Molecular characterization and functional analysis of the vitellogenin receptor in the rice stem borer,Chilo suppressalis

As a member of the low-density lipoprotein receptor (LDLR) superfamily, vitellogenin (Vg) receptor (VgR) is responsible for the uptake of Vg into developing oocytes and is a potential target for pest control. Here, a full-length VgR complementary DNA (named as CsVgR) was isolated and characterized in the rice stem borer, Chilo suppressalis. The composite CsVgR gene contained an open reading frame of 5,484 bp encoding a protein of 1,827 amino acid residues. Structural analysis revealed that CsVgR contained two ligand-binding domains (LBDs) with four Class A (LDLR_A) repeats in LBD1 and seven in LBD2, which was structurally different from most non-Lepidopteran insect VgRs having five repeats in LBD1 and eight in LBD2. The developmental expression analysis showed that CsVgR messenger RNA expression was first detectable in 3-day-old pupae, sharply increased in newly emerged female adults, and reached a peak in 2-day-old female adults. Consistent with most other insects VgRs, CsVgR was exclusively expressed in the ovary. Notably, injection of dsCsVgR into late pupae resulted in fewer follicles in the ovarioles as well as reduced fecundity, suggesting a critical role of CsVgR in female reproduction. These results may contribute to the development of RNA interference-mediated disruption of reproduction as a control strategy of C. suppressalis.     

Molecular characterization of a cDNA encoding vitellogenin in the banana shrimp, Penaeus (Litopenaeus) merguiensis and sites of vitellogenin mRNA expression

In order to determine the primary structure of banana shrimp, Penaeus merguiensis, vitellogenin (Vg), we previously purified vitellin (Vt) from the ovaries of vitellogenic females, and chemically analyzed the N-terminal amino acid sequence of its 78 kDa subunit. In this study, a cDNA from this species encoding Vg was cloned based on the N-terminal amino acid sequence of the major 78 kDa subunit of Vt and conserved sequences of Vg/Vt from other crustacean species. The complete nucleotide sequence of Vg cDNA was achieved by RT-PCR and 5' and 3' rapid amplification of cDNA ends (RACE) approaches. The full-length Vg cDNA consisted of 7,961 nucleotides. The open reading frame of this cDNA encoding a precursor peptide was comprised of 2,586 amino acid residues, with a putative processing site, R-X-K/R-R, recognized by subtilisin-like endoproteases. The deduced amino acid sequence was obtained from the Vg cDNA and its amino acid composition showed a high similarity to that of purified Vt. The deduced primary structure, of P. merguiensis Vg was 91.4% identical to the Vg of Penaeus semisulcatus and was also related to the Vg sequences of six other crustacean species with identities that ranged from 86.9% to 36.6%. In addition, the amino acid sequences corresponding to the signal peptide, N-terminal region and C-terminal region of P. merguiensis Vg were almost identical to the same sequences of the seven other reported crustacean species. Results from RT-PCR analysis showed that Vg mRNA expression was present in both the ovary and hepatopancreas of vitellogenic females but was not detected in other tissues including muscle, heart, and intestine of females or in the hepatopancreas of mature males. These results indicate that the Vg gene may be expressed only by mature P. merguiensis females and that both the ovary and hepatopancreas are possible sites for Vg synthesis in this species of shrimp.     

Molecular evidence for two vitellogenin genes and processing of vitellogenins in the American cockroach, Periplaneta americana

The American cockroach, Periplaneta americana has two vitellins (Vn1 and Vn2) and corresponding vitellogenins (Vg1 and Vg2). Vns/Vgs were separated on the SDS-PAGE as three major polypeptide bands [170, 100 (multisubunits), and 50 kD] and a minor polypeptide band (150 kD) both in the egg (mature terminal oocyte) extract and in the female hemolymph. We previously cloned one Vg (Vg1) cDNA and showed that the 170-kD polypeptide originated from the C-terminus of the Vg1. In the present study, we cloned the other Vg (Vg2) cDNA. It is 5,826 bp long encoding 1,876 amino acid residues (including 16 residues for putative signal peptide) in a single ORF. The deduced amino acid sequences of both Vgs (Vg1 and Vg2) of P. americana showed 30% identity. The GL/ICG motif is followed by eight cysteine residues at conserved locations near the C-terminal and the DGXR motif starts 18 residues upstream of the GL/ICG motif. The chemically determined N-terminal amino acid sequences of the 150-kD and of the 50-kD polypeptides matched exactly with each other and with the deduced N-terminal amino acid sequence of the Vg2 cDNA. The pattern of processing in P. americana Vns/Vgs is discussed.     

The relationship between nutrition,vitellogenin, vitellin and ovarian development in the housefly, Musca domestica L.

The effect of adult nutrition on oögenesis during the first gonotropic cycle was studied in three strains of the housefly, Musca domestica. Two of the strains were anautogenous and the third was autogenous. In these strains, three subunits (51, 43 and 42 kdaltons) of vitellogenin and vitellin were electrophoretically identical using SDS-PAGE electrophoresis for haemolymph proteins of vitellogenic females and for egg extracts. Each developmental stage of the ovary in individual females flies of both autogenous and anautogenous strains fed on either sugar or protein clearly reflected the appearance of electrophoretic bands for vitellogenin and vitellin. Using immunological analysis, a very small amount of vitellogenin was detectable in the haemolymph of previtellogenic flies. The highest level of vitellogenin appeared in the haemolymph at the middle of vitellogenic phase and reached about 25% of the total haemolymph protein. There were differences in vitellogenin concentration in females with mature eggs between the two anautogenous strains: vitellogenin was not detectable in one strain, and the other showed 30% of the maximal level.     

The gamma-chain cytokine/receptor system in fish: more ligands and receptors

The mammalian gamma-chain (γC) cytokine family consists of interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15 and IL-21. They signal through a receptor complex containing the common γC and a private alpha chain, and in the case of IL-2 and IL-15 an additional common IL-2/15Rβ chain. Deficiency of γC signalling in mammals prevents CD4⁺ T cells from developing effector functions and CD8⁺ T cells from developing immunological memory. Thus γC cytokines are critical for the generation and peripheral homeostasis of naïve and memory T cells. This review will give an update on the γC ligands and receptor subunits in fish, and also present some new data on the cloning and expression of a second γC and two IL-2Rβ chains in rainbow trout Oncorhynchus mykiss. In recent years, aided by the availability of sequenced fish genomes and expressed sequence tag databases, five of the six mammalian γC cytokines and their cognate receptors have been discovered in fish, with only the IL-9/IL-9R homologues apparently absent. Paralogues have been discovered in diploid fish and all the receptors described in the tetraploid rainbow trout, including γC itself, IL-2Rβ, IL-4Rα, IL-13Rα1, IL-13Rα2 and IL-2/15Rα, have duplicates. As a consequence of the teleost and salmonid whole genome duplications, even more paralogues may yet be discovered. Some of the paralogues have changes in domain structures and show differential expression and modulation, suggesting the potential for a change in function. Functional characterisation of fish γC cytokines is beginning but made more difficult by the co-existence of so many paralogues of the ligands and their receptors. Initial functional studies have shown that fish γC cytokines can modulate the expression of key cytokines (e.g. interferon-γ, IL-10 and IL-22) of the adaptive immune response, and may thus have promise as adjuvants to improve vaccination efficiency in fish.     

Patterns of haemolymph vitellogenin and ovarian vitellin in the German cockroach, and the role of Juvenile Hormone

Abstract. The concentrations of fat body and haemolymph vitellogenin and ovarian vitellin during the first gonadotropic cycle of the cockroach Blattella germanica (L.) (Dictyoptera, Blattellidae) have been studied. For these purposes, a polyclonal antibody against B. germanica vitellogenin and vitellin has been obtained, and an ELISA to quantify these proteins has been developed. Ovarian vitellin levels follow a pattern which parallels those of basal oocyte growth and Juvenile Hormone production by the corpora allata. This suggests that Juvenile Hormone regulates vitellogenin uptake into oocytes. Fat body and haemolymph vitellogenin levels give cyclic and parallel patterns. However, the cycle of Juvenile Hormone appears delayed with respect to that of vitellogenin. We suggest that the production of Juvenile Hormone, although cyclic in profile, does not modulate alone the cycle of vitellogenin. At least a supplementary mechanism, apparently independent of Juvenile Hormone, may be involved in the decline of vitellogenin production at the end of the vitellogenic cycle.     

黑尾叶蝉卵黄原蛋白受体基因cDNA的克隆、序列分析及表达模式

【目的】卵黄原蛋白受体(vitellogenin receptor,VgR)属于低密度脂蛋白受体,通过介导内吞作用为发育中的卵母细胞摄取卵黄原蛋白,为胚胎发育提供营养物质,在昆虫生殖过程中发挥关键作用。为研究黑尾叶蝉Nephotettix cincticeps VgR(NcVgR)基因的生理功能及其在生殖中的作用,本研究克隆并解析了NcVgR基因的序列,并对其时空表达进行了研究。【方法】根据黑尾叶蝉转录组数据信息,利用RT-PCR克隆了NcVgR基因,并进行了生物信息学分析;利用实时荧光定量PCR研究了不同发育时期、成虫不同组织NcVgR的表达水平。【结果】NcVgR c DNA序列全长6 676 bp,开放阅读框长度5 568 bp,编码1 855个氨基酸,预测编码蛋白的分子量为206 k D,N端前17个氨基酸为信号肽。序列分析显示,NcVgR具有低密度脂蛋白家族的5个经典保守域,即:配体结合域(ligand-binding domain,LBD)、表皮生长因子前体同源域(EGF-precursor homology domain,EGFP)、O-糖链结构域(O-linked sugar domain,OLSD)、跨膜域(transmembrane domain,TMD)和胞质尾域(cytoplasmic domain)。系统发育分析表明,NcVgR与褐飞虱N.lugens VgR亲缘关系最近。实时荧光定量PCR结果显示,NcVgR转录起始时间为5龄若虫,羽化后转录水平逐渐上升,至羽化后8 d达到峰值,随后下降。有意思的是,随着黑尾叶蝉产卵,NcVgR转录水平再次上升,至羽化后16 d达到最高水平。组织定位结果显示,NcVgR在黑尾叶蝉雌成虫卵巢中特异性高表达,而在雌成虫脂肪体和肠道中微量表达,在雌成虫脑及雄成虫中均未检测到表达。【结论】NcVgR在黑尾叶蝉雌成虫卵巢中特异性表达,并且不同发育时期具有不同的表达量,这为研究黑尾叶蝉的生殖调控机理提供了分子信息。     

烟粉虱MEAM1隐种卵黄原蛋白受体基因cDNA的克隆、 序列分析及在不同发育时期的表达

卵黄原蛋白受体（vitellogenin receptor, VgR）是卵黄原蛋白被卵母细胞摄取的关键因子, 在卵黄发生和卵母细胞发育等生理过程中发挥着重要作用。为探讨烟粉虱Bemisia tabaci VgR的功能, 我们采用RT-PCR和RACE等技术扩增了烟粉虱MEAM1隐种B. tabaci Middle East-Asia Minor 1 (MAEM1) 的VgR基因cDNA 全长序列。生物信息学分析表明, 烟粉虱MEAM1隐种的VgR基因cDNA全长5 774 bp, 编码1 919个氨基酸, 推测分子量约201 kDa, N-端前31个氨基酸为信号肽。烟粉虱MEAM1隐种的VgR属于低密度脂蛋白受体（low density lipoprotein receptor, LDLR）家族, 蛋白质三维结构预测分析表明, 该受体具有LDLR家族基因典型的保守功能结构域。通过实时荧光定量PCR技术研究了烟粉虱MEAM1隐种VgR基因不同发育时期的表达, 结果表明VgR基因在伪蛹期开始表达, 并在羽化后1 d达到高峰, 此后逐渐降低, 3 d后又逐渐升高, 直至羽化后7 d达到峰值。研究结果丰富了卵黄原蛋白受体家族基因的数据库, 为今后深入研究并揭示烟粉虱卵黄发生的调控机制奠定了基础。     

Diversity in mammalian tachykinin peptidergic neurons: multiple peptides, receptors, and regulatory mechanisms

The tachykinins comprise a family of closely related peptides that participate in the regulation of diverse biological processes. The tachykinin peptides substance P, neurokinin A, neurokinin A(3-10), neuropeptide K, and neuropeptide gamma are produced from a single preprotachykinin gene as a result of differential RNA splicing and differential posttranslational processing. Another tachykinin, neurokinin B, is produced from a separate preprotachykinin gene. These preprotachykinin mRNAs and peptide products are differentially distributed throughout the nervous system. Three distinct G protein-coupled tachykinin receptors exist for these tachykinin peptides. The three receptors interact differentially with the tachykinin peptides and are uniquely distributed throughout the nervous system. The NK-1 receptor preferentially interacts with substance P, the NK-2 receptor prefers neurokinin A, neuropeptide K, and neuropeptide gamma, and the NK-3 receptor interacts best with neurokinin B. Examples of the roles of tachykinin peptidergic neuronal systems are taken from the spinal cord sensory system and the nigrostriatal extrapyramidal motor system. Analysis of the functional significance of multiple tachykinin peptide systems, receptor-second messenger coupling mechanisms, and developmental and regulatory mechanisms underlying peptide mRNA and receptor expression represent areas of current and future investigation.     

Molecular and pharmacological characterization of muscarinic receptors in retinal pigment epithelium: role in light-adaptive pigment movements

Muscarinic receptors are the predominant cholinergic receptors in the central and peripheral nervous systems. Recently, activation of muscarinic receptors was found to elicit pigment granule dispersion in retinal pigment epithelium isolated from bluegill fish. Pigment granule movement in retinal pigment epithelium is a light-adaptive mechanism in fish. In the present study, we used pharmacological and molecular approaches to identify the muscarinic receptor subtype and the intracellular signaling pathway involved in the pigment granule dispersion in retinal pigment epithelium. Of the muscarinic receptor subtype-specific antagonists used, only antagonists specific for M1 and M3 muscarinic receptors were found to block carbamyl choline (carbachol)-induced pigment granule dispersion. A phospholipase C inhibitor also blocked carbachol-induced pigment granule dispersion, and a similar result was obtained when retinal pigment epithelium was incubated with an inositol trisphosphate receptor inhibitor. We isolated M2 and M5 receptor genes from bluegill and studied their expression. Only M5 was found to be expressed in retinal pigment epithelium. Taken together, pharmacological and molecular evidence suggest that activation of an odd subtype of muscarinic receptor, possibly M5, on fish retinal pigment epithelium induces pigment granule dispersion.     

Structure,haemolymph titre,and regulatory effects of juvenile homone during ovarian maturation in Leucophaea maderae

Juvenile hormone (JH) synthesized and secreted in vitro by the corpora allata of mated adult Leucophaea maderae females was determined to be JH III (methyl-10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate).The haemolymph titre of JH was determined during maturation of the terminal oöcytes in the first reproductive cycle of L. maderae. In virgin females, JH is not detectable in the haemolymph during the first eight days following adult emergence; however, by 10 days after emergence, trace quantities of JH are apparent. Mating stimuli induce a dramatic increase in the concentration of haemolymph JH, with a peak occurring approximately 12 days after mating; thereafter, the JH concentration declines until it has reached an undetectable level 19 days after mating, at the time of chorion deposition.During ovarian maturation, changes in the rates of synthesis of vitellogenin by the fat body and DNA by the ovary correlate closely with the haemolymph titre of JH. However, no such correlation exists between the JH titre and the extensive ovarian protein synthesis that occurs in L. maderae coincident with chorion formation.The effects of JH I and JH III on both vitellogenin synthesis and ovarain DNA synthesis are statistically similar.     

Potent family-18 chitinase inhibitors: x-ray structures, affinities, and binding mechanisms

Six novel inhibitors of Vibrio harveyi chitinase A (VhChiA), a family-18 chitinase homolog, were identified by in vitro screening of a library of pharmacologically active compounds. Unlike the previously identified inhibitors that mimicked the reaction intermediates, crystallographic evidence from 14 VhChiA-inhibitor complexes showed that all of the inhibitor molecules occupied the outer part of the substrate-binding cleft at two hydrophobic areas. The interactions at the aglycone location are well defined and tightly associated with Trp-397 and Trp-275, whereas the interactions at the glycone location are patchy, indicating lower affinity and a loose interaction with two consensus residues, Trp-168 and Val-205. When Trp-275 was substituted with glycine (W275G), the binding affinity toward all of the inhibitors dramatically decreased, and in most structures two inhibitor molecules were found to stack against Trp-397 at the aglycone site. Such results indicate that hydrophobic interactions are important for binding of the newly identified inhibitors by the chitinase. X-ray data and isothermal microcalorimetry showed that the inhibitors occupied the active site of VhChiA in three different binding modes, including single-site binding, independent two-site binding, and sequential two-site binding. The inhibitory effect of dequalinium in the low nanomolar range makes this compound an extremely attractive lead compound for plausible development of therapeutics against human diseases involving chitinase-mediated pathologies.     

Modification of agonist binding moiety in hybrid derivative 5/7-{[2-(4-aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-naphthalen-1-ol/-2-amino versions: Impact on functional activity and selectivity for dopamine D2/D3 receptors

The goal of the present study was to explore, in our previously developed hybrid template, the effect of introduction of additional heterocyclic rings (mimicking catechol hydroxyl groups as bioisosteric replacement) on selectivity and affinity for the D₃ versus D₂ receptor. In addition, we wanted to explore the effect of derivatization of functional groups of the agonist binding moiety in compounds developed by us earlier from the hybrid template. Binding affinity (K_i) of the new compounds was measured with tritiated spiperone as the radioligand and HEK-293 cells expressing either D₂ or D₃ receptors. Functional activity of selected compounds was assessed in the GTPγS binding assay. In the imidazole series, compound 10a exhibited the highest D₃ affinity whereas the indole derivative 13 exhibited similar high D₃ affinity. Functionalization of the amino group in agonist (+)-9d with different sulfonamides derivatives improved the D₃ affinity significantly with (+)-14f exhibiting the highest affinity. However, functionalization of the hydroxyl and amino groups of 15 and (+)-9d, known agonist and partial agonist, to sulfonate ester and amide in general modulated the affinity. In both cases loss of agonist potency resulted from such derivatization.     

Colour receptors in the eye of the digger wasp,Sphex cognatus Smith: Evaluation by selective adaptation

Summary Pigment granule migration in pigment cells and retinula cells of the digger wasp Sphex cognatus Smith was analysed morphologically after light adaptation to natural light, dark adaptation and after four selective chromatic adaptations in the range between 358 nm and 580 nm and used as the index of receptor cell sensitivity. The receptor region of each ommatidium consists of nine retinula cells which form a centrally located rhabdom. Two morphologically and physiologically different visual units can be described, defined by the arrangement of the rhabdomeric microvilli, the topographical relationship of the receptor cells with respect to the eye axes and the unique retinula cell screening pigmentation. These two different sets of ommatidia (type A and B) are randomly distributed in a ratio of 13 throughout the eye (Ribi, 1978b). Chromatic adaptation experiments with wavelengths of 358 nm, 443 nm, 523 nm and 580 nm and subsequent histological examination reveal two UV receptors, two blue receptors and four yellow-green receptors in type A ommatidia and two UV receptors and six green to yellow-green receptors in type B ommatidia. The pigments in cells surrounding each ommatidium (two primary pigment cells, 20 secondary pigment cells and four pigmented cone extensions) were not affected significantly by the adaptation experiments.     

An enzyme-linked immunosorbent assay (ELISA) for measuring vitellogenin in English sole (Pleuronectes vetulus): development, validation and cross-reactivity with other pleuronectids

Vitellogenin (Vtg) is a yolk protein produced in the liver of oviparous animals in response to estrogen. Vitellogenesis is normally observed only in sexually mature females, but it can be induced in male and juvenile animals by exposure to exogenous estradiol (E₂) or substances that mimic estrogens. The abnormal production of Vtg by males can, therefore, be used as a biological indicator for exposure to xenoestrogens. In this study, an enzyme-linked immunosorbent assay (ELISA) for measuring Vtg in English sole (Pleuronectes vetulus) was developed and validated. Plasmatic Vtg was purified from E₂-injected male English sole using DEAE ion-exchange and Sepharose size-exclusion chromatography, and polyclonal antibodies against the purified Vtg protein were generated in rabbits. In this assay, a competition for the Vtg antibody was established between Vtg coated onto microtiter plate wells and free Vtg. Detection of adsorbed antigen–antibody complexes was achieved using a horseradish peroxidase conjugated anti-rabbit secondary antibody whose enzyme activity was revealed with 3,3′,5,5′-tetramethyl benzidine (TMB) substrate. Assay conditions provided a detectable Vtg range of 10–450 ng ml⁻¹ (85–20% of binding) of diluted sample. Plasma dilution curves from vitellogenic female and E₂-treated male English sole showed parallelism with the standard dilution curve. We are presently conducting field and laboratory studies to investigate estrogenic and anti-estrogenic activity resulting from exposure to contaminants.     

Opioid receptors: Some perspectives from early studies of their role in normal physiology,stress responsivity,and in specific addictive diseases

The early history of research on the possible existence of specific opioid receptors and on developing a new form of pharmacotherapy for the treatment of heroin addiction in New York City, from 1960–1973, along with the special relationships between two leading scientists conducting these research efforts, Dr. Eric Simon and Dr. Vincent P. Dole Jr., are presented in a historical perspective. The linkage of these early efforts and the subsequent identification and the elucidation of the effects of exogenous opiates acting at specific opiate receptors in human physiology, including some findings from perspective studies of heroin addicts at time of entry to and during methadone maintenance treatment, are presented in the context of the important clues which thereby were provided concerning the possible roles of the endogenous opioids in normal mammalian physiology. From many of these early clinical research findings and studies in animal models, the hypothesis that the endogenous opioids system may play an important role in stress responsivity was formulated along with the related hypothesis, first presented in the early 1970s, that an atypical responsivity to stress and stressors might be involved in the acquisition and persistence of, and relapse to specific addictive diseases, including heroin addiction, cocaine dependency and alcoholism. More recent studies of the possible involvement of the specific opioid receptors in these three addictive diseases—heroin addiction, cocaine addiction and alcoholism—from our laboratory are discussed in a historical perspective of the development of these ideas from the early research findings of not only Dr. Eric Simon, but his numerous colleagues in opioid research in the United States and throughout the world. Special issue dedicated to Dr. Eric J. Simon.