Effects of mutations in the helix G region of horseradish peroxidase

Horseradish peroxidase (HRP) has long attracted intense research interest and is used in many biotechnological fields, including diagnostics, biosensors and biocatalysis. Enhancement of HRP catalytic activity and/or stability would further increase its usefulness. Based on prior art, we substituted solvent-exposed lysine and glutamic acid residues near the proximal helix G (Lys 232, 241; Glu 238, 239) and between helices F and F' (Lys 174). Three single mutants (K232N, K232F, K241N) demonstrated increased stabilities against heat (up to 2-fold) and solvents (up to 4-fold). Stability gains are likely due to improved hydrogen bonding and space-fill characteristics introduced by the relevant substitution. Two double mutants showed stability gains but most double mutations were non-additive and non-synergistic. Substitutions of Lys 174 or Glu 238 were destabilising. Unexpectedly, notable alterations in steady-state V(m)/E values occurred with reducing substrate ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)), despite the distance of the mutated positions from the active site.     

The genetic basis for indole-diterpene chemical diversity in filamentous fungi

Indole-diterpenes are a structurally diverse group of secondary metabolites with a common cyclic diterpene backbone derived from geranylgeranyl diphosphate and an indole group derived from indole-3-glycerol phosphate. Different types and patterns of ring substitutions and ring stereochemistry generate this structural diversity. This group of compounds is best known for their neurotoxic effects in mammals, causing syndromes such as ‘ryegrass staggers’ in sheep and cattle. Because many of the fungi that synthesise these compounds form symbiotic relationships with plants, insects, and other fungi, the synthesis of these compounds may confer an ecological advantage to these associations. Considerable recent progress has been made on understanding indole-diterpene biosynthesis in filamentous fungi, principally through the cloning and characterisation of the genes and gene products for paxilline biosynthesis in Penicillium paxilli. Important insights into how the indole-diterpene backbone is synthesised and decorated have been obtained using P. paxilli mutants in this pathway. This review provides an overview of these recent developments.     

Maternal cigarette smoking, metabolic gene polymorphisms, and preterm delivery: new insights on G×E interactions and pathogenic pathways

Preterm delivery (PTD, <37 weeks of gestation) is a significant clinical and public health problem. Previously, we reported that maternal smoking and metabolic gene polymorphisms of CYP1A1 MspI and GSTT1 synergistically increase the risk of low birth weight. This study investigates the relationship between maternal smoking and metabolic gene polymorphisms of CYP1A1 MspI and GSTT1 with preterm delivery (PTD) as a whole and preterm subgroups. This case–control study included 1,749 multi-ethnic mothers (571 with PTD and 1,178 controls) enrolled at Boston Medical Center. After adjusting covariates, regression analyses were performed to identify individual and joint associations of maternal smoking, two functional variants of CYP1A1 and GSTT1 with PTD. We observed a moderate effect of maternal smoking on PTD (OR = 1.6; 95% CI: 1.1–2.2). We found that compared to non-smoking mothers with low-risk genotypes, there was a significant joint association of maternal smoking, CYP1A1 (Aa/aa) and GSTT1 (absent) genotypes with gestational age (β = −3.37; SE = 0.86; P = 9 × 10⁻⁵) and with PTD (OR = 5.8; 95% CI: 2.0–21.1), respectively. Such joint association was particularly strong in certain preterm subgroups, including spontaneous PTD (OR = 8.3; 95% CI: 2.7–30.6), PTD < 32 weeks (OR = 11.1; 95% CI: 2.9–47.7), and PTD accompanied by histologic chorioamnionitis (OR = 15.6; 95% CI: 4.1–76.7). Similar patterns were observed across ethnic groups. Taken together, maternal smoking significantly increased the risk of PTD among women with high-risk CYP1A1 and GSTT1 genotypes. Such joint associations were strongest among PTD accompanied by histologic chorioamnionitis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Prior hypoglycemia enhances glucose responsiveness in some ventromedial hypothalamic glucosensing neurons

Antecedent insulin-induced hypoglycemia (IIH) reduces adrenomedullary responses (AMR) to subsequent bouts of hypoglycemia. The ventromedial hypothalamus [VMH: arcuate (ARC) + ventromedial nuclei] contains glucosensing neurons, which are thought to be mediators of these AMR. Since type 1 diabetes mellitus often begins in childhood, we used juvenile (4- to 5-wk-old) rats to demonstrate that a single bout of IIH (5 U/kg sc) reduced plasma glucose by 24% and peak epinephrine by 59% 1 day later. This dampened AMR was associated with 46% higher mRNA for VMH glucokinase, a key mediator of neuronal glucosensing. Compared with neurons from saline-injected rats, ventromedial nucleus glucose-excited neurons from insulin-injected rats demonstrated a leftward shift in their glucose responsiveness (EC50 = 0.45 and 0.10 mmol/l for saline and insulin, respectively, P = 0.05) and a 31% higher maximal activation by glucose (P = 0.05), although this maximum occurred at a higher glucose concentration (saline, 0.7 vs. insulin, 1.5 mmol/l). Although EC50 values did not differ, ARC glucose-excited neurons had 19% higher maximal activation, which occurred at a lower glucose concentration in insulin- than saline-injected rats (saline, 2.5 vs. insulin, 1.5 mmol/l). In addition, ARC glucose-inhibited neurons from insulin-injected rats were maximally inhibited at a fivefold lower glucose concentration (saline, 2.5 vs. insulin, 0.5 mmol/l), although this inhibition declined at >0.5 mmol/l glucose. These data suggest that the increased VMH glucokinase after IIH may contribute to the increased responsiveness of VMH glucosensing neurons to glucose and the associated blunting of the AMR.     

The role of eNOS, iNOS, and NF-kappaB in upregulation and activation of cyclooxygenase-2 and infarct size reduction by atorvastatin

Pretreatment with atorvastatin (ATV) reduces infarct size (IS) and increases myocardial expression of phosphorylated endothelial nitric oxide synthase (p-eNOS), inducible NOS (iNOS), and cyclooxygenase-2 (COX2) in the rat. Inhibiting COX2 abolished the ATV-induced IS limitation without affecting p-eNOS and iNOS expression. We investigated 1) whether 3-day ATV pretreatment limits IS in eNOS(-/-) and iNOS(-/-) mice and 2) whether COX2 expression and/or activation by ATV is eNOS, iNOS, and/or NF-kappaB dependent. Male C57BL/6 wild-type (WT), University of North Carolina eNOS(-/-) and iNOS(-/-) mice received ATV (10 mg.kg(-1).day(-1); ATV(+)) or water alone (ATV(-)) for 3 days. Mice underwent 30 min of coronary artery occlusion and 4 h of reperfusion, or hearts were harvested and subjected to ELISA, immunoblotting, biotin switch, and electrophoretic mobility shift assay. As a result, ATV reduced IS only in the WT mice. ATV increased eNOS, p-eNOS, iNOS, and COX2 levels and activated NF-kappaB in WT mice. It also increased myocardial COX2 activity. In eNOS(-/-) mice, ATV increased COX2 expression but not COX2 activity or iNOS expression. NF-kappaB was not activated by ATV in the eNOS(-/-) mice. In the iNOS(-/-) mice, eNOS and p-eNOS levels were increased but not iNOS and COX2 levels; however, NF-kappaB was activated. In conclusion, both eNOS and iNOS are essential for the IS-limiting effect of ATV. The expression of COX2 by ATV is iNOS, but not eNOS or NF-kappaB, dependent. Activation of COX2 is dependent on iNOS.     

Role of TNF-alpha-induced reactive oxygen species in endothelial dysfunction during reperfusion injury

We hypothesized that neutralization of TNF-alpha at the time of reperfusion exerts a salubrious role on endothelial function and reduces the production of reactive oxygen species. We employed a mouse model of myocardial ischemia-reperfusion (I/R, 30 min/90 min) and administered TNF-alpha neutralizing antibodies at the time of reperfusion. I/R elevated TNF-alpha expression (mRNA and protein), whereas administration of anti-TNF-alpha before reperfusion attenuated TNF-alpha expression. We detected TNF-alpha expression in vascular smooth muscle cells, mast cells, and macrophages, but not in the endothelial cells. I/R induced endothelial dysfunction and superoxide production. Administration of anti-TNF-alpha at the onset of reperfusion partially restored nitric oxide-mediated coronary arteriolar dilation and reduced superoxide production. I/R increased the activity of NAD(P)H oxidase and of xanthine oxidase and enhanced the formation of nitrotyrosine residues in untreated mice compared with shams. Administration of anti-TNF-alpha before reperfusion blocked the increase in activity of these enzymes. Inhibition of xanthine oxidase (allopurinol) or NAD(P)H oxidase (apocynin) improved endothelium-dependent dilation and reduced superoxide production in isolated coronary arterioles following I/R. Interestingly, I/R enhanced superoxide generation and reduced endothelial function in neutropenic animals and in mice treated with a neutrophil NAD(P)H oxidase inhibitor, indicating that the effects of TNF-alpha are not through neutrophil activation. We conclude that myocardial ischemia initiates TNF-alpha expression, which induces vascular oxidative stress, independent of neutrophil activation, and leads to coronary endothelial dysfunction.     

Mouse Ae1 E699Q mediates SO42-i/anion-o exchange with [SO42-]i-dependent reversal of wild-type pHo sensitivity

The SLC4A1/AE1 gene encodes the electroneutral Cl(-)/HCO(3)(-) exchanger of erythrocytes and renal type A intercalated cells. AE1 mutations cause familial spherocytic and stomatocytic anemias, ovalocytosis, and distal renal tubular acidosis. The mutant mouse Ae1 polypeptide E699Q expressed in Xenopus oocytes cannot mediate Cl(-)/HCO(3)(-) exchange or (36)Cl(-) efflux but exhibits enhanced dual sulfate efflux mechanisms: electroneutral exchange of intracellular sulfate for extracellular sulfate (SO(4)(2-)(i)/SO(4)(2-)(o) exchange), and electrogenic exchange of intracellular sulfate for extracellular chloride (SO(4)(2-)(i)/Cl(-)(o) exchange). Whereas wild-type AE1 mediates 1:1 H(+)/SO(4)(2-) cotransport in exchange for either Cl(-) or for the H(+)/SO(4)(2-) ion pair, mutant Ae1 E699Q transports sulfate without cotransport of protons, similar to human erythrocyte AE1 in which the corresponding E681 carboxylate has been chemically converted to the alcohol (hAE1 E681OH). We now show that in contrast to the normal cis-stimulation by protons of wild-type AE1-mediated SO(4)(2-) transport, both SO(4)(2-)(i)/Cl(-)(o) exchange and SO(4)(2-)(i)/SO(4)(2-)(o) exchange mediated by mutant Ae1 E699Q are inhibited by acidic pH(o) and activated by alkaline pH(o). hAE1 E681OH displays a similarly altered pH(o) dependence of SO(4)(2-)(i)/Cl(-)(o) exchange. Elevated [SO(4)(2-)](i) increases the K(1/2) of Ae1 E699Q for both extracellular Cl(-) and SO(4)(2-), while reducing inhibition of both exchange mechanisms by acid pH(o). The E699Q mutation also leads to increased potency of self-inhibition by extracellular SO(4)(2-). Study of the Ae1 E699Q mutation has revealed the existence of a novel pH-regulatory site of the Ae1 polypeptide and should continue to provide valuable paths toward understanding substrate selectivity and self-inhibition in SLC4 anion transporters.     

Recognition of a common rDNA target site in archaea and eukarya by analogous LAGLIDADG and His-Cys box homing endonucleases

The presence of a homing endonuclease gene (HEG) within a microbial intron or intein empowers the entire element with the ability to invade genomic targets. The persistence of a homing endonuclease lineage depends in part on conservation of its DNA target site. One such rDNA sequence has been invaded both in archaea and in eukarya, by LAGLIDADG and His–Cys box homing endonucleases, respectively. The bases encoded by this target include a universally conserved ribosomal structure, termed helix 69 (H69) in the large ribosomal subunit. This region forms the ‘B2a’ intersubunit bridge to the small ribosomal subunit, contacts bound tRNA in the A- and P-sites, and acts as a trigger for ribosome disassembly through its interactions with ribosome recycling factor. We have determined the DNA-bound structure and specificity profile of an archaeal LAGLIDADG homing endonuclease (I-Vdi141I) that recognizes this target site, and compared its specificity with the analogous eukaryal His–Cys box endonuclease I-PpoI. These homodimeric endonuclease scaffolds have arrived at similar specificity profiles across their common biological target and analogous solutions to the problem of accommodating conserved asymmetries within the DNA sequence, but with differences at individual base pairs that are fine-tuned to the sequence conservation of archaeal versus eukaryal ribosomes.     

Genetic variants in the 8q24 locus and risk of testicular germ cell tumors

Much evidence supports the premise that population genetic variation contributes significantly to the risk of testicular germ-cell tumor (TGCT). However, investigations of the association between genomic markers and TGCT susceptibility are scarce. Single nucleotide polymorphisms (SNPs) at the locus 8q24 have recently been found to be associated with prostate, colorectal and breast cancer. The US Servicemen’s testicular tumor environmental and endocrine determinants (STEED) study was used to investigate the association of 15 specific 8q24 SNPs with TGCT and its two main histologic groups of seminoma and nonseminoma. Conditional and unconditional logistic regression models, adjusted for the matching variables of year of birth, race/ethnicity and serum date, were utilized to produce odds ratios (OR) and 95% confidence intervals (95%CI). The analysis included 680 controls and 568 TGCT cases. In the TGCT analysis, no SNP was associated with risk in both heterozygotes and variant homozygotes. When stratified by histology the seminoma analysis also showed no association with the 8q24 SNPs. Conversely, the analysis of nonseminomas had three tentative associations (rs6470494, OR_{genotype AG} = 1.15, 95%CI: 0.86–1.54; OR_{genotype GG} = 1.68, 95%CI: 1.04–2.73; p for trend = 0.04) (rs13254738, OR_{genotype GT} = 1.04, 95%CI: 0.77–1.40; OR_{genotype TT} = 1.62, 95%CI: 1.06–2.49; p for trend = 0.07) (rs10505476, OR_{genotype CT} = 0.67, 95%CI: 0.50, 0.91; OR_{genotype TT} = 0.81, 95%CI: 0.47–1.39; p for trend = 0.04). There was no linkage disequilibrium between any of the 8q24 SNPs analyzed in this population. In conclusion, this study has found little evidence for an association between the reported 8q24 SNPs and TGCTs, although the findings for nonseminoma may be worth further investigation.     

Discovery of novel 4-amino-6-arylaminopyrimidine-5-carbaldehyde oximes as dual inhibitors of EGFR and ErbB-2 protein tyrosine kinases

We herein disclose a novel series of 4-aminopyrimidine-5-carbaldehyde oximes that are potent and selective inhibitors of both EGFR and ErbB-2 tyrosine kinases, with IC(50) values in the nanomolar range. Structure-activity relationship (SAR) studies elucidated a critical role for the 4-amino and C-6 arylamino moieties. The X-ray co-crystal structure of EGFR with 37 was determined and validated our design rationale.