The choice of controls in a case-control study on WBC-DNA adducts and metabolic polymorphisms

The choice of the control group is a key issue in case-control studies, particularly in studies of molecular epidemiology. We discuss the potential bias introduced by different options. To exemplify the consequences of different choices, we have analysed two sets of controls in the context of a case-control study on bladder cancer: 55 were patients with urological conditions (cystitis, prostate hypertrophy), while 49 had a miscellany of medical or surgical conditions. We measured DNA adducts in white blood cells (WBC) by ³²P-postlabelling and a series of metabolic polymorphisms (GSTM1, GSTT1, GSTP1, NAT2, NQO1). While no statistically significant differences were found for metabolic polymorphisms, the two series of controls showed different concentrations of DNA adducts, suggesting that conditions related to bladder cancer or intermediate steps leading to bladder cancer, such as chronic cystitis, may be associated with higher adduct levels. An association between DNA adduct levels and infection has been noted before in experimental animals: both in lung and in the skin, an inflammatory response increased the biologically effective doses of polycyclic aromatic hydrocarbons. An alternative explanation is confounding; in fact, after adjustment for the level of consumption of fruit and vegetables (but not for smoking) the difference between the two control groups was no longer statistically significant. In conclusion, the choice of controls in studies of molecular epidemiology has subtle methodological implications, including confounding of metabolic/molecular measurements by complex exposures such as diet.     

Carnitine is a pharmacological allosteric chaperone of the human lysosomal α-glucosidase

Pompe disease is an inherited metabolic disorder due to the deficiency of the lysosomal acid α-glucosidase (GAA). The only approved treatment is enzyme replacement therapy with the recombinant enzyme (rhGAA). Further approaches like pharmacological chaperone therapy, based on the stabilising effect induced by small molecules on the target enzyme, could be a promising strategy. However, most known chaperones could be limited by their potential inhibitory effects on patient’s enzymes. Here we report on the discovery of novel chaperones for rhGAA, L- and D-carnitine, and the related compound acetyl-D-carnitine. These drugs stabilise the enzyme at pH and temperature without inhibiting the activity and acted synergistically with active-site directed pharmacological chaperones. Remarkably, they enhanced by 4-fold the acid α-glucosidase activity in fibroblasts from three Pompe patients with added rhGAA. This synergistic effect of L-carnitine and rhGAA has the potential to be translated into improved therapeutic efficacy of ERT in Pompe disease.     

Evidence for two separate beta-ketoacyl CoA reductase components of the hepatic microsomal fatty acid chain elongation system in the rat

The hepatic microsomal fatty acid chain elongation system can utilize either NADPH or NADH. Elongation activity, measured as the rate of malonyl CoA incorporation into palmitoyl CoA, was enhanced by a fat-free diet and by bovine serum albumin (BSA) when either cofactor was employed. When the intermediate products were determined, it was observed that in the presence of BSA and NADPH, the predominant product was the saturated elongated fatty acid, whereas in the presence of BSA and NADH, the major intermediate was the beta-ketoacyl derivative. Employing beta-ketostearoyl CoA as substrate, BSA markedly inhibited NADH-supported beta-ketoacyl CoA reductase activity and stimulated NADPH-supported activity. Furthermore, the sum of the NADH-dependent and NADPH-dependent beta-ketoreductase activities approximated the activity obtained when both cofactors were present in the incubation medium, suggesting the existence of two beta-ketoacyl CoA reductases, one using NADH and the other, NADPH.     

Long,Processive Enzymatic DNA Synthesis Using 100% Dye-Labeled Terminal Phosphate-Linked Nucleotides

We demonstrate the efficient synthesis of DNA with complete replacement of the four deoxyribonucleoside triphosphate (dNTP) substrates with nucleotides carrying fluorescent labels. A different, spectrally separable fluorescent dye suitable for single molecule fluorescence detection was conjugated to each of the four dNTPs via linkage to the terminal phosphate. Using these modified nucleotides, DNA synthesis by φ29 DNA polymerase was observed to be processive for products thousands of bases in length, with labeled nucleotide affinities and DNA polymerization rates approaching unmodified dNTP levels. Results presented here show the compatibility of these nucleotides for single-molecule, real-time DNA sequencing applications.     

Polymorphisms of estrogen receptors alpha and beta in idiopathic, infertile Brazilian men: a case-control study

Estrogen plays an important role in the human reproductive system and its action is mediated mainly by two specific receptors: α (ERα) and β (ERβ). There are polymorphic variants in both ER genes, and studies showed their association with reproductive outcomes. We aimed to determine the distribution of ERα and ERβ gene polymorphisms in idiopathic, infertile Brazilian patients in a case–control study comprising 187 idiopathic, infertile Brazilian men with nonobstructive azoospermia (NOA, n = 78) or severe oligozoospermia (SO, n = 109) and 216 fertile men. Detection of ERα (PvuII and XbaI) and ERβ (AluI and RsaI) gene polymorphisms were performed using TaqMan PCR. The results were analyzed statically, and a P‐value < 0.05 was considered significant. Single‐marker analysis revealed that neither PvuII nor XbaI polymorphisms of the ERα gene were associated either with NOA group (P = 0.662 and P = 0.527, respectively) or SO group (P = 0.777 and P = 1.0, respectively). Regarding ERβ polymorphisms, no statistical difference was observed between the AluI polymorphism and NOA group compared to controls (P = 1.0) or between SO group and controls (P = 0.423). We found similar results with the RsaI polymorphism. Statistical analysis did not reveal a difference between NOA (P = 0.740) and SO (P = 0.920) groups compared to controls. Combined genotypes of ERα and ERβ polymorphisms did not identify a haplotype associated with idiopathic infertility. Thus, in the Brazilian population, genetic variations in both estrogen receptors alpha (PvuII and XbaI) and beta (AluI and RsaI) were not relevant to idiopathic infertility. Mol. Reprod. Dev. 78:665–672, 2011. © 2011 Wiley‐Liss, Inc.     

The Novel Mas agonist,CGEN-856S,Attenuates Isoproterenol-Induced Cardiac Remodeling and Myocardial Infarction Injury in Rats

CGEN-856S is a novel Mas agonist. Herein, we examined the effects of this peptide on isoproterenol (ISO)-induced cardiac remodeling and myocardial infarction (MI) injury. We also sought to determine whether CGEN-856S activates the underlying mechanisms related to Mas receptor activation. Heart hypertrophy and fibrosis were induced by ISO (2 mg·kg⁻¹·day⁻¹) in Wistar rats. After a 7-day treatment period with CGEN-856S (90 µg·kg⁻¹·day⁻¹) or vehicle, the cardiomyocyte diameter was evaluated in left ventricular sections stained with hematoxylin and eosin, and immunofluorescence labeling and quantitative confocal microscopy were used to quantify the deposition of type I and III collagen and fibronectin in the left ventricles. MI was induced by coronary artery ligation, and CGEN-856S (90 µg·kg⁻¹·day⁻¹) or saline was administered for 14 days. The Langendorff technique was used to evaluate cardiac function, and left ventricular sections were stained with Masson’s trichrome dye to quantify the infarct area. Using Chinese hamster ovary cells stably transfected with Mas cDNA, we evaluated whether CGEN-856S alters AKT and endothelial nitric oxide synthase (eNOS) phosphorylation. CGEN-856S reduced the degree of ISO-induced hypertrophy (13.91±0.17 µm vs. 12.41±0.16 µm in the ISO+CGEN-856S group). In addition, the Mas agonist attenuated the ISO-induced increase in collagen I, collagen III, and fibronectin deposition. CGEN-856S markedly attenuated the MI-induced decrease in systolic tension, as well as in +dT/dt and -dT/dt. Furthermore, CGEN-856S administration significantly decreased the infarct area (23.68±2.78% vs. 13.95±4.37% in the MI+CGEN-856S group). These effects likely involved the participation of AKT and NO, as CGEN-856S administration increased the levels of p-AKT and p-eNOS. Thus, our results indicate that CGEN-856S exerts cardioprotective effects on ISO-induced cardiac remodeling and MI-mediated heart failure in rats through a mechanism likely involving the eNOS/AKT pathway.     

Mutant huntingtin regulates EGF receptor fate in non-neuronal cells lacking wild-type protein

Huntingtin (htt) is a scaffold protein localized at the subcellular level and is involved in coordinating the activity of several protein for signaling and intracellular transport. The emerging properties of htt in intracellular trafficking prompted us to study the role of mutant htt (polyQ-htt) in the intracellular fate of epidermal growth factor receptor (EGFR), whose activity seems to be strictly regulated by htt. In particular, to evaluate whether protein trafficking dysfunction occurs in non-neuronal cells in the absence of functional htt, we monitored the EGFR protein in fibroblasts from homozygotic HD patients and their healthy counterpart. We found that polyQ-htt controls EGFR degradation and recycling. Lack of wild‐type htt caused alteration of the ubiquitination cycle, formation of EGFR-incorporating high-molecular weight protein aggregates and abnormal EGFR distribution in endosomes of the degradation and recycling pathways after EGF stimulation. PolyQ-htt-induced alteration of EGFR trafficking affected cell migration and proliferation, at least in part, through inhibition of ERK signaling. To our knowledge the data here reported represent the first signaling and phenotypic characterization of polyQ-htt involvement in the modulation of growth factor stimulation in non-neuronal cells.     

Progesterone signaling mediated through progesterone receptor membrane component-1 in ovarian cells with special emphasis on ovarian cancer

Various ovarian cell types including granulosa cells and ovarian surface epithelial cells express the progesterone (P4) binding protein, progesterone receptor membrane component-1 (PGRMC1). PGRMC1 is also expressed in ovarian tumors. PGRMC1 plays an essential role in promoting the survival of both normal and cancerous ovarian cell in vitro. Given the clinical significance of factors that regulate the viability of ovarian cancer, this review will focus on the role of PGRMC1 in ovarian cancer, while drawing insights into the mechanism of PGRMC1's action from cell lines derived from healthy ovaries as well as ovarian tumors.Studies using PGRMC1siRNA demonstrated that P4's ability to inhibit ovarian cells from undergoing apoptosis in vitro is dependent on PGRMC1. To confirm the importance of PGRMC1, the ability of PGRMC1-deplete ovarian cancer cell lines to form tumors in intact nude mice was assessed. Compared to PGRMC1-expressing ovarian cancer cells, PGRMC1-deplete ovarian cancer cells formed tumors in fewer mice (80% compared to 100% for controls). Moreover, the number of tumors derived from PGRMC1-deplete ovarian cancer cells was 50% of that observed in controls. Finally, the tumors that formed from PGRMC1-deplete ovarian cancer cells were about a fourth the size of tumors derived from ovarian cancer cells with normal levels of PGRMC1. One reason for PGRMC1-deplete tumors being smaller is that they had a poorly developed microvasculature system. How PGRMC1 regulates cell viability and in turn tumor growth is not known but part of the mechanism likely involves the regulation of genes that promote cell survival and inhibit apoptosis.