lymphocytes whereas 11.1% and 4.8% of splenocytes expressing the PRL-R were CD4 positive T-helper (T_H) and CD8 positive T-cytotoxic (T_C) lymphocytes, respectively. B lymphocytes also stained more brightly than T lymphocytes. This distribution of PRL-R expression did not show significant alterations on total splenocytes or T_H and T_C lymphocytes during various hormonal stages. However, the percentage of PRL-R-positive B lymphocytes increased markedly in OVX rats (twofold), compared to rats at oestrus. In summary, no correlation was found between the responsiveness to prolactin as a mitogen and levels of PRL-R expression by lymphocytes from rats at different hormonal states. This result suggests that sex steroid hormones may control prolactin responsiveness of lymphocytes by affecting the signal transduction pathway through PRL-R rather than by altering the level of the cell surface receptor expression.     

Visually Mediated ‘Paratrooper Copulations’ in the Mating Behavior of <Emphasis Type="Italic">Agrilus planipennis</Emphasis> (Coleoptera: Buprestidae), a Highly Destructive Invasive Pest of North American Ash Trees

The emerald ash borer, Agrilus planipennis, is a serious invasive pest of North American ash (Fraxinus) trees. In captivity, mating is initiated by beetles at least 10 days old, and appears to be based simply on random contact with a member of the opposite sex. In the field, male A. planipennis search the tree during flight, and attempt to copulate with dead beetles of both sexes pinned to leaves, after descending rapidly straight down onto the pinned beetles from a height of from 30 to 100 cm. All evidence suggests that males find potential mates using visual cues. Equal numbers of feral males approach all ‘dummy’ beetles; however, considerably more time is spent attempting copulation with dead females rather than males, suggesting a contact chemical cue. Sticky traps prepared from dead, pinned EAB capture crawling insects as well as male A. planipennis, at a rate similar to that at which small purple sticky traps of similar overall area capture crawling insects and both sexes of feral EAB.     

Genetic diversity among eight <Emphasis Type="Italic">Dendrolimus</Emphasis> species in Eurasia (Lepidoptera: Lasiocampidae) inferred from mitochondrial COI and COII,and nuclear ITS2 markers

Background

Moths of genus Dendrolimus (Lepidoptera: Lasiocampidae) are among the major pests of coniferous forests worldwide. Taxonomy and nomenclature of this genus are not entirely established, and there are many species with a controversial taxonomic position. We present a comparative evolutionary analysis of the most economically important Dendrolimus species in Eurasia.

Results

Our analysis was based on the nucleotide sequences of COI and COII mitochondrial genes and ITS2 spacer of nuclear ribosomal genes. All known sequences were extracted from GenBank. Additional 112 new sequences were identified for 28 specimens of D. sibiricus, D. pini, and D. superans from five regions of Siberia and the Russian Far East to be able to compare the disparate data from all previous studies. In total, 528 sequences were used in phylogenetic analysis. Two clusters of closely related species in Dendrolimus were found. The first cluster includes D. pini, D. sibiricus, and D. superans; and the second, D. spectabilis, D. punctatus, and D. tabulaeformis. Species D. houi and D. kikuchii appear to be the most basal in the genus.

Conclusion

Genetic difference among the second cluster species is very low in contrast to the first cluster species. Phylogenetic position D. tabulaeformis as a subspecies was supported. It was found that D. sibiricus recently separated from D. superans. Integration of D. sibiricus mitochondrial DNA sequences and the spread of this species to the west of Eurasia have been established as the cause of the unjustified allocation of a new species: D. kilmez. Our study further clarifies taxonomic problems in the genus and gives more complete information on the genetic structure of D. pini, D. sibiricus, and D. superans.

     

Comparative efficacy of two controlled-release gypsy moth mating disruption formulations

The effects of aerial applications of the gypsy moth sex pheromone, disparlure, on mating disruption and suppression of growth of populations of the gypsy moth, Lymantria dispar (L.), were investigated. Two formulations of disparlure, plastic laminate flakes applied in a single application and polymethacrylate beads applied in two applications, were compared in two separate tests conducted in 1993 and 1994. The beads were applied in two applications spaced 2 weeks apart because preliminary tests had indicated that they released pheromone too rapidly to maintain adequate emission rates throughout the period of male flight. In 1993, the flakes were applied at a rate of 50 g a.i./ha, and the beads were applied at a rate of 15 g a.i./ha for each application. In 1994, the flakes were applied at a rate of 75 g a.i./ha and the beads were applied at rates of 32.5 and 42.5 g a.i./ha for the two applications. Beads with larger average particle size were used in 1994 to prolong disparlure release. The treatments applied in 1993 resulted in >97% reduction in mating and >82% suppression of population growth in the following year. Because of a 1995 collapse of gypsy moth populations in the vicinity of the tests, reliable population growth data were not available for the treatments applied in 1994, but significant mating disruption did occur under both treatments. Based on measurements of residual disparlure after field aging, the flakes released 32 and 48% of their disparlure content during the 6 weeks of male moth flight in 1993 and 1994, respectively. The smaller beads used in 1993 released 75% of their disparlure content, and the larger beads used in 1994 released 52% of their disparlure content, during the 6 weeks of male flight. The biological efficacy data suggest that the bead and flake formulations, as applied in these tests, have similar effects on gypsy moth mating disruption and subsequent population growth. Based on the observed release rates from both 1993 and 1994, a single application of the beads would provide emission rates equal to or greater than those provided by the flakes when applied at an equal dose.     

The use of direct cDNA selection to rapidly and effectively identify genes in the fungus Aspergillus fumigatus

Aspergillus fumigatus is one of the causes of invasive lung disease in immunocompromised individuals. To rapidly identify genes in this fungus, including potential targets for chemotherapy, diagnostics, and vaccine development, we constructed cDNA libraries. We began with non-normalized libraries, then to improve this approach we constructed a normalized cDNA library using direct cDNA selection. Normalization resulted in a reduction of the frequency of clones with highly expressed genes and an enrichment of underrepresented cDNAs. Expressed sequence tags generated from both the original and the normalized libraries were compared with the genomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans, indicating that a large proportion of A. fumigatus genes do not have orthologs in these fungal species. This method allowed the expeditious identification of genes in a fungal pathogen. The same approach can be applied to other human or plant pathogens to rapidly identify genes without the need for genomic sequence information.     

Expression of Lex antigen in Schistosoma japonicum and S.haematobium and immune responses to Lex in infected animals: lack of Lex expression in other trematodes and nematodes

Adults of the human parasitic trematode Schistosoma mansoni, which causes hepatosplenic/intestinal complications in humans, synthesize glycoconjugates containing the Lewis x (Lex) Galbeta1-->4(Fucalpha1-- >3)GlcNAcbeta1-->R, but not sialyl Lewis x (sLex), antigen. We now report on our analyses of Lexand sLexexpression in S.haematobium and S.japonicum, which are two other major species of human schistosomes that cause disease, and the possible autoimmunity to these antigens in infected individuals. Antigen expression was evaluated by both ELISA and Western blot analyses of detergent extracts of parasites using monoclonal antibodies. Several high molecular weight glycoproteins in both S. haematobium and S. japonicum contain the Lexantigen, but no sialyl Lexantigen was detected. In addition, sera from humans and rodents infected with S.haematobium and S.japonicum contain antibodies reactive with Lex. These results led us to investigate whether Lexantigens are expressed in other helminths, including the parasitic trematode Fasciola hepatica , the parasitic nematode Dirofilaria immitis (dog heartworm), the ruminant nematode Haemonchus contortus , and the free-living nematode Caenorhabditis elegans . Neither Lexnor sialyl-Lexis detectable in these other helminths. Furthermore, none of the helminths, including schistosomes, express Lea, Leb, Ley, or the H- type 1 antigen. However, several glycoproteins from all helminths analyzed are bound by Lotus tetragonolobus agglutinin , which binds Fucalpha1-->3GlcNAc, and Wisteria floribunda agglutinin, which binds GalNAcbeta1-->4GlcNAc (lacdiNAc or LDN). Thus, schistosomes may be unique among helminths in expressing the Lexantigen, whereas many different helminths may express alpha1,3-fucosylated glycans and the LDN motif.      

[3H]thymidine incorporation in bovine lymphocytes treated with the tumor promoter, 12-O-tetradecanoyl phorobol-13-acetate

Treatment of bovine lymph node lymphocytes with the tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA) leads to depressed [³H]thymidine incorporation in response to phytohemagglutinin (PHA). Radioautographic and morphological analyses showed that depression was at the level of blast-cell formation. Isotope-dilution experiments, and the use of [³H]deoxycytidine to label DNA indicated that the inhibition was not due to a block in thymidine transport in the treated cells. These experiments, as well as a bioassay designed to measure thymidine in the culture medium, showed that the apparent inhibition of [³H]thymidine incorporation and DNA synthesis was not the result of production of cold thymidine in the cultures. The results taken together support the idea that most TPA-treated cells are inhibited from responding to the mitogenic lectins. Those cells which do respond appear to form blast cells and synthesize DNA at the same rate as do untreated cells.     

Cytokines secreted by bone-metastatic breast cancer cells alter the expression pattern of f-actin and reduce focal adhesion plaques in osteoblasts through PI3K

Breast cancer frequently metastasizes to bone, resulting in osteolytic lesions. These lesions, formed by activated osteoclasts, cause pain, an increased susceptibility to fractures, and hypercalcemia. It has been shown that breast cancer cells communicate with osteoblasts and subsequently stimulate osteoclast activity; however, little research has focused on understanding the interaction between breast cancer cells and osteoblasts. We recently reported that conditioned medium from MDA-MB-231 breast cancer cells inhibited the differentiation of MC3T3-E1 osteoblasts through the secretion of transforming growth factor beta (TGFbeta). In addition, the breast cancer conditioned medium altered MC3T3-E1 morphology, the pattern of actin stress fibers, and reduced focal adhesion plaques. In the current study, we identified the mechanism used by MDA-MB-231 cells to cause these effects. When MC3T3-E1 osteoblasts were cultured with MDA-MB-231 conditioned medium preincubated with neutralizing antibodies to platelet derived growth factor (PDGF), insulin-like growth factorII (IGFII), and TGFbeta, focal adhesion plaques and actin stress fiber formation were restored. These cytokines were further found to signal through PI3Kinase and Rac. In conclusion, TGFbeta, PDGF, and IGFII might be good therapeutic targets for treating breast cancer-induced osteolytic lesions.