Effect of Ethanol Extract of Whole Plant of Trichosanthes cucumerina var. cucumerina L. on Gonadotropins, Ovarian Follicular Kinetics and Estrous Cycle for Screening of Antifertility Activity in Albino Rats

Ethanol extract of whole plant of Trichosanthes cucumerina L. var. cucumerina was evaluated for antiovulatory activity in adult rats. The ethanol extract at the doses 200 and 400mg/kg body weight (orally) affected the normal estrous cycle showing a significant increase in estrus and metestrus phases and decrease in diestrus and proestrus phases. The extract also significantly reduced the number of healthy follicles (Class I-Class VI) and corpora lutea and increased the number of regressing follicles (Stage IA, Stage IB, Stage IIA, and Stage IIB). The protein and glycogen content in the ovaries were significantly reduced in treated rats. The cholesterol level was significantly increased, whereas, the enzyme activities like 3b-HSD and 17b-HSD were significantly inhibited in the ovary of treated rats. Serum FSH and LH levels were significantly reduced in the treated groups were measured by RIA. In acute toxicity test, neither mortality nor change in the behavior or any other physiological activities in mice were observed in the treated groups. In chronic toxicity studies, no mortality was recorded and there were no significant differences in the body and organ weights were observed between controls and treated rats. Hematological analysis showed no significant differences in any of the parameters examined (RBC, WBC count and Hemoglobin estimation). These observations showed the antiovulatory activity of ethanol extract of whole plant of Trichosanthes cucumerina L. var. cucumerina in female albino rats.

Estimation of red blood cell lifespan from alveolar carbon monoxide measurements

Measurement of alveolar carbon monoxide (CO) presents a facile technique to estimate the lifespan, L, of red blood cells (RBCs) in vivo. Several recent studies employ this technique and calculate L (in days) using the expression, L = 13.8 (Hb)/P-CO(end), where (Hb) is the concentration (in g/dL) of hemoglobin in blood, and P-CO(end) is the endogenous production of CO (in ppm). Implicit in this calculation is the assumption that the fraction, f, of endogenous CO production due to RBC turnover is a constant equal to 0.7, which yields the expected RBC lifespan, L approximate to 120 days, in normal controls. In anemic patients, however, enhanced RBC turnover may increase f substantially above 0.7. The above expression then overestimates L. Here, we deriv an alternative tive expression, L = 3390[Hb]/322P(CO (end)-110, that accounts explicitly for the dependence of f on the rate of RBC turnover and thereby provides more accurate estimates of L without requiring additional measurements. Using the latter expression, we recalculate L from recent measurements on hepatitis C virus infected patients undergoing treatment with ribavirin. We find that our estimates of L in these patients (39 +/- 13 days) are significantly lower than current estimates (46 +/- 14 days), indicating that ribavirin affects RBC survival more severely than expected from current studies. Our expression for L is simple to employ in a clinical setting and would render the broadly applicable technique of alveolar CO measurement for the estimation of RBC lifespan more accurate.

Glycodelin A triggers T cell apoptosis through a novel calcium-independent galactose-binding lectin activity

Glycodelin A (GdA) is one of the progesterone inducible endometrial factors that protect the fetal semiallograft from maternal immune rejection. The immumoregulatory effects of GdA are varied, with diverse effects on the fate and function of most immune cell types. Its effects on T cells are particularly relevant as it is capable of regulating T cell activation, differentiation, as well as apoptosis. We have previously reported that GdA triggers mitochondrial stress and apoptosis in activated T cells by a mechanism that is distinct and independent of its effects on T cell activation. In this study we describe the characterization of a cell surface receptor for GdA on T cells. Our results reveal a novel calcium-independent galactose-binding lectin activity of GdA, which is responsible for its apoptogenic function. This discovery adds GdA to a select group of soluble immunoregulatory lectins that operate within the feto-placental compartment, the only other members being the galectin family proteins. We also report for the first time that both CD4(+) and CD8(+) T cell subsets are equally susceptible to inhibition with GdA, mediated by its novel lectin activity. We demonstrate that GdA selectively recognizes complex-type N-linked glycans on T cell surface glycoproteins. and propose that the galectin-1 glycoprotein receptor CD7 maybe a novel target for GdA on T cells. This study, for the first time, links the lectin activity of GdA to its biological function.

The role of glycodelin as an immune-modulating agent at the feto-maternal interface

Glycodelin A is a progesterone-induced endometrial glycoprotein which has been amply documented to play a role in down-modulation of the maternal immune response to fetal allo-antigens and to be indispensable for the maintenance and progression of pregnancy. Earlier studies from our laboratory have focused on the effect of glycodelin on T cells, key regulators of both the antibody and cell-mediated arms of the acquired immune system. Glycodelin-induced apoptosis inactivated T cells occurs through a caspase-dependant intrinsic mitochondrial pathway. Interestingly, glycodelin inhibited the proliferation of B cells but did not induce apoptosis. More recently, we have studied the effect of glycodelin on the cells of the innate immune system, namely monocytes and NK cells. We have found that glycodelin induced apoptosis in monocytic cells before their differentiation to macrophages, via the mitochondrial pathway, but did not affect their phagocytic capacity after differentiation. Glycodelin induced apoptosis in NK cells but this activity was independent of caspases. In conclusion, glycodelin is observed to affect many cells of the immune system, although the nature of the effect and signaling mechanisms involved in each cell type may be distinct.

Growth inhibition of human promonocytic leukaemic U937 cells by interferon gamma is irreversible and not cell cycle phase-specific.

Growth of human promonocytic leukaemic U937 cells was found arrested within 24 h upon exposure to interferon gamma (IFN-gamma). Removal of the interferon did not result in the resumption of growth, as is evident from the absence of doubling of viable cell count and(3)H-thymidine incorporation. 5-Bromo-2'-deoxyuridine-based flow cytometric analysis of the growth-arrested cells, 24 h subsequent to the removal of IFN-gamma, showed absence of DNA synthesis, confirming the irreversible nature of the growth inhibition. Propidium iodide-based flow cytometric analysis of the growth-arrested cells showed a distribution which is typical of a growth inhibition without resulting in the accumulation of cells in any specific phase of the cell cycle. These results indicated that IFN-gamma arrested growth of U937 cells in an irreversible and cell cycle phase-independent manner. These observations were in contrast to our earlier report on the reversible and cell cycle phase-specific growth inhibition of human amniotic (fetal epithelial) WISH cells by the interferon. Copyright 1999 Academic Press.

Ultrasound-Triggered Controlled Drug Delivery and Biosensing Using Silica Nanotubes

Silica nanotubes (SNTs) have been demonstrated here as a versatile host for controlled drug delivery and biosensing. The sol-gel template synthesized SNTs have a slow rate of drug release. Application of an external stimulus in the form of ultrasound to or chemical functionalization of synthesized SNT results in higher yield of drug release as well as yield of drug release varying linearly with time. In case of controlled drug delivery triggered by ultrasound, drug yield as function of time is found to be heavily dependent on the ultrasound impulse protocol. Impulses of shorter duration (similar to 0.5 min) and shorter time intervals between successive impulses resulted in higher drug yields. Confinement of hemoglobin (Hb) inside nanometer sized channels of SNT does not have any detrimental effect on the native protein structure and function. Observance of significant enhancement in direct electron transfer of Hb makes the SNTs also promising for application in biosensors.

Action of N-bromosuccinimide on sperm whale myoglobin

Previously, it was reported from this laboratory that the heme groups of hemoglobin are “buried” within globin at pH 4.0 and not dissociated, on the basis of the obiligatory requirement of urea for the reaction of N-bromosuccinimide with the heme groups of hemoglobin at pH4.0, and also on the basis of the “normalization” of the spectrum of hemoglobin at this pH in the presence of urea or sucrose. In the present study, it has been shown that the behaviour of sperm whale myoglobin with respect to its reaction with N-bromosuccinimide and with respect to spectral “normalization” in urea or sucrose are essentially similar to that of hemoglobin. It has also been demonstrated that the spectral “normalization” obtained with crystalline hemin is not identical with that obtained with either hemoglobin or myoglobin. The bearing of the results of the present study on the earlier work on hemoglobin is indicated.

Early pregnancy termination in rats immunized with denatured chicken riboflavin carrier protein

Immunoneutralization of the maternal riboflavin carrier protein in the pregnant rat with antibodies to chicken egg vitamin carrier has earlier been shown to terminate their pregnancies. In order to understand the nature of the epitopic conformations capable of eliciting antibodies bioneutralizing the endogenous riboflavin carrier protein in the pregnant rat, we compared pregnancy progression in the fertile rodents following active immunization with either the native, SDS-denatured, reduced-carboxymethylated or SDS-treated reduced carboxymethylated avian egg white riboflavin carrier protein. The data revealed that despite the total antibody titers being higher in the animals immunized with the native protein, the antibodies elicited against the denatured avian vitamin carrier exhibited relatively better potencies to bioneutralize the endogenous maternal protein as evidenced by higher rates of early fetal resorption.

devovo biosynthesis of heme offers a new chemotherapeutic target in the human malarial parasite

The human malarial parasite, Image , has been found to synthesize heme Image , despite the accumulation of large quantities of polymeric heme derived from the hemoglobin of the red cell host. The parasite δ-aminolevulinate dehydrase level is significantly lower than that of the host and its inhibition by succinylacetone leads to inhibition of parasite protein synthesis and viability.

Developmental regulation of cytochrome P-450 (b+e) and glutathione transferase (Ya+Yc) gene expression in rat liver

The expression of cytochrome P-450 (b+e) and glutathione transferase (Ya+Yc) genes has been studied as a function of development in rat liver. The levels of cytochrome P-450 (b+e) mRNAs and their transcription rates are too low for detection in the 19-day old fetal liver before or after phenobarbitone treatment. However, glutathione transferase (Ya+Yc) mRNAs can be detected in the fetal liver as well as their induction after phenobarbitone treatment can be demonstrated. These mRNAs contents as well as their inducibility with phenobarbitone are lower in maternal liver than that of adult nonpregnant female rat liver. Steroid hormone administration to immature rats blocks substantially the phenobarbitone mediated induction of the two mRNA families as well as their transcription. It is suggested that steroid hormones constitute one of the factors responsible for the repression of the cytochrome P-450 (b+e) and glutathione transferase (Ya+Yc) genes in fetal liver.

Ribavirin-Induced Anemia in Hepatitis C Virus Patients Undergoing Combination Therapy

The current standard of care for hepatitis C virus (HCV) infection - combination therapy with pegylated interferon and ribavirin - elicits sustained responses in only similar to 50% of the patients treated. No alternatives exist for patients who do not respond to combination therapy. Addition of ribavirin substantially improves response rates to interferon and lowers relapse rates following the cessation of therapy, suggesting that increasing ribavirin exposure may further improve treatment response. A key limitation, however, is the toxic side-effect of ribavirin, hemolytic anemia, which often necessitates a reduction of ribavirin dosage and compromises treatment response. Maximizing treatment response thus requires striking a balance between the antiviral and hemolytic activities of ribavirin. Current models of viral kinetics describe the enhancement of treatment response due to ribavirin. Ribavirin-induced anemia, however, remains poorly understood and precludes rational optimization of combination therapy. Here, we develop a new mathematical model of the population dynamics of erythrocytes that quantitatively describes ribavirin-induced anemia in HCV patients. Based on the assumption that ribavirin accumulation decreases erythrocyte lifespan in a dose-dependent manner, model predictions capture several independent experimental observations of the accumulation of ribavirin in erythrocytes and the resulting decline of hemoglobin in HCV patients undergoing combination therapy, estimate the reduced erythrocyte lifespan during therapy, and describe inter-patient variations in the severity of ribavirin-induced anemia. Further, model predictions estimate the threshold ribavirin exposure beyond which anemia becomes intolerable and suggest guidelines for the usage of growth hormones, such as erythropoietin, that stimulate erythrocyte production and avert the reduction of ribavirin dosage, thereby improving treatment response. Our model thus facilitates, in conjunction with models of viral kinetics, the rational identification of treatment protocols that maximize treatment response while curtailing side effects.

Angelman Syndrome Protein UBE3A Interacts with Primary Microcephaly Protein ASPM, Localizes to Centrosomes and Regulates Chromosome Segregation

Many proteins associated with the phenotype microcephaly have been localized to the centrosome or linked to it functionally. All the seven autosomal recessive primary microcephaly (MCPH) proteins localize at the centrosome. Microcephalic osteodysplastic primordial dwarfism type II protein PCNT and Seckel syndrome (also characterized by severe microcephaly) protein ATR are also centrosomal proteins. All of the above findings show the importance of centrosomal proteins as the key players in neurogenesis and brain development. However, the exact mechanism as to how the loss-of-function of these proteins leads to microcephaly remains to be elucidated. To gain insight into the function of the most commonly mutated MCPH gene ASPM, we used the yeast two-hybrid technique to screen a human fetal brain cDNA library with an ASPM bait. The analysis identified Angelman syndrome gene product UBE3A as an ASPM interactor. Like ASPM, UBE3A also localizes to the centrosome. The identification of UBE3A as an ASPM interactor is not surprising as more than 80% of Angelman syndrome patients have microcephaly. However, unlike in MCPH, microcephaly is postnatal in Angelman syndrome patients. Our results show that UBE3A is a cell cycle regulated protein and its level peaks in mitosis. The shRNA knockdown of UBE3A in HEK293 cells led to many mitotic abnormalities including chromosome missegregation, abnormal cytokinesis and apoptosis. Thus our study links Angelman syndrome protein UBE3A to ASPM, centrosome and mitosis for the first time. We suggest that a defective chromosome segregation mechanism is responsible for the development of microcephaly in Angelman syndrome.

CNT loading into cationic cholesterol suspensions show improved DNA binding and serum stability and ability to internalize into cancer cells

Methods which disperse single-walled carbon nanotubes (SWNTs) in water as `debundled', while maintaining their unique physical properties are highly useful. We present here a family of cationic cholesterol compounds (Chol(+)) {Cholest-5en-3 beta-oxyethyl pyridinium bromide (Chol-PB+), Cholest-5en-3 beta-oxyethyl N-methyl pyrrolidinium bromide (Chol-MPB+), Cholest-5en-3 beta-oxyethyl N-methyl morpholinium bromide (Chol-MMB+) and Cholest-5en-3 beta-oxyethyl diazabicyclo octanium bromide (Chol-DOB+)}. Each of these could be easily dispersed in water. The resulting cationic cholesterol (Chol(+)) suspensions solubilized single-walled carbon nanotubes (SWCNTs) by the non-specific physical adsorption of Chol(+) to form stable, transparent, dark aqueous suspensions at room temperature. Electron microscopy reveals the existence of highly segregated CNTs in these samples. Zeta potential measurements showed an increase in potential of cationic cholesterol aggregates on addition of CNTs. The CNT-Chol(+) suspensions were capable of forming stable complexes with genes (DNA) efficiently. The release of double-helical DNA from such CNT-Chol(+) complexes could be induced upon the addition of anionic micellar solution of SDS. Furthermore, the CNT-based DNA complexes containing cationic cholesterol aggregates showed higher stability in fetal bovine serum media at physiological conditions. Confocal studies confirm that CNT-Chol(+) formulations adhere to HeLa cell surfaces and get internalized more efficiently than the cationic cholesterol suspensions alone (devoid of any CNTs). These cationic cholesterol-CNT suspensions therefore appear to be a promising system for further use in biological applications.

Glycodelin-A interferes with IL-2/IL-2R signalling to induce cell growth arrest, loss of effector functions and apoptosis in T-lymphocytes

The progesterone-regulated glycoprotein glycodelin-A (GdA), secreted by the decidualized endometrium at high concentrations in primates, inhibits the maternal immune response against fetal antigens and thereby contributes to the tolerance of the semi-allogenic fetus during a normal pregnancy. Our earlier studies demonstrated the ability of GdA to induce an intrinsic apoptotic cascade in CD4 T-lymphocytes and suppress the cytolytic effector function of CD8 T-lymphocytes. In this report, we investigated further into the mechanism of action of GdA controlling perforin and granzyme B expression in CD8 T-lymphocytes and the mechanism of action of GdA leading to lymphocyte death. Flow cytometry analysis was performed to check for the surface expression of interleukin-2 receptor (IL-2R) and intracellular eomesodermin (Eomes) in activated T-lymphocytes, whereas quantitative RTPCR analysis was used to find out their mRNA profile upon GdA treatment. Western analysis was carried out to confirm the protein level of Bax and Bcl-2. GdA reduces the surface expression of the high-affinity IL-2R complex by down-regulating the synthesis of IL-2R (CD25). This disturbs the optimal IL-2 signalling and decreases the Eomes expression, which along with IL-2 directly regulates perforin and granzymes expression. Consequently, the CD8 T-lymphocytes undergo growth arrest and are unable to mature into competent cytotoxic T-lymphocytes. In the CD4 T-lymphocytes, growth factor IL-2 deprivation leads to proliferation inhibition, decreased Bcl-2/enhanced Bax expression, culminating in mitochondrial stress and cell death. GdA spurs cell cycle arrest, loss of effector functions and apoptosis in different T-cell subsets by making T-lymphocytes unable to respond to IL-2.

Early protein malnutrition disrupts cerebellar development and impairs motor coordination

Maternal malnutrition affects every aspect of fetal development. The present study asked the question whether a low-protein diet of the mother could result in motor deficits in the offspring. Further, to examine whether cerebellar pathology was correlated with motor deficits, several parameters of the postnatal development of the cerebellum were assayed. This is especially important because the development of the cerebellum is unique in that the time scale of development is protracted compared with that of the cortex or hippocampus. The most important result of the study is that animals born to protein-deficient mothers showed significant delays in motor development as assessed by rotarod and gait analysis. These animals also showed reduced cell proliferation and reduced thickness in the external granular layer. There was a reduction in the number of calbindin-positive Purkinje cells (PC) and granular cells in the internal granular layer. However, glial fibrillary acidic protein-positive population including Bergmann glia remained unaffected. We therefore conclude that the development of the granular cell layer and the PC is specifically prone to the effects of protein malnutrition potentially due to their protracted developmental period from approximately embryonic day 11 to 13 until about the third postnatal week.