Elucidating Critical Proteinopathic Mechanisms and Potential Drug Targets in Neurodegeneration.

Neurodegeneration entails progressive loss of neuronal structure as well as function leading to cognitive failure, apathy, anxiety, irregular body movements, mood swing and ageing. Proteomic dysregulation is considered the key factor for neurodegeneration. Mechanisms involving deregulated processing of proteins such as amyloid beta (Aß) oligomerization; tau hyperphosphorylation, prion misfolding; α-synuclein accumulation/lewy body formation, chaperone deregulation, acetylcholine depletion, adenosine 2A (A2A) receptor hyperactivation, secretase deregulation, leucine-rich repeat kinase 2 (LRRK2) mutation and mitochondrial proteinopathies have deeper implications in neurodegenerative disorders. Better understanding of such pathological mechanisms is pivotal for exploring crucial drug targets. Herein, we provide a comprehensive outlook about the diverse proteomic irregularities in Alzheimer's, Parkinson's and Creutzfeldt Jakob disease (CJD). We explicate the role of key neuroproteomic drug targets notably Aß, tau, alpha synuclein, prions, secretases, acetylcholinesterase (AchE), LRRK2, molecular chaperones, A2A receptors, muscarinic acetylcholine receptors (mAchR), N-methyl-D-aspartate receptor (NMDAR), glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) and mitochondrial/oxidative stress-related proteins for combating neurodegeneration and associated cognitive and motor impairment. Cross talk between amyloidopathy, synucleinopathy, tauopathy and several other proteinopathies pinpoints the need to develop safe therapeutics with ability to strike multiple targets in the aetiology of the neurodegenerative disorders. Therapeutics like microtubule stabilisers, chaperones, kinase inhibitors, anti-aggregation agents and antibodies could serve promising regimens for treating neurodegeneration. However, drugs should be target specific, safe and able to penetrate blood-brain barrier.

Role of inflammatory mediators (TNF-α, IL-6, CRP), biochemical and hematological parameters in type 2 diabetes mellitus patients of Kashmir, India.

Background: Type II Diabetes mellitus (T2DM) is a multifactorial disease and a leading cause of premature deaths. Inflammatory cytokines are reported that they have potential to enhance insulin resistance and hence T2DM. Assessment of immunological profile in T2DM patients of Kashmir valley is unclear. So, detection of cytokines is relevant to determine the extent and direction of immune responses. The current research was taken to study the role of inflammatory mediators in T2DM along with insulin sensitivity, biochemical and hematological parameters in mountainous valley of Kashmiri population. Methods: A total of 340 subjects were selected in this study among them 160 were T2DM cases and 180 were healthy controls. Serum expression of inflammatory mediators (TNF-α and IL-6 ) were quantified by ELISA technique, WBC count was measured on Sysmax (Germany) hematology analyzer, biochemical and Immunoassay parameters were done on Abbott c4000 (USA) and Abbott C1000 (USA) fully automatic analyzer. Data was analyzed using statistical 'software SPSS 16.1' (Chicago, IL). For all assessments, p<0.05 were considered statistically significant. Results: The expressions of candidate cytokines (TNF-α, IL-6, CRP, and WBC) were highly significant (p<0.001) in T2DM. Among inflammatory mediators, TNF-α shows a positive correlation (p<0.001) with glycemic profile and insulin sensitivity in T2DM cases in comparison with healthy normal. Biochemical (fasting sugar, HbA1c, insulin resistance, lipid profile) and anthropometric (BMI) parameters were highly significant (p<0.001) in T2DM cases as compared to non-diabetic normal. Conclusion: Low grade inflammation and up regulation of inflammatory mediators has been purported to play a significant role in pathogenesis of T2DM. Our findings confirm that positive correlation of TNF-α and IL-6 with T2DM and insulin sensitivity. These can act as early prediction biomarkers of T2DM. Further studies on wider range of pro and anti- inflammatory cytokines i.e. mediators, in association with other biochemical, immunoassay and hematological parameters are needed to help clinicians manage and treat T2DM effectively.

Promoter Hypermethylation and Its Impact on Expression of MGMT Gene in the GIT Malignant Patients of Kashmiri Origin.

Epigenetic alterations, in addition to multiple gene abnormalities, are involved in the genesis and progression of human cancers. Gastrointestinal tract (GIT) cancer is a major medical and economic burden worldwide. Aberrant methylation of CpG islands within promoter regions is associated with transcriptional inactivation of various tumor suppressor genes. Although a number of cancer-associated genes have been found to be hypermethylated in GIT cancer, valuable methylation markers for early diagnosis and prognostic evaluation of this cancer remain largely unknown. O6-methyguanine DNA methyltransferase (MGMT) is a DNA-repair gene that removes mutagenic and cytotoxic adducts from the O6 position of guanine induced by alkylating agents. MGMT promoter hypermethylation and reduced expression have been found in some primary human carcinomas. We studied DNA methylation of CpG islands of the MGMT gene and its relation with MGMT protein expression in human GIT carcinomas. A total of 210 GIT tumor samples and 90 adjacent normal tissues were analyzed for MGMT promoter methylation by methylation-specific polymerase chain reaction after bisulfite modification of DNA and same samples were analyzed for MGMT protein expression by Western blotting. The methylation frequencies of MGMT gene promoter were 41.4%, 34.2%, and 44.2% in stomach, esophageal, and colorectal cancer cases while as 16.6, 13.3, and 13.3 in respective controls. MGMT protein was found downregulated in controls of all GIT. The results suggest that methylation at CpG islands of MGMT may be responsible for the downregulation of MGMT protein expression in GIT cancers.

Association of Pro12Ala Polymorphism of Peroxisome Proliferator-Activated Receptor gamma 2 (PPARγ2) Gene with Type 2 Diabetes Mellitus in Ethnic Kashmiri Population.

Type 2 diabetes mellitus (T2DM) is characterized by chronic hyperglycemia associated with insulin resistance and relative insulin deficiency. T2DM is believed to be attributable to the combined effect of genetic and environmental factors. Peroxisome proliferator-activated receptor gamma 2 (PPARγ2) is one of the main candidate genes that are implicated in T2DM. A common proline 12 alanine (Pro12Ala) polymorphism in PPARγ2 has been shown to be associated with T2DM. The aim of this work was to investigate the possible role of PPARγ2 gene polymorphism, as a genetic risk factor for T2DM. The study comprised 200 ethnic unrelated subjects (100 T2DM patients and 100 controls). PCR-RFLP technique was used for genotyping analysis. The frequency of the Pro allele was 79 and 91.5 % for controls and cases, respectively (P < 0.05; OR 3.2; 95 % CI 1.64-6.3). The Pro12Ala polymorphism was in Hardy-Weinberg equilibrium in both patients and controls (χ 2 = 0.13, P > 0.05). We found a significant association of Pro12Ala polymorphism of PPARγ2 gene with T2DM, however the genotypes showed statistically significant association only with few clinical parameters including body mass index, total cholesterol, and low-density lipoprotein (P < 0.05). The study signifies that Pro allele in PPARγ2 may be a genotypic risk factor that confers susceptibility to T2DM in ethnic Kashmiri population.

Anti-inflammatory and immuno-modulatory studies on LC-MS characterised methanol extract of Gentiana kurroo Royle.

BACKGROUND: In ayurvedic traditional medicine Gentiana kurroo Royle (family; Gentianaceae) is used to treat several metabolic diseases. This plant is rich in various compounds belonging to flavonoids and glycosides. Till now little work has been carried out on immunomodulatory and anti-inflammatory potential of this plant. This study confirms the presence of bioactive compounds and evaluates the anti-inflammatory and immunomodulatory effect of this plant. METHODS: To carry out this work, the methanol extract was investigated in different doses using in vivo and in vitro models. In vivo study involved haemagglutination titre and DTH methods, and in vitro study was done using splenocyte proliferation assay and LPS stimulated macrophage culture. TNF-α, IL-6 and NO were assayed using ELISA kit methods, while NF-κB was evaluated by western blotting. LC-ESI-MS/MS was used for the characterization of the methanol extract. RESULTS: The results showed suppression of both humoral and cell mediated immunity in vivo. This effect was also observed by inhibition of B and T cell proliferation in splenocyte proliferation assay. TNF-α, IL-6 and NO concentrations were also less in extract treated macrophage cultures. The NF-κB expression was also lowered in treated macrophages as compared to untreated macrophages. All these observations were found to be dose dependent. LC-MS characterization of this extract showed the presence of known compounds which are glycosides, alkaloids and flavonoids in nature. CONCLUSION: The methanol extract of this plant was found to be rich in glycoside, alkaloid and flavonoid compounds. These compounds are probably responsible for the suppression of immune response and anti-inflammatory activity. The extract as such and identified bioactive compounds can be useful for the treatment of inflammatory disorders.

Potential risk of esophageal squamous cell carcinoma due to nucleotide excision repair XPA and XPC gene variants and their interaction among themselves and with environmental factors.

The association of nucleotide excision repair (NER) gene polymorphisms with esophageal squamous cell carcinoma (ESCC) is inconclusive. The aim of the current study was to assess the association of repair gene xeroderma pigmentosum A (XPA) (rs-1800975) and xeroderma pigmentosum C (XPC) (rs-2228000) polymorphisms with ESCC risk as well as modifying effects of environmental factors. The genotyping was done in 450 confirmed ESCC cases and equal number of individually matched controls by the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) and direct sequencing methods. Conditional logistic regression models were used to assess the genotypic associations and interactions. A high ESCC risk was found in subjects who carried the homozygous minor allele of XPA (odds ratio (OR) = 3.57; 95 % confidence interval (CI) = 1.76-7.23), and the risk was higher when analysis was limited to participants who were ever smokers (OR = 4.22; 95 % CI = 2.01-8.88), lived in adobe houses (OR = 8.42; 95 % CI = 3.74-18.95), consumed large volumes of salt tea (OR = 7.42; 95 % CI = 3.30-16.69), or had a positive family history of cancer (FHC) (OR = 9.47; 95 % CI = 4.67-19.20). In case of XPC, a homozygous minor allele also showed strong association with ESCC risk (OR = 4.43; 95 % CI = 2.41-8.16). We again observed a very strong effect of the above environmental factors in elevating the risk of ESCC. Further, the variant genotypes of both genes in combination showed an increased risk towards ESCC (OR = 7.01; 95 % CI = 3.14-15.64) and such association was synergistically significant. Salt tea consumption showed an interaction with genotypes of XPA and XPC. However, an interaction with FHC was significant in the case of XPA genotype only. XPA and XPC genotypes are associated with an increased risk of ESCC, and such association was reasonably modulated by different exposures.

Polymorphism of Metastasis Suppressor Genes MKK4 and NME1 in Kashmiri Patients with Breast Cancer.

Genetic polymorphisms in metastatic suppressor genes like MKK4 and NME1 are not well studied in breast cancer. Hence, we analyzed the relationship between MKK4 and NME1 polymorphisms and breast cancer risk in Kashmir, India. The different genotypes of NME1 and MKK4 genes were analyzed by polymerase chain reaction and restriction fragment length polymorphism in 130 breast cancer cases and 200 age- and sex-matched controls. Conditional logistic regression models were used to assess the association of various genotypes with breast cancer. In this study, we found an inverse association between MKK4 promoter polymorphism and breast cancer risk. As compared to TT (wild) genotype, individuals with TG (heterozygous) (OR = 0.32; 95% CI = (0.17-0.58) and GG (mutant) (OR = 0.13; CI = 0.04-0.40) genotypes showed decreased risk of breast cancer. When participants were classified on the basis of lymph node involvement, a strong association between NME1 heterozygous genotype (OR = 3.82; CI = (1.54-9.44) and breast cancer was found.

Crataegus songarica methanolic extract accelerates enzymatic status in kidney and heart tissue damage in albino rats and its in vitro cytotoxic activity.

CONTEXT: Crataegus songarica K. Koch (Rosaceae) has been used in folk medicine to treat various diseases. OBJECTIVE: This study evaluates the effect of C. songarica methanol extract on the kidney and heart tissue damage of albino rats, and to determine cytotoxic activity of various extracts of songarica on various human cancer cell lines. MATERIALS AND METHODS: Rats were divided into six groups, Group I received water only; Group II received CCl4 (1 mL/kg b wt) intraperitoneal; C. songarica extract (at doses of 100, 200 and 300 mg/kg b wt) orally for 15 days. Cytotoxic activity was determined by SRB method using MCF-7, HeLa, HepG2, SF-295, SW480 and IMR-32 cell lines. RESULTS: Compared with CCl4 group, administration of C. songarica extract at the dose of 300 mg/kg b wt, significantly decreases serum creatinine (59.74%), urea (40.23%) and cholesterol (54 mg/dL), MDA (0.007 nmol/mg protein) in kidney and (0.025 nmol/mg protein) in heart tissue, along with evaluation of GSH (209.79 ± 54.6), GR (111.45 ± 2.84), GPx (94.01 ± 14.80), GST (201.71) in kidney tissue and GSH (51.47 ± 1.47), GR (45.42 ± 6.69), GPx (77.19 ± 10.94), GST (49.89) in heart tissue. In addition, methanol, ethanol and ethyl acetate extracts exhibited potent anticancer activity on six cancer cell lines with IC50 values ranging from 28.57 to 85.106 µg/mL. DISCUSSION AND CONCLUSION: Crataegus songarica methanol extract has a potential antioxidant effect as it protects the kidney and heart tissue against CCl4-induced toxicity, prevents DNA damage and showed strong anticancer activity.

Protein expression and methylation of MGMT, a DNA repair gene and their correlation with clinicopathological parameters in invasive ductal carcinoma of the breast.

Epigenetic mechanisms such as DNA methylation are being increasingly recognized to play an important role in cancer and may serve as a cancer biomarker. The aim of this study was to evaluate the promoter methylation status of MGMT (O6-methylguanine-DNA methyltransferase) and a possible correlation with the expression of MGMT and standard clinicopathological parameters in invasive ductal breast carcinoma patients (IDC) of Kashmir. Methylation-specific PCR was carried out to investigate the promoter methylation status of MGMT in breast tumors paired with the corresponding normal tissue samples from 128 breast cancer patients. The effect of promoter methylation on protein expression in the primary breast cancer and adjacent normal tissues was evaluated by immunohistochemistry (n = 128) and western blotting (n = 30). The frequency of tumor hypermethylation was 39.8 % and a significant difference in methylation frequency among breast tumors were found (p < 0.001) when compared with the corresponding normal tissue. Immunohistochemical analysis showed no detectable expression of MGMT in 68/128 (53.1 %) tumors. MGMT promoter methylation mediated gene silencing was associated with loss of its protein expression (rs = -0.285, p = 0.001, OR = 3.38, 95 % CI = 1.59-7.17). A significant correlation was seen between loss of MGMT and lymph node involvement (p = 0.030), tumor grade (p < 0.0001), loss of estrogen receptors (ER; p = 0.021) and progesterone receptors (PR) (p = 0.016). Also, MGMT methylation was found to be associated with tumor grade (p = 0.011), tumor stage (p = 0.009), and loss of ER (p = 0.003) and PR receptors (p = 0.009). To our knowledge, our findings, for the first time, in Kashmiri population, indicate that MGMT is aberrantly methylated in breast cancer and promoter hypermethylation could be attributed to silencing of MGMT gene expression in breast cancer. Our data suggests that MGMT promoter hypermethylation could have a potential function as molecular biomarker of breast oncogenesis. Also, based on their predictive value of response to therapy, the immunohistochemical evaluation and interpretation of MGMT may also help in future to establish therapeutic strategies for patients with breast cancer.

CYP1A1 and CYP2E1 genotypes and risk of esophageal squamous cell carcinoma in a high-incidence region, Kashmir.

The study analyzed the relationship between genetic polymorphisms of phase I xenobiotic metabolizing enzymes, cytochromes P450 (CYP) 1A1 and CYP2E1 and esophageal squamous cell carcinoma (ESCC) in Kashmir, India. The different genotypes of CYP1A1 and CYP2E1 were analyzed by polymerase chain reaction and restriction fragment length polymorphism in 526 ESCC cases and equal number of matched controls. Conditional logistic regression models were used to assess the association of various genotypes with ESCC, gene-gene, and gene-environment interactions. High risk of ESCC was found in participants who carried CYP1A1 Val/Val genotype (OR=2.87; 95 % CI=1.00-8.44) and the risk increased in such individuals when c1/c1 of CYP2E1 genotype was also present (OR=5.68; 95 % CI=1.09-29.52). Risk due to CYP1A1 Val/Val genotype was further enhanced (OR=8.55; 95 % CI=1.86-42.20) when the analysis was limited to ever smokers. Participants who carried CYP2E1 c1/c2 genotype showed an inverse relation (OR=0.27; 95 % CI=0.17-0.43) with ESCC. The inverse association of CYP2E1 c1/c2 genotype was retained when CYP1A1 Ile/Ile was also present (OR=0.18; 95 % CI=0.09-0.32), as well as when analysis was limited to ever smokers (OR=0.45; 95 % CI=0.23-0.90). Significant interaction was found between CYP1A1 (Val/Val) and CYP2E1 (c1/c1) genotypes (OR=1.30; 95 % CI=1.12-1.51; P=0.001) and between CYP1A1 (Val/Val) and smoking (OR=1.31; 95 % CI=1.01-1.69; P=0.043). The study suggests CYP1A1 Val/Val and CYP2E1 c1/c1 genotypes are significantly associated with ESCC risk.

Purification and partial characterization of a fructose-binding lectin from the leaves of Euphorbia helioscopia.

A lectin was purified from leaves of Euphorbia helioscopia, by a combination of ion-exchange and gel filtration chromatography. On ion exchange using a DEAE- cellulose column in 0.2 M phosphate buffer, pH 7.2, the bound protein was eluted with a linear sodium chloride gradient of 0.1 M to 0.5 M. Further purification of the lectin was achieved by gel filtration on Sephadex G-100. Euphorbia helioscopia lectin (EHL) agglutinates only chick erythrocytes, showing no agglutination of all human blood group erythrocytes. The EHL induced hemagglutination is inhibited by fructose. The purified protein showed one band, both in non-denaturing PAGE and SDS-PAGE establishing the charge and size homogeneities of the lectin preparation. The molecular mass of the lectin as indicated by SDS-PAGE was approximately 31 kDa and that estimated from G-100 gel filtration chromatography was about 65 kDa establishing that the lectin is a homodimer. The lectin was stable within a temperature range of 0°C-40°C and exhibited a narrow range of pH stability, being optimally active at around pH 7. EHL also possesses antimicrobial activity and is an inhibitor of bacterial growth particularly Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli.

Socioeconomic status and esophageal squamous cell carcinoma risk in Kashmir, India.

Studies have persistently associated esophageal squamous cell carcinoma (ESCC) risk with low socioeconomic status (SES), but this association is unexplored in Kashmir, an area with a high incidence of ESCC in the northernmost part of India. We carried out a case-control study to assess the association of multiple indicators of SES and ESCC risk in the Kashmir valley. A total number of 703 histologically confirmed ESCC cases and 1664 controls matched to the cases for age, sex, and district of residence were recruited from October 2008 to January 2012. Conditional logistic regression models were used to calculate unadjusted and adjusted odds ratios and 95% confidence intervals. Composite wealth scores were constructed based on the ownership of several appliances using multiple correspondence analyses. Higher education, living in a kiln brick or concrete house, use of liquefied petroleum gas and electricity for cooking, and higher wealth scores all showed an inverse association with ESCC risk. Compared to farmers, individuals who had government jobs or worked in the business sector were at lower risk of ESCC, but this association disappeared in fully adjusted models. Occupational strenuous physical activity was strongly associated with ESCC risk. In summary, we found a strong relationship of low SES and ESCC in Kashmir. The findings need to be studied further to understand the mechanisms through which such SES parameters increase ESCC risk.

Hepatoprotective and antioxidant activity of rhizome of Podophyllum hexandrum against carbon tetra chloride induced hepatotoxicity in rats.

OBJECTIVE: To test possible antioxidant activity of n-hexane extract of Podophyllum hexandrum under in vitro and in vivo conditions. METHODS: The in vitro antioxidant activity was evaluated by the ability of the extract to interact with the stable free radical DPPH, Superoxide (O2-), Hydroxyl (OH-), Hydrogen peroxide (H2O2) radicals, and reducing power ability of the extract was also evaluated. Under in vivo conditions the extract was evaluated for its hepatoprotective activity by measuring different biochemical parameters, such as serum alanine aminotransaminase, serum aspartate aminotransaminase and serum lactate dehydrogenase and antioxidant enzymes. Antioxidant status was estimated by determining the activities of antioxidative enzymes, glutathione reductase (GR), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and superoxide dismutase (SOD), and by determining the levels of reduced glutathione (GSH) and thiobarbituric acid reactive substances (TBARS). RESULTS: Hexane extract of P. hexandrum exhibited good radical scavenging capacity in neutralization of DPPH, O2-, OH-, and H2O2 radicals in a dose dependent manner. n-hexane extract of Podophyllum hexandrum at the doses of 20, 30, and 50 mg/kg-day produced hepatoprotective effect by decreasing the activity of serum marker enzymes, while it significantly increased the levels of glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GR), super oxide dismutase (SOD), and glutathione-S-transferase (GST) in a dose dependant manner. The effect of n-hexane extract was comparable to that of standard antioxidant vitamin E. CONCLUSION: The extract of Podophyllum hexandrum possess free radical scavenging activity under in vitro conditions and could protect the liver tissue against CCl(4) induced oxidative stress probably by increasing antioxidant defense activities.

Variation in bioactive principles of Artemisia amygdalina Decne. in wild and tissue culture regenerants.

Wild and tissue culture raised regenerants of Artemisia amygdalina, a critically endangered and endemic plant of Kashmir and North West Frontier Provinces of Pakistan were screened for the amount of bioactive principles and in particular antimalarial compound artemesinin. Phytochemical screening of extracts revealed the presence of terpenes, alkaloids, phenolics, tannins (polyphenolics), cardiac glycosides and steroids in wild (aerial, inflorescence) and tissue culture regenerants (in vitro grown plant, callus and green house acclimatized plants). HPLC of Artemisia amygdalina revealed the presence of artemesinin in petroleum ether extracts of wild aerial part, tissue culture raised plant and green house acclimatized plants. Acetonitrile and water in 70:30 ratios at flow rate of 1ml/min was standardised as mobile phase. Retention time for standard chromatogram was 6.7. Wild inflorescences and callus does not produce artemesinin. This is the first report of phytochemical screening and artemesinin estimation of wild and tissue culture raised regenerants of Artemisia amygdalina.

Comparison of hematological parameters in untreated and treated subclinical hypothyroidism and primary hypothyroidism patients.

BACKGROUNDS: Thyroid hormones play an important physiological role in human metabolism. Erythrocyte abnormalities are frequently associated with thyroid disorder. However, they are rarely investigated and related to the subclinical and primary hypothyroidism in Kashmiri Patients. In this study an attempt was made to study hematological parameters in untreated and treated subclinical hypothyroidism and primary hypothyroidism patients. METHODS: This retrospective study included 600 subjects, among which were untreated subclinical hypothyroid (n=110), treated subclinical hypothyroid (n=110), untreated primary hypothyroid (n=100), treated primary hypothyroid (n=100) and euthyroid (n=180). This study was carried out at Department of Biochemistry, Government Medical College Srinagar. The hematological parameters and thyroid profile of the subjects were assessed by the Sysmex (Italy) and ECLIA (Germany) 2010 automatic analyzer. Mean, standard deviation (SD), analysis of variance (Two-way ANOVA), and multiple comparisons were used to report our results, with p<0.05 or p<0.01 considered as statistically significant. RESULTS: In this study group we compared the hematological parameters in these groups, untreated subclinical hypothyroid, treated subclinical hypothyroid, untreated primary hypothyroid, treated primary hypothyroid and euthyroid. We found that hematological parameters like Hb, RBC, MCV, HCT, RDW,RBC% were significantly increased in untreated subclinical hypothyroidism and untreated primary hypothyroidsm, with the p value being less than 0.05 whereas, in treated SCH & Pr. Hypothyroid, results were insignificant. The results reported in these groups as mean±SD, were statistically tested by ANOVA and multiple comparison tests. In untreated subclinical hypothyroid the values were: Hb (10.83±1.33 g/dl), RBC (4.21±0.66 10(6)/µl), MCV (84.56±6.84 fL), HCT (38.5±2.2%), RDW (17.91±2.37 fL), RBC% (84.36±13.2%) and in untreated primary hypothyroid, Hb (10.73±0.86 g/dl), RBC (4.63±0.51 10(6)/µl), MCV (83.34±6.92 fL), HCT (38.6±2.6%), RDW (14.93±5.47 fL), RBC% (92.63±10.30%) suggesting that these patients were at risk of anemia and other erythrocyte abnormalities. MCV is an inexpensive approach to study the types of anemia and explore related information like production, destruction, loss and morphological changes of RBC'S. CONCLUSION: The thyroid dysfunction is frequently associated with anemia in subclinical hypothyroidism and primary hypothyroidism. Subclinical hypothyroidism (SCH) is associated with serious complications. Substantial numbers of patients with the risk of SCH could be getting converted into primary hypothyroidism. Such conditions should be identified and corrected. On the other hand, their presence could move to a thyroid dysfunction, allowing its early management.

CYP2D6 rs35742686 and rs3892097 Gene Polymorphisms and Childhood Acute Lymphoblastic Leukemia: Relation to Disease Susceptibility in Kashmiri Children.

CYP2D6 is one of the most widely investigated CYPs in relation to gene polymorphism. This study analyzed the relationship between CYP2D6 rs35742686 and rs3892097 single-nucleotide polymorphisms (SNPs) and potential risk factors in the development of acute lymphoblastic leukemia (ALL) in Kashmiri children. We recruited 300 cases and 600 controls for genotyping and risk factors assessment. Genotypes of rs35742686 and rs3892097 were analyzed by polymerase chain reaction-restriction fragment length polymorphism method. CYP2D6 expression analysis was done by quantitative reverse transcription polymerase chain reaction in ALL cases. Conditional logistic regression models were used to calculate odds ratios (OR) and 95% confidence intervals (CI). High risk of ALL was observed in cases who carried the mutant genotypes of rs35742686 (OR = 18.15; 95% CI = 4.13-79.66, p < 0.0001) or rs3892097 (OR = 24.06; 95% CI = 10.23-56.53, p < 0.0001). Significant interaction was observed between rs35742686 and rs3892097 SNPs (P interaction = 0.001). The risk associated with the variant genotypes of rs35742686 and rs3892097 was retained in the cases whose fathers were smokers or had maternal X-ray exposure ( p < 0.001). Relative messenger ribonucleic acid expression across genotypes was significantly decreased in cases carrying rs35742686 3 (*3/*3) ( n -fold = 0.37 ± 0.156, p < 0.0079) and rs3892097 SNPs (*4/*4) ( n -fold = 0.02 ± 0.0075, p < 0.0001) suggesting these two events are independent in ALL cases. The study concluded that rs35742686 and rs3892097 SNPs are significantly associated with ALL risk in Kashmiri children.

Antioxidant and protective effect of ethyl acetate extract of podophyllum hexandrum rhizome on carbon tetrachloride induced rat liver injury.

The antioxidant and hepatoprotective activities of ethyl acetate extract was carefully investigated by the methods of DPPH radical scavenging activity, Hydroxyl radical scavenging activity, Superoxide radical scavenging activity, Hydrogen peroxide radical scavenging activity and its Reducing power ability. All these in vitro antioxidant activities were concentration dependent which were compared with standard antioxidants such as BHT, α-tocopherol. The hepatoprotective potential of Podophyllum hexandrum extract was also evaluated in male Wistar rats against carbon tetrachloride (CCl(4))-induced liver damage. Pre-treated rats were given ethyl acetate extract at 20, 30 and 50 mg/kg dose prior to CCl(4) administration (1 ml/kg, 1:1 in olive oil). Rats pre-treated with Podophyllum hexandrum extract remarkably prevented the elevation of serum AST, ALT, LDH and liver lipid peroxides in CCl(4)-treated rats. Hepatic glutathione levels were significantly increased by the treatment with the extract in all the experimental groups. The extract at the tested doses also restored the levels of liver homogenate enzymes (glutathione peroxidase, glutathione reductase, superoxide dismutase and glutathione-S- transferase) significantly. This study suggests that ethyl acetate extract of P. hexandrum has a liver protective effect against CCl(4)-induced hepatotoxicity and possess in vitro antioxidant activities.

Carbon tetrachloride induced kidney and lung tissue damages and antioxidant activities of the aqueous rhizome extract of Podophyllum hexandrum.

BACKGROUND: The present study was conducted to evaluate the in vitro and in vivo antioxidant properties of aqueous extract of Podophyllum hexandrum. The antioxidant potential of the plant extract under in vitro situations was evaluated by using two separate methods, inhibition of superoxide radical and hydrogen peroxide radical. Carbon tetrachloride (CCl4) is a well known toxicant and exposure to this chemical is known to induce oxidative stress and causes tissue damage by the formation of free radicals. METHODS: 36 albino rats were divided into six groups of 6 animals each, all animals were allowed food and water ad libitum. Group I (control) was given olive oil, while the rest groups were injected intraperitoneally with a single dose of CCl4 (1 ml/kg) as a 50% (v/v) solution in olive oil. Group II received CCl4 only. Group III animals received vitamin E at a concentration of 50 mg/kg body weight and animals of groups IV, V and VI were given extract of Podophyllum hexandrum at concentration dose of 20, 30 and 50 mg/kg body weight. Antioxidant status in both kidney and lung tissues were estimated by determining the activities of antioxidative enzymes, glutathione reductase (GR), glutathione peroxidase (GPX), glutathione-S-transferase (GST) and superoxide dismutase (SOD); as well as by determining the levels of reduced glutathione (GSH) and thiobarbituric acid reactive substances (TBARS). In addition, superoxide and hydrogen peroxide radical scavenging activity of the extract was also determined. RESULTS: Results showed that the extract possessed strong superoxide and hydrogen peroxide radical scavenging activity comparable to that of known antioxidant butylated hydroxy toluene (BHT). Our results also showed that CCl4 caused a marked increase in TBARS levels whereas GSH, SOD, GR, GPX and GST levels were decreased in kidney and lung tissue homogenates of CCl4 treated rats. Aqueous extract of Podophyllum hexandrum successfully prevented the alterations of these effects in the experimental animals. CONCLUSION: Our study demonstrated that the aqueous extract of Podophyllum hexandrum could protect the kidney and lung tissue against CCl4 induced oxidative stress probably by increasing antioxidant defense activities.

Promoter hypermethylation regulates vitamin D receptor (VDR) expression in colorectal cancer-A study from Kashmir valley.

BACKGROUND: Colorectal carcinogenesis (CRC) is a multistep process, involving both genetic and epigenetic modifications of genes involved in diverse pathways ranging from tumor suppression to DNA mismatch repair. PURPOSE: This study was undertaken to assess the role of promoter methylation of vitamin D receptor (VDR) gene, a transcription factor with myriad biological functions, in relation to its expression and clinicopathological parameters. METHODS: Tissue specimens were taken from a total of 75 colorectal cancer cases paired with their normal surrounding epithelium and analyzed by Real-time RT-PCR for assessing the expression profile and MS-PCR for analyzing the promoter methylation status of the VDR gene. Blood sample from the same patients was drawn for vitamin D estimation. RESULTS: The frequency of promoter methylation in cancerous tissue was 37.33% against 9.33% in normal tissues (p<0.001). The hypermethylated status of VDR promoter showed significantly inverse association with its expression (p=0.008). Furthermore, when compared with the clinical parameters, methylation status of VDR promoter was significantly associated with tumor staging (p=0.008), grading (p<0.001), depth of invasion (p=0.002) and lymph node metastases (p<0.001). Univariate and multivariate analysis indicated patients with increased VDR expression (p<0.001) and decreased methylation status (p=0.012) exhibited longer overall survival. Additionally, serum 25(OH)D3 levels were not significantly associated with any of the patient characteristics. CONCLUSION: Our study, first of its kind from Kashmir, indicated that VDR shows aberrant methylation pattern in CRC with consequent loss in its expression.

Phytochemical screening of Prunella vulgaris l. - an important medicinal plant of Kashmir.

Kashmiri medicinal plant (Prunella vulgaris) was analyzed for its chemical composition and amount of bioactive constituents. The results showed that the herb contains on an average alkaloid (1120 mg %), saponins (350 mg %), phenolics (55.785 mg %) and tannins (52.25 mg %). The medicinal plant contained carbohydrates (375 mg %), proteins (441.6 mg %) and lipids (2403.8 mg %). Role of these bioactive principles are discussed according to their folkloric use in Kashmir valley. Besides the herb is of great importance as far as its other clinical application are concerned. This quantitative estimation can be used for comparative evaluation of bioactive constituents with other populations of Prunella vulgaris present in different parts of the world and can be used for selection of superior quality of this herb to use in pharmaceutical industries.