Induction of apoptosis by methanolic extract of Rubia cordifolia Linn in HEp-2 cell line is mediated by reactive oxygen species

Rubia cordifolia Linn, which belongs to the Rubiaceae family, is a well-known herb used in Ayurvedic medicine. In the present study, we investigated the influence of a methanolic extract (RC) on the induction of apoptosis in HEp-2 (human laryngeal carcinoma) cell line, as evidenced by cytotoxicity, morphological changes and modification in the levels of pro-oxidants. Inhibition of cell proliferation and lactate dehydrogenase (LDH) release increased in a time and dose-dependent manner. Further, reduced glutathione (GSH), glutathione transferase (GST) and protein levels decreased and lipid peroxidation increased significantly on RC treatment in a dose dependent manner when compared to controls. Based on the results we determined the optimal dose as 30 mg/ ml and the apoptotic effect of RC extract (30 mg/ml) on HEp-2 cells was confirmed by fluorescent microscopy and transmission electron microscopy (TEM) based on morphological and ultrastructural changes. RC extract suppressed the proliferation of HEp-2 oral cancer cells inducing apoptotic cell death in vitro. These results point to potential of RC extract as an agent for the treatment of laryngeal squamous cell carcinoma.     

TO STUDY INCIDENCE OF CHLAMYDIAL GENITAL TRACT INFECTIONS USING ENZYME IMMUNO ASSAY-ANTIGEN DETECTION AND CELL CULTURE METHODS

Endocervical swabs from 315 patients were screened for chlamydial infection by using Enzyme Immuno Assay technique for antigen detection. Of these, 190 patients were of infertility and 125 patients were with history suggestive of pelvic inflammatory disease (PID). 100 age matched controls were also screened for the detection of chlamydial antigen by using EIA. The overall incidence of chlamydial infection in this study group was 15.2%. 21 (11.05%) of the infertility patients and 27 (21.6%) of the pelvic inflammatory disease cases were found to be positive for chlamydial antigen. The prevalence rate was found to be high in the age group of 31–40 years in both study groups i.e. infertility group (14.7%) and PID group (50%). All the ELISA positive cases (48) and randomly selected (10) age matched controls were screened by tissue culture using McCoy cell line. In the tissue culture, 44 of the 48 samples were found to be positive and none of the controls groups were found positive. 4 samples showed discordant results possibly due to the presence of non-viable organism or inhibitory material present at the sample site. The sensitivity and specificity of ELISA with respect to tissue culture are 100% and 71% respectively. The positive predictive value and the negative predictive value of the ELISA are 91.6% and 100% respectively. The efficiency of the test was found to be 93.1%.Key Words: Chlamydia trachomatis, Infertility, PID     

MUC1 expression and its association with other aetiological factors and localization to mitochondria in preneoplastic and neoplastic gastric tissues

BackgroundThe molecular events that underlie the conversion of normal human gastric epithelium into adenocarcinoma are poorly understood. MUC1 overexpression and localization in mitochondria might confer cancer cells with attenuation of stress induced apoptosis. We studied MUC1 expression pattern, interaction with HSP70 and localization in mitochondria in preneoplastic and neoplastic human gastric tissues.MethodsImmunohistochemistry and Western blot were used to study MUC1 expression pattern and localization in mitochondria. Coimmunoprecipitation was used to study MUC1 interaction with HSP70. MUC1 expression was correlated with other causative features including erbB2 expression.ResultsMUC1 was expressed in 75.8% (147/194). MUC1 overexpression was detected in 50.0% (19/38 cases) dysplasia and 58.2% (32/55 cases) adenocarcinoma tissues. MUC1-CT–HSP70 interaction was seen in 71.66% (43/60 cases) and MUC1 localized to mitochondria in 33.33% (5/15) dysplasia samples and in 47.05% (8/17) adenocarcinoma samples. MUC1 expression showed significant association with smoking (χ² = 5.945; p < 0.015), alcohol consumption (χ² = 4.055; p < 0.044) and erbB2 positivity (χ² = 10.75; p < 0.001). MUC1 expression did not show appreciable association with age (χ² = 0.15; p < 0.698), sex (χ² = 0.22; p < 0.640) or Helicobacter pylori infection (χ² = 3.06; p < 0.080).ConclusionsSignificant correlation was found between MUC1 expression and smoking, alcohol and erbB2 expression. MUC1 showed aberrant expression in dysplasia and adenocarcinoma stages. MUC1 cytosolic tail was bound by HSP70 in all the stages but MUC1-CT was found to localize in mitochondria only in dysplasia and adenocarcinoma. MUC1-CT localization to mitochondria in dysplasia and adenocarcinoma might aid in the attenuation of epithelial stress response induced loss of polarity.     

Divergence between LDL oxidative susceptibility and urinary F(2)-isoprostanes as measures of oxidative stress in type 2 diabetes.

BACKGROUND: Oxidative stress is pivotal in atherogenesis. Although the most widely used indirect assay to quantify oxidative stress is LDL oxidative susceptibility, direct assays such as urinary F(2)-isoprostanes have shown great promise. METHODS: We evaluated the utility of both a direct measure of oxidative stress (urinary F(2)-isoprostanes) and an indirect measure of copper-catalyzed, LDL oxidation in a model of increased oxidative stress (diabetes). We also evaluated an enzyme immunoassay (EIA) method for urinary F(2)-isoprostanes with a gas chromatography-mass spectrometry method. RESULTS: Excellent intraassay and interassay CVs of <4% were obtained with our EIA method. A good correlation was obtained between the two methods (r = 0.80; n = 68) of F(2)-isoprostane measurement. An excellent correlation for F(2)-isoprostane concentrations was obtained between a timed collection vs 24-h urine (r = 0.96; n = 46). Baseline F(2)-isoprostane concentrations by EIA were significantly higher in both type 2 diabetics with and without macrovascular complications compared with controls (P <0.001). Supplementation with alpha-tocopherol led to a significant reduction in F(2)-isoprostane concentrations in all diabetic patients compared with baseline values (2.51 +/- 1.76 compared with 1.69 +/- 1.32 ng/mg creatinine; P <0.001). There were no significant differences in LDL oxidation in both diabetic groups compared with controls. alpha-Tocopherol supplementation led to significant increases in the lag phase of oxidation as measured by 3 indices in all groups. CONCLUSIONS: The measurement of urinary F(2)-isoprostanes provides a direct measure of in vivo lipid peroxidation and oxidative stress and appears to be superior to an indirect measure, e.g., LDL oxidative susceptibility, in type 2 diabetes.     

Effects of low-dose aspirin on serum C-reactive protein and thromboxane B2 concentrations: a placebo-controlled study using a highly sensitive C-reactive protein assay.

OBJECTIVES

We performed a placebo-controlled study to evaluate the effect of low-dose aspirin on serum C-reactive protein (CRP) levels.

BACKGROUND

Elevated circulating concentrations of CRP, an inflammatory marker, increase the risk of thrombotic cardiovascular diseases such as myocardial infarction (MI). Moreover, low-dose aspirin therapy has been reported to be more effective in preventing MI in men with higher CRP levels than it is in those with lower levels, raising the possibility that aspirin prevents thrombosis by reducing vascular inflammation. The effect of low-dose aspirin therapy on serum CRP levels in men has been addressed recently, but the results of the two studies conflict.

METHODS

Effects of aspirin (81 mg every day or 325, 81 or 40 mg every-third-day given for 31 days) on serum CRP, using a highly-sensitive assay, and on serum platelet-cyclo-oxygenase (COX)-1-derived thromboxane (Tx) B₂ concentrations were studied simultaneously in 57 healthy volunteers (30 men and 27 women).

RESULTS

Trough platelet COX-1-derived serum Tx B₂ concentrations decreased by 100% with daily aspirin and by 90%, 84% and 78% with 325, 81 and 40 mg aspirin every-third-day (p < 0.001). However, there were no significant changes in serum CRP levels from baseline with daily low-dose aspirin therapy, with any of the every-third-day aspirin regimens or with placebo treatment.

CONCLUSIONS

Low doses of aspirin that markedly inhibit platelet COX-1 activity, as manifested by a profound decline in platelet-derived serum Tx B₂ concentrations, have no detectable effect on serum CRP levels in healthy men and women.     

Heterozygosity for the mouse Apex gene results in phenotypes associated with oxidative stress.

Apurinic/apyrimidinic endonuclease is a key enzyme in the process of base excision repair, required for the repair of spontaneous base damage that arises as a result of oxidative damage to DNA. In mice, this endonuclease is coded by the Apex gene, disruption of which is incompatible with embryonic life. Here we confirm the embryonic lethality of Apex-null mice and report the phenotypic characterization of mice that are heterozygous mutants for the Apex gene (Apex+/-). We show that Apex heterozygous mutant cells and animals are abnormally sensitive to increased oxidative stress. Additionally, such animals manifest elevated levels of oxidative stress markers in serum, and we show that dietary supplementation with antioxidants restores these to normal levels. Apex+/- embryos and pups manifest reduced survival that can also be partially rescued by dietary supplementation with antioxidants. These results are consistent with a proposed role for this enzyme in protection against the deleterious effects of oxidative stress and raise the possibility that humans with heterozygous mutations in the homologous HAP1 gene may be at increased risk for the phenotypic consequences of oxidative stress in cells.     

Red blood cell transfusion does not increase oxygen consumption in critically ill septic patients

Background  

Red blood cell (RBC) transfusion is commonly used to increase oxygen transport in patients with sepsis. However it does not consistently increase oxygen uptake at either the whole-body level, as calculated by the Fick method, or within individual organs, as assessed by gastric intra-mucosal pH.     

Metabolic syndrome: an appraisal of the pro-inflammatory and procoagulant status.

Inflammation and hypercoagulability predispose to atherothrombosis and seem to be important features of the metabolic syndrome.The most convincing evidence is the association with increased levels of C-reactive protein. The hemostatic abnormality that has been most consistently associated with insulin resistance is an elevated plasminogen activator inhibitor-1 level. In contrast, markers of hypercoagulability have been associated inconsistently with hyperinsulinemia and glucose intolerance. Fibrinogen clusters with inflammatory factors, which suggests involvement of adipose tissue-generated inflammatory cytokines. Elevated von Willebrand's factor and factor VIII levels aggregate with indicators of endothelial injury,whereas vitamin K-dependent coagulation proteins correlate with triglyceride levels.