Cloning of insertion sequence IS1485 from Enterococcus species.

We have cloned and identified an insertion sequence, IS1485, that was present in several members of the genus Enterococcus. IS1485 exists in varying copy numbers with at least 12 copies in E. durans (ATCC 11576), 3 copies in E. faecium (ATCC 19434), and one copy each in E. faecalis (ATCC 19433) and E. avium (ATCC 14024). It was also detected in clinical strains of E. gallinarum, E. casseliflavus, and E. saccharolyticus. IS1485 is 1366 bp in length, it has imperfect terminal inverted repeats with 25 of the terminal 39 residues matched, and it contains three open reading frames exceeding 50 codons, designated orfA, orfB, and orfC. The largest, orfB, was located 36 bp downstream and in the -1 reading frame relative to orfA; orfC is oriented in the opposite direction and overlaps orfA. The genetic organization of IS1485 resembles that of members of the IS3 family of transposable elements. Sequence homology exists with several members of the IS3 family especially with IS199 from Streptococcus mutans.     

Effect of STI-571 (imatinib mesylate) in combination with retinoic acid and gamma-irradiation on viability of neuroblastoma cells

Neuroblastoma (NB) expresses the tyrosine kinase receptors c-Kit, PDGFR-alpha and -beta-targets for STI-571.We investigated a possible combination therapy of STI-571 with retinoic acid (RA) and gamma-irradiation on NB cell viability in vitro. Expression of tyrosine kinase receptors and their ligands was examined in 6 NB cell lines by RT-PCR and FACS. The effect on cell viability was determined by MTT assay. Cell viability of all 6 NB cell lines was significantly inhibited after treatment with 20 microM STI-571 for 72h, two cell lines responding already to 10 microM. Cell lines responded irrespective of their mRNA status or cell surface expression of c-Kit, PDGFR-alpha and -beta. Co-incubation with 9-cis RA sensitized cells to the inhibitory effects of STI-571. However, pre-treatment with 9-cis RA resulted in resistance of NB cell lines to STI-571 and gamma-irradiation. Treatment of NB with STI-571 in combination with 9-cis RA might be a therapeutic strategy for patients in consolidation therapy who have completed gamma-irradiation therapy.     

C-Terminal Determinants of Kir4.2 Channel Expression

Inward rectifier potassium (Kir) channels serve important functional and modulatory roles in a wide variety of cells. While the activity of several members of this channel family are tightly regulated by intracellular messengers such as adenosine triphosphate, G proteins, protein kinases and pH, other members are tonically active and activity is controlled only by the expression level of the protein. In a number of Kir channels, sequence motifs have been identified which determine how effectively the channel is trafficked to and from the plasma membrane. In this report, we identify a number of trafficking determinants in the Kir4.2 channel. Using mutational analysis, we found that truncation of the C terminus of the protein increased current density in Xenopus oocytes, although multiple mutations of the C terminus had no effect on current density. Instead, mutation of a unique region of the channel significantly increased current density. Selective mutation of a putative tyrosine phosphorylation site within this region mimicked the increase in current, suggesting that tyrosine phosphorylation of the protein increases channel retrieval from the membrane (or prevents trafficking to the membrane). Mutation of a previously identified trafficking determinant, K110N, also caused an increase in current density, and combining these mutations caused a multiplicative increase in current, suggesting that these two mutations increase current by independent mechanisms. These data demonstrate novel determinants of Kir4.2 channel expression.     

Behavioural differences during host selection between alate virginoparae of generalist and tobacco-specialist Myzus persicae

Host plant selection and acceptance by aphids involves four consecutive steps: (1) prealighting behaviour, (2) leaf surface exploration and probing of subepidermal tissues, (3) deep probing of plant tissues, and (4) evaluation of the phloem sap. Host specialisation in aphids may involve not only different performances on potential hosts, but also different strategies for host selection and acceptance. Myzus persicae s.s. (Sulzer) (Homoptera: Aphididae) is one of the most polyphagous aphid species, although a tobacco‐adapted subspecies, M. persicae nicotianae, has been described. These two taxa constitute a good system for studying the effect of host range on host selection strategies. We studied the first two steps in the host selection process by alate virginoparae of M. persicae s.s. and M. persicae nicotianae on host and non‐host plants, using three types of behavioural assays: wind tunnel, olfactometry, and video‐recording. Alate virginoparae of M. persicae nicotianae recognised and chose their host plant more efficiently than M. persicae s.s., on the basis of olfactory and visual cues, and factors residing at cuticular and subcuticular levels. Host recognition was evident before phloem tissues were contacted. Olfactory cues were apparently not involved in host selection by M. persicae s.s.     

Wnt-4 Expression Induces a Pregnancy-like Growth Pattern in Reconstituted Mammary Glands in Virgin Mice

Expression of Wnt-4, a member of the Wnt gene family, is induced during early pregnancy in the mouse mammary gland. To investigate the function of Wnt-4, we used a recombinant retrovirus to constitutively express the gene in transplanted mammary epithelium grown in virgin animals. In fully grown glands, Wnt-4 expression resulted in ducts that were more highly branched than normal and caused some premature alveolar development. These changes resembled those seen during pregnancy, suggesting that endogenous Wnt-4 expression may regulate epithelial branching in early pregnancy. The modified growth pattern induced by Wnt-4 expression was similar to that induced by Wnt-1, one of the members of the Wnt gene family activated by mouse mammary tumour virus. As Wnt-1 is not normally expressed in the mammary gland, it may exert its effect on the mammary gland by activating a developmental pathway normally regulated by Wnt-4.     

Hepatitis C Virus Induced Endothelial Inflammatory Response Depends on the Functional Expression of TNFα Receptor Subtype 2

In hepatitis C virus (HCV) infection, morbidity and mortality often result from extrahepatic disease manifestations. We provide evidence for a role of receptors of the innate immune system in virally induced inflammation of the endothelium in vitro and in vivo. Corresponding to the in vitro finding of an HCV-dependent induction of proinflammatory mediators in endothelial cells, mice treated with poly (I:C) exhibit a significant reduction in leukocyte rolling velocity, an increase in leukocyte adhesion to the vessel wall and an increased extravasation of leukocytes. HCV directly promotes activation, adhesion and infiltration of inflammatory cells into the vessel wall by activation of endothelial viral receptors. Poly (I:C) induces the expression of TLR3 in vivo and hereby allows for amplification of all of the aforementioned responses upon viral infection. Proinflammatory effects of viral RNA are specifically mediated by TLR3 and significantly enhanced by tumor necrosis factor alpha (TNFα). HCV-RNA induces the endothelial expression of TNFα and TNFα receptor subtype 2 and we provide evidence that leucocyte adhesion and transmigration in response to activation of viral RNA receptors seem to depend on expression of functional TNFR2. Our results demonstrate that endothelial cells actively participate in immune mediated vascular inflammation caused by viral infections.     

Tracking the Development of Muscular Myoglobin Stores in Mysticete Calves

For marine mammals, the ability to tolerate apnea and make extended dives is a defining adaptive trait, facilitating the exploitation of marine food resources. Elevated levels of myoglobin within the muscles are a consistent hallmark of this trait, allowing oxygen collected at the surface to be stored in the muscles and subsequently used to support extended dives. In mysticetes, the largest of marine predators, details on muscular myoglobin levels are limited. The developmental trajectory of muscular myoglobin stores has yet to be documented and any physiological links between early behavior and the development of muscular myoglobin stores remain unknown. In this study, we used muscle tissue samples from stranded mysticetes to investigate these issues. Samples from three different age cohorts and three species of mysticetes were included (total sample size = 18). Results indicate that in mysticete calves, muscle myoglobin stores comprise only a small percentage (17–23%) of conspecific adult myoglobin complements. Development of elevated myoglobin levels is protracted over the course of extended maturation in mysticetes. Additionally, comparisons of myoglobin levels between and within muscles, along with details of interspecific differences in rates of accumulation of myoglobin in very young mysticetes, suggest that levels of exercise may influence the rate of development of myoglobin stores in young mysticetes. This new information infers a close interplay between the physiology, ontogeny and early life history of young mysticetes and provides new insight into the pressures that may shape adaptive strategies in migratory mysticetes. Furthermore, the study highlights the vulnerability of specific age cohorts to impending changes in the availability of foraging habitat and marine resources.     

Karyotype variation in the South American aphid genus Neuquenaphis (Hemiptera,Aphididae, Neuquenaphidinae)

The endemic South American aphid genus Neuquenaphis (Hemiptera, Aphididae, Neuquenaphidinae) forms an important component of the phytophagous insect fauna associated with southern beeches, Nothofagus (Nothofagaceae), but has not previously been studied cytologically. As part of ongoing studies of the taxonomy, evolution and host relationships of this genus, the karyotypes of 12 species are described and illustrated. Species are mostly distinguishable by differences in number and/or relative lengths of chromosomes, with 2n (female) numbers ranging from 6 to 16. The taxonomic and evolutionary significance of the karyotype variation in this group are discussed.     

Genomic deletion marking an emerging subclone of Francisella tularensis subsp. holarctica in France and the Iberian Peninsula

Francisella tularensis subsp. holarctica is widely disseminated in North America and the boreal and temperate regions of the Eurasian continent. Comparative genomic analyses identified a 1.59-kb genomic deletion specific to F. tularensis subsp. holarctica isolates from Spain and France. Phylogenetic analysis of strains carrying this deletion by multiple-locus variable-number tandem repeat analysis showed that the strains comprise a highly related set of genotypes, implying that these strains were recently introduced or recently emerged by clonal expansion in France and the Iberian Peninsula.