Reaction of chromium (VI) with hydrogen peroxide in the presence of glutathione: reactive intermediates and resulting DNA damage

The reaction of chromium(VI) with hydrogen peroxide was studied in the presence of glutathione. In vitro, reaction of chromium(VI) with hydrogen peroxide alone led to production of hydroxyl radical as the significant reactive intermediate, while reaction of chromium(VI) with glutathione led to formation of two chromium(V)-glutathione complexes and the glutathione thiyl radical. Incubation of chromium(VI) with glutathione prior to addition of hydrogen peroxide led to formation of peroxochromium(V) species and a dramatic increase in hydroxyl radical production over that detected in the reaction of chromium(VI) with hydrogen peroxide alone. In contrast, addition of chromium(VI) to a preincubated mixture of glutathione and hydrogen peroxide led to a decrease in hydroxyl radical production over that obtained in the reaction of chromium(VI) with hydrogen peroxide. When pBR322 DNA was added to the above reactions, the extent of chromium(VI)-induced DNA strand breakage correlated with the relative amount of hydroxyl radical formed. Reaction of chromium(VI) with calf thymus DNA in the presence of a preincubated mixture of glutathione and hydrogen peroxide led to detection of the 8-hydroxydeoxyguanosine adduct, whose formation correlated with that of hydroxyl radical production. No significant chromium-DNA adduct formation was detected. The results suggest that, in the cellular metabolism of chromium(VI), preformed chromium(V)-glutathione complexes may react with hydrogen peroxide in a Fenton-type manner to produce hydroxyl radical as the DNA-damaging agent. However, if glutathione reacts with hydrogen peroxide prior to exposure to chromium(VI), the amount of hydroxyl radical generated may not be sufficient to cause significant DNA damage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological evidence for complex and multiple site interaction of CXCR4 with SDF-1alpha: implications for development of selective CXCR4 antagonists

The C-X-C chemokine SDF-1 and its receptor CXCR4, mediate a pivotal role in the pathophysiology of HIV-1 infection and vascular inflammatory diseases. In this study, we investigated the pharmacological properties of SDF-1alpha interaction with CXCR4 in human leukemia cell lines. Our data, based on [125I]-SDF-1alpha radioligand binding, SDF-1alpha-induced [35S]-GTPgammaS binding and use of specific CXCR4 antagonist AMD3100 reveals the complex nature of SDF-1alpha-CXCR4 interaction. Firstly, homologous competition with cold SDF-1alpha revealed a bimodal ligand displacement curve and secondly, although AMD3100 inhibited both SDF-1alpha-mediated chemotaxis (IC(50)=4.7 nM) and [35S]-GTPgammaS binding (IC(50)=7.4 nM) with high affinity, it was intriguingly up to 3000-fold less potent (IC(50)=15.2 microM) in the radioligand binding assay. These results provide pharmacological evidence for the recently described two-site model for SDF-1alpha-CXCR4 interaction. Accordingly, inhibition of SDF-1alpha binding to one of the receptor sites is sufficient to antagonize function, without causing its complete displacement from the receptor. Furthermore, these findings have important implications in the development and evaluation of CXCR4-selective small molecule antagonists for therapeutic use.     

Hollow microneedles for intradermal injection fabricated by sacrificial micromolding and selective electrodeposition

Limitations with standard intradermal injections have created a clinical need for an alternative, low-cost injection device. In this study, we designed a hollow metal microneedle for reliable intradermal injection and developed a high-throughput micromolding process to produce metal microneedles with complex geometries. To fabricate the microneedles, we laser-ablated a 70 μm?×?70 μm square cavity near the tip of poly(lactic acid) (PLA) microneedles. The master structure was a template for multiple micromolded poly(lactic acid-co-glycolic acid) (PLGA) replicas. Each replica was sputtered with a gold seed layer with minimal gold deposited in the cavity due to masking effects. In this way, nickel was electrodeposited selectively outside of the cavity, after which the polymer replica was dissolved to produce a hollow metal microneedle. Force-displacement tests showed the microneedles, with 12 μm thick electrodeposition, could penetrate skin with an insertion force 9 times less than their axial failure force. We injected fluid with the microneedles into pig skin in vitro and hairless guinea pig skin in vivo. The injections targeted 90 % of the material within the skin with minimal leakage onto the skin surface. We conclude that hollow microneedles made by this simple microfabrication method can achieve targeted intradermal injection.     

Molecular and pharmacological characterization of genes encoding urotensin-II peptides and their cognate G-protein-coupled receptors from the mouse and monkey

Urotensin-II (U-II) and its receptor (UT) represent novel therapeutic targets for management of a variety of cardiovascular diseases. To test such hypothesis, it will be necessary to develop experimental animal models for the manipulation of U-II/UT receptor system. The goal of this study was to clone mouse and primate preproU-II and UT for pharmacological profiling. Monkey and mouse preproU-II genes were identified to encode 123 and 125 amino acids. Monkey and mouse UT receptors were 389, and 386 amino acids, respectively. Genomic organization of mouse genes showed that the preproU-II has four exons, while the UT receptor has one exon. Although initially viewed by many exclusively as cardiovascular targets, the present study demonstrates expression of mouse and monkey U-II/UT receptor mRNA in extra-vascular tissue including lung, pancreas, skeletal muscle, kidney and liver. Ligand binding studies showed that [125I]h U-II bound to a single sites to the cloned receptors in a saturable/high affinity manner (Kd 654+/-154 and 214+/-65 pM and Bmax of 1011+/-125 and 497+/-68 fmol mg-1 for mouse and monkey UT receptors, respectively). Competition binding analysis demonstrated equipotent, high affinity binding of numerous mammalian, amphibian and piscine U-II isopeptides to these receptors (Ki=0.8 - 3 nM). Fluorescein isothiocyanate (FITC) labelled U-II, bound specifically to HEK-293 cells expressing mouse or monkey UT receptor, confirming cell surface expression of recombinant UT receptor. Exposure of these cells to human U-II resulted in an increase in intracellular [Ca2+] concentrations (EC50 3.2+/-0.8 and 1.1+/-0.3 nM for mouse and monkey UT receptors, respectively) and inositol phosphate (Ip) formation (EC50 7.2+/-1.8 and 0.9+/-0.2 nM for mouse and monkey UT receptors, respectively) consistent with the primary signalling pathway for UT receptor involving phospholipase C activation.     

Serum Ornithine Carbamoyl Transferase as a Surrogate Marker in Malaria

Ornithine carbamoyl transferase (OCT) activity and other liver function tests were studied in a total of 50 patients of clinical malaria and 15 controls. They were grouped as group I (positive for malarial parasite on peripheral blood smear, n=18), group II (negative for malarial parasite on peripheral blood smear (PBS) but responded to antimalarials, n=17) and group III (peripheral blood smear negative and did not respond to antimalarial therapy, n=15). The mean OCT levels were significantly raised in group I (6.79 ± 1.84 IU/L, p value = 0.006) and group II (5.0 ± 1.15 IU/L, p value = 0.014) as compared to controls (2.5 ± 1.13 IU/L) and returned to normal after treatment In contrast, group III had normal levels except in a case of kala azar and septicemia where OCT levels were high and increased further on treatment. Taking PBS positivity as a gold standard of diagnostic criteria, OCT had a sensitivity of 83% and specificity of 86% with a high positive predictive value of 88% as compared to ALT which had a lower sensitivity of 55% and specificity of 80%. The clinical response rate in PBS negative cases of fever having high OCT level was 83% as compared to 35% in cases with normal OCT level, making OCT a good surrogate marker of malaria. OCT levels could also be of prognostic significance as 2 cases of cerebral malaria had high OCT levels of 11.1 UAL and 10.7 IU/L, respectively.Key Words: Malaria, Ornithine carbamoyl transferase     

G protein-coupled receptors in drug discovery

G protein-coupled receptors (GPCRs) represent one of the most important drug discovery targets such that compounds targeted against GPCRs represent the single largest drug class currently on the market. With the revolutionary advances in human genome sciences and the identification of numerous orphan GPCRs, it is even more important to identify ligands for these orphan GPCRs so that their physiological and pathological roles can be delineated. To this end, major pharmaceutical industries are investing enormous amounts of time and money to achieve this object. This review is a bird's eye view on the various aspects of GPCRs in drug discovery.