Regulation of Reproduction in the Primitively Eusocial Wasp Ropalidia marginata: on the Trail of the Queen Pheromone

Queens and workers are not morphologically differentiated in the primitively eusocial wasp, Ropalidia marginata. Upon removal of the queen, one of the workers becomes extremely aggressive, but immediately drops her aggression if the queen is returned. If the queen is not returned, this hyper-aggressive individual, the potential queen (PQ), will develop her ovaries, lose her hyper-aggression, and become the next colony queen. Because of the non-aggressive nature of the queen, and because the PQ loses her aggression by the time she starts laying eggs, we hypothesized that regulation of worker reproduction in R marginata is mediated by pheromones rather than by physical aggression. Based on the immediate loss of aggression by the PQ upon return of the queen, we developed a bioassay to test whether the queen's Dufour's gland is, at least, one of the sources of the queen pheromone. Macerates of the queen's Dufour's gland, but not that of the worker's Dufour's gland, mimic the queen in making the PQ decrease her aggression. We also correctly distinguished queens and workers of R. marginata nests by a discriminant function analysis based on the chemical composition of their respective Dufour's glands.

Detoxication of benzoate by glycine conjugation in the silkworm, Bombyx mori L

The role of hippuric acid formation as a mechanism for detoxication of benzoic acid in the silkworm has been investigated. Benzoate is inhibitory to the growth of the silkworm and excreted as hippuric acid, which is not toxic. Hippuric acid is not a normal constituent of excreta. Synthesis of hippuric acid has been shown to occur in the intestines of the silkworm. Hippuricase activity is present in the fat body and silk-gland tissue.

Mechanism of glycine uptake by the silk glands of the silkworm Bombyx mori L.

The transport of glycine in vitro into the silk glands of the silkworm has been studied. Glycine accumulates inside the tissue to a concentration higher than that present outside, indicating an active transport mechanism. The kinetics of uptake show a biphasic curve and two apparent Km values for accumulation, 0.33 mM and 5.00 mM. The effect of inhibitors on the energy metabolism of glycine transport is inconclusive. Exchange studies indicate the existence of two pools inside the gland, one that is easily removed by exchange and osmotic shock, and the other which is not. The results obtained conform with the carrier model of Britten and McClure concerning the amino-acid pool in E. coli.

DNA Polymerase4 from the Silk Glands of Bombyx mori

The silk gland of Bombyx mori is a terminally differentiated tissue in which DNA replication continues without cell or nuclear division during larval development. DNA polymerase-delta activity increases in the posterior and middle silk glands during the development period, reaching maximal levels in the middle of the fifth instar larvae. The enzyme has been purified to homogeneity by a series of column chromatographic and affinity purification steps. It is a multimer comprising of three heterogeneous subunits, M(r) 170,000, 70,000, and 42,000. An auxiliary protein from B. mori silk glands, analogous to the proliferating cell nuclear antigen, enhances the processivity of the enzyme and stimulates catalytic activity by 3-fold. This auxiliary protein has also been purified to homogeneity. It is a dimer comprised of a single type M(r) 40,000 subunit. Polymerase-delta possesses an intrinsic 3' --> 5' exonuclease activity which participates in proofreading by mismatch match repair during DNA synthesis and is devoid of any primase activity. DNA polymerase-delta activity could be further distinguished from polymerase-alpha from the same tissue based on its sensitivity to various inhibitors and polyclonal antibodies to the individual enzymes. Like DNA polymerase-alpha, polymerase-delta is also tightly associated with the nuclear matrix. The polymerase alpha-primase complex could be readily separated from polymerase-delta (exonuclease) in the purification protocol adopted. DNA polymerase-delta from B. mori silk glands resembles the mammalian delta-polymerases. Considering that both DNA polymerase-delta and -alpha are present in nearly equal amounts in this highly replicative tissue and their close association with the nuclear matrix, the involvement of both the enzymes in the chromosomal endoreplication process in B. mori is strongly implicated.

Differential transcription of multiple copies of a silk worm gene encoding tRNA

Ten different tRNAGly1 genes from the silk worm, Bombyx mori, have been cloned and characterized. These genes were transcribed in vitro in homologous nuclear extracts from the posterior silk gland (PSG) or nuclear extracts derived from the middle silk gland or ovarian tissues. Although the transcription levels were much higher in the PSG nuclear extracts, the transcriptional efficiency of the individual genes followed a similar pattern in all the extracts. Based on the levels of in vitro transcription, the ten tRNAGly1 genes could be divided into three groups, viz., those which were transcribed at very high levels (e.g., clone pR8), high to medium levels (e.g., pBmil, pBmpl, pBmhl, pBmtl) and low to barely detectable levels (e.g., pBmsl, pBmjl and pBmkl). The coding sequences of all these tRNA genes being identical, the differential transcription suggested that the flanking sequences modulate their transcriptional efficiency. The presence of positive and negative regulatory elements in the 5' flanking regions of these genes was confirmed by transcription competition experiments. A positive element was present in the immediate upstream A + T-rich sequences in all the genes, but no consensus sequences correlating to the transcriptional status could be generated. The presence of negative elements on the other hand was indicated only in some of the genes and therefore may have a role in the differential transcription of these tRNAGly genes in vivo.

Isolation and characterization of DNA polymerase epsilon from the silk glands of Bombyx mori

The silk gland of Bombyx mori, an endomitotically replicative tissue shows high levels of DNA polymerases alpha, delta, and epsilon activities. The ratio of polymerase alpha to that of delta plus epsilon is maintained at 1.1 to 1.3 in both the posterior and middle silk glands for the entire duration of late larval development. The three activities copurify in the initial stages of fractionation through phosphocellulose and DE52 but polymerase alpha gets resolved from the others on hydroxylapatite column. Separation between polymerase delta and epsilon is achieved by chromatography on QAE-Sephadex. DNA polymerase epsilon is a heterodimer comprising of 215- and 42-kDa subunits. The activity is maximum at pH 6.5 and the Km values for dNTPs vary between 3-9 microM. The enzyme possesses an intrinsically associated exonuclease activity which functions in the mismatch repair during DNA synthesis. Both polymerase and 3'-->5' exonuclease activities are associated with the 215-kDa subunit. By itself, DNA polymerase epsilon is processive and the catalytic activity is not enhanced by externally added bPCNA (Bombyx-proliferating cell nuclear antigen, an auxiliary protein for DNA polymerase delta). The enzyme resembles polymerase delta in having the exonuclease activity and in its response to aphidicolin or substrate analogs, but could be distinguished from the latter by its lack of response to the bPCNA and sensitivity to dimethyl sulfoxide. The two enzymes show partial immunological cross-reactivity with each other but no immunological relatedness to polymerase alpha. The absence of the repair enzyme DNA polymerase beta and the presence of substantial levels of polymerase epsilon in the silk glands suggest a possible role for the latter in DNA repair in that tissue.

Structure and activity cyclase activated of OK-GC: A kidney receptor-guanylate cyclase activated by guanylin peptides

Uroguanylin, guanylin, and lymphoguanylin are small peptides that activate renal and intestinal receptor guanylate cyclases (GC). They are structurally similar to bacterial heat-stable enterotoxins (ST) that cause secretory diarrhea. Uroguanylin, guanylin, and ST elicit natriuresis, kaliuresis, and diuresis by direct actions on kidney GC receptors. A 3,762-bp cDNA characterizing a uroguanylin/guanylin/ST receptor was isolated from opossum kidney (OK) cell RNA/cDNA. This kidney cDNA (OK-GC) encodes a mature protein containing 1,049 residues sharing 72.4�75.8% identity with rat, human, and porcine forms of intestinal GC-C receptors. COS or HEK-293 cells expressing OK-GC receptor protein were activated by uroguanylin, guanylin, or ST13 peptides. The 3.8-kb OK-GC mRNA transcript is most abundant in the kidney cortex and intestinal mucosa, with lower mRNA levels observed in urinary bladder, adrenal gland, and myocardium and with no detectable transcripts in skin or stomach mucosa. We propose that OK-GC receptor GC participates in a renal mechanism of action for uroguanylin and/or guanylin in the physiological regulation of urinary sodium, potassium, and water excretion. This renal tubular receptor GC may be a target for circulating uroguanylin in an endocrine link between the intestine and kidney and/or participate in an intrarenal paracrine mechanism for regulation of kidney function via the intracellular second messenger, cGMP.

Structure and activity of OK-GC: a kidney receptor guanylate cyclase activated by guanylin peptides

Uroguanylin, guanylin, and lymphoguanylin are small peptides that activate renal and intestinal receptor guanylate cyclases (GC). They are structurally similar to bacterial heat-stable enterotoxins (ST) that cause secretory diarrhea. Uroguanylin, guanylin, and ST elicit natriuresis, kaliuresis, and diuresis by direct actions on kidney GC receptors. A 3,762-bp cDNA characterizing a uroguanylin/guanylin/ST receptor was isolated from opossum kidney (OK) cell RNA/cDNA. This kidney cDNA (OK-GC) encodes a mature protein containing 1,049 residues sharing 72.4-75.8% identity with rat, human, and porcine forms of intestinal GC-C receptors. COS or HEK-293 cells expressing OK-GC receptor protein were activated by uroguanylin, guanylin, or ST13 peptides. The 3.8-kb OK-GC mRNA transcript is most abundant in the kidney cortex and intestinal mucosa, with lower mRNA levels observed in urinary bladder, adrenal gland, and myocardium and with no detectable transcripts in skin or stomach mucosa. We propose that OK-GC receptor GC participates in a renal mechanism of action for uroguanylin and/or guanylin in the physiological regulation of urinary sodium, potassium, and water excretion. This renal tubular receptor GC may be a target for circulating uroguanylin in an endocrine link between the intestine and kidney and/or participate in an intrarenal paracrine mechanism for regulation of kidney function via the intracellular second messenger, cGMP.

Systems biology of tuberculosis

Systems biology seeks to study biological systems as a whole, by adopting an integrated approach to study and understand the function of biological systems, particularly, the response of such systems to various perturbations. In this article, we focus on the Indian efforts towards systems-level studies of Mycobacterium tuberculosis and its interaction with the host. Availability of a variety of genome-scale experimental data, providing first level `omics' descriptions of the pathogen, render it feasible to study it at a systems level. Various aspects of the pathogen, from metabolic pathways to protein-protein interaction networks have been modelled and simulated, while host-pathogen interactions have been studied experimentally using siRNA-based techniques. These studies have been useful in obtaining a global perspective of the pathogen and its interactions with the host in many ways. For example, significant insights have been gained about different aspects such as proteins essential for bacterial survival, proteins that are highly influential in the network, pathways that are highly connected, host factors responsible for maintaining the TB infection and key factors involved in autophagy and pathogenesis. A rational pipeline developed for drug target identification incorporating analyses of the interactome, reactome, genome, pocketome and the transcriptome is discussed. Finally, exploring host factors as drug targets and insights about the emergence of drug resistance are also discussed. (C) 2011 Elsevier Ltd. All rights reserved.

Queen signal should be honest to be involved in maintenance of eusociality: chemical correlates of fertility in Ropalidia marginata

Queens of many social insect species are known to maintain reproductive monopoly by pheromonal signalling of fecundity. Queens of the primitively eusocial wasp Ropalidia marginata appear to do so using secretions from their Dufour's glands, whose hydrocarbon composition is correlated with fertility. Solitary nest foundresses of R. marginata are without nestmates; hence expressing a queen signal can be redundant, since there is no one to receive the signal. But if queen pheromone is an honest signal inextricably linked with fertility, it should correlate with fertility and be expressed irrespective of the presence or absence of receivers of the signal, by virtue of being a byproduct of the state of fertility. Hence we compared the Dufour's gland hydrocarbons and ovaries of solitary foundresses with queens and workers of post-emergence nests. Our results suggest that queen pheromone composition in R. marginata is a byproduct of fertility and hence can honestly signal fertility. This provides important new evidence for the honest signalling hypothesis.

Road to Royalty - Transition of Potential Queen to Queen in the Primitively Eusocial Wasp Ropalidia marginata

Ropalidia marginata, a primitively eusocial wasp, is different from typical primitively eusocial species in having docile queens who cannot be using dominance to maintain reproductive monopoly and instead appear to use a pheromone from the Dufour's gland to do so. When a docile queen is removed from her colony, one of the workers (potential queen, PQ) becomes highly aggressive, and if the queen is not returned, gradually loses her aggression and becomes the new docile queen within a few days. We hypothesized that the decrease in aggression of the PQ with time since queen removal should be correlated with her change in ovaries and pheromone profile. Because the Dufour's gland hydrocarbon composition in R.marginata can be correlated with fertility, this also gave us an opportunity to test whether PQ is different from workers in her Dufour's gland hydrocarbons. In this study, we therefore trace the road to royalty in R.marginata, that is, the transition of the PQ during queen establishment, in terms of her ovaries, aggression, and Dufour's gland hydrocarbons. Our study focuses on queen establishment, which is important for understanding how reproductive conflict can be manifested and resolved.

Detailed investigation of a gamma-cyclodextrin inclusion complex with L-thyroxine for improved pharmaceutical formulations

Thyroxine is a naturally occurring human hormone produced by the thyroid gland. Clinical applications of thyroxine to treat several chronic disorders are limited by poor water solubility and instability under physiological conditions. An inclusion complex of levo-thyroxine (l-thyroxine), the active form of the hormone with gamma cyclodextrin (gamma-CD) has been obtained and studied with the aim of improving oral delivery rather than the injection formulation of the sodium salt. In addition to greater patient acceptability, inclusion complexes often improve aqueous solubility and bioavailability, stability, and reduce toxicity of drugs, thus providing enhanced pharmaceutical formulations. Physicochemical characterization of the inclusion complex was carried out using Fourier transform infrared spectroscopy, X-ray diffractometry, differential scanning calorimetry, scanning electron microscopy and proton nuclear magnetic resonance spectroscopy. Intermolecular dipolar interactions for the inclusion complex were also studied using 2 dimensional ROESY experiments. Formation of the inclusion complex between the protons H3 and H5 of cyclodextrin with aromatic protons of thyroxine was confirmed by their dipolar interaction. Molecular modelling was used to understand the basis for the complex formation and predict the formation of other complexes. Interestingly, we found that l-thyroxine forms an inclusion complex only with the larger gamma-CD and not with other available alpha and beta forms.

A new species of Duttaphrynus (Anura : Bufonidae) from Northeast India

A new species of montane toad Duttaphrynus is described from Nagaland state of Northeast India. The new species is diagnosable based on following combination of characters: absence of preorbital, postorbital and orbitotympanic ridges, elongated and broad parotid gland, first finger longer than second and presence of a mid-dorsal line. The tympanum is hidden under a skin fold (in male) or absent (in female). The species is compared with its congers from India and Indo-China. We propose to consider Duttaphrynus wokhaensis as junior synonym of Duttaphrynus melanostictus.

Regulation of protumorigenic pathways by Insulin like growth factor binding protein2 and its association along with beta-catenin in breast cancer lymph node metastasis

Background: Insulin like growth factor binding proteins modulate the mitogenic and pro survival effects of IGF. Elevated expression of IGFBP2 is associated with progression of tumors that include prostate, ovarian, glioma among others. Though implicated in the progression of breast cancer, the molecular mechanisms involved in IGFBP2 actions are not well defined. This study investigates the molecular targets and biological pathways targeted by IGFBP2 in breast cancer. Methods: Transcriptome analysis of breast tumor cells (BT474) with stable knockdown of IGFBP2 and breast tumors having differential expression of IGFBP2 by immunohistochemistry was performed using microarray. Differential gene expression was established using R-Bioconductor package. For validation, gene expression was determined by qPCR. Inhibitors of IGF1R and integrin pathway were utilized to study the mechanism of regulation of beta-catenin. Immunohistochemical and immunocytochemical staining was performed on breast tumors and experimental cells, respectively for beta-catenin and IGFBP2 expression. Results: Knockdown of IGFBP2 resulted in differential expression of 2067 up regulated and 2002 down regulated genes in breast cancer cells. Down regulated genes principally belong to cell cycle, DNA replication, repair, p53 signaling, oxidative phosphorylation, Wnt signaling. Whole genome expression analysis of breast tumors with or without IGFBP2 expression indicated changes in genes belonging to Focal adhesion, Map kinase and Wnt signaling pathways. Interestingly, IGFBP2 knockdown clones showed reduced expression of beta-catenin compared to control cells which was restored upon IGFBP2 re-expression. The regulation of beta-catenin by IGFBP2 was found to be IGF1R and integrin pathway dependent. Furthermore, IGFBP2 and beta-catenin are co-ordinately overexpressed in breast tumors and correlate with lymph node metastasis. Conclusion: This study highlights regulation of beta-catenin by IGFBP2 in breast cancer cells and most importantly, combined expression of IGFBP2 and beta-catenin is associated with lymph node metastasis of breast tumors.

Antithyroid drugs and their analogues: synthesis, structure, and mechanism of action

Thyroid hormones are essential for the development and differentiation of all cells of the human body. They regulate protein, fat, and carbohydrate metabolism. In this Account, we discuss the synthesis, structure, and mechanism of action of thyroid hormones and their analogues. The prohormone thyroxine (14) is synthesized on thyroglobulin by thyroid peroxidase (TPO), a heme enzyme that uses iodide and hydrogen peroxide to perform iodination and phenolic coupling reactions. The monodeiodination of T4 to 3,3',5-triiodothyronine (13) by selenium-containing deiodinases (ID-1, ID-2) is a key step in the activation of thyroid hormones. The type 3 deiodinase (ID-3) catalyzes the deactivation of thyroid hormone in a process that removes iodine selectively from the tyrosyl ring of T4 to produce 3,3',5'-triiodothyronine (rT3). Several physiological and pathological stimuli influence thyroid hormone synthesis. The overproduction of thyroid hormones leads to hyperthyroidism, which is treated by antithyroid drugs that either inhibit the thyroid hormone biosynthesis and/or decrease the conversion of T4 to T3. Antithyroid drugs are thiourea-based compounds, which indude propylthiouracil (PTU), methimazole (MM I), and carbimazole (CBZ). The thyroid gland actively concentrates these heterocyclic compounds against a concentration gradient Recently, the selenium analogues of PTU, MMI, and CBZ attracted significant attention because the selenium moiety in these compounds has a higher nucleophilicity than that of the sulfur moiety. Researchers have developed new methods for the synthesis of the selenium compounds. Several experimental and theoretical investigations revealed that the selone (C=Se) in the selenium analogues is more polarized than the thione (C=S) in the sulfur compounds, and the selones exist predominantly in their zwitterionic forms. Although the thionamide-based antithyroid drugs have been used for almost 70 years, the mechanism of their action is not completely understood. Most investigations have revealed that MMI and PTU irreversibly inhibit TPO. PTU, MTU, and their selenium analogues also inhibit ID-1, most likely by reacting with the selenenyl iodide intermediate. The good ID-1 inhibitory activity of Pill and its analogues can be ascribed to the presence of the -N(H)-C(=O)- functionality that can form hydrogen bonds with nearby amino add residues in the selenenyl sulfide state. In addition to the TPO and ID-1 inhibition, the selenium analogues are very good antioxidants. In the presence of cellular reducing agents such as GSH, these compounds catalytically reduce hydrogen peroxide. They can also efficiently scavenge peroxynitrite, a potent biological oxidant and nitrating agent.