Modulation of glucose transporter (GLUT4) by vanadate and Trigonella in alloxan-diabetic rats

Oral administration of vanadate is an effective treatment for diabetes in animal models. However, vanadate exerts these effects at high doses and several toxic effects are produced. Low doses of vanadate are relatively safe but are unable to elicit any antidiabetic effect. The present study explored the prospect of using low doses of vanadate in combination with Trigonella seed powder (TSP) to evaluate their antidiabetic effect in alloxan-diabetic rats. Alloxan-diabetic rats were treated with insulin, vanadate, TSP and vanadate and TSP in combination for 3 weeks. The effect of these antidiabetic compounds was examined on general physiological parameters and distribution of glucose transporter (GLUT4) in skeletal muscle by immunoblotting and immunohistochemistry. Treatment of alloxan-diabetic rats with insulin, vanadate, TSP and vanadate in combination with TSP revived normoglycemia and restored the disturbances in the distribution of GLUT4 in skeletal muscle. TSP treatment was only partially effective in the restoration of diabetic alterations. The treatment of diabetic rats with combined doses of vanadate and TSP was most effective in the normalization of plasma glucose levels and correction of altered GLUT4 distribution.     

Bioenergetic properties of human sarcoma cells help define sensitivity to metabolic inhibitors

Sarcomas represent a diverse group of malignancies with distinct molecular and pathological features. A better understanding of the alterations associated with specific sarcoma subtypes is critically important to improve sarcoma treatment. Renewed interest in the metabolic properties of cancer cells has led to an exploration of targeting metabolic dependencies as a therapeutic strategy. In this study, we have characterized key bioenergetic properties of human sarcoma cells in order to identify metabolic vulnerabilities between sarcoma subtypes. We have also investigated the effects of compounds that inhibit glycolysis or mitochondrial respiration, either alone or in combination, and examined relationships between bioenergetic parameters and sensitivity to metabolic inhibitors. Using 2-deoxy-D-glucose (2-DG), a competitive inhibitor of glycolysis, oligomycin, an inhibitor of mitochondrial ATP synthase, and metformin, a widely used anti-diabetes drug and inhibitor of complex I of the mitochondrial respiratory chain, we evaluated the effects of metabolic inhibition on sarcoma cell growth and bioenergetic function. Inhibition of glycolysis by 2-DG effectively reduced the viability of alveolar rhabdomyosarcoma cells vs. embryonal rhabdomyosarcoma, osteosarcoma, and normal cells. Interestingly, inhibitors of mitochondrial respiration did not significantly affect viability, but were able to increase sensitivity of sarcomas to inhibition of glycolysis. Additionally, inhibition of glycolysis significantly reduced intracellular ATP levels, and sensitivity to 2-DG-induced growth inhibition was related to respiratory rates and glycolytic dependency. Our findings demonstrate novel relationships between sarcoma bioenergetics and sensitivity to metabolic inhibitors, and suggest that inhibition of metabolic pathways in sarcomas should be further investigated as a potential therapeutic strategy.     

Proton Beam Therapy for Localized Prostate Cancer 101: Basics,Controversies, and Facts

Proton beam therapy for prostate cancer has become a source of controversy in the urologic community, and the rapid dissemination and marketing of this technology has led to many patients inquiring about this therapy. Yet the complexity of the technology, the cost, and the conflicting messages in the literature have left many urologists ill equipped to counsel their patients regarding this option. This article reviews the basic science of the proton beam, examines the reasons for both the hype and the controversy surrounding this therapy, and, most importantly, examines the literature so that every urologist is able to comfortably discuss this option with inquiring patients.Key words: Prostate cancer, Proton beam therapy, External beam radiation therapy, Intensity modulated radiation therapyProton beam therapy (PBT) has become a source of controversy in the urologic community. It is not uncommon to hear mixed messages regarding the issue, from zealous advocates to cost-conscious skeptics, leaving many urologists unsure what to tell their patients with prostate cancer. What is clear, however, is that the technology is disseminating across the nation, and as our patients turn to the internet to learn more about their diagnosis, they are going to encounter increasingly more information about PBT, both scientific and promotional in nature. Hence, it is necessary for every urologist to understand the basics of PBT to help guide our patients through treatment options. This article reviews and compares the basic science of conventional external beam radiation therapy (EBRT) with PBT, examines the reasons for both the hype and the controversy surrounding this therapy, and, most importantly, examines the literature so that all urologists are adequately equipped to counsel their patients on this subject.     

Unconventional interactions between water and heterocyclic nitrogens in protein structures

We report an unusual interaction in which a water molecule approaches the heterocyclic nitrogen of tryptophan and histidine along an axis that is roughly perpendicular to the aromatic plane of the side chain. The interaction is distinct from the well-known conventional aromatic hydrogen-bond, and it occurs at roughly the same frequency in protein structures. Calculations indicate that the water-indole interaction is favorable energetically, and we find several cases in which such contacts are conserved among structural orthologs. The indole-water interaction links side chains and peptide backbone in turn regions, connects the side chains in beta-sheets, and bridges secondary elements from different domains. We suggest that the water-indole interaction can be indirectly responsible for the quenching of tryptophan fluorescence that is observed in the folding of homeodomains and, possibly, many other proteins. We also observe a similar interaction between water and the imidazole nitrogens of the histidine side chain. Taken together, these observations suggest that the unconventional water-indole and water-imidazole interactions provide a small but favorable contribution to protein structures.     

Expedient liquid chromatographic method with fluorescence detection for montelukast sodium in micro-samples of plasma

This study describes an expedient assay for the analysis of the asthma medication, montelukast sodium (Singulair, MK-0476), in human plasma samples. After a simple extraction of the plasma, the drug and internal standard, quinine bisulfate, were measured by HPLC. The chromatographic system consisted of a single pump, a refrigerated autosampler, a C₈ 4-μm particle size radial compression cartridge at 40°C and a fluorescence detector with the excitation and emission wavelengths set at 350 and 400 nm, respectively. The mobile phase which was delivered at 1.0 ml/min, was prepared by adding 200 ml of 0.025 M sodium acetate, pH adjusted to 4.0 with acetic acid, to 800 ml of acetonitrile, with 50 μl triethylamine. With a run time of only 10 min per sample, this assay had an overall recovery of >97% with a detection limit of 1 ng/ml. The inter- and intra-run relative standard deviations at 0.05, 0.2 and 1.0 μg/ml were all <9.2%, while the analytical recovery at the same concentrations were within 7.7% of the amount added.     

Angiotensin AT2 receptor deficiency after myocardial infarction

Hearts of normotensive angiotensin II type 2 receptor (AT₂)-deficient mice do not develop fibrosis after angiotensin II-induced chronic hypertension. Thus, the goal of our study was to clarify whether AT₂ knockouts (KOs) are also characterized by altered left ventricular (LV) function and modified remodeling of the extracellular matrix (ECM) after induction of myocardial infarction (MI). MI was induced in 5-mo-old female AT₂-deficient mice and controls by occlusion of the left coronary artery. Time-matched sham-operated animals served as controls. After 48 h, the first sets of mice were hemodynamically characterized using a pressure-tip catheter (n=8/group). We also obtained pressure volume loops using a microconductance catheter in additional sets of animals 3 wk after induction of MI (n=7/group). Finally, the collagen index was illustrated by Sirius red staining and quantified by digital analysis. Whereas the LV function of sham-operated animals did not differ between both genotypes, the collagen index was 44% lower in KO animals. Forty-eight hours and 3 wk post-MI, systolic and diastolic LV function were impaired in both AT₂-deficient and wild-type (WT) animals to the same extent by approx 45%. No differences were found between the two genotypes with respect to LV hypertrophy and the fibrosis index in the infarcted and noninfarcted areas 3 wk post-MI. While AT₂-KO mice had less cardiac collagen content under basal conditions, the receptor deficiency had no significant influence on LV function at the two investigated time points after induction of MI or on the remodeling of ECM at the latter time point. Thus, hypetension-induced fibrosis is probably triggered by other control mechanisms than fibrosis induced by MI.     

A combined NMR and molecular dynamics study of the transmembrane solubility and diffusion rate profile of dioxygen in lipid bilayers

The transmembrane profile of oxygen solubility and diffusivity in a lipid bilayer was assessed by (13)C NMR of the resident lipids (sn-2-perdeuterio-1-myristelaidoyl-2-myristoyl-sn-glycero-3-phosphocholine) in combination with molecular dynamics (MD) simulations. At an oxygen partial pressure of 50 atm, distinct chemical shift perturbations of a paramagnetic origin were observed, spanning a factor of 3.2 within the sn-1 chain and an overall factor of 10 from the headgroup to the hydrophobic interior. The distinguishing feature of the (13)C NMR shift perturbation measurements, in comparison to ESR and fluorescence quenching measurements, is that the local accessibility of oxygen is achieved for nearly all carbon atoms in a single experiment with atomic resolution and without the use of a probe molecule. MD simulations of an oxygenated and hydrated lipid bilayer provided an immersion depth distribution of all carbon nuclei, in addition to the distribution of oxygen concentration and diffusivity with immersion depth. All oxygen-induced (13)C NMR chemical shift perturbations could be reasonably approximated by simply accounting for the MD-derived immersion depth distribution of oxygen in the bilayer, appropriately averaged according to the immersion depth distribution of the (13)C nuclei. Second-order effects in the paramagnetic shift are attributed to the collisionally accessible solid angle or to the propensity of the valence electrons in the vicinity of a given nuclear spin to be polarized or delocalized by oxygen. A method is presented to measure such effects. The excellent agreement between MD and NMR provides an important cross-validation of the two techniques.     

Involvement of p53 in gemcitabine mediated cytotoxicity and radiosensitivity in breast cancer cell lines

Gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdCyd) is one of the anti-metabolites drugs that target DNA replication. We evaluated dFdCyd cytotoxicity and its radiosensitizing ability in human breast cancer cell lines, MCF-7 (wild-type p53) and MDA-MB-231 (mutant-type p53) along with normal mammary epithelial cell line (MCF-12) for comparison. Radiosensitivity and cytotoxicity were measured by the clonogenic survival assays. DNA DSBs was studied by Pulse Field Gel Electrophoresis (PFGE) and cell cycle distribution was analyzed by flow cytometry. MDA-MB-231 cells were the most sensitive to the cytotoxicity of dFdCyd (IC(50) 5 nM) then MCF-7 (IC(50) 10nM), whereas MCF-12 cells were the most resistant to the cytotoxicity of dFdCyd (IC(50) 70 nM). MCF-12 and MCF-7 cell lines did not show any radiosensitization to dFdCyd, whereas the MDA-MB-231 cells showed significantly increased radioresistant to dFdCyd at equimolar concentration (p=0.002) and at IC(50) concentration (p<0.001). The DNA double strand breaks (DSBs) repair showed that dFdCyd neither increases DNA DSBs nor decreases the rate of their repair in MCF-12 and MCF-7 cell lines, while the same treatment in MDA-MB-231 cell line led to decrease the rate of DSBs or increase the rate of DNA repair (p=0.034). Therefore, dFdCyd is a cytotoxic agent, especially in the cancer cells irrespective of having wild-type or mutated p53 protein, but it is not effective as radiosensitizer in the cell lines used in this study. dFdCyd combined with radiation reduces the efficacy of chemo-radiotherapy in p53 mutated cells. Therefore, p53-mutated cancer could be a counter-indication for radiation-gemcitabine combined treatment.