Carbon monoxide exposure in rat heart: glutathione depletion is prevented by antioxidants

Rat hearts were perfused for 15min with buffer equilibrated with 0.01% or 0.05% CO. The buffer was equilibrated with 21% O(2) throughout. The ventricular glutathione content decreased by 76% and 84%, 90min post-exposure to 0.01% and 0.05% CO, respectively, compared with 0% CO controls (0.45+/-0.01 micromol/g wet tissue; +/-SEM, n=3). Both reduced and oxidised glutathione contributed to this decline. When ascorbate and Trolox C were included during exposure to 0.05% CO the glutathione pool was partly protected; here the glutathione decrease was 46%. In most hearts additional creatine kinase activity in the perfusate indicated minor tissue injury occurring immediately after the start and/or about 10min after the end of exposure to 0.01% CO or 0.05% CO. Ventricle lactate levels were unaffected by exposure to 0.01% CO. This evidence supports a role for oxidative stress in CO cardiotoxicity.     

The gain of rod phototransduction: reconciliation of biochemical and electrophysiological measurements

We have resolved a central and long-standing paradox in understanding the amplification of rod phototransduction by making direct measurements of the gains of the underlying enzymatic amplifiers. We find that under optimized conditions a single photoisomerized rhodopsin activates transducin molecules and phosphodiesterase (PDE) catalytic subunits at rates of 120-150/s, much lower than indirect estimates from light-scattering experiments. Further, we measure the Michaelis constant, Km, of the rod PDE activated by transducin to be 10 microM, at least 10-fold lower than published estimates. Thus, the gain of cGMP hydrolysis (determined by kcat/Km) is at least 10-fold higher than reported in the literature. Accordingly, our results now provide a quantitative account of the overall gain of the rod cascade in terms of directly measured factors.     

Differential Sensitivities of Fast- and Slow-Cycling Cancer Cells to Inosine Monophosphate Dehydrogenase 2 Inhibition by Mycophenolic Acid

As uncontrolled cell proliferation requires nucleotide biosynthesis, inhibiting enzymes that mediate nucleotide biosynthesis constitutes a rational approach to the management of oncological diseases. In practice, however, results of this strategy are mixed and thus elucidation of the mechanisms by which cancer cells evade the effect of nucleotide biosynthesis restriction is urgently needed. Here we explored the notion that intrinsic differences in cancer cell cycle velocity are important in the resistance toward inhibition of inosine monophosphate dehydrogenase (IMPDH) by mycophenolic acid (MPA). In short-term experiments, MPA treatment of fast-growing cancer cells effectively elicited G0/G1 arrest and provoked apoptosis, thus inhibiting cell proliferation and colony formation. Forced expression of a mutated IMPDH2, lacking a binding site for MPA but retaining enzymatic activity, resulted in complete resistance of cancer cells to MPA. In nude mice subcutaneously engrafted with HeLa cells, MPA moderately delayed tumor formation by inhibiting cell proliferation and inducing apoptosis. Importantly, we developed a lentiviral vector–based Tet-on label-retaining system that enables to identify, isolate and functionally characterize slow-cycling or so-called label-retaining cells (LRCs) in vitro and in vivo. We surprisingly found the presence of LRCs in fast-growing tumors. LRCs were superior in colony formation, tumor initiation and resistance to MPA as compared with fast-cycling cells. Thus, the slow-cycling compartment of cancer seems predominantly responsible for resistance to MPA.     

Functional PAK-2 knockout and replacement with a caspase cleavage-deficient mutant in mice reveals differential requirements of full-length PAK-2 and caspase-activated PAK-2p34

p21-Activated protein kinase 2 (PAK-2) has both anti- and pro-apoptotic functions depending on its mechanism of activation. Activation of full-length PAK-2 by the monomeric GTPases Cdc42 or Rac stimulates cell survival, whereas caspase activation of PAK-2 to the PAK-2p34 fragment is involved in the apoptotic response. In this study we use functional knockout of PAK-2 and gene replacement with the caspase cleavage-deficient PAK-2D212N mutant to differentiate the biological functions of full-length PAK-2 and caspase-activated PAK-2p34. Knockout of PAK-2 results in embryonic lethality at early stages before organ development, whereas replacement with the caspase cleavage-deficient PAK-2D212N results in viable and healthy mice, indicating that early embryonic lethality is caused by deficiency of full-length PAK-2 rather than lack of caspase activation to the PAK-2p34 fragment. However, deficiency of caspase activation of PAK-2 decreased spontaneous cell death of primary mouse embryonic fibroblasts and increased cell growth at high cell density. In contrast, stress-induced cell death by treatment with the anti-cancer drug cisplatin was not reduced by deficiency of caspase activation of PAK-2, but switched from an apoptotic to a nonapoptotic, caspase-independent mechanism. Homozygous PAK-2D212N primary mouse embryonic fibroblasts that lack the ability to generate the proapoptotic PAK-2p34 show less activation of the effector caspase 3, 6, and 7, indicating that caspase activation of PAK-2 amplifies the apoptotic response through a positive feedback loop resulting in more activation of effector caspases.     

Benzo[c]phenanthrene adducts and nogalamycin inhibit DNA transesterification by vaccinia topoisomerase

Vaccinia DNA topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a specific target site 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p downward arrow N(-1) in duplex DNA. Here we study the effects of position-specific DNA intercalators on the rate and extent of single-turnover DNA transesterification. Chiral C-1 R and S trans-opened 3,4-diol 1,2-epoxide adducts of benzo[c]phenanthrene (BcPh) were introduced at single N2-deoxyguanosine and N6-deoxyadenosine positions within the 3'-G(+5)G(+4)G(+3)A(+2)A(+1)T(-1)A(-2) sequence of the nonscissile DNA strand. Transesterification was unaffected by BcPh intercalation between the +6 and +5 base pairs, slowed 4-fold by intercalation between the +5 and +4 base pairs, and virtually abolished by BcPh intercalation between the +4 and +3 base pairs and the +3 and +2 base pairs. Intercalation between the +2 and +1 base pairs by the +2R BcPh dA adduct abolished transesterification, whereas the overlapping +1S BcPh dA adduct slowed the rate of transesterification by a factor of 2700, with little effect upon the extent of the reaction. Intercalation at the scissile phosphodiester (between the +1 and -1 base pairs) slowed transesterification by a factor of 450. BcPh intercalation between the -1 and -2 base pairs slowed cleavage by two orders of magnitude, but intercalation between the -2 and -3 base pairs had little effect. The anthracycline drug nogalamycin, a non-covalent intercalator with preference for 5'-TG dinucleotides, inhibited the single-turnover DNA cleavage reaction of vaccinia topoisomerase with an IC50 of 0.7 microM. Nogalamycin was most effective when the drug was pre-incubated with DNA and when the cleavage target site was 5'-CCCTT/G instead of 5'-CCCTT/A. These findings demarcate upstream and downstream boundaries of the functional interface of vaccinia topoisomerase with its DNA target site.     

Novel IL10 gene family associations with systemic juvenile idiopathic arthritis

Juvenile idiopathic arthritis (JIA) is the most common cause of chronic childhood disability and encompasses a number of disease subgroups. In this study we have focused on systemic JIA (sJIA), which accounts for approximately 11% of UK JIA cases. This study reports the investigation of three members of the IL10 gene family as candidate susceptibility loci in children with sJIA. DNA from 473 unaffected controls and 172 patients with sJIA was genotyped for a single nucleotide polymorphism (SNP) in IL19 and IL20 and two SNPs in IL10. We examined evidence for association of the four SNPs by single marker and haplotype analysis. Significant differences in allele frequency were observed between cases and controls, for both IL10-1082 (p = 0.031) and IL20-468 (p = 0.028). Furthermore, examination of the haplotypes of IL10-1082 and IL20-468 revealed greater evidence for association (global p = 0.0006). This study demonstrates a significant increased prevalence of the low expressing IL10-1082 genotype in patients with sJIA. In addition, we show a separate association with an IL20 polymorphism, and the IL10-1082A/IL20-468T haplotype. The two marker 'A-T' haplotype confers an odds ratio of 2.24 for sJIA. This positive association suggests an important role for these cytokines in sJIA pathogenesis.     

Glp-1 analog, liraglutide, ameliorates hepatic steatosis and cardiac hypertrophy in C57BL/6J mice fed a Western diet

The aims of this study were designed to determine whether liraglutide, a long-acting glucagon-like peptide, could reverse the adverse effects of a diet high in fat that also contained trans-fat and high-fructose corn syrup (ALIOS diet). Specifically, we examined whether treatment with liraglutide could reduce hepatic insulin resistance and steatosis as well as improve cardiac function. Male C57BL/6J mice were pair fed or fed ad libitum either standard chow or the ALIOS diet. After 8 wk the mice were further subdivided and received daily injections of either liraglutide or saline for 4 wk. Hyperinsulinemic-euglycemic clamp studies were performed after 6 wk, revealing hepatic insulin resistance. Glucose tolerance and insulin resistance tests were performed at 8 and 12 wk prior to and following liraglutide treatment. Liver pathology, cardiac measurements, blood chemistry, and RNA and protein analyses were performed. Clamp studies revealed hepatic insulin resistance after 6 wk of ALIOS diet. Liraglutide reduced visceral adiposity and liver weight (P < 0.001). As expected, liraglutide improved glucose and insulin tolerance. Liraglutide improved hypertension (P < 0.05) and reduced cardiac hypertrophy. Surprisingly, liver from liraglutide-treated mice had significantly higher levels of fatty acid binding protein, acyl-CoA oxidase II, very long-chain acyl-CoA dehydrogenase, and microsomal triglyceride transfer protein. We conclude that liraglutide reduces the harmful effects of an ALIOS diet by improving insulin sensitivity and by reducing lipid accumulation in liver through multiple mechanisms including, transport, and increase β-oxidation.     

Revising Berg-Purcell for finite receptor kinetics

From nutrient uptake to chemoreception to synaptic transmission, many systems in cell biology depend on molecules diffusing and binding to membrane receptors. Mathematical analysis of such systems often neglects the fact that receptors process molecules at finite kinetic rates. A key example is the celebrated formula of Berg and Purcell for the rate that cell surface receptors capture extracellular molecules. Indeed, this influential result is only valid if receptors transport molecules through the cell wall at a rate much faster than molecules arrive at receptors. From a mathematical perspective, ignoring receptor kinetics is convenient because it makes the diffusing molecules independent. In contrast, including receptor kinetics introduces correlations between the diffusing molecules because, for example, bound receptors may be temporarily blocked from binding additional molecules. In this work, we present a modeling framework for coupling bulk diffusion to surface receptors with finite kinetic rates. The framework uses boundary homogenization to couple the diffusion equation to nonlinear ordinary differential equations on the boundary. We use this framework to derive an explicit formula for the cellular uptake rate and show that the analysis of Berg and Purcell significantly overestimates uptake in some typical biophysical scenarios. We confirm our analysis by numerical simulations of a many-particle stochastic system.     

Interictal Neurocognitive Processing of Visual Stimuli in Migraine: Evidence from Event-Related Potentials

Research has established decreased sensory habituation as a defining feature in migraine, while decreased cognitive habituation has only been found with regard to cognitive assessment of the relative probability of the occurrence of a stimulus event. Our study extended the investigation of interictal habituation in migraine to include cognitive processing when viewing of a series of visually-complex images, similar to those we encounter on the internet everyday. We examined interictal neurocognitive function in migraine from a habituation perspective, using a novel paradigm designed to assess how the response to a series of images changes over time. Two groups of participants--migraineurs (N = 25) and non-migraine controls (N = 25)--were asked to view a set of 232 unfamiliar logos in the context of a target identification task as their brain electrical responses were recorded via event-related potentials (ERPs). The set of logos was viewed serially in each of 10 separate trial blocks, with data analysis focusing on how the ERP responses to the logos in frontal electrodes from 200-600 ms changed across time within each group. For the controls, we found that the amplitude of the late positive potential (LPP) ERP component elicited by the logos had no significant change across trial blocks. In contrast, in migraineurs we found that the LPP significantly increased in amplitude across trial blocks, an effect consistent with a lack of habituation to visual stimuli seen in previous research. Our findings provide empirical support abnormal cognitive processing of complex visual images across time in migraineurs that goes beyond the sensory-level habituation found in previous research.