Relative ability of ovine follicle stimulating hormone and itsβ-subunit to generate antibodies having bioneutralization potential in nonhuman primates

The relative ability of ovine follicle stimulating hormone and itsβ-subunit, two potential candidates for male contraceptive vaccine, to generate antibodies in monkeys capable of bioneutralizing follicle stimulating hormone was assessed usingin vitro model systems. Antiserum against native ovine follicle stimulating hormone was found to be highly specific to the intact form with no cross-reactivity with either of the two subunits while the antiserum againstβ-subunit of follicle stimulating hormone could bind to theβ-subunit in its free form as well as when it is combined withα-subunit to form the intact hormone. Both antisera could block the binding of the hormone to the receptor if the hormone was preincubated with the antibody. However, the follicle stimulating hormoneβ-antisera could only inhibit the binding of the hormone partially (33% inhibition) if the antibody and receptor were mixed prior to the addition of the hormone, while antisera to the native follicle stimulating hormone could block the binding completely (100% inhibition) in the same experiment. Similarly antisera to the native follicle stimulating hormone was significantly effective in blocking (100%) response to follicle stimulating hormone but not theβ-subunit antisera (0%) as checked using anin vitro granulosa cell system. Thus the probability of obtaining antibodies of greater bioneutralization potential is much higher if intact hormone is used as an antigen rather than itsβ-subunit as a vaccine. Majority of the work reported here was carried out during the tenure of Visiting Scientist fellowship awarded by the MRC Canada to the first author.     

Mechanism of down regulation of luteinizing hormone receptors and steroidogenesis in corpora lutea

The mechanism of ‘down regulation’ of luteinizing hormone receptors was investigated in pseudopregnant rats using a modified radioimmunoassay capable of measuring endogenous tissue-bound hormone. Treatment of pseudopregnant animals with a desensitizing dose (desensitization treatment) of human chorionic gonadotropin resulted in a decrease in receptor concentration. This decrease was prevented if the animals were treated prior to the desensitization treatment with indomethacin, an inhibitor of prostaglandin biosynthesis, suggesting a role for prostaglandins in down regulation. The desensitization treatment resulted in a time-dependent decrease in subsequent responsiveness of the tissue to luteinizing hormone. Basal progesterone production rate was also decreased following desensitization. Total tissue cholesterol was found to be decreased following desensitization treatment, without any change in the ratio of free to esterified cholesterol. Mitochondrial cholesterol was significantly reduced and pregnenolone production by the mitochondria of desensitized corpora lutea was also markedly reduced. However, when cholesterol was added to the mitochondria of desensitized corpora lutea, pregnenolone production was increased, reaching values almost equal to that shown by the control mitochondria. These results show that decrease in the responsiveness following desensitization treatment is due to, besides receptor loss, decrease in tissue cholesterol, in particular mitochondrial cholesterol. The cholesterol side chain cleavage activity, although low, appears to be functionally intact; the low activity could be attributed to low levels of mitochondrial cholesterol.     

Attenuation of visible light by phytoplankton in a vertically structured ocean: solutions and applications

To find the solution to problems in applied marine optics, wedo not always need to compute the submarine irradiance at alldepths; in many cases, it is sufficient to know the averageattenuation of light for finite layers of arbitrary thickness.If multiple scattering can be neglected, the average attenuationof light in a finite layer of the water column depends onlyon the vertical distribution of the attenuating substances;in open-ocean waters, the most important of these is phytoplankton.It is shown how the average attenuation of monochromatic lightin an arbitrary layer can be determined when the vertical pigmentprofile takes one of two standardized forms: a shifted Gaussianor a triangle. The choice of efficient algorithms to computethe attenuation in a layer using these models is discussed.The extension to polychromatic light involves the selectionof a function to represent the spectral distribution of thespecific absorption coefficient for phytoplankton, as determinedby observation. Chebyshev polynomial representation is shownto be convenient for applications which require the calculationof a weighted wavelength integral. An efficient procedure forthe evaluation of these integrals, Gauss-Chebyshev quadrature,is presented and evaluated. The extension to the computationof bulk properties for discrete layers is straightforward.     

Polydnaviral Ankyrin Proteins Aid Parasitic Wasp Survival by Coordinate and Selective Inhibition of Hematopoietic and Immune NF-kappa B Signaling in Insect Hosts

Polydnaviruses are mutualists of their parasitoid wasps and express genes in immune cells of their Lepidopteran hosts. Polydnaviral genomes carry multiple copies of viral ankyrins or vankyrins. Vankyrin proteins are homologous to IκB proteins, but lack sequences for regulated degradation. We tested if Ichnoviral Vankyrins differentially impede Toll-NF-κB-dependent hematopoietic and immune signaling in a heterologous in vivo Drosophila, system. We first show that hematopoiesis and the cellular encapsulation response against parasitoid wasps are tightly-linked via NF-κB signaling. The niche, which neighbors the larval hematopoietic progenitors, responds to parasite infection. Drosophila NF-κB proteins are expressed in the niche, and non cell-autonomously influence fate choice in basal and parasite-activated hematopoiesis. These effects are blocked by the Vankyrin I²-vank-3, but not by P-vank-1, as is the expression of a NF-κB target transgene. I²-vank-3 and P-vank-1 differentially obstruct cellular and humoral inflammation. Additionally, their maternal expression weakens ventral embryonic patterning. We propose that selective perturbation of NF-κB-IκB interactions in natural hosts of parasitic wasps negatively impacts the outcome of hematopoietic and immune signaling and this immune deficit contributes to parasite survival and species success in nature.     

‘What Do I Know? Should I Participate?’ Considerations on Participation in HIV Related Research among HIV Infected Adults in Bangalore,South India

Background

India has the highest number of HIV infected persons in the world after South Africa. Much HIV related behavioral, clinical and laboratory based research is ongoing in India. Yet little is known on Indian HIV patients'' knowledge of research, their processes of decision making and motives for participation. We aimed to explore these areas among HIV infected individuals to understand their reasons for participating in research.

Methodology/Principal Findings

This is a cross sectional survey among 173 HIV infected adults at a tertiary level hospital in Bangalore, India, done between October 2010 and January 2011. A pre-tested questionnaire was administered to the participants by trained research assistants to assess their knowledge regarding research, willingness to participate, decision making and determinants of participation. Participants were presented with five hypothetical HIV research studies. Each study had a different level of intervention and time commitment. Of respondents, 103(60%), said that research meant ‘to discover something new’ and 138(80%) were willing to participate in research. A third of the respondents were unaware of their right to refuse participation. Willingness to participate in research varied with level of intervention. It was the lowest for the hypothetical study involving sensitive questions followed by the hypothetical drug trial; and was the highest for the hypothetical cross sectional questionnaire based study (p<0.0015). Individual health benefits and altruism were the primary motives for participation in research and indicate the presence of therapeutic misconception. Women were less likely to make autonomous decisions for participation in interventional studies.

Conclusions/Significance

Despite a majority willing to participate, over a third of respondents did not have any knowledge of research or the voluntary nature of participation. This has ethical implications. Researchers need to focus on enabling potential research participants understand the concepts of research, promote autonomous decisions, especially by women and restrict therapeutic misconception.     

A new high performance liquid chromatographic method for quantification of atomoxetine in human plasma and its application for pharmacokinetic study

Atomoxetine is the first, non-stimulant alternative to other stimulant medications used for the treatment of Attention-Deficit/Hyperactivity Disorder (ADHD). Reported methods for the determination of atomoxetine include expensive liquid chromatography tandem mass spectrometry (LCMS) and high performance liquid chromatography (HPLC) with liquid scintillation counting (LSC) detection. Till date, no method has been reported in literature to determine atomoxetine using HPLC with UV detection. In this paper, we describe a new HPLC method for the determination of atomoxetine using liquid-liquid extraction with tertiary butyl methyl ether and UV detector. This method was found to be linear over the concentration range of 0.05-3.0 microg/ml. The limit of quantification was 0.05 microg/ml. Intra- and inter-day precision was <15% and accuracy was in the range of 95.67-108.80%. Stability studies showed that atomoxetine was stable in human plasma for short- and long-term period for sample preparation and analysis. This method was used for sample analysis in a pharmacokinetic study of atomoxetine (25mg) in five healthy adult female volunteers. The observed mean+/-S.D. pharmacokinetic parameters Cmax, Tmax and AUC(0-t) were 0.40+/-0.06 microg/ml, 3.40+/-0.42 h and 1.34+/-0.52 microg h/ml, respectively.     

Quantitative analysis of phenol oxidase activity in insect hemolymph

We describe a simple, inexpensive, and robust protocol for the quantification of phenol oxidase activity in insect hemolymph. Discrete volumes of hemolymph from Drosophila melanogaster larvae are applied to pieces of filter paper soaked in an L-3, 4-dihydroxyphenylalanine (L-DOPA) solution. Phenol oxidase present in the samples catalyzes melanin synthesis from the L-DOPA precursor, resulting in the appearance of a roughly circular melanized spot on the filter paper. The filter paper is then scanned and analyzed with image-processing software. Each pixel in an image is assigned a grayscale value. The mean of the grayscale values for a circular region of pixels at the center of the image of each spot is used to compute a melanization index (MI) value, the computation is based on a comparison to an external standard (India ink). Numerical MI values for control and experimental larvae can then be pooled and subjected to statistical analysis. This protocol was used to evaluate phenol oxidase activity in larvae of different backgrounds: wild-type, lozenge, hopscotch(Tumorous-lethal) (which induces the formation of large melanotic tumors), and body-color mutations ebony and yellow. Our results demonstrate that this assay is sensitive enough for use in genetic screens with D. melanogaster and could conceivably be used for evaluation of MI from hemolymph of other insects.