Jyotdeep Kaur

Kinetic characteristics of folate binding to rat renal brush border membrane in chronic alcoholism.

The binding of folic acid to the plasma membrane is an important event for its reabsorption and conservation by renal epithelium. We studied [3H] folic acid binding to the renal cortical brush border membrane vesicles (BBMV) in rats after 12 weeks of chronic ethanol ingestion. Our results demonstrated that after chronic ethanol intake, the binding of folic acid to the membrane vesicles: (1) was decreased by a mechanism that decreased Bmax without affecting Kd, (2) was insensitive to Na+ ions in the medium (3) behaved differently to divalent cations in the medium in contrast to control group. However under such conditions there was no significant effect of ethanol on pH optimum of the process. Binding was reduced at pH less than 6 but there was no effect of ethanol on binding in pH range from 6 to 8. Increasing the osmolarity at pH 5.5 had no effect on the binding of folate to BBMV, thus confirming that the observed changes in Bmax values were due to site-specific binding in the two groups. Moreover, ethanol does not impart its effect on involvement of sulfhydryl group at the binding site of folate transport system. These findings highlight the possible mechanism of perturbed renal conservation of folate during chronic alcoholism. (Mol Cell Biochem xxx: 219–225, 2005)

Epigenetic mechanisms regulate placental c-myc and hTERT in normal and pathological pregnancies; c-myc as a novel fetal DNA epigenetic marker for pre-eclampsia

Placental development is known for its resemblance with tumor development, such as in the expression of oncogenes (c-myc) and telomerase (hTERT). The expression of c-myc and hTERT is up-regulated during early pregnancy and gestational trophoblastic diseases (GTDs). To determine the role of DNA methylation [via methylation-sensitive high resolution melting (MS-HRM)] and histone modifications [via chromatin immunoprecipitation (ChIP assay)] in regulating the differential expression of c-myc and hTERT during normal gestation and their dysregulation during placental disorders, we obtained placental samples from 135 pregnant women, in five groups: normal first, second and third trimester (n = 30 each), pre-eclamptic pregnancy (n = 30) and molar pregnancy (n = 15). Two placental cell lines (JEG-3 and HTR-8/SVneo) and isolated first-trimester cytotrophoblasts were also studied. Quantitative RT-PCR revealed decreased mRNA expression levels of c-myc and hTERT, which were associated with a higher level of H3K9me3 (1.5-fold, P < 0.05) and H3K27me3 (1.9-fold, P < 0.05), respectively, in third-trimester placental villi versus first-trimester villi. A significantly lower level of H3K27me3 in molar placenta was associated with a higher mRNA expression of c-myc and hTERT. The development of pre-eclampsia (PE) was associated with increased methylation (P < 0.001) and H3K27me3 (P < 0.01) at the c-myc promoter and reduced H3K9me3 (P < 0.01) and H3K27me3 (P < 0.05) at the hTERT promoter. Further, mRNA expression of c-myc and hTERT was strongly correlated in molar villi (r = 0.88, P < 0.01) and JEG-3 cells (r = 0.99, P < 0.02). Moreover, on the basis of methylation data, we demonstrate the potential of c-myc as a fetal DNA epigenetic marker for pre-eclamptic pregnancies. Thus we suggest a role for epigenetic mechanisms in regulating differential expression of c-myc and hTERT during placental development and use of the c-myc promoter region as a potential fetal DNA marker in the case of PE.

Association of Graded Folic Acid Supplementation and Total Plasma Homocysteine Levels With Hematological Toxicity During First-line Treatment of Nonsquamous NSCLC Patients With Pemetrexed-based Chemotherapy.

Background: Pemetrexed is the preferred treatment of nonsquamous non–small cell lung cancer (ns-NSCLC). Folic acid supplementation (FAS) (350 to 1000 &mgr;g daily PO) is recommended to minimize hematological toxicity (HTox). Elevated total plasma homocysteine (tpHcy) predicts increased risk of HTox with pemetrexed in absence of FAS. The current study aimed to assess prevalence of elevated tpHcy levels at baseline and after pemetrexed treatment. Association of graded tpHcy levels/FAS with toxicity was also assessed. Materials and Methods: Retrospective analysis of all ns-NSCLC patients undergoing first-line treatment with pemetrexed-containing platinum doublet over 3½ years was carried out. All eligible patients received pemetrexed (500 mg/m2) and cisplatin (65 mg/m2) each on D1 of a 3-week cycle. FAS was 400 &mgr;g for tpHcy< upper limit of normal (ULN), 700 &mgr;g for tpHcy 1 to 2 ULN, and 1000 &mgr;g for tpHcy>2 ULN. All patients also received oral ferrous sulphate and injectable vitamin B12. Exact 95% confidence intervals (CI) were calculated for comparison with previously published studies. Results: 75.7% of 111 patients had stage IV disease. Prevalence of tpHcy levels 2 ULN were 47.8%, 41.4%, and 10.8% pretreatment and 78.9%, 21.1%, and 0% posttreatment, respectively (P<0.0001). Incidence of any grade and grade 3/4 HTox was 87.4% and 17.1% (anemia), 53.2% and 7.2% (leukopenia), 36.9% and 10.8% (neutropenia), and 39.6% and 7.2% (thrombocytopenia), respectively. HTox, non-HTox, and radiologic responses did not differ among patient groups based upon baseline tpHcy levels or upon graded baseline FAS. Incidence of grade 3/4 anemia was higher in current (17.1%; 95% CI, 11.3%-25.2%) as compared with previous studies. Conclusions: Prevalence of elevated tpHcy levels posttreatment as compared with baseline was reduced significantly with FAS. Among ns-NSCLC patients treated with pemetrexed and with FAS of 400 to 1000 &mgr;g daily, HTox was not associated with either baseline tpHcy levels or with graded baseline FAS.

Imbalance between matrix metalloproteinases and their tissue inhibitors in preeclampsia and gestational trophoblastic diseases

The invasion cascade exhibited by placental trophoblasts and cancerous cells bears many similarities, and it is attributed to extracellular matrix degradation mediated by matrix metalloproteinases (MMPs). Although proper and controlled invasion by trophoblasts into the maternal uterus is an essential requirement for maintenance of normal pregnancy, any abnormality in this phenomenon results in the development of invasion-related disorders such as gestational trophoblastic diseases (GTDs) and preeclampsia. We studied the epigenetic basis of differential expression of two placental MMPs (MMP2 and MMP9) and tissue inhibitors of metalloproteinases (TIMP2 and TIMP1) during normal gestation and invasion-related disorders, i.e., preeclampsia and GTDs. Our study suggests the association of H3K9/27me3 with differential expression of these MMPs and their inhibitors, which regulate the placental invasion during normal pregnancy, whereas no role of CpG methylation was observed in the differential expression of MMPs/TIMPs. Further, development of GTDs was associated with abnormally higher expression of these MMPs and lower levels of their inhibitors, whereas the reverse trends were observed for MMPs and their TIMPs in case of preeclampsia, in association with abnormal changes in H3K9/27me3. These results suggest the involvement of higher levels of MMPs in an aggressive invasive behavior depicted by GTDs, whereas lower levels of these MMPs in shallow and poor invasive phenotype associated with preeclampsia. Thus, our study shows the significance of a proper balance regulated by histone trimethylation between differential expression of MMPs and their TIMPs for maintaining normal pregnancy and its deregulation as a contributing factor for pathogenesis of invasive disorders during pregnancy.

Identification of regulatory mechanisms of intestinal folate transport in condition of folate deficiency

Folic acid is an essential micronutrient, deficiency of which can lead to disturbance in various metabolic processes of cell. Folate transport across intestine occurs via the involvement of specialized folate transporters viz. proton coupled folate transporter (PCFT) and reduced folate carrier (RFC), which express at the membrane surfaces. The current study was designed to identify the regulatory mechanisms underlying the effects of folate deficiency (FD) on folate transport in human intestinal cell line as well as in rats and to check the reversibility of such effects. Caco-2 cells were grown for five generations in control and FD medium. Following treatment, one subgroup of cells was shifted on folate sufficient medium and grown for three more generations. Similarly, rats were fed an FD diet for 3 and 5 months, and after 3 months of FD treatment, one group of rats were shifted on normal folate-containing diet. Increase in folate transport and expression of folate transporters were observed on FD treatment. However, when cells and rats were shifted to control conditions after treatment, transport and expression of these genes restored to the control level. FD was found to have no impact on promoter methylation of PCFT and RFC; however, messenger RNA stability of transporters was found to be decreased, suggesting some adaptive response. Overall, increased expression of transporters under FD conditions can be attributed to enhanced rate of transcription of folate transporters and also to the increased binding of specificity protein 1 transcription factor to the RFC promoter only.

Gene specific epigenetic regulation of hepatic folate transport system is responsible for perturbed cellular folate status during aging and exogenous modulation.

SCOPE The present study was designed to identify the molecular mechanism of folate modulation and aging on aberrant liver folate transporter system. METHODS AND RESULTS An in vivo rat model was used, in which weanling, young and adult rats were given folate deficient diet for 3 and 5 months and after 3 months of folate deficiency, one group received physiological folate repletion (2 mg/kg diet) and another group received over supplemented folate diet (8 mg/kg diet) for another 2 months. In adult group, 3 and 5 months of folate deficiency decreased serum and tissue folate levels with decreased uptake of folate, further associated with decreased expression levels of reduced folate carrier (RFC) and increased expression levels of folate exporter (ABCG2) at both mRNA and protein levels, which in turn regulated by promoter hypermethylation of RFC and promoter hypomethylation of ABCG2 gene. CONCLUSION Promoter hypermethylation of RFC and promoter hypomethylation of ABCG2 may be attributed to the down regulation of RFC and up regulation of ABCG2 at mRNA and protein levels in conditions of 3 and 5 months of folate deficiency in the adult group.

Association of aberrant methylation at promoter regions of tumor suppressor genes with placental pathologies.

AIM The resemblance between invasive behavior of cancer cells and placental trophoblasts and the role of aberrant epigenetic regulation in cancer development is well known. METHODS We analyzed the role of promoter region CpG-methylation and H3K9/27me3 of tumor suppressor genes in normal and pathological pregnancies and utilized their CpG-methylation data to search for fetal DNA epigenetic marker in maternal blood. RESULTS CpG and H3K9/27-methylation associated decreased expression of RASSF1A and APC and increased expression of P16, RB1 and PRKCDBP was observed with advancing normal gestation. Gestational trophoblastic diseases and preeclampsia revealed gene-specific epigenetic deregulation of candidate tumor suppressor genes. Furthermore, APC and PRKCDBP showed the potential to act as fetal DNA epigenetic markers, similar to RASSF1A. CONCLUSION Deregulation of methylation of tumor suppressor genes contributes to the development of preeclampsia and gestational trophoblastic diseases. APC and PRKCDBP may act as fetal DNA epigenetic markers for prenatal diagnosis.

Epigenetic modifications at DMRs of placental genes are subjected to variations in normal gestation, pathological conditions and folate supplementation.

Invasive placentation and cancer development shares many similar molecular and epigenetic pathways. Paternally expressed, growth promoting genes (SNRPN, PEG10 and MEST) which are known to play crucial role in tumorogenesis, are not well studied during placentation. This study reports for the first time of the impact of gestational-age, pathological conditions and folic acid supplementation on dynamic nature of DNA and histone methylation present at their differentially methylated regions (DMRs). Here, we reported the association between low DNA methylation/H3K27me3 and higher expression of SNRPN, PEG10 and MEST in highly proliferating normal early gestational placenta. Molar and preeclamptic placental villi, exhibited aberrant changes in methylation levels at DMRs of these genes, leading to higher and lower expression of these genes, respectively, in reference to their respective control groups. Moreover, folate supplementation could induce gene specific changes in mRNA expression in placental cell lines. Further, MEST and SNRPN DMRs were observed to show the potential to act as novel fetal DNA markers in maternal plasma. Thus, variation in methylation levels at these DMRs regulate normal placentation and placental disorders. Additionally, the methylation at these DMRs might also be susceptible to folic acid supplementation and has the potential to be utilized in clinical diagnosis.

Association of rno-miR-183-96-182 cluster with diethyinitrosamine induced liver fibrosis in Wistar rats

Chronic liver injury due to various etiological factors including environmental carcinogens results in development of liver fibrosis. Numerous studies showed role of miRNAs in liver fibrosis. In the present study, we determined the rno‐miR‐183‐96‐182 cluster expression during hepatic fibrosis induced by diethylnitrosamine (DEN) treated Wistar rats and its association with plasma levels of circulating rno‐miR‐96, rno‐miR‐182, rno‐miR‐183, liver function test and lipid profile, aiming to identify their potential for histological stratification and early diagnosis of liver fibrosis. We found significant upregulation in the hepatic expression of rno‐miR‐183‐96‐182 cluster upon development of fibrosis in a DEN treated rats. Interestingly, the hepatic expression of this miRNA cluster correlates positively with the progression of fibrosis. Univariate analysis showed that hepatic expression of rno‐miR‐182‐5p and rno‐miR‐183‐5p and plasma activity of ALT are significant predictors of fibrosis. Multivariate logistic regression analysis revealed a panel of rno‐miR‐182‐5p and ALT that can discriminate F2‐F3 from F0‐F1 (AUC = 0.87; P‐value < 0.001), F4‐F5‐F6 from F0 to F1 (AUC = 0.981; P‐value < 0.001), and F4‐F5‐F6 from F2 to F3 (AUC = 0.824; P‐value < 0.001). A significant positive correlation of rno‐miR‐183‐96‐182 cluster members was also observed with plasma activities of ALT, AST, ALP, and levels of total cholesterol, HDLc and LDLc during fibrosis progression in DEN treated Wistar rats. Thus, it can be concluded that rno‐miR‐183‐96‐182 cluster being significantly up regulated and associated with chronic liver disease might play a role in fibrosis maintenance and progression. A panel of rno‐miR‐182‐5p and ALT being significant predictors of fibrosis might improve histological stratification of fibrosis staging.

Serial changes in morphology and biochemical markers in platelet preparations with storage

Background: This study was designed to perform serial assessment of alterations in platelet (PLT) count, morphology and biochemical markers of PLT activation during storage of platelet concentrates (PCs) and to correlate morphological changes with these activation markers. Materials and Methods: Our study included the platelet-rich plasma (PRP)-PC and buffy coat reduced PC (BC-PC) prepared from whole blood (WB) donations and the apheresis platelets (AP-PC). Routinely evaluated in vitro PLT parameters were followed. Morphology score (MS) was performed using the light microscopy, glucose and lactate concentration and soluble P-selectin (sP-selectin) level were determined using commercial kits. Results: The fall in mean pH from day 0 to the last day of storage was significant (P < 0.001) in all the groups. Glucose utilization was less in PRP-PC prepared from WB donations at Blood Donation Centre [PRP-PC (BDC)] when compared to PRP-PC prepared from WB donations at mobile blood drives [PRP-PC (M)] and BC-PC. Lactate accumulation was almost similar in these groups on day 3 of storage, but it was significantly lower in the AP-PC (67.54 mg/dl) except on day 5. The deterioration in MS (out of 200) was similar for PRP-PC and BC-PC on day 3 (145/144 and 145 respectively), whereas the AP-PC had a score of 161 and 147 on days 4 and 5 respectively. sP-selectin level was significantly higher in PRP-PC (BDC) in comparison to BC-PC (P = 0.001) from day 1 to day 3 and in AP-PC it was not so high (P = 0.067) even on day 5. A negative correlation existed between the MS and sP-selectin level on all days of storage within each group of PC (r = −0.351; P < 0.001) and a positive correlation was found between the MS and pH from day 0 to day 3 (r = 0.680; P = 0.004). Conclusion: The AP-PCs are superior to the BC-PC and PRP-PC with respect to in vitro quality control parameters, morphological changes and biochemical markers of PLT activation. The PRP-PCs prepared from WB donations at outstation exhibiting more rapid changes should be utilized earlier for transfusion.