A novel bioassay system for evaluating the toxicity of Bacillus thuringiensis israelensis against mosquito larvae.

A bioassay system employing acutely toxic concentrations of a spore-crystal mixture of Bacillus thuringiensis subspecies israelensis against fourth instar larvae of Aedes aegypti (L.) is described. Individual larvae are separately exposed to toxin in glass-lined miniature wells or scintillation vials. This method is free from the deaths due to predation among larvae. Such larval deaths are commonly encountered in bioassay groups of 25 larvae as currently specified in the World Health Organization guidelines. Our method offers shortened testing time, increased accuracy, and improved statistical precision.     

Posttranslational modifications of tubulin in teleost photoreceptor cytoskeletons

1. Posttranslational modifications of tubulin by acetylation and detyrosination have been correlated previously with microtubule stability in numerous cell types. 2. In this study, posttranslational modifications of tubulin and their regional distribution within teleost photoreceptor cones and rods are demonstrated immunohistochemically using antibodies specific for acetylated, detyrosinated, or tyrosinated tubulin. 3. Immunolocalization was carried out on isolated whole cones and mechanically detached rod and cone inner/outer segments. 4. Acetylated tubulin within rods and cones is found only in microtubules of the ciliary axoneme of the outer segment. Detyrosinated tubulin is also enriched in axonemes of both rod and cone outer segments. 5. Distributions of tyrosinated and detyrosinated cytoplasmic microtubules differ within cones and rods. In cones, detyrosinated and tyrosinated tubulins are both abundant throughout the cell body. In rods, the ellipsoid and myoid contain much more tyrosinated tubulin than detyrosinated tubulin. Comparisons between whole cones and cone fragments suggest that detyrosinated microtubules are more stable than tyrosinated microtubules in teleost photoreceptors. 6. Our findings provide further evidence that microtubules of teleost cones differ from rod microtubules in their stabilities and rapidity of turnover within the photoreceptor inner segment.     

Evidence for D4 Receptor Regulation of Retinomotor Movement in Isolated Teleost Cone Inner-Outer Segments

Abstract: In the retinas of teleost fish, cone photoreceptors change shape in response to light and circadian signals. They elongate in the dark, contract in the light, and under conditions of constant darkness undergo appropriate movements at expected dusk and dawn. Dopamine induces cones to contract, thus mimicking the effect of light or expected dawn. To identify the receptor subtype responsible for mediating dopamine regulation of cone retinomotor movements, we have carried out pharmacological studies using isolated fragments of teleost cones consisting of cone inner segments-cone outer segments (CIS-COS). Isolated CIS-COS retain the ability to elongate in dark culture and contract when subsequently exposed to light or dopamine. We report that dark-induced elongation of CIS-COS was inhibited by dopamine and its agonists with an effectiveness ranking of dopamine = quinpirole > bromocriptine ⋙SKF-38393. After 60 min of elongation in dark culture, CIS-COS myoids contracted when subsequently cultured in the dark with dopamine or quinpirole. Quinpirole-induced inhibition of elongation and quinpirole-induced contraction were completely blocked by clozapine at 1 µ M or by sulpiride at 100 µ M . These effectiveness profiles for dopamine agonists and antagonists suggest that dopamine regulation of cone retinomotor movement is mediated by a D₄-like receptor.     

Dopamine Induces Light-Adaptive Retinomotor Movements in Bullfrog Cones via D2 Receptors and in Retinal Pigment Epithelium via D1 Receptors

In the eyes of lower vertebrates, retinal photoreceptors and melanin pigment granules of the retinal pigment epithelium (RPE) exhibit characteristic retinomotor movements in response to changes in ambient illumination and to signals from an endogenous circadian clock. We previously reported that 3,4-dihydroxyphenylethylamine (dopamine) mimicked the effect of light on these movements in photo-receptors and RPE cells of green sunfish, Lepomis cyanellus, by interacting with D2 dopaminergic receptors. Here, we report that dopamine also mimics the effect of light on cone and RPE retinomotor movements in bullfrogs, Rana catesbeiana, i.e., dopamine induces cone contraction and RPE pigment dispersion. Dopamine induced cone contraction in isolated dark-adapted bullfrog retinas incubated in constant darkness in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). This effect of dopamine was inhibited by a D2 but not a D1 antagonist and mimicked by a D2 but not a D1 agonist. These results suggest that induction of cone contraction by dopamine is mediated by D2 dopaminergic receptors and that cone adenylate cyclase activity is inhibited. Thus, dopamine acts via the same type of receptor in both bullfrog and green sunfish retinas to induce cone contraction. In contrast, dopamine influences RPE retinomotor movement via different receptors in fish and bullfrog. Dopamine induced light-adaptive pigment dispersion in isolated dark-adapted bullfrog RPE-eyecups incubated in constant darkness in normal Ringer's solution. Because the retina was not present, these experiments demonstrate a direct effect of dopamine on bullfrog RPE. This effect of dopamine on bullfrog RPE was inhibited by a D1 but not a D2 antagonist and mimicked by a D1 but not a D2 agonist. Furthermore, agents that increase the concentration of intracellular cyclic AMP also induced pigment dispersion in dark-adapted bullfrog RPE-eyecups incubated in the dark. These results suggest that dopamine induces pigment dispersion in bullfrog RPE via D1 dopaminergic receptors. Thus, dopamine acts via different receptors on bullfrog (D1) versus green sunfish (D2) RPE to induce pigment dispersion. In addition, inhibitor studies indicate that pigment dispersion is actin dependent in teleost but not in bullfrog RPE. Dopamine-induced pigment dispersion was inhibited by cytochalasin D in isolated RPE sheets of green sunfish but not in RPE-eyecups of bullfrogs. Together, these observations indicate that dopamine mimics the effect of light on cone and RPE retinomotor movements in both fish and bullfrogs. However, in the RPE, different receptors mediate the effect of dopamine, and different cytoskeletal mechanisms are used to affect pigment transport.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparative migration strategies of wild and captive‐bred Asian Houbara Chlamydotis macqueenii

For migratory species, the success of population reintroduction or reinforcement through captive‐bred released individuals depends on survivors undertaking appropriate migrations. We assess whether captive‐bred Asian Houbara Chlamydotis macqueenii from a breeding programme established with locally sourced individuals and released into suitable habitat during spring or summer undertake similar migrations to those of wild birds. Using satellite telemetry, we compare the migrations of 29 captive‐bred juveniles, 10 wild juveniles and 39 wild adults (including three birds first tracked as juveniles), examining migratory propensity (proportion migrating), timing, direction, stopover duration and frequency, efficiency (route deviation), and wintering and breeding season locations. Captive‐bred birds initiated autumn migration an average of 20.6 (±4.6 se) days later and wintered 470.8 km (±76.4) closer to the breeding grounds, mainly in Turkmenistan, northern Iran and Afghanistan, than wild birds, which migrated 1217.8 km (±76.4), predominantly wintering in southern Iran and Pakistan (juveniles and adults were similar). Wintering locations of four surviving captive‐bred birds were similar in subsequent years (median distance to first wintering site = 70.8 km, range 6.56–221.6 km), suggesting that individual captive‐bred birds (but not necessarily their progeny) remain faithful to their first wintering latitude. The migratory performance of captive‐bred birds was otherwise similar to that of wild juveniles. Although the long‐term fitness consequences for captive‐bred birds establishing wintering sites at the northern edge of those occupied by wild birds remain to be quantified, it is clear that the pattern of wild migrations established by long‐term selection is not replicated. If the shorter migration distance of young captive‐bred birds has a physiological rather than a genetic basis, then their progeny may still exhibit wild‐type migration. However, as there is a considerable genetic component to migration, captive breeding management must respect migratory population structure as well as natal and release‐site fidelity.     

A novel interferon regulatory factor (IRF), IRF-10, has a unique role in immune defense and is induced by the v-Rel oncoprotein

The cloning and functional characterization of a novel interferon regulatory factor (IRF), IRF-10, are described. IRF-10 is most closely related to IRF-4 but differs in both its constitutive and inducible expression. The expression of IRF-10 is inducible by interferons (IFNs) and by concanavalin A. In contrast to that of other IRFs, the inducible expression of IRF-10 is characterized by delayed kinetics and requires protein synthesis, suggesting a unique role in the later stages of an antiviral defense. Accordingly, IRF-10 is involved in the upregulation of two primary IFN-gamma target genes (major histocompatibility complex [MHC] class I and guanylate-binding protein) and interferes with the induction of the type I IFN target gene for 2',5'-oligo(A) synthetase. IRF-10 binds the interferon-stimulated response element site of the MHC class I promoter. In contrast to that of IRF-1, which has some of the same functional characteristics, the expression of IRF-10 is not cytotoxic for fibroblasts or B cells. The expression of IRF-10 is induced by the oncogene v-rel, the proto-oncogene c-rel, and IRF-4 in a tissue-specific manner. Moreover, v-Rel and IRF-4 synergistically cooperate in the induction of IRF-10 in fibroblasts. The level of IRF-10 induction in lymphoid cell lines by Rel proteins correlates with Rel transformation potential. These results suggest that IRF-10 plays a role in the late stages of an immune defense by regulating the expression some of the IFN-gamma target genes in the absence of a cytotoxic effect. Furthermore, IRF-10 expression is regulated, at least in part, by members of the Rel/NF-kappa B and IRF families.     

Induction of host gene expression following infection of chicken embryo fibroblasts with oncogenic Marek's disease virus

Microarrays containing 1,126 nonredundant cDNAs selected from a chicken activated T-cell expressed sequence tag database (http://chickest.udel.edu) were used to examine changes in host cell gene expression that accompany infection of chicken embryo fibroblasts (CEF) with Marek's disease virus (MDV). Host genes that were reproducibly induced by infection of CEF with the oncogenic RB1B strain of MDV included macrophage inflammatory protein, interferon response factor 1, interferon-inducible protein, quiescence-specific protein, thymic shared antigen 1, major histocompatibility complex (MHC) class I, MHC class II, beta(2)-microglobulin, clusterin, interleukin-13 receptor alpha chain, ovotransferrin, a serine/threonine kinase, and avian leukosis virus subgroup J glycoprotein.     

Computational and functional analysis of the putative SH2 domain in Janus Kinases

Src homology 2 (SH2) domains interact in a highly specific manner with phosphorylated tyrosine residues on other signaling molecules. Protein tyrosine kinases (PTK) frequently contain SH2 domains, which often control signaling specificity. The Janus Kinases (JAKs) are a family of PTKs involved in signal transduction pathways mediated by various cytokines. Initial characterization of JAKs showed no identifiable SH2 domain. However, we have found substantial evidence supporting the existence of an SH2 domain in JAKs through the use of various web-based computational analysis programs. Predictive secondary and tertiary structures recognize an SH2 domain in JAKs. In addition, a three-dimensional homology model was constructed using the SH2 domains of Src tyrosine kinase and Syp tyrosine phosphatase as templates. These results, in conjunction with preliminary binding studies showing interactions with tyrosine phosphorylated proteins in activated splenocytes, suggest a functional role for this domain in JAKs.     

Species differences in the efficacy of compounds at the nociceptin receptor (ORL1)

Recent studies have identified compounds with reduced efficacy relative to nociceptin/orphanin FQ at the opioid-like receptor ORL1. Utilizing stimulation of [(35)S]GTPgammaS binding as in vitro assays, it was determined that both [Phe(1)psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2) and the hexapeptide Ac-RYYRIK-NH(2) act as partial agonists in CHO cells transfected with either human or mouse ORL1. Maximal activity for both [Phe(1)psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2) and Ac-RYYRIK-NH(2) was significantly greater in cells transfected with the human receptor (90% and 73% in a high expressing clone, 76% and 68% in low expressing clone) rather than the mouse receptor (37.5 and 33%), regardless of receptor number in individual clones. In vitro studies in cells transfected with exaggerated receptor numbers can lead to unreliable estimates of agonist and antagonist activity, however, these studies suggest that animal experiments on the activity of novel compounds may not always be better predictors of the ultimate activity in humans.