Novel reproductive mode in a torrent frog Micrixalus saxicola (Jerdon) from the Western Ghats, India

Reproductive modes in anurans are highly diverse despite external fertilization being a constraint. There are 39 reproductive modes documented so far (Wells, 2007). An apparently new reproductive mode is reported in a torrent frog, Micrixalus saxicola, an endemic and ancient anuran frog of the Western Ghats, considering the type of cavity made inside the lotic water body, involvement of the female in digging the cavity and concealing the eggs.

Synthetic Modifications of Estrogens and Androgens

Estrogens the female sex hormones have numerous biological actions. Estradiol is the most abundant estrogen in women before menopause. It influences the development, maturation and function of the female reproductive tract. It also plays a role in mammary cancer. Accordingly determinations of estradiol level in body fluids assist in the evaluation of ovarian function and diagnosis for malignancies. Estriol is the primary estrogen in pregnant women and secreted from the fetoplacental unit. Measurement of estriol in maternal body fluids is the basis of fetoplacental monitoring test. Concentration of estrogens in body fluids is determined by immunoassay. Accuracy of this measurement depends on the availability of a specific antibody. As estrogens are not antigenic, their derivatives (haptens) are coupled with a carrier and this hapten-protein conjugate is used to generate antibodies. Specificity of the generated antibody largely depends on the structure of hapten. Therefore the synthesis of a hapten with a right structure is crucial for the accurate measurement of a steroid. We have synthesised new haptens for estradiol and estriol by adding an alkyl or alkoxy side chain at the C-7 of estrane skeleton. The side chains carry a terminal amino group, which can be used for conjugation with a carrier molecule. Estrogens and their biosynthetic precursor androgens both exist as fatty acid esters. They are known to act as hormone storage but their physiological role is not completely known yet. Our collaborator is studying their effect in cardiovascular diseases. We synthesised fatty acid ester derivatives of several steroids in high yield by a very rapid procedure (in 1 min) under microwave irradiation in an ionic liquid (IL). An expedient regioselective hydrolysis at C-3 of estradiol diesters is also reported. 8-Isoestrogens are compounds of pharmaceutical interests, their synthesis, structure, conformation and biological activity studies are ongoing. 7-Hydroxy-8-isoestradiol and 7-alkyl ether of it were synthesised as well. During this study we have developed a selective O-debenzylation method. A mild route for selective removal of benzylic protection on phenol in presence of benzyl protected alcohol was explored.

Cyclin dependent protein kinases as drug targets – potential in cardiovascular diseases

Complications of atherosclerosis such as myocardial infarction and stroke are the primary cause of death in Western societies. The development of atherosclerotic lesions is a complex process, including endothelial cell dysfunction, inflammation, extracellular matrix alteration and vascular smooth muscle cell (VSMC) proliferation and migration. Various cell cycle regulatory proteins control VSMC proliferation. Protein kinases called cyclin dependent kinases (CDKs) play a major role in regulation of cell cycle progression. At specific phases of the cell cycle, CDKs pair with cyclins to become catalytically active and phosphorylate numerous substrates contributing to cell cycle progression. CDKs are also regulated by cyclin dependent kinase inhibitors, activating and inhibitory phosphorylation, proteolysis and transcription factors. This tight regulation of cell cycle is essential; thus its deregulation is connected to the development of cancer and other proliferative disorders such as atherosclerosis and restenosis as well as neurodegenerative diseases. Proteins of the cell cycle provide potential and attractive targets for drug development. Consequently, various low molecular weight CDK inhibitors have been identified and are in clinical development. Tylophorine is a phenanthroindolizidine alkaloid, which has been shown to inhibit the growth of several human cancer cell lines. It was used in Ayurvedic medicine to treat inflammatory disorders. The aim of this study was to investigate the effect of tylophorine on human umbilical vein smooth muscle cell (HUVSMC) proliferation, cell cycle progression and the expression of various cell cycle regulatory proteins in order to confirm the findings made with tylophorine in rat cells. We used several methods to determine our hypothesis, including cell proliferation assay, western blot and flow cytometric cell cycle distribution analysis. We demonstrated by cell proliferation assay that tylophorine inhibits HUVSMC proliferation dose-dependently with an IC50 value of 164 nM ± 50. Western blot analysis was used to determine the effect of tylophorine on expression of cell cycle regulatory proteins. Tylophorine downregulates cyclin D1 and p21 expression levels. The results of tylophorine’s effect on phosphorylation sites of p53 were not consistent. More sensitive methods are required in order to completely determine this effect. We used flow cytometric cell cycle analysis to investigate whether tylophorine interferes with cell cycle progression and arrests cells in a specific cell cycle phase. Tylophorine was shown to induce the accumulation of asynchronized HUVSMCs in S phase. Tylophorine has a significant effect on cell cycle, but its role as cell cycle regulator in treatment of vascular proliferative diseases and cancer requires more experiments in vitro and in vivo.

Patents, Ethics and Stem Cell Research : The Case of hESC Innovation in Australia, Europe and the United States

Embryonic stem cells offer potentially a ground-breaking insight into health and diseases and are said to offer hope in discovering cures for many ailments unimaginable few years ago. Human embryonic stem cells are undifferentiated, immature cells that possess an amazing ability to develop into almost any body cell such as heart muscle, bone, nerve and blood cells and possibly even organs in due course. This remarkable feature, enabling embryonic stem cells to proliferate indefinitely in vitro (in a test tube), has branded them as a so-called miracle cure . Their potential use in clinical applications provides hope to many sufferers of debilitating and fatal medical conditions. However, the emergence of stem cell research has resulted in intense debates about its promises and dangers. On the one hand, advocates hail its potential, ranging from alleviating and even curing fatal and debilitating diseases such as Parkinson s, diabetes, heart ailments and so forth. On the other hand, opponents decry its dangers, drawing attention to the inherent risks of human embryo destruction, cloning for research purposes and reproductive cloning eventually. Lately, however, the policy battles surrounding human embryonic stem cell innovation have shifted from being a controversial research to scuffles within intellectual property rights. In fact, the ability to obtain patents represents a pivotal factor in the economic success or failure of this new biotechnology. Although, stem cell patents tend to more or less satisfy the standard patentability requirements, they also raise serious ethical and moral questions about the meaning of the exclusions on ethical or moral grounds as found in European and to an extent American and Australian patent laws. At present there is a sort of a calamity over human embryonic stem cell patents in Europe and to an extent in Australia and the United States. This in turn has created a sense of urgency to engage all relevant parties in the discourse on how best to approach patenting of this new form of scientific innovation. In essence, this should become a highly favoured patenting priority. To the contrary, stem cell innovation and its reliance on patent protection risk turmoil, uncertainty, confusion and even a halt on not only stem cell research but also further emerging biotechnology research and development. The patent system is premised upon the fundamental principle of balance which ought to ensure that the temporary monopoly awarded to the inventor equals that of the social benefit provided by the disclosure of the invention. Ensuring and maintaining this balance within the patent system when patenting human embryonic stem cells is of crucial contemporary relevance. Yet, the patenting of human embryonic stem cells raises some fundamental moral, social and legal questions. Overall, the present approach of patenting human embryonic stem cell related inventions is unsatisfactory and ineffective. This draws attention to a specific question which provides for a conceptual framework for this work. That question is the following: how can the investigated patent offices successfully deal with patentability of human embryonic stem cells? This in turn points at the thorny issue of application of the morality clause in this field. In particular, the interpretation of the exclusions on ethical or moral grounds as found in Australian, American and European legislative and judicial precedents. The Thesis seeks to compare laws and legal practices surrounding patentability of human embryonic stem cells in Australia and the United States with that of Europe. By using Europe as the primary case study for lessons and guidance, the central goal of the Thesis then becomes the determination of the type of solutions available to Europe with prospects to apply such to Australia and the United States. The Dissertation purports to define the ethical implications that arise with patenting human embryonic stem cells and intends to offer resolutions to the key ethical dilemmas surrounding patentability of human embryonic stem cells and other morally controversial biotechnology inventions. In particular, the Thesis goal is to propose a functional framework that may be used as a benchmark for an informed discussion on the solution to resolving ethical and legal tensions that come with patentability of human embryonic stem cells in Australian, American and European patent worlds. Key research questions that arise from these objectives and which continuously thread throughout the monograph are: 1. How do common law countries such as Australia and the United States approach and deal with patentability of human embryonic stem cells in their jurisdictions? These practices are then compared to the situation in Europe as represented by the United Kingdom (first two chapters), the Court of Justice of the European Union and the European Patent Office decisions (Chapter 3 onwards) in order to obtain a full picture of the present patenting procedures on the European soil. 2. How are ethical and moral considerations taken into account at patent offices investigated when assessing patentability of human embryonic stem cell related inventions? In order to assess this part, the Thesis evaluates how ethical issues that arise with patent applications are dealt with by: a) Legislative history of the modern patent system from its inception in 15th Century England to present day patent laws. b) Australian, American and European patent offices presently and in the past, including other relevant legal precedents on the subject matter. c) Normative ethical theories. d) The notion of human dignity used as the lowest common denominator for the interpretation of the European morality clause. 3. Given the existence of the morality clause in form of Article 6(1) of the Directive 98/44/EC of the European Parliament and of the Council of 6 July 1998 on the legal protection of biotechnological inventions which corresponds to Article 53(a) European Patent Convention, a special emphasis is put on Europe as a guiding principle for Australia and the United States. Any room for improvement of the European morality clause and Europe s current manner of evaluating ethical tensions surrounding human embryonic stem cell inventions is examined. 4. A summary of options (as represented by Australia, the United States and Europe) available as a basis for the optimal examination procedure of human embryonic stem cell inventions is depicted, whereas the best of such alternatives is deduced in order to create a benchmark framework. This framework is then utilised on and promoted as a tool to assist Europe (as represented by the European Patent Office) in examining human embryonic stem cell patent applications. This method suggests a possibility of implementing an institution solution. 5. Ultimately, a question of whether such reformed European patent system can be used as a founding stone for a potential patent reform in Australia and the United States when examining human embryonic stem cells or other morally controversial inventions is surveyed. The author wishes to emphasise that the guiding thought while carrying out this work is to convey the significance of identifying, analysing and clarifying the ethical tensions surrounding patenting human embryonic stem cells and ultimately present a solution that adequately assesses patentability of human embryonic stem cell inventions and related biotechnologies. In answering the key questions above, the Thesis strives to contribute to the broader stem cell debate about how and to which extent ethical and social positions should be integrated into the patenting procedure in pluralistic and morally divided democracies of Europe and subsequently Australia and the United States.

Reproductive behavior and health in consanguineous marriages

In many regions of Asia and Africa, consanguineous marriages currently account for approximately 20 to 50% of all unions, and preliminary observations indicate that migrants from these areas continue to contract marriages with close relatives when resident in North America and Western Europe. Consanguinity is associated with increased gross fertility, due at least in part to younger maternal age at first livebirth. Morbidity and mortality also may be elevated, resulting in comparable numbers of surviving offspring in consanguineous and nonconsanguineous families. With advances in medicine and public health, genetic disorders will account for an increased proportion of disease worldwide. Predictably, this burden will fall more heavily on countries and communities in which consanguinity is strongly favored, as the result of the expression of deleterious recessive genes. However, studies conducted in such populations indicate that the adverse effects associated with inbreeding are experienced by a minority of families.

Differential reproductive success in Cassia fistula in different habitants-A case of pollinator limitations?

Differences in flower success patterns in two habitat types that differed drastically with respect to rainfall, tree density and species composition were studied at Mudumalai wildlife sanctuary, India. Observations on phenological patterns of two species, Cassia fistula and Gmelina arborea, were made from April 1988 through June 1990. Quantitative data on flower-fruit ratio, insect visitation rates, pollen grain per stigma and the number of fruits per tree were recorded. Data were also collected on the number of pollen deposited on the stigma after different types of bees visited the flower. The data suggested that only carpenter bees (Xylocopa spp) effect pollination in C. fistula. The differences in fruit-flower ratios were attributed to the differences in insect visitation rates to inflorescences between sites. The low pollen number per stigma and the resultant reduction in reproductive success in C. fistula are attributed to the competing species G. arborea receiving more visitations from pollinators in the wetter site. These results suggest that pollinator limitation is another constraint in reproductive success of plants.

Reproductive phenology of a tropical dry forest in Mudumalai, Southern India

1 Flowering and fruiting phenologies of a tropical dry forest in Mudumalai, southern India, were studied between April 1988 and August 1990. Two sites, a wetter site I receiving 1100mm and a drier site II receiving 600mm of rainfall annually, are compared. A total of 286 trees from 38 species at site I and 167 trees from 27 species at site II was marked for phenological observations. There were 11 species common to the two sites. Several hypotheses relating to the evolution of reproductive phenology are tested. 2 Frequency of species flowering attained a peak at site I during the dry season but at site II, where soil moisture may be limiting during the dry months, the peak was during the wet season. At both sites a majority of species flushed leaves and flowered simultaneously. Among various guilds, the bird-pollinated guild showed distinct dry season flowering, which may be related to better advertisement of large flowers to pollinators during the leafless dry phase. The wind-pollinated guild flowered mainly during the wet season, when wind speeds are highest and favourable for pollen transport. The insect-pollinated guild showed no seasonality in flowering in site I but a wet season flowering in site II. 3 Fruiting frequency attained a peak in site I during the late wet season extending into the early dry season; a time-lag correlation showed that fruiting followed rainfall with a lag of about two months. Site II showed a similar fruiting pattern but this was not statistically significant. The dispersal guilds (animal, wind, and explosive passively-dispersed) did not show any clear seasonality in fruiting, except for the animal-dispersed guild which showed wet season fruiting in site I. 4 Hurlbert's overlap index was also calculated in order to look at synchrony in flowering and fruiting irrespective of climatic (dry and wet month) seasonality. In general, overlap in flowering and fruiting guilds was high because of seasonal aggregation. Among the exceptions, at site II the wind-pollinated flowering guild did not show significant overlap between species although flowering aggregated in the wet season. This could be due to the need to avoid heterospecific pollen transfer. 5 Rarer species tended to flower earlier in the dry season and this again could be an adaptation to avoid the risk of heterospecific pollen transfer or competition for pollinators. The more abundant species flowered mainly during the wet season. Species which flower earlier have larger flowers and, having invested more energy in flowers, also have shorter flower to fruit durations.

Effective drug dosage design for treatment of infectious diseases using dynamic inversion

A generic nonlinear mathematical model describing the human immunological dynamics is used to design an effective automatic drug administration scheme. Even though the model describes the effects of various drugs on the dynamic system, this work is confined to the drugs that kill the invading pathogen and heal the affected organ. From a system theoretic point of view, the drug inputs can be interpreted as control inputs, which can be designed based on control theoretic concepts. The controller is designed based on the principle of dynamic inversion and is found to be effective in curing the �nominal model patient� by killing the invading microbes and healing the damaged organ. A major advantage of this technique is that it leads to a closed-form state feedback form of control. It is also proved from a rigorous mathematical analysis that the internal dynamics of the system remains stable when the proposed controller is applied. A robustness study is also carried out for testing the effectiveness of the drug administration scheme for parameter uncertainties. It is observed from simulation studies that the technique has adequate robustness for many �realistic model patients� having off-nominal parameter values as well.

Expression of the receptor guanylyl cyclase C and its ligands in reproductive tissues of the rat: A potential role for a novel signaling pathway in the epididymis

Guanylyl cyclase C (GC-C) is a membrane-associated form of guanylyl cyclase and serves as the receptor for the heat-stable enterotoxin (ST) peptide and endogenous ligands guanylin, uroguanylin, and lymphoguanylin. The major site of expression of GC-C is the intestinal epithelial cell, although GC-C is also expressed in extraintestinal tissue such as the kidney, airway epithelium, perinatal liver, stomach, brain, and adrenal glands. Binding of ligands to GC-C leads to accumulation of intracellular cGMP, the activation of protein kinases G and A, and phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that regulates salt and water secretion. We examined the expression of GC-C and its ligands in various tissues of the reproductive tract of the rat. Using reverse transcriptase and the polymerase chain reaction, we demonstrated the presence of GC-C, uroguanylin, and guanylin mRNA in both male and female reproductive organs. Western blot analysis using a monoclonal antibody to GC-C revealed the presence of differentially glycosylated forms of GC-C in the caput and cauda epididymis. Exogenous addition of uroguanylin to minced epididymal tissue resulted in cGMP accumulation, suggesting an autocrine or endocrine activation of GC-C in this tissue. Immunohistochemical analyses demonstrated expression of GC-C in the tubular epithelial cells of both the caput epididymis and cauda epididymis. Our results suggest that the GC-C signaling pathway could converge on CFTR in the epididymis and perhaps control fluid and ion balance for optimal sperm maturation and storage in this tissue.

Reproductive queue without overt conflict in the primitively eusocial wasp Ropalidia marginata

Colonies of the primitively eusocial wasp Ropalidia marginata consist of a single egg layer (queen) and a number of non-egg-laying workers. Although the queen is a docile individual, not at the top of the behavioral dominance hierarchy of the colony, she maintains complete reproductive monopoly. If the queen is lost or removed, one and only one of the workers potential queen (PQ)] becomes hyperaggressive and will become the next queen of the colony. The PQ is almost never challenged because she first becomes hyperaggressive and then gradually loses her aggression, develops her ovaries, and starts laying eggs. Although we are unable to identify the PQ when the queen is present, she appears to be a ``cryptic heir designate.'' Here, we show that there is not just one heir designate but a long reproductive queue and that PQs take over the role of egg-laying, successively, without overt conflict, as the queen or previous PQs are removed. The dominance rank of an individual is not a significant predictor of its position in the succession hierarchy. The age of an individual is a significant predictor, but it is not a perfect predictor because PQs often bypass older individuals to become successors. We suggest that such a predesignated reproductive queue that is implemented without overt conflict is adaptive in the tropics, where conspecific usurpers from outside the colony, which can take advantage of the anarchy prevailing in a queenless colony and invade it, are likely to be present throughout the year.

NMR studies of preimplantation embryo metabolism in human assisted reproductive techniques: a new biomarker for assessment of embryo implantation potential

There has been growing interest in understanding energy metabolism in human embryos generated using assisted reproductive techniques (ART) for improving the overall success rate of the method. Using NMR spectroscopy as a noninvasive tool, we studied human embryo metabolism to identify specific biomarkers to assess the quality of embryos for their implantation potential. The study was based on estimation of pyruvate, lactate and alanine levels in the growth medium, ISM1, used in the culture of embryos. An NMR study involving 127 embryos from 48 couples revealed that embryos transferred on Day 3 (after 72 h in vitro culture) with successful implantation (pregnancy) exhibited significantly (p < 10(-5)) lower pyruvate/alanine ratios compared to those that failed to implant. Lactate levels in media were similar for all embryos. This implies that in addition to lactate production, successfully implanted embryos use pyruvate to produce alanine and other cellular functions. While pyruvate and alanine individually have been used as biomarkers, the present study highlights the potential of combining them to provide a single parameter that correlates strongly with implantation potential. Copyright (C) 2012 John Wiley & Sons, Ltd.