Measurement of FSH in the ovarian tissue by radioimmunoassay: Correlation to serum FSH levels and follicular development in the hamster

A method is described for monitoring the concentration of endogenous receptor-bound gonadotropin in the ovarian tissue. This involved development of a radioimmunoassay procedure, the validity of which for measuring all of the tissue-bound hormone has been established. The specificity of the method of measurement was indicated by the fact that high levels of FSH could be measured only in target tissue such as follicles, while non-target organs showed little FSH. Using this method, the amount of FSH in the non-luteal ovarian tissue of the hamster at different stages of the estrous cycle was quantitated and compared with serum FSH levels found at these times. No correlation could be found between serum and tissue FSH levels at all times. On the morning of estrus, for example, when the serum level of FSH was high, the ovarian concentration was low, and on the evening of diestrus-2 the ovary exhibited high concentration of FSH, despite the serum FSH concentration being low at this time. The highest concentration of FSH in the ovary during the cycle was found on the evening of proestrus. Although a large amount of this was found in the Graafian follicles, a considerable amount could still be found in the �growing� follicles. Ovarian FSH concentration could be considered to be a reflection of FSH receptor content, since preventing the development of FSH receptors by blocking initiation of follicular development during the cycle resulted in a decrease in the concentration of FSH in the ovary. The high concentration of FSH in the ovary seen on the evening of diestrus-2 was not influenced either by varying the concentration of estrogen or by neutralization of LH. Neutralization of FSH on diestrus-2, on the other hand, caused a drastic reduction in the ovarian LH concentration on the next day (i.e. at proestrus), thus suggesting the importance of FSH in the induction of LH receptors.

Temporally distinct roles of ATM and ROS in genotoxic- stress-dependent induction and maintenance of cellular senescence

Cells exposed to genotoxic stress induce cellular senescence through a DNA damage response (DDR) pathway regulated by ATM kinase and reactive oxygen species (ROS). Here, we show that the regulatory roles for ATM kinase and ROS differ during induction and maintenance of cellular senescence. Cells treated with different genotoxic agents were analyzed using specific pathway markers and inhibitors to determine that ATM kinase activation is directly proportional to the dose of the genotoxic stress and that senescence initiation is not dependent on ROS or the p53 status of cells. Cells in which ROS was quenched still activated ATM and initiated the DDR when insulted, and progressed normally to senescence. By contrast, maintenance of a viable senescent state required the presence of ROS as well as activated ATM. Inhibition or removal of either of the components caused cell death in senescent cells, through a deregulated ATM-ROS axis. Overall, our work demonstrates existence of an intricate temporal hierarchy between genotoxic stress, DDR and ROS in cellular senescence. Our model reports the existence of different stages of cellular senescence with distinct regulatory networks.

Effect of pregnant mare serum gonadotropin on the induction and degradation of FSH and LH receptors in the granulosa cell of the immature rat

The relative induction of FSH and LH receptors in the granulosa cells of immature rat ovary by pregnant mare serum gonadotropin (PMSG) has been studied. A single injection of PMSG (15 IU) brought about a 3- and 12-fold increase in FSH and LH receptor concentration,respectively, in the granulosa cells. Maximal concentration was reached by 72 h but the receptor levels showed a sharp decline during the next 24–48 h. The kinetic properties of the newly formed FSH receptors were indistinguishable from the pre-existing ones. The induced FSH receptors were functional as demonstrated by an increase in the in vitro responsiveness of the cells to exogenous FSH in terms of progesterone production. Treatment of immature rats with cyanoketone, an inhibitor of Δ5,3β-hydroxysteroid dehydrogenase, prior to PMSG injection effectively reduced the PMSG-stimulated increase in the serum estradiol, uterine weight and LH receptors but had no effect on the FSH receptor induction. The ability of PMSG to induce gonadotropin receptors can be arrested at any given time by injecting its antibody, thereby suggesting a continuous need for the hormonal inducer. Estrogen in the absence of the primary inducer was unable to maintain the induced LH and FSH receptor concentration. Inhibition of prostaglandin synthesis using indomethacin also had no effect on either the induction or degradation of gonadotropin receptors. Administration of PMSG antiserum, 48 h after PMSG injection, brought about a rapid decline in the induced receptors over the next 24 h, with a rate constant and \iota 1/2 of 0.078 h−1 and 8.9 h for FSH receptors and 0.086 h−1 and 8.0 h for the LH receptors, respectively.

Hormones and Cuscuta development: IAA uptake transport and metabolism in relation to growth in the absence and presence of applied cytokinin

Transport of 1-14C-IAA in successive stem segments of Cuscuta was strictly basipetal in growing and non growing regions of the vine with a flux velocity of 10-12 mm/h (intercept method). This transport showed a distinct peaked profile, increasing from a low value at 10 mm from the apex to a maximum between 50 and 90 mm before declining to a low value again around 160 mm at which elongation growth ceased. The IAA transport profile paralleled the in vivo growth rate profile, though the latter peaked ahead of transport. A better correlation was observed between the profile of growth responsiveness of the vine to exogenous IAA application and the profile of IAA transport. Growth responsiveness was determined as the differential in growth rate of stem segments in vitro in the absence and presence of growth optimal concentration of IAA (10 μm). Retention of exogenous IAA in the stem was maximal where transport decreased, and this coincided with the region of maximal conjugation of applied 1-14C-IAA to aspartic acid to form indoleacetylaspartate (IAAsp). In addition to aspartate, IAA was conjugated to a small extent to an unidentified compound. IAA destruction by decarboxylation was greatest where transport was low, particularly in the nongrowing region, where lignification occurred (i.e., beyond 180 mm). At concentrations up to 20 μM, a pulse of 1-14C-IAA chased by "cold" IAA moved as a peak (with a peak displacement velocity of 12-18 mm/h) in the "growth" region of the vine, but became diffusionlike where growth either fell off steeply or ceased. At a higher (50 μM) IAA concentration, though uptake was not saturated, transport in the growth region became diffusionlike, indicating saturation of the system. Reduced IAA flux in the region where growth responsiveness to IAA declined coincided with the region of increased IAA conjugation. However, it cannot be concluded whether increased IAA conjugation was the cause or effect of decreased IAA flux. Application of benzyladenine to the vines in vivo, a treatment that elicited haustoria formation by 72 h, resulted in the inhibition of both IAA transport and elongation growth rate in the subapical region. In vitro treatment of vine segments with BA similarly increased IAA retention and decreased IAA transport. IAA loss was suppressed, and conjugation to IAAsp was enhanced. © 1989 Springer-Verlag New York Inc.

Structural requirements for the induction and inhibition of 2,3-dihydroxybenzoic acid decarboxylase ofAspergillus niger

2,3-Dihydroxybenzoic acid decarboxylase inAspergillus niger was induced by many substrate analogs including salicylate and gentisate. Catechol, which is the product, induced the enzyme tenfold. The purified enzyme was competitively inhibited by manyortho substituted benzoic acids. The Ki values for salicylate,o-fluoro ando-chloro benzoic acids were 0.12 mM, 0.12 mM, and 0.13 mM respectively; these values were lower than the Km value for the substrate. As the size of the group in theortho position increased, as in the case of bromo- and iodo-derivatives, there was an increase in their Ki values. The C-2 hydroxyl group was essential both for the induction and for interaction with the enzyme. The C-3 hydroxyl group was not necessary for induction or inhibition, but it might be essential for the catalysis.

Densification and microstructure development in spark plasma sintered WC–6 wt% ZrO2 nanocomposites

In this paper, we report the results of a transmission electron microscopy investigation on WC–6 wt% ZrO2nanocomposite, spark plasma sintered at 1300 °C, for varying times of up to 20 min. The primary aim of this work was to understand the evolution of microstructure during such a sintering process. The investigation revealed the presence of nanocrystalline ZrO2particles (30–50 nm) entrapped within submicron WC grains. In addition, relatively coarser ZrO2(60–100 nm) particles were observed to be either attached to WC grain boundaries or located at WC triple grain junctions. The evidence of the presence of a small amount of W2C, supposed to have been formed due to sintering reaction between WC and ZrO2, is presented here. Detailed structural investigation indicated that ZrO2in the spark plasma sintered nanocomposite adopted an orthorhombic crystal structure, and the possible reasons for o-ZrO2formation are explained. The increase in kinetics of densification due to the addition of ZrO2is believed to be caused by the enhanced diffusion kinetics in the presence of nonstoichiometric nanocrystalline ZrO2.

Characterization of phase transformation behaviour and microstructural development of electroless Ni-B coating

Phase transformation behaviour of amorphous electroless Ni-B coating with a targeted composition of Ni-6wt% B is characterized in conjunction with microstructural development and hardness. Microscopic observations of the as-deposited coating display a novel microstructure which is already phase separated at multiple length scales. Spherical colonies of similar to 5 mu m consist of 2-3 mu m nodular regions which are surrounded by similar to 2-3 mu m region that contains fine bands ranging from 10 to 70 nm in width. The appearance of three crystalline phases in this binary system at different stages of heat treatment and the concomitant variation in hardness are shown to arise from nanoscale fluctuations in the as-deposited boron content from 4 to 8 wt%. High temperature annealing reveals continuous crystallization up to 430 degrees C, overlapping with the domain of B loss due to diffusion into the substrate. The implications of such a microstructure for optimal heat treatment procedures are discussed. (C) 2011 Elsevier B.V. All rights reserved.

Stress and texture development during sputtering of yttria, zirconia, and yttria stabilized zirconia films on Si substrates

Understanding and controlling growth stress is a requisite for integrating oxides with Si. Yttria stabilized zirconia (YSZ) is both an important functional oxide and a buffer layer material needed for integrating other functional oxides. Stress evolution during the growth of (100) and (111) oriented YSZ on Si (100) by radio frequency and reactive direct current sputtering has been investigated with an in-situ monitor and correlated with texture evolution. Films nucleated at rates <5 nm/min are found to be (111) oriented and grow predominantly under a compressive steady state stress. Films nucleated at rates >20 nm/min are found to be (100) oriented and grow under tension. A change in growth rate following the nucleation stage does not change the orientation. The value of the final steady state stress varies from -4.7 GPa to 0.3 GPa. The in-situ studies show that the steady state stress generation is a dynamic phenomenon occurring at the growth surface and not decided at film nucleation. The combination of stress evolution and texture evolution data shows that the adatom injection into the grain boundaries is the predominant source of compressive stress and grain boundary formation at the growth surface is the source of tensile stress. (C) 2012 American Institute of Physics. http://dx.doi.org/10.1063/1.4757924]

Antibody-mediated catalysis: Induction and therapeutic relevance

Abzymes are immunoglobulins endowed with enzymatic activities. The catalytic activity of an abzyme resides in the variable domain of the antibody, which is constituted by the close spatial arrangement of amino acid residues involved in catalysis. The origin of abzymes is conferred by the innate diversity of the immunoglobulin gene repertoire. Under deregulated immune conditions, as in autoimmune diseases, the generation of abzymes to self-antigens could be deleterious. Technical advancement in the ability to generate monoclonal antibodies has been exploited in the generation of abzymes with defined specificities and activities. Therapeutic applications of abzymes are being investigated with the generation of monoclonal abzymes against several pathogenesis-associated antigens. Here, we review the different contexts in which abzymes are generated, and we discuss the relevance of monoclonal abzymes for the treatment of human diseases.

THE LEGACY OF GN RAMACHANDRAN AND THE DEVELOPMENT OF STRUCTURAL BIOLOGY IN INDIA

G.N. Ramachandran is among the founding fathers of structural molecular biology. He made pioneering contributions in computational biology, modelling and what we now call bioinformatics. The triple helical coiled coil structure of collagen proposed by him forms the basis of much of collagen research at the molecular level. The Ramachandran map remains the simplest descriptor and tool for validation of protein structures. He has left his imprint on almost all aspects of biomolecular conformation. His contributions in the area of theoretical crystallography have been outstanding. His legacy has provided inspiration for the further development of structural biology in India. After a pause, computational biology and bioinformatics are in a resurgent phase. One of the two schools established by Ramachandran pioneered the development of macromolecular crystallography, which has now grown into an important component of modern biological research in India. Macromolecular NMR studies in the country are presently gathering momentum. Structural biology in India is now poised to again approach heights of the kind that Ramachandran conquered more than a generation ago.