Roles for two aminopeptidases in vacuolar hemoglobin catabolism in Plasmodium falciparum

During the erythrocytic stage of its life cycle, the human malaria parasite Plasmodium falciparum catabolizes large quantities of host-cell hemoglobin in an acidic organelle, the food vacuole. A current model for the catabolism of globin-derived oligopeptides invokes peptide transport out of the food vacuole followed by hydrolysis to amino acids by cytosolic aminopeptidases. To test this model, we have examined the roles of four parasite aminopeptidases during the erythrocytic cycle. Localization of tagged aminopeptidases, coupled with biochemical analysis of enriched food vacuoles, revealed the presence of amino acid-generating pathways in the food vacuole as well as the cytosol. Based on the localization data and in vitro assays, we propose a specific role for one of the plasmodial enzymes, aminopeptidase P, in the catabolism of proline-containing peptides in both the vacuole and the cytosol. We establish an apparent requirement for three of the four aminopeptidases (including the two food vacuole enzymes) for efficient parasite proliferation. To gain insight into the impact of aminopeptidase inhibition on parasite development, we examined the effect of the presence of amino acids in the culture medium of the parasite on the toxicity of the aminopeptidase inhibitor bestatin. The ability of bestatin to block parasite replication was only slightly affected when 19 of 20 amino acids were withdrawn from the medium, indicating that exogenous amino acids cannot compensate for the loss of aminopeptidase activity. Together, these results support the development of aminopeptidase inhibitors as novel chemotherapeutics directed against malaria.     

Inactivation of metabolic enzymes by photo-treatment with zinc meta N-methylpyridylporphyrin

Cell proliferation is notably dependent on energy supply and generation of reducing equivalents in the form of NADPH for reductive biosynthesis. Blockage of pathways generating energy and reducing equivalents has proved successful for cancer treatment. We have previously reported that isomeric Zn(II) N-methylpyridylporphyrins (ZnTM-2(3,4)-PyP⁴⁺) can act as photosensitizers, preventing cell proliferation and causing cell death in vitro. The present study demonstrates that upon illumination, ZnTM-3-PyP inactivates glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, NADP⁺ -linked isocitrate dehydrogenase, aconitase, and fumarase in adenocarcinoma LS174T cells. ZnTM-3-PyP⁴⁺ was significantly more effective than hematoporphyrin derivative (HpD) for inactivation of all enzymes, except aconitase and isocitrate dehydrogenase. Enzyme inactivation was accompanied by aggregation, presumably due to protein cross-linking of some of the enzymes tested. Inactivation of metabolic enzymes caused disruption of cancer cells' metabolism and is likely to be one of the major reasons for antiproliferative activity of ZnTM-3-PyP.     

Preparation and characterization of combi-CLEAs catalyzing multiple non-cascade reactions

A novel cross-linked enzyme aggregates (CLEA) concept called combi-CLEA has been described. It is based upon the fact that CLEA can be made from heterogeneous populations of proteins/enzymes. Porcine pancreatic acetone powder crude extract was used for preparing CLEA in such a way that lipase, -amylase, phospholipase A₂ activities were retained upto 100%. The lipase present in the CLEA showed greater thermal stability at 50 °C as compared to free enzyme. For lipase and phospholipase A₂, V_max/K_m showed no significant change upon combi-CLEA formation but decreased significantly for -amylase activity from 190 to 114 min⁻¹. The lipase activity and -amylase activity in CLEA were completely retained upto three cycles of use. The scanning electron microscopic (SEM) studies showed that morphology of CLEA changed upon inactivation by reuses.     

Impact of hyperglycemia on the expression of GLUT1 during oral carcinogenesis in rats

Molecular Biology Reports - On the background of the epidemiological link between diabetes and oral cancer, the present study aimed to analyze the potential involvement of selected glucose...     

Novel diarylheptanoids as inhibitors of TNF-α production

Synthesis and anti-inflammatory activity of novel diarylheptanoids [5-hydroxy-1-phenyl-7-(pyridin-3-yl)-heptan-3-ones and 1-phenyl-7-(pyridin-3-yl)hept-4-en-3-ones] as inhibitors of tumor necrosis factor-α (TNF-α) production is described in the present article. The key reactions involve the formation of a β-hydroxyketone by the reaction of substituted 4-phenyl butan-2-ones with pyridine-3-carboxaldehyde in presence of LDA and the subsequent dehydration of the same to obtain the α,β-unsaturated ketones. Compounds 4i, 5b, 5d, and 5g significantly inhibit lipopolysaccharide (LPS)-induced TNF-α production from human peripheral blood mononuclear cells in a dose-dependent manner. Of note, the in vitro TNF-α inhibition potential of 5b and 5d is comparable to that of curcumin (a naturally occurring diarylheptanoid). Most importantly, oral administration of 4i, 5b, 5d, and 5g (each at 100 mg/kg) but not curcumin (at 100 mg/kg) significantly inhibits LPS-induced TNF-α production in BALB/c mice. Collectively, our findings indicate that these compounds may have potential therapeutic implications for TNF-α-mediated auto-immune/inflammatory disorders.     

Prognostic significance of cyclooxygenase-2 and response to chemotherapy in invasive ductal breast carcinoma patients by real time surface plasmon resonance analysis

Cyclooxygenase-2 (COX-2), an inducible enzyme, has been implicated in the progression and angiogenesis of breast cancer. The aim of the study is to quantify the concentration of COX-2 and its association with clinico-pathological parameters and response to treatment in patients with invasive ductal carcinoma receiving both neo-adjuvant and adjuvant chemotherapy. The level of COX-2 was estimated using a novel biosensor-based surface plasmon resonance technique in serum of 84 patients with breast cancer (48 patients of neo-adjuvant chemotherapy and 36 patients of adjuvant chemotherapy) and 40 age- and gender-matched normal individuals. A significant increase in COX-2 level was observed in patients compared with normal individuals (p>0.0001). The COX-2 level in serum was found to be significantly higher in patients with lymph node involvement (p<0.0061). 68% (33/48) of the patients receiving neo-adjuvant chemotherapy showed significantly (p<0.0025) reduced COX-2 levels. This study shows significant decrease of COX-2 level in patients with breast cancer treated with both neo-adjuvant and adjuvant chemotherapy. Estimation of COX-2 level in serum may serve as a tumor biomarker in patients with breast cancer.     

Comparative proteomic analysis of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis>for the identification of key proteins in bile tolerance

Background  

Lactic acid bacteria are commonly marketed as probiotics based on their putative or proven health-promoting effects. These effects are known to be strain specific but the underlying molecular mechanisms remain poorly understood. Therefore, unravelling the determinants behind probiotic features is of particular interest since it would help select strains that stand the best chance of success in clinical trials. Bile tolerance is one of the most crucial properties as it determines the ability of bacteria to survive in the small intestine, and consequently their capacity to play their functional role as probiotics. In this context, the objective of this study was to investigate the natural protein diversity within the Lactobacillus plantarum species with relation to bile tolerance, using comparative proteomics.     

ABL/BCR gene variant with two-step mechanism: Unusual localization and rare/novel chromosomal rearrangements in CML patients

Aims: Variant translocations involving 9q, 22q and at least one additional genomic locus occur in 5-10% of the patients with chronic myeloid leukemia (CML). The mechanisms for the formation of these variant translocations are not fully characterized. Here we report CML cases presenting a variant translocation indicating two-step mechanism with rare/novel chromosomal rearrangement. Methods: Karyotype analysis was performed on metaphases obtained through short-term cultures of bone marrow and blood. Detection of BCR-ABL fusion gene was performed using dual-color dual-fusion (D-FISH) and extra signal (ES) translocation probes. BAC-FISH was also carried out. Results: In Patient 1, the third partner chromosome was der(11)(p15) with a 2F2G1R signal pattern, which is an unusual signal pattern with the two-step mechanism. Patients 2 and 3 showed typical positive (2F1G1R) signal pattern. In Patient 2, both the chromosome 22s were involved in variant formation. The second fusion was observed below the BCR gene of the second homologue. In Patient 3 the third chromosome was der(13)(q14). The fourth patient showed a variant pattern with BCR/ABL-ES probe involving der(X)(q13) region. Conclusion: The presence of different rearrangements of both 9q34 and 22q11 regions highlights the genetic heterogeneity of this subgroup of CML. In each case with variants, further studies with FISH, BAC-FISH or more advanced technique such as microarray should be performed. Future studies should be performed to confirm the presence of true breakpoint hot spots and assess their implications in CML with variant Ph.     

Coffee, alcohol, smoking, physical activity and QT interval duration: results from the Third National Health and Nutrition Examination Survey

Background

Abnormalities in the electrocardiographic QT interval duration have been associated with an increased risk of ventricular arrhythmias and sudden cardiac death. However, there is substantial uncertainty about the effect of modifiable factors such as coffee intake, cigarette smoking, alcohol consumption, and physical activity on QT interval duration.

Methods

We studied 7795 men and women from the Third National Health and Nutrition Survey (NHANES III, 1988–1994). Baseline QT interval was measured from the standard 12-lead electrocardiogram. Coffee and tea intake, alcohol consumption, leisure-time physical activities over the past month, and lifetime smoking habits were determined using validated questionnaires during the home interview.

Results

In the fully adjusted model, the average differences in QT interval comparing participants drinking ≥6 cups/day to those who did not drink any were −1.2 ms (95% CI −4.4 to 2.0) for coffee, and −2.0 ms (−11.2 to 7.3) for tea, respectively. The average differences in QT interval duration comparing current to never smokers was 1.2 ms (−0.6 to 2.9) while the average difference in QT interval duration comparing participants drinking ≥7 drinks/week to non-drinkers was 1.8 ms (−0.5 to 4.0). The age, race/ethnicity, and RR-interval adjusted differences in average QT interval duration comparing men with binge drinking episodes to non-drinkers or drinkers without binge drinking were 2.8 ms (0.4 to 5.3) and 4.0 ms (1.6 to 6.4), respectively. The corresponding differences in women were 1.1 (−2.9 to 5.2) and 1.7 ms (−2.3 to 5.7). Finally, the average differences in QT interval comparing the highest vs. the lowest categories of total physical activity was −0.8 ms (−3.0 to 1.4).

Conclusion

Binge drinking was associated with longer QT interval in men but not in women. QT interval duration was not associated with other modifiable factors including coffee and tea intake, smoking, and physical activity.     

The D246V mutant of DNA polymerase beta misincorporates nucleotides: evidence for a role for the flexible loop in DNA positioning within the active site

DNA polymerase beta, a member of the X family of DNA polymerases, is known to be involved in base excision repair. A key to determining the biochemical properties of this DNA polymerase is structure-function studies of site-specific mutants that result in substitution of particular amino acids at critical sites. In a previous genetic screen, we identified three 3'-azido-2',3'-dideoxythymidine 5'-triphosphate-resistant mutants, namely E249K, D246V, and R253M, of polymerase beta in the flexible loop of the palm domain. In this work, we perform an extensive kinetic analysis to investigate the role of the D246V mutant on polymerase fidelity. We find that D246V misincorporates T opposite template bases G and C. The mechanistic basis of misincorporation appears to be altered DNA positioning within the active site. We provide evidence that the fidelity of D246V is greatly affected by the base that is 5' of the templating base. We propose that the Asp residue at position 246 helps to maintain the proper positioning of the DNA within the polymerase active site and maintains the fidelity of polymerase beta. Altogether, the results suggest that the flexible loop domain of polymerase beta plays a major role in its fidelity.