DNA binding properties of the ecdysteroid receptor in the salivary gland of the female ixodid tick, Amblyomma hebraeum

Salivary gland degeneration in the female tick, Amblyomma hebraeum Koch (Acari: Ixodidae) is controlled by an ecdysteroid hormone. In an earlier study (Mao, H., McBlain, W.A., Kaufman, W.R., 1995. Some properties of the ecdysteroid receptor in the salivary gland of the ixodid tick, Amblyomma hebraeum. Gen. Comp. Endocrinol. 99, 340–348), we demonstrated that a protein component of a salivary gland extract binds to ponasterone A (Pon A) with high affinity (Kd1 nM), suggesting a tick ecdysteroid receptor (EcR). In this study, the Pon A binding protein bound to calf thymus DNA; this binding could be dissociated by Drosophila hsp27 EcRE. The binding protein shifted the [³²P]hsp27 EcRE band on a gel mobility shift assay; formation of the complex with hsp27 EcRE required KCl (optimal concentration was approximately 75 mM). A number of physiologically effective ecdysteroids enhanced the binding with the following order of potency: Pon A>Mur A>Mak A>20E>ecdysone, whereas vertebrate steroids (estradiol, cholesterol, corticosterone, progesterone, testosterone) had no such effect. Using monoclonal antibodies against Drosophila EcR and USP, we found that AG10.2 recognized three bands (90.5, 87.3 and 84 kDa for EcR) and AB11 recognized at least two major bands (50.3 and 47.1 kDa for USP) in the salivary gland extract by western blot analysis. In addition, AB11 supershifted the tick EcR-hsp27 EcRE band on a gel mobility shift assay, indicating that the tick EcR heterodimerized with a USP-like protein for DNA binding. Furthermore, selective mutations to the 15-basepair palindrome of hsp27 EcRE at positions −5, +2, or adding a base to the spacer, resulted in considerably reduced affinity to the tick EcR/USP. We thus propose a sequence similarity of EcREs between A. hebraeum and its insect counterpart.     

Reinforcement selection acting on the European house mouse hybrid zone

Behavioural isolation may lead to complete speciation when partial postzygotic isolation acts in the presence of divergent‐specific mate‐recognition systems. These conditions exist where Mus musculus musculus and M. m. domesticus come into contact and hybridize. We studied two mate‐recognition signal systems, based on urinary and salivary proteins, across a Central European portion of the mouse hybrid zone. Introgression of the genomic regions responsible for these signals: the major urinary proteins (MUPs) and androgen binding proteins (ABPs), respectively, was compared to introgression at loci assumed to be nearly neutral and those under selection against hybridization. The preference of individuals taken from across the zone regarding these signals was measured in Y mazes, and we develop a model for the analysis of the transition of such traits under reinforcement selection. The strongest assortative preferences were found in males for urine and females for ABP. Clinal analyses confirm nearly neutral introgression of an Abp locus and two loci closely linked to the Abp gene cluster, whereas two markers flanking the Mup gene region reveal unexpected introgression. Geographic change in the preference traits matches our reinforcement selection model significantly better than standard cline models. Our study confirms that behavioural barriers are important components of reproductive isolation between the house mouse subspecies.     

Modulation of inwardly rectifying Na+-K+ channels by serotonin and cyclic nucleotides in salivary gland cells of the leech,Haementeria

Summary The electrically excitable salivary cells of the giant Amazon leech, Haementeria, display a time-dependent inward rectification. Under voltage clamp, hyperpolarizing steps to membrane potentials negative to about –70 mV were associated with the activation of a slow inward current (I _h) which showed no inactivation with time. The time course of activation of I _hwas described by a single-exponential function and was strongly voltage dependent. The activation curve of_hranged from –72 to –118 mV, with half-activation occurring at –100 mV. Ion-substitution experiments indicated that I _his carried by both Na⁺ and K⁺ ions. 5-Hydroxytryptamine (5-HT) increased the amplitude of I _hand its rale of activation. It also produced a positive shift of the activation curve of the conductance underlying I _h G_hwithout altering the slope factor, thus indicating that the voltage dependence of I _hwas modulated by 5-HT. Cs⁺ blocked both I _hand the 5-HT-polentiated current in a voltage-independent manner, whereas Ba²⁺ had little effect. It is concluded that 5-HT increases I _hby modulating the inwardly rectifying Na⁺-K⁺ channels in the salivary cells. The effect of 5-HT may be mediated by an increase in adenylate cyclase activity since I _hwas increased by 8-bromocyclic AMP and by the phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine. In contrast, I _hwas reduced by 8-bromo-cyclic GMPand by zaprinast (an inhibitor of cyclic GMP-scnsitive phosphodieslerase). Cyclic GMP itself also reduced I _h, and the effect was specific to the 3,5 form; 2,3-cyclic GMP was inactive. The results suggest that the inward-rectifier channel may be modulated in opposite directions by cyclic AMP and cyclic GMPThis work was supported by a grant from the Science and Engineering Research Council (no. GR/F/17087). We are grateful to the SmithKline (1982) Foundation for provision of a pulse generator     

5′-Nitro-indirubinoxime induces G1 cell cycle arrest and apoptosis in salivary gland adenocarcinoma cells through the inhibition of Notch-1 signaling

Background

5′-Nitro-indirubinoxime (5′-NIO) is a new derivative of indirubin that exhibits anti-cancer activity in a variety of human cancer cells. However, its mechanism has not been fully clarified.

Methods

Human salivary gland adenocarcinoma (SGT) cells were used in this study. Western blot and RT-PCR analyses were performed to determine cellular Notch levels. The cell cycle stage and level of apoptosis were analyzed using flow cytometry analysis.

Results

5′-NIO significantly inhibited the mRNA levels of Notch-1 and Notch-3 and their ligands (Delta1, 2, 3, and Jagged-2) in SGT cells. Immunocytochemistry analysis showed that 5′-NIO specifically decreased the level of Notch-1 in the nucleus. In addition, 5′-NIO induced G1 cell cycle arrest by reducing levels of CDK4 and CDK6 in SGT cells. Using flow cytometry and immunoblotting analysis, we found that 5′-NIO induces apoptosis following the secretion of cytochrome c and the activation of caspase-3 and caspase-7. Intracellular Notch-1 overexpression led to a decrease in G1 phase arrest and an inhibition of 5′-NIO-induced apoptosis.

Conclusion

These observations suggest that 5′-NIO induces cell cycle arrest and apoptosis by down-regulating Notch-1 signaling.

General significance

This study identifies a new mechanism of 5′-NIO-mediated anti-tumor properties. Thus, 5′-NIO could be used as a candidate for salivary gland adenocarcinoma therapeutics.     

Extracellular matrix is required for the survival and differentiation of transplanted hepatic progenitor cells

Engelbreth-Holm-Swarm (EHS) gel has been reported to maintain the mature hepatocyte phenotypes in primary cultured hepatocytes. We investigated the effect of EHS gel on the differentiation of fetal liver cells, which contain stem/progenitor cells. The isolated fetal liver cells cultured on EHS gel formed a spherical shape and increased liver-specific gene expressions compared with cells cultured on collagen. The hepatic progenitor cells that were transplanted subcutaneously to BALB/c nude mice could survive and express hepatocyte marker alpha-fetoprotein when the cells were suspended with EHS gel. These findings demonstrate that EHS gel supports cytodifferentiation from immature progenitor cells to hepatocytes and maintain its differentiated phenotypes in vitro and in vivo.     

Acute oxidative stress is associated with cell proliferation in the mouse liver

Oxidative stress is known to produce tissue injury and to activate various signaling pathways. To investigate the molecular events linked to acute oxidative stress in mouse liver, we injected a toxic dose of paraquat. Liver necrosis was first observed, followed by histological marks of cell proliferation. Concomitantly, activation of the MAP kinase pathway and increased levels of the anti-apoptotic protein Bcl-XL were observed. Gene expression profiles revealed that the differentially expressed genes were potentially involved in cell proliferation. These data suggest that paraquat-induced acute oxidative stress triggers the activation of regeneration-related events in the liver.     

Growth of the male accessory gland in adult locusts: Roles of juvenile hormone,JH esterase,and JH binding proteins

The participation of juvenile hormone (JH) in the regulation of growth and protein synthesis in the accessory reproductive gland of male Locusta migratoria has been investigated. After elimination of endogenous JH with ethoxyprecocene, the accessory gland failed to grow, but growth was restored by a single application of the JH analog, pyriproxyfen. Pyriproxyfen appeared to stimulate total protein synthesis by 3 h, with a significant effect by 12 h, in contrast to 24 h observed in fat body. The dose curve for stimulation of protein synthesis 12 h after applying pyriproxyfen gave an ED₅₀ of 0.1 μg; the dose curve for gland growth at 72 h was biphasic, with steps at about 0.01 μg and 10 μg, suggesting two phases in JH action. SDS-PAGE analysis showed several components that were stimulated by pyriproxyfen, the effect being strongest in an 11 kDa band. A 5 kDa component was enhanced in the soluble and reduced in the particulate fraction after precocene treatment. The accessory gland contained JH esterase activity at levels about 100 times those in fat body or hemolymph, and was higher in precocene treated locusts. Binding activity for [³H]10R -JH III was high in cytosolic and nuclear fractions, and was identified immunologically as due to the previously described hemolymph JH binding protein. The results indicate that the mode of action of JH in the accessory gland may differ from that in the fat body. The presence of intracellular JH binding protein suggests a direct action of JH within the gland, that may be modulated by JH esterase. © 1995 Wiley-Liss, Inc.     

Clonal proliferation of multipotent stem/progenitor cells in the neonatal and adult salivary glands

Salivary gland stem/progenitor cells are thought to be present in intercalated ductal cells, but the fact is unclear. In this study, we sought to clarify if stem/progenitor cells are present in submandibular glands using colony assay, which is one of the stem cell assay methods. Using a low-density culture of submandibular gland cells of neonatal rats, we developed a novel culture system that promotes single cell colony formation. Average doubling time for the colony-forming cells was 24.7 (SD=+/-7.02)h, indicating high proliferative potency. When epidermal growth factor (EGF) and hepatocyte growth factor (HGF) were added to the medium, the number of clonal colonies increased greater than those cultured without growth factors (13.2+/-4.18 vs. 4.5+/-1.73). The RT-PCR and immunostaining demonstrated expressing acinar, ductal, and myoepithelial cell lineage markers. This study demonstrated the presence of the salivary gland stem/progenitor cells that are highly proliferative and multipotent in salivary glands.     

The Anopheles gambiae cE5 salivary protein: a sensitive biomarker to evaluate the efficacy of insecticide-treated nets in malaria vector control

Evaluation of vector control is crucial for improving malaria containment and, according to World Health Organization, new complementary indicators would be very valuable. In this study the IgG response to the Anopheles-specific cE5 salivary protein was tested as a tool to evaluate the efficacy of insecticide-treated nets in reducing human exposure to malaria vectors. Sera collected during a longitudinal study carried out in Angola, and including entomological and parasitological data, were used to assess the IgG response to the Anopheles gambiae cE5 in both children and adults, before and after the application of insecticide-treated nets. Seasonal fluctuation of specific IgG antibody levels according to exposure was only found in children (up to ≈14 years old) whose anti-cE5 IgG response dropped after bed nets installation. These results were fully consistent with previous findings obtained with the same set of sera and indicating a substantial reduction of human-vector contact shortly after nets implementation. Overall, children IgG response to the cE5 protein appeared a very sensitive biomarker, which allowed for the detection of even weak exposure to Anopheles bites, indicating it may represent a reliable additional tool to evaluate the efficacy of vector control interventions.     

New insights into the autoinhibition mechanism of glycogen synthase kinase-3beta

It has been suggested that phosphorylation at serine 9 near the N-terminus of glycogen synthase kinase-3β (GSK-3β) mimics the prephosphorylation of its substrate and, therefore, the N-terminus functions as a pseudosubstrate. The molecular basis for the pseudosubstrate's binding to the catalytic core and autoinhibition has not been fully defined. Here, we combined biochemical and computational analyses to identify the potential residues within the N-terminus and the catalytic core engaged in autoinhibition of GSK-3β. Bioinformatic analysis found Arg4, Arg6, and Ser9 in the pseudosubstrate sequence to be extremely conserved through evolution. Mutations at Arg4 and Arg6 to alanine enhanced GSK-3β kinase activity and impaired its ability to autophosphorylate at Ser9. In addition, and unlike wild-type GSK-3β, these mutants were unable to undergo autoinhibition by phosphorylated Ser9. We further show that Gln89 and Asn95, located within the catalytic core, interact with the pseudosubstrate. Mutation at these sites prevented inhibition by phosphorylated Ser9. Furthermore, the respective mutants were not inhibited by a phosphorylated pseudosubstrate peptide inhibitor. Finally, computational docking of the pseudosubstrate into the catalytic active site of the kinase suggested specific interactions between Arg6 and Asn95 and of Arg4 to Asp181 (apart from the interaction of phosphorylated serine 9 with the “phosphate binding pocket”). Altogether, our study supports a model of GSK-3-pseudosubstrate autoregulation that involves phosphorylated Ser9, Arg4, and Arg6 within the N-terminus and identified the specific contact sites within the catalytic core.     

Cytoplasmic TSC-22 (transforming growth factor-beta-stimulated clone-22) markedly enhances the radiation sensitivity of salivary gland cancer cells

We transfected a salivary gland cancer cell line, TYS, with three different forms of TSC-22 (transforming growth factor-beta-stimulated clone-22) gene: full-length TSC-22 (TSC-22FL) containing nuclear export signal, TSC-box and leucine zipper, truncated TSC-22 (TSC-22LZ) containing only TSC-box and leucine zipper, and truncated TSC-22 with nuclear localization signal (NLS-TSC-22LZ). High expression of TSC-22FL in the cytoplasm markedly enhanced the radiation-sensitivity of TYS cells, while, moderate expression of TSC-22FL marginally affected the radiation-sensitivity. TSC-22LZ, which was expressed in the cytoplasm and the nucleus, enhanced the radiation-sensitivity of TYS cells irrespective to its expression level. NLS-TSC-22LZ, which was expressed only in the nucleus, marginally affected the radiation-sensitivity of the cells even at high expression level. Interestingly, cytoplasmic TSC-22 translocates to nucleus concomitant with radiation-induced apoptosis. These results suggest that cytoplasmic localization of TSC-22 and translocation of TSC-22 from cytoplasm to nucleus is important for regulating the cell death signal after irradiation-induced DNA damage.     

Production and secretion in CHO cells of the extracellular domain of AMOGβ2, a type-II membrane protein

A hybrid gene consisting of the sequences coding for the signal peptide and N terminus of a type-I membrane protein, the neural cell adhesion molecule (N-CAM), and the extracellular domain of the adhesion molecule on glia (AMOG/β2), a type-II membrane protein, was constructed. The sequence was inserted into a eukaryotic expression vector containing the human cytomegalovirus promoter and the glutamine synthetase selection marker, and used to transfect Chinese hamster ovary cells. The resulting stably transformed cell lines produced large amounts of soluble recombinant AMOG/β2 (reAMOG/β2), which was secreted into the culture medium as a heavily glycosylated 40-55-kDa protein. N-terminal sequence analysis revealed that the protein is not cleaved at the natural signal peptide cleavage site of N-CAM, but two amino acids (aa) further downstream. Treatment of reAMOG/β2 with N-glycosidase F (GlycoF) reduced the molecular mass to 27 kDa, corresponding to the calculated mass of the unglycosylated form. In contrast to AMOG/β2 isolated from mouse brain, which is sensitive to endoglycosidase H, the immuno affinity-purified re-protein is more resistant to this treatment, indicating that the sugars attached to reAMOG/β2 are mainly of the complex type. Our results demonstrate the feasibility of secreting the extracellular domain of a type-II membrane protein, which is usually inserted into the membrane with the C terminus facing the extracellular side.     

Immunofluorescence localization of the regulatory subunit type II of cAMP-dependent protein kinase in PC12 and 3T3 cells in different proliferative states

Localization of the regulatory subunit of cAMP-dependent protein kinase type II was studied in proliferating and quiescent fibroblasts 3T3 and in a cell line of neural origin pheochromocytoma PC12. In actively proliferating PCl2 cells the regulatory subunit was found to be localized in the nucleus. Transition of these cells into a quiescent state was accompanied by a regulatory subunit translocation to the cytoplasm. In 3T3 cells the regulatory subunit was localized in the cytoplasm both in the quiescent and proliferating (though less actively than PC12 cells) states. Similar results were obtained both with monoclonal antibodies and with rabbit monospecific antiserum raised against the regulatory subunit type II from pig brain.     

Dermacentor variabilis: regulation of fibroblast migration by tick salivary gland extract and saliva

We examined the effects of tick SGx and saliva on basal- and platelet-derived growth factor (PDGF)-stimulated cell migration and extracellular signal-regulated kinase (ERK) signaling in fibroblasts. Repair of injured monolayers was delayed by SGx pretreatment and was not associated with reductions in cell number. In migration assays, SGx suppressed both basal- and PDGF-stimulated fibroblast movement. Furthermore, SGx and saliva reduced PDGF-stimulated ERK activity. Thus, the delayed repair of monolayer injuries resulted from SGx inhibiting fibroblast migratory responses to chemotactic signals. SGx also suppressed injury- and growth factor-induced ERK activation in renal epithelial OK cells. Our data suggest that maintenance of the tick feeding lesion results, in part, from suppressing ERK signaling and fibroblast migration, events playing integral roles in the wound healing response. The effects of SGx on cells not involved in wound healing suggest that a constituent(s) in tick saliva has global effects on the ERK signaling pathway.