Study of Spectrum of Chromosomal Rearrangements in Recurrent Pregnancy Loss.

INTRODUCTION: Recurrent pregnancy losses (RPLs) are seen in up to 15-20% of all clinically recognized pregnancies, 1-2% of women in general population. Repeated losses are seen in 5-10% of women. The prevalence of chromosomal rearrangements is 6.65% in couples with repeated pregnancy losses. Two to 4% of RPL are associated with parental balanced reciprocal and Robertsonian translocations. MATERIALS AND METHODS: The study was conducted at a tertiary care hospital in New Delhi, and in total, 204 couples with RPL enrolled in the study. RESULTS: In total, 4490 couples presented to the obstetric clinic, of which 204 (4.5%) couples had repeated pregnancy losses. Cytogenetic analysis was done in 198 couples. Out of total 198 patients, 14 patients (7.1%) had cytogenetic alterations. Most common aberrations observed were structural rearrangements, of which reciprocal translocations were more common. In our study cohort, all the couples had maternal age of ≤ 35 years and all the alterations were seen either in mother or in both parents. DISCUSSION: Our study highlights that cytogenetic alterations not only are common in first trimester miscarriages, but are an important event in miscarriages presenting at later period of gestation and in young mothers as well.

Drought stress induced changes in lipid peroxidation and antioxidant system in genus Avena.

Seven species of genus Avena viz., Avena sativa, Avena strigosa, Avena brevis, Avena vaviloviana, Avena abyssinica, Avena marocana and Avena sterilis were used to study the impact of drought stress on lipid peroxidation and other antioxidant enzymes. Maximum increase in the catalase activity was recorded in A. vaviloviana (129.97%) followed by A. sativa (122.82%) and A. brevis (83.38%) at vegetative stage; however at flowering stage the maximum increase was reported in A. sativa (25.62%) followed by A. sterilis (20.46%) and A. brevis (18.53%). At vegetative stage drought, maximum increase in peroxidase activity was recorded in A. sativa (122.82%) followed by A. brevis (83.38%) and A. sterilis (49.78%). Flowering stage drought, showed maximum increase in A. Sativa (27.09%) followed by A. marocana (23.50%) and A. sterilis (20.46%). A. sativa and A. sterilis showed stress tolerance at both the stages by accumulating higher percentage of peroxidase followed by A. brevis at vegetative and A. marocana at flowering stage. Level of lipid peroxidation in terms of Malondialdehyde (MDA) content was increased in the leaves when plants were subjected to moisture stress. The rate of increase in lipid peroxidation occurs irrespective of stage however; maximum increase was recorded in A. strigosa at both the stages. Avena species which showed high level of MDA content, indicates more lipid peroxidation and more membrane permeability and are comparatively more susceptible for water stress than those which produce less Malondialdehyde (MDA) content at higher magnitude of water stress such species have better capability for moisture stress tolerance.

Growth, yield and photosynthesis of Panicum maximum and Stylosanthes hamata under elevated CO2.

Plant height, biomass production, assimilatory functions and chlorophyll accumulation of Panicum maximum and Stylosanthes hamata in intercropping systems was influenced significantly under elevated CO2 (600 +/- 50 ppm) in open top chambers (OTCs). The plant height increased by 32.0 and 49.0% over the control in P. maximum and S. hamata respectively in intercropping system under elevated CO2 over open field grown crops (Ca). P. maximum and S. hamata produced 67 and 85% higher fresh and dry biomass respectively under elevated CO2. Rates of photosynthesis and stomatal conductance increased in both the crop species in intercropping systems under elevated CO2. The canopy photosynthesis (photosynthesis x leaf area index) of these crop species increased significantly under elevated CO2 over the open grown crops. The chlorophyll a and b accumulation were also higher in the leaves of both the crop species as grown in OTC with elevated CO2. The increased chlorophyll content, leaf area index and canopy photosynthesis led to higher growth and biomass production in these crop species under elevated CO2. The total carbon sequestration in crop biomass and soils during the three years was 21.53 Mg C/ha under elevated CO2. The data revealed that P. maximum and S. hamata intercropping system is the potential as a sink for the increasing level of CO2 in the atmosphere in the semi-arid tropics.

Growth, biomass production and photosynthesis of Cenchrus ciliaris L. under Acacia tortilis (Forssk.) Hayne based silvopastoral systems in semi arid tropics.

The growth, biomass production and photosynthesis of Cenchrus ciliaris was studied under the canopies of 17 yr old Acacia tortilis trees in semi arid tropical environment. On an average the full grown canopy of A. tortilis at the spacing of 4 x 4 m allowed 55% of total Photosynthetically Active Radiation (PAR) which in turn increased Relative Humidity (RH) and reduced under canopy temperature to -1.75 degrees C over the open air temperature. C. ciliaris attained higher height under the shade of A. tortilis. The tiller production and leaf area index decreased marginally under the shade of tree canopies as compared to the open grown grasses. C. ciliaris accumulated higher chlorophyll a and b under the shade of tree canopies indicating its shade adaptation potential. The assimilatory functions such as rate of photosynthesis, transpiration, stomatal conductance, photosynthetic water use efficiency (PN/TR) and carboxylation efficiency (PN/CINT) decreased under the tree canopies due to low availability of PAR. The total biomass production in term of fresh and dry weight decreased under the tree canopies. On average of 2 yr C. ciliaris had produced 12.78 t ha(-1) green and 3.72 -t ha(-1) dry biomass under the tree canopies of A. tortilis. The dry matter yield reduced to 38% under the tree canopies over the open grown grasses. The A. tortilis + C. ciliaris maintained higher soil moisture, organic carbon content and available N P K for sustainable biomass production for the longer period. The higher accumulation of crude protein, starch, sugar and nitrogen in leaves and stem of C. ciliaris indicates that this grass species also maintained its quality under A. tortilis based silvopastoral system. The photosynthesis and dry matter accumulation are closely associated with available PAR indicating that for sustainable production of this grass species in the silvopasture systems for longer period about 55% or more PAR is required.

Wavelet based ST-segment analysis.

A novel algorithm for ST-segment analysis is developed using the multi-resolution wavelet approach. The system detects the QRS complexes and analyses each beat using the wavelet transform to identify the characteristic points (fiducial points). These fiducial points are, iso-electric level, the J point, and onsets and offsets of the QRS complex and T wave. The algorithm determines the T onset by looking for a point of inflection between the J point and the T peak. Furthermore, detection of characteristic points by the wavelet technique reduces the effect of noise. The results show that the proposed approach gives very accurate ST levels, as compared to the conventional (empirical) technique, at higher heart rates and with different morphologies. The algorithm detects the ST-segment length in 92.3% beats with an error of 4 ms, and in 97.3% beats the error is within 8 ms. The algorithm has been implemented on a TMS320C25 based add-on DSP card connected to a PC to provide the on-line analysis and display of ST-segment data.

Determination of 14,15-epoxyeicosatrienoic acid and 14,15-dihydroxyeicosatrienoic acid by fluoroimmunoassay.

A fluoroimmunoassay (FIA) for 14,15-epoxyeicosatrienoic acid (14,15-EET) and 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), cytochrome P450 epoxygenase products of arachidonic acid, was developed using fluorescence polarization. 14-15-EET was hydrolyzed and analyzed as 14,15-DHET. 14,15-DHET was conjugated to thyroglobulin and a specific antibody was raised in rabbits. Both [3H8]14,15-DHET in radioimmunoassay or fluorescein-labeled 14,15-DHET (14, 15-DHET*) in FIA bound to this antibody and were competitively displaced by 14,15-DHET. The binding activity and cross-reactivity of 14,15-DHET antibody were also studied by RIA compared to FIA. The antibody cross-reacted < or = 1% with 11,12-DHET and 14,15-EET and < 0.1% with other regioisomeric DHETs and arachidonic acid metabolites. The detection limit of 14,15-DHET was 2 pg/0.6 ml by FIA. Using this method, we found that A23187 stimulated the production of 14,15-EET by endothelial cells by angiotensin II stimulated 14,15-EET release from zona glomerulosa cells. The production of 14,15-EET in these samples was confirmed by gas chromatography/mass spectrometry. These studies demonstrate a sensitive and specific FIA for 14,15-EET and 14,15-DHET and that agonists stimulate the release of these eicosanoids in two cell types, bovine coronary artery endothelial cells and bovine zona glomerulosa cells.

Beat by beat QT interval detection and characterization.

A novel algorithm for detection and analysis of QT interval, a risk factor for sudden cardiac death, is developed using the multiresolution wavelet approach. The characteristic points for detection of QT interval, i.e., the onset and offsets of the QRS complex and the T wave are detected by analyzing the wavelet transform of the ECG at particular scales. The results of the detailed study using standard data base indicate that proposed technique can be used to monitor critical heart patients for localization of problems in the duration of ventricular activation. The algorithm has been implemented on TMS320C25 based add-on DSP card to PC to provide the beat by beat analysis and display of QT interval data.

A DSP based real time system for analysis of bundle of His and late potentials using wavelet transforms.

A new system for analysis of Bundle of HIS and Late Potentials has been developed using the wavelet approach. The objective of the present research work is to develop a real-time system which does not rely on averaged data and has the capability to detect beat to beat variations in the cardiac micro-volt signals from the body surface recordings. Multiresolution wavelet analysis gives better time and frequency resolution of the signal and its implementation on DSP hardware makes the system real time. The clinical applicability of the system developed is currently being investigated with initial success on pre clinical data.

Production of 3R-hydroxy-polyenoic fatty acids by the yeast Dipodascopsis uninucleata.

Various fatty acids were fed to the yeast Dipodascopsis uninucleata UOFS Y 128, and the extracted samples were analyzed for the accumulation of 3-hydroxy metabolites with the help of electron impact gas chromatography-mass spectrometry. Fatty acids containing of 5Z,8Z-diene system (5Z,8Z,11Z-eicosatrienoic, 5Z,8Z,11Z,14Z-eicosatetraenoic, and 5Z,8Z,11Z,14Z,17Z-eicosapentaenoic acids) yielded the corresponding 3-hydroxy-all-Z-eicosapolyenoic acids. Moreover, linoleic acid (9Z,12Z-octadecadienoic acid) and 11Z,14Z,17Z-eicosatrienoic acid were converted to the 3-hydorxylated metabolites of shorter chain length, e,g., 3-hydroxy-5Z,8Z-tetradecadienoic acid and 3-hydroxy-5Z,8Z,11Z-tetradecatrienoic acid, respectively. In contrast, no accumulation of a 3-hydroxy metabolite was observed with oleic acid (9Z-octadecenoic acid), linolelaidic acid (9E,12E-octadecadienoic acid), gamma-linolenic acid (6Z,9Z,12Z-octadecatrienoic acid), and eicosanoic acid as substrate. These findings pinpoint that the 3-hydroxylation of a fatty acid in Dipodascopsis uninucleata requires a 5Z,8Z-diene system either directly or following initial incomplete beta-oxidation. Following analysis of the enantiomer composition, the arachidonic acid metabolite was identified as 3R-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid, which rules out a normal beta-oxidation as biosynthetic route to this new class of oxylipins.

Quantitative analysis of errors due to power-line interference and base-line drift in detection of onsets and offsets in ECG using wavelets.

Timing characterisation of the ECG using wavelet transforms is a new technique in which multiscale analysis reduces the influence of noise. This technique issued to investigate the effect of noise and to estimate the errors involved in the detection of onsets and offsets of ECG waves. With appropriate choice of scales of analysis, the study shows that the errors involved in the measurement of QRS width in the presence of base-line wander are negligible. The 50 Hz power-line interference introduces a maximum error of 6.25% if it is greater than 50% of the signal amplitude. The P and T complexes are not affected by power-line interference, but the base-line wander introduces a maximum error of 9.6%. In situations with the simultaneous presence of both types of noise, the use of an optimised scale restricts the errors to within clinically acceptable limits.

Effects of metabolites of leukotriene B4 on human neutrophil migration and cytosolic calcium levels.

Leukotriene B4 (LTB4) is metabolized by beta-oxidation, omega-oxidation and the 12-hydroxyeicosanoid dehydrogenase/delta 10-reductase pathway. We have investigated the effects of metabolites formed by the latter pathway on calcium mobilization and migration in human neutrophils and have compared their potencies with those of other LTB4 derivatives. 12-Oxo-LTB4 and 10,11-dihydro-LTB4 were 60 to 100 times less potent than LTB4 in stimulating neutrophils, whereas 10,11-dihydro-12-oxo-LTB4 and 10,11-dihydro-12-epi-LTB4 exhibited still lower potencies. The 6-trans isomers of 12-oxo-LTB4 and 10,11-dihydro-12-oxo-LTB4 were much less potent than the 6-cis compounds. The EC50 values for biologically and chemically (6-cis) synthesized 12-oxo-LTB4 were similar, indicating that the 6,7-double bond is retained in the cis configuration in the biologically formed compound. Methylation of LTB4 markedly reduced its effect on cytosolic calcium levels, whereas addition of a 3-hydroxyl group had a much more modest effect. Modifications of the omega end of the molecule also resulted in lower potencies for calcium mobilization. Nearly all of the compounds tested desensitized neutrophils to LTB4-induced calcium mobilization, which suggests that their effects were mediated by receptors for the latter compound. However, modifications in the carboxyl end of the molecule had smaller effects on desensitization than on calcium mobilization, whereas the reverse was true for modifications in the omega end of the molecule. This suggests that the structural requirements for agonist-induced desensitization to LTB4 may differ to some extent from the requirements for calcium mobilization.

Stereochemical analysis and biological activity of 3-hydroxy-leukotriene B4: a metabolite from ethanol-treated rat hepatocytes.

Leukotriene B4 (LTB4), a biologically active metabolite derived from arachidonic acid by the 5-lipoxygenase cascade, is inactivated by cytochrome P-450-dependent omega-hydroxylation followed by second oxidation into a omega-carboxyl group. In many tissues, this second step is mediated by alcohol dehydrogenase. Isolated rat hepatocytes metabolized LTB4 in the presence of ethanol and ethoxyresorufin into substantial quantities of 3-hydroxy-LTB4 as determined by mass spectrometry. The absolute configuration of this metabolite was found to be greater than 98% 3(S)-hydroxy-LTB4 by comparison to synthetic standards. Investigation of the pharmacologic properties of the 3(S)- and 3(R)-hydroxy-LTB4 revealed that both caused a significant increase in intracellular free calcium in human neutrophils at 1 microM. Both enantiomers also induced thromboxane A2 release from the isolated guinea pig lung in a dose-dependent manner. This activity was fully blocked by a specific LTB4 receptor antagonist, LY223982, with an IC50 of 0.21 microM for LTB4. These results suggested that activation of the LTB4 receptor does not involve significant recognition of the carbon atoms close to the carboxyl moiety of LTB4. The failure of the hepatocyte to metabolically inactivate LTB4 in the presence of ethanol may be of importance to humans, particularly because the bioactive metabolite 3(S)-hydroxy-LTB4 was further metabolized by human neutrophils significantly more slowly than LTB4.

Prevalence and pattern of major neurological disorders in rural Kashmir (India) in 1986.

In 1986 in the Kuthar Valley in the Anantnag District of south Kashmir (northwestern India), we studied the population to ascertain the prevalence and pattern of various neurological diseases. A house-to-house survey was done in a rural population of 63,645 (according to a World Health Organization protocol, 1981). 616 cases of major neurological disorders were detected, yielding a prevalence of ratio of 9.67/1,000 as of prevalence day November 1, 1986. The prevalence ratios for various common neurological disorders were: epilepsy 2.47/1,000; stroke 1.43/1,000; paralytic poliomyelitis 2.18/1,000; mental retardation 2.09/1,000; deaf mutism 1.63/1,000, and cerebral palsy 1.24/1,000. Persons with these conditions constituted 92% of all neurological cases. Patients with motor neuron disease, Alzheimer's dementia or multiple sclerosis were not found.

Metabolism of arachidonic acid to epoxyeicosatrienoic acids by human granulosa cells may mediate steroidogenesis.

Epoxyeicosatrienoic acids (EETs), cytochrome P-450 metabolites of arachidonic acid, have attracted attention because of their effects on stimulus-response coupling in endocrine, renal, and vascular cells. To investigate a possible role for EETs in ovarian physiology, we conducted a series of experiments using human luteinized granulosa cells. Granulosa cell microsomes produce EETs, which are identified by their comigration with known standards using reverse phase high pressure liquid chromatography. EET synthesis by granulosa cells is NADPH dependent and inhibited by ketoconazole, suggesting an enzymatic mechanism of production. Intact granulosa cells synthesize EETs from exogenous arachidonic acid, and EET production is increased by hCG stimulation of the cells. To investigate whether EETs have a role in ovarian steroidogenesis, they were added to cultures of granulosa cells. Varying concentrations of 14,15-EET differentially affected estradiol secretion; 0.001-0.05 microM stimulated estradiol production, whereas 14,15-EET concentrations of 10-50 microM inhibited estradiol production. hCG-stimulated estradiol secretion was also inhibited by 10-50 microM 14,15-EET. In contrast, progesterone secretion was not affected by any concentration of 14,15-EET tested. The cellular concentration of cAMP was not affected by the addition of EETs. These findings suggest that hCG stimulates granulosa cell production of EETs via an NADPH-supported, cytochrome P-450-dependent enzymatic mechanism. EETs may have an important autocrine or paracrine role in regulating ovarian granulosa cell estrogen synthesis.

14(R),15(S)-epoxyeicosatrienoic acid (14(R),15(S)-EET) receptor in guinea pig mononuclear cell membranes.

A high affinity binding site for 14(R),15(S)-EET, one of the major cytochrome P-450 metabolites of arachidonic acid (AA) in blood vessels, liver, kidney and urine of patients with pregnancy-induced hypertension, has been identified in a membrane preparation from guinea pig mononuclear (GPM) cells. Using a radioligand assay, binding of 14(R),15(S)-[3H]EET to its receptor site was saturable, specific and reversible. Scatchard analysis of saturation binding studies yielded a dissociation constant (Kd) of 5.7 x 10(-9) M, and maximum number of binding sites (Bmax) of 2.4 pmol/mg membrane protein. The specificity of the binding site was determined by competition studies. 14(S),15(R)-EET and 8,9-EET had a Ki of 6.3 and 8.8 nM, respectively, followed by 12(R)-HETE and LTD4. 12(S)-HETE and 5,6-EET were even less effective as a competitive inhibitor of radioligand and binding with Ki values from 2 to 20 microM. Receptor antagonists for TxA2, LTB4, LTD4 and PAF failed to displace 14(R),15(S)-[3H]EET from its binding site on GPM cell membranes. The results correlate well with the reported biological functions of 14,15-EET. In view of its potent biological activities, 14,15-EET may exert its cellular function through the binding and activation of its stereo-specific cell surface binding sites or receptor.

Resolution of dihydroxyeicosanoates and of dihydroxyeicosatrienoates by chiral phase chromatography.

A chromatographic method is described for the direct enantiomeric characterization of 5,6-, 8,9-, 11,12-, and 14,15-vic-dihydroxyeicosatrienoic acids (DHETs), metabolites of the cytochrome P-450 arachidonate epoxygenase pathway, and of their corresponding saturated vic-dihydroxyeicosanoic acids. Following esterification, the individual methyl or pentafluorobenzyl esters are resolved by chiral-phase chromatography utilizing a Chiralcel OC or OD column. This methodology will find analytical and preparative applications since it is simple and efficient and preserves, intact, the diol functionality.

Metabolism of 12(R)-hydroxyeicosatetraenoic acid by rat liver microsomes.

The in vitro metabolism of 12(R)-hydroxyeicosatetraenoic acid was studied using freshly isolated rat liver microsomes. Ten metabolites were isolated and identified by a combination of ultraviolet spectroscopy and gas chromatography/mass spectrometry. The two major metabolites were dihydroxyeicosatetraenoic acids generated by omega/omega-1 hydroxylation. Oxidation at C-5 resulted in the formation of four leukotriene-like compounds, two of which differed from leukotriene B4 in double-bond geometry alone. The other two differed from leukotriene B4 in olefin geometry and C-5 configuration. Epoxidation at the 14,15-olefin resulted in the formation of two diastereomeric epoxy alcohols, while C-16 hydroxylation gave two diastereomeric dihydroxyeicosatetraenoic acids.