Biomedical subjects
Publications and source records attributed to A Palit.
We present the first reported case of endometroid adenocarcinoma of the uterine cervix in a young girl. The differential diagnosis of a vaginal mass in this age group is usually rhabdomyosarcoma, although other, rarer tumours also occur.
This paper examines the transient kinetics of substrate binding to the Na+/K(+)-ATPase labelled with iodoacetamidofluorescein (IAF) using fluorescence quenching by trinitrophenyl-ATP (TNP-ATP). Earlier work (E.H. Hellen, P.R. Pratap, 1996, Fluorescence quenching of IAF-Na+/K(+)-ATPase via energy transfer to TNP-labelled nucleotide, Proceedings of the VIIIth International Conference on the Na+/K(+)-ATPase, in press) has shown that TNP-nucleotide binds to specific sites (from which unlabelled nucleotide can displace it) and nonspecific sites (from which unlabelled nucleotide cannot displace it). Under stopped-flow conditions, quenching of IAF-enzyme fluorescence was well described by a stretched exponential (F(t) = F infinity + delta F exp[-Bt alpha]). Physically, this function may be interpreted in terms of its inverse Laplace transform phi (k), which describes a distribution of rate-constants; alpha reflects the width of this distribution. As TNP-ATP concentration increased, alpha decreased, reflecting TNP-ATP binding to sites with higher energy barriers. alpha decreased by about the same amount with increasing [TNP-ATP] in the presence of saturating ATP, indicating that the distribution of rate-constants is largely associated with the nonspecific binding sites. However, alpha was significantly less than 1 for ATP-induced fluorescence recovery in the presence of TNP-ATP, indicating that rate-constants associated with specific binding site are also distributed. The distribution of rate-constants for binding to the specific site indicates a distribution in the energy of the transition state for substrate binding. These results suggest that the specific binding site (in either the empty or the full state) may exist in a series of conformations separated by small energy barriers. However, the energy barriers for binding associated with these conformations are significantly distributed.
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The Na+/K(+)-ATPase functions in cells to couple energy from the hydrolysis of ATP to the transport Na+ out and K+ in. The fluorescent probe IAF (iodoacetamidofluorescein) covalently binds to this enzyme, reporting conformational changes without inhibiting enzyme activity. This paper describes experiments using dog kidney enzyme labeled with IAF to examine kinetics of conformational changes resulting from added Na+ and K+, measured in terms of steady-state and stopped-flow fluorescence changes. Kinetics of these fluorescence changes were examined as a function of temperature from two initial conditions: (a) enzyme in the high-fluorescence form (E(high)) was rapidly mixed with varying [K+]; and (b) enzyme in the low-fluorescence form (E(low)) was rapidly mixed with varying [ATP]. These experiments showed: (1) The rate constant for the fluorescence change from E(high) to E(low) was much larger than that for the opposite transition, E(low) to E(high); (2) the apparent free energy of activation (Ea(app)) for the two transitions were different (as estimated from Arrhenius plots); (3) under steady-state conditions, IAF fluorescence did not change when ATP was added to E(low)(K+) in the absence of Na+; (4) the apparent free energy of activation was independent of [K+] for the E(high) to E(low) transition (at 16.4 kcal/mol) but increased with [ATP] for the E(low) to E(high) transition; (5) Ea(app) for the E(low) to E(high) transition with 1 mM ATP was approximately the same as that in the absence of ATP (34 kcal/mol). These results can be interpreted as: (i) in the transition from E(low) to E(high), IAF reported a conformational change that occurred after K+ release to the intracellular side and which is involved in Na+ binding; (ii) Ea(app) increased with [ATP], while increasing the entropy of the transition state. Thus, ATP appeared to destabilize the enzyme during the transition from E(low) to E(high).
An experimental, trivalent, bovine, leptospiral vaccine, containing inactivated Leptospira interrogans serovars pomona and copenhageni and L. borgpetersenii serovar hardjo Hardjobovis, was developed. The experimental vaccine was shown to protect hamsters against virulent challenge with each of the component serovars. In a serological efficacy test in cattle, the experimental vaccine was compared for bioequivalence with a similar product, registered in New Zealand for veterinary use. The experimental vaccine induced higher titres in cattle than the latter mentioned product.
A pilot study for the control of Ph. argentipes, a known vector of kala-azar in India, was carried out using an ecological approach. Of the 15 houses selected for the study 10, including the cattle sheds and latrines, were plastered with a mixture of mud and lime, up to a height of 1.22 m taking care to seal all cracks and crevices. The remaining five houses were left unplastered and were considered as control areas. The pre-treatment and post-treatment resting densities of the sandfly were monitored both in treated and untreated houses. A sudden drop in the sandfly density was noticed in the treated houses, whereas there was no significant reduction in the check houses, suggesting an effective control.
Placental blood was harvested using a novel collection system into packs containing citrate-phosphate-dextrose-adenine and then stored for 28 d. Before and during storage, sterility, adequacy of anticoagulation, blood chemistry, and red cell viability were assessed weekly. The average volume of blood collected was 65 mL (range 30-110 mL) with a 0.42 hematocrit once diluted in anticoagulant. All blood culture specimens were sterile at collection and during storage. Adequate anticoagulation (activated partial thromboplastin time > 90 s) on the day of collection was achieved in 15 specimens, all less than 65 mL of blood volume. Ten specimens, in blood volumes ranging from 65 to 110 mL, had mean activated partial thromboplastin time values of 65.3 +/- 10.2 s. Activated partial thromboplastin time values for all 25 specimens at 1, 2, 3, and 4 wk storage were > 90 s, presumably because of the decline in labile clotting factors after 24 h of storage. Hematocrit, red cell ATP, and red cell shape were maintained during storage. As expected, red cell 2,3-diphosphoglycerate declined and potassium levels rose significantly but were not different than levels reported for adult cells similarly preserved. Based on our results, it appears that placental blood represents a potential source of autologous transfusion for the sick neonate over the first month of life.
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Twelve seronegative cows were vaccinated with an experimental bivalent Leptospira interrogans serovars hardjo and pomona vaccine late in their first pregnancy. Calves born of these dams were divided into 4 equal groups that received this vaccine at 4, 6, 10 and 18 weeks of age, respectively. Before vaccination the group geometric mean titres of maternally-derived circulating antibodies ranged from 2 to 25 for the microscopic agglutination (MA) test and 3 to 35 for enzyme-linked immunosorbent assay (ELISA) using a serovar hardjo outer envelope antigen. Post-vaccination peak titres were 645 to 1612 for MA and 562 to 1037 for ELISA, respectively. Calves vaccinated at the youngest age, had the highest pre-vaccination circulating maternal antibody titres, but showed the smallest rise in post-vaccination antibody titres. Circulating maternal antibody was detected in calves up to 13 weeks of age. All immunised calves were protected against a virulent challenge with serovar hardjo type Hardjobovis, regardless of their age or maternally-derived antibody titres. These findings indicate that calves as young as 4 weeks old, vaccinated in the presence of maternally-derived antibody, can be fully protected against homologous virulent challenge.
The sum-of-sinusoids method of nonlinear system identification has been applied to the light-growth response of the Phycomyces sporangiophore. Experiments were performed on the Phycomyces tracking machine with the wild-type strain with single and double mutants affected in genes madA, madB, and madC. The sum-of-sinusoids test stimuli were applied to the logarithm of the light intensity. The log-mean intensity level was 10(-1) W m-2 and the wavelength was 477 nm. The system identification results are in the form of first- and second-order frequency kernels, which are related to temporal kernels that appear in the Wiener functional series. The first-order kernels agree well with those obtained previously by the white noise method. In particular, the madA madB and madB madC double mutants show very weak responses. With the superior precision of the sum-of-sinusoids methods, we have achieved sufficient resolution to measure and analyze their second-order kernels. The first- and second-order frequency kernels were interpreted by system analysis methods involving a nonlinear parametric model. In addition a nonparametric hypothesis concerning interactions of gene products was tested. Results from the interaction tests confirm the earlier conclusion that the madB and madC gene products interact. In addition, with the enhanced precision and with the extension to nonlinear analysis, we have found evidence of interaction of the madA gene product with the madB and madC gene products. Thus all three genes appear to have mutual interactions, presumably because of their close physical association in a photoreceptor complex.
The light-growth response of Phycomyces has been studied further with the sum-of-sinusoids method in the framework of the Wiener theory of nonlinear system identification. The response was treated as a black box with the logarithm of light intensity as the input and elongation rate as the output. The nonlinear input-output relation of the light-growth response can be represented mathematically by a set of weighting functions called kernels, which appear in the Wiener intergral series. The linear (first-order) kernels of wild type, and of single and double mutants affected in genes madA to madG were determined previously with Gaussian white noise test stimuli, and were used to investigate the interactions among the products of these genes (R.C. Poe, P. Pratap, and E.D. Lipson. 1986. Biol. Cybern. 55:105.). We have used the more precise sum-of-sinusoids method to extend the interaction studies, including both the first- and second-order kernels. Specifically, we have investigated interactions of the madH ("hypertropic") gene product with the madC ("night blind") and madG ("stiff") gene products. Experiments were performed on the Phycomyces tracking machine. The log-mean intensity of the stimulus was 6 x 10(-2) W m-2 and the wavelength was 477 nm. The first- and second-order kernels were analyzed in terms of nonlinear kinetic models. The madH gene product was found to interact with those of madC and madG. This result extends previous findings that themadH gene product is associated with the input and the ouput of the sensory transduction complex for the lightgrowth response.
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The light-growth response of Phycomyces has been studied with the sum-of-sinusoids method of nonlinear system identification (Victor, J.D., and R.M. Shapley, 1980, Biophys. J., 29:459). This transient response of the sporangiophore has been treated as a black-box system with one input (logarithm of the light intensity, I) and one output (elongation rate). The light intensity was modulated so that log I, as a function of time, was a sum of sinusoids. The log-mean intensity was 10(-4) W m-2 and the wavelength was 477 nm. The first- and second-order frequency kernels, which represent the linear and nonlinear behavior of the system, were obtained from the Fourier transform of the response at the appropriate component and combination frequencies. Although the first-order kernel accounts for most of the response, there remains a significant nonlinearity beyond the logarithmic transducer presumed to occur at the input of the sensory transduction chain. From the analysis of the frequency kernels, we have derived a dynamic nonlinear model of the light-growth response system. The model consists of a nonlinear subsystem followed by a linear subsystem. The model parameters were estimated from a combined nonlinear least-squares fit to the first- and second-order frequency kernels.
The light-growth response of the Phycomyces sporangiophore was studied further with the sum-of-sinusoids method of nonlinear system identification. The first- and second-order frequency kernels, which represent the input-output relation of the system, were determined at 12 wavelengths (383-529 nm) and 4 temperatures (17 degrees, 20 degrees, 23 degrees, and 26 degrees C). The parametric model of the light-growth response system, introduced in the preceding paper, consists of nonlinear and linear dynamic subsystems in cascade. The model parameters were analyzed as functions of wavelength and temperature. At longer wavelengths, the system becomes more nonlinear. The latency and the bandwidth (cutoff frequency) of the system also vary significantly with wavelength. In addition, the latency decreases progressively with temperature (Q10 = 1.6). At low temperature (17 degrees C), the bandwidth is reduced. The results indicate that about half of the latency is due to physical processes such as diffusion, and the other half to enzymatic reactions. The dynamics of the nonlinear subsystem also vary with wavelength. The dependence of various model components on wavelength supports the hypothesis that the light-growth response, as well as phototropism, are mediated by multiple interacting photoreceptors.
The light-growth responses of Phycomyces behavioral mutants, defective in genes madB, madC, and madH, were studied with the sum-of-sinusoids method of system identification. Modified phototropic action spectra of these mutants have indicated that they have altered photoreceptors (P. Galland and E.D. Lipson, 1985, Photochem. Photobiol. 41:331). In the two preceding papers, a kinetic model of the light-growth response system was developed and applied to wild-type frequency kernels at several wavelengths and temperatures. The present mutant studies were conducted at wavelength 477 nm. The log-mean intensity was 6 X 10(-2)W m-2 for the madB and madC night-blind mutants, and 10(-4)W m-2 for the madH hypertropic mutant. The prolonged light-growth responses of the madB and madC mutants are reflected in the reduced dynamic order of their frequency kernels. The linear response of the hypertropic mutant is essentially normal, but its nonlinear behavior shows modified dynamics. The behavior of these mutants can be accounted for by suitable modifications of the parametric model of the system. These modifications together support the hypothesis that an integrated complex mediates sensory transduction in the light responses and other responses of the sporangiophore.
The virulence and viability of various serovars of Leptospira interrogans were successfully preserved by storage in liquid nitrogen. Dimethyl sulphoxide at a final concentration of 2.5% (v/v) was added as cryoprotectant to a culture of leptospires grown in Ellinghausen-McCullough-Johnson-Harris medium. Ampoules were cooled at a controlled rate of 1 degree-3 degrees C/min to -70 degrees C, then transferred to the liquid phase of a liquid nitrogen storage unit. Glycerol was discounted as a cryoprotectant as it was found to be approximately 10 times more toxic than dimethyl sulphoxide to four of five serovars used in this study. The viability of nine strains has so far been observed over a period of 8-22 months storage in liquid nitrogen and full viability of all strains has been preserved over this period. Virulence of strains of serovars pomona and hardjo was well preserved, as demonstrated by challenge tests in guinea pigs and domestic pigs.
In the District of Burdwan, a rural area of West Bengal State, India, Japanese encephalitis (JE) virus is endemic. In one village a longitudinal survey was conducted in order to find out whether associations could be established between the density of the vector mosquito Culex vishnui in two types of resting places, the incidence of infections in sentinel chicks exposed at monthly intervals, the prevalence of antibodies in wild birds and the occurrence of clinical infections in the human population. The experiment lasted from August 1981 till August 1982. Meteorologically a summer season (March-June), a rainy season (July-October) and a winter season (November-February) are distinguished. In the sentinel chicks infections were observed in all three seasons; in wild birds antibodies were prevalent throughout the year; these observations suggest perennial transmission of the virus in its maintenance cycle. Human infections were observed periodically with, in August 1982, a pronounced peak; this may point to fluctuations in the level of circulation of the virus in its maintenance cycle and spillover to the human population at times of peak circulation. The peaks may be related to the influx of young non-immune birds and newborn mammals into the animal population in summer. Further studies, including virus isolation attempts from mosquitoes and nestling birds, are required to prove this hypothesis.
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