In thrombin stimulated human platelets Citalopram, Promethazine, Risperidone, and Ziprasidone, but not Diazepam, may exert their pharmacological effects also through intercalation in membrane phospholipids in a receptor-independent manner

Intercalation of drugs in the platelet membrane affects phospholipid-requiring enzymatic processes according to the drugs’ intercalation capability. We investigated effects of Promethazine, Citalopram, Ziprasidone, Risperidone, and Diazepam on phospholipase A₂ (PLA₂) and polyphosphoinositide (PPI) metabolism in thrombin-stimulated human platelets. We also examined effects of the drugs on monolayers of glycerophospholipids using the Langmuir technique. Diazepam did not influence PLA ₂ activity, had no effects on PPI cycle, and caused no change in mean molecular area of phospholipid monolayers. The remaining psychotropic drugs affected these parameters in different ways and levels of potency suggesting that they act by being intercalated between the molecules of adjacent membrane phospholipids, thus causing changes in substrate availability for phospholipid-hydrolyzing enzymes (PLA₂ and Phospholipase C). We show that several psychotropic drugs can also have other cellular effects than receptor antagonism. These effects may be implicated in the psychotropic effects of the drugs and/or their side effects.     

A Genome-Wide Association Study Confirms VKORC1, CYP2C9, and CYP4F2 as Principal Genetic Determinants of Warfarin Dose

We report the first genome-wide association study (GWAS) whose sample size (1,053 Swedish subjects) is sufficiently powered to detect genome-wide significance (p<1.5×10⁻⁷) for polymorphisms that modestly alter therapeutic warfarin dose. The anticoagulant drug warfarin is widely prescribed for reducing the risk of stroke, thrombosis, pulmonary embolism, and coronary malfunction. However, Caucasians vary widely (20-fold) in the dose needed for therapeutic anticoagulation, and hence prescribed doses may be too low (risking serious illness) or too high (risking severe bleeding). Prior work established that ~30% of the dose variance is explained by single nucleotide polymorphisms (SNPs) in the warfarin drug target VKORC1 and another ~12% by two non-synonymous SNPs (*2, *3) in the cytochrome P450 warfarin-metabolizing gene CYP2C9. We initially tested each of 325,997 GWAS SNPs for association with warfarin dose by univariate regression and found the strongest statistical signals (p<10⁻⁷⁸) at SNPs clustering near VKORC1 and the second lowest p-values (p<10⁻³¹) emanating from CYP2C9. No other SNPs approached genome-wide significance. To enhance detection of weaker effects, we conducted multiple regression adjusting for known influences on warfarin dose (VKORC1, CYP2C9, age, gender) and identified a single SNP (rs2108622) with genome-wide significance (p=8.3×10⁻¹⁰) that alters protein coding of the CYP4F2 gene. We confirmed this result in 588 additional Swedish patients (p<0.0029) and, during our investigation, a second group provided independent confirmation from a scan of warfarin-metabolizing genes. We also thoroughly investigated copy number variations, haplotypes, and imputed SNPs, but found no additional highly significant warfarin associations. We present power analysis of our GWAS that is generalizable to other studies, and conclude we had 80% power to detect genome-wide significance for common causative variants or markers explaining at least 1.5% of dose variance. These GWAS results provide further impetus for conducting large-scale trials assessing patient benefit from genotype-based forecasting of warfarin dose.     

Defective reproductive organ morphology and function in domestic rooster embryonically exposed to o,p'-DDT or ethynylestradiol

Environmental pollutants with estrogenic activity have a potential to disrupt estrogen-dependent developmental processes. The objective of this study was to investigate if embryonic exposure to the environmental estrogens o,p'-DDT (1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2,2-trichloroethane; 37, 75, 150 or 300 microg/g egg) and EE2 (17alpha-ethynyl estradiol; 60 ng/g egg) affects the reproductive system in domestic roosters. Following egg injection on Embryonic Day 4, the newly hatched chicks were sexed by cloacal inspection. A skewed phenotypic sex ratio with overrepresentation of chicks deemed as females was observed in the groups exposed to the three highest doses of o,p'-DDT but not in the EE2-exposed group. Normal sex ratios were observed in all groups at adulthood. However, a cloacal deformation seemed to remain in the adult roosters, causing an abnormal semen flow upon semen collection. Semen yield was significantly reduced in both o,p'-DDT-exposed and EE2- exposed birds, whereas semen quality was unaffected. When killed, deformations of the left testis were found in all treatment groups. Image analysis revealed a reduced seminiferous tubular area in the roosters exposed to the two highest doses of o,p'-DDT. Embryonic exposure to o,p'-DDT caused decreased comb weight and right-spur diameter, while EE2 only affected right-spur diameter. In conclusion, this study shows that embryonic exposure to estrogenic compounds can induce permanent effects in male birds. The effects of the two studied compounds were partly similar but o,p'-DDT also induced alterations not seen in the EE2-treated birds.     

Characterization of Foxp3+CD4+CD25+ and IL-10-secreting CD4+CD25+ T cells during cure of colitis

CD4+CD25+ regulatory T cells can prevent and resolve intestinal inflammation in the murine T cell transfer model of colitis. Using Foxp3 as a marker of regulatory T cell activity, we now provide a comprehensive analysis of the in vivo distribution of Foxp3+CD4+CD25+ cells in wild-type mice, and during cure of experimental colitis. In both cases, Foxp3+CD4+CD25+ cells were found to accumulate in the colon and secondary lymphoid organs. Importantly, Foxp3+ cells were present at increased density in colon samples from patients with ulcerative colitis or Crohn's disease, suggesting similarities in the behavior of murine and human regulatory cells under inflammatory conditions. Cure of murine colitis was dependent on the presence of IL-10, and IL-10-producing CD4+CD25+ T cells were enriched within the colon during cure of colitis and also under steady state conditions. Our data indicate that although CD4+CD25+ T cells expressing Foxp3 are present within both lymphoid organs and the colon, subsets of IL-10-producing CD4+CD25+ T cells are present mainly within the intestinal lamina propria suggesting compartmentalization of the regulatory T cell response at effector sites.     

Decrease in intramuscular lipid droplets and translocation of HSL in response to muscle contraction and epinephrine

A better understanding of skeletal muscle lipid metabolism is needed to identify the molecular mechanisms relating intramuscular triglyceride (IMTG) to muscle metabolism and insulin sensitivity. An increasing number of proteins have been reported to be associated with intracellular triglyceride (TG), among them the PAT family members: perilipin, ADRP (for adipocyte differentiation-related protein), and TIP47 (for tail-interacting protein of 47 kDa). Hormone-sensitive lipase (HSL) is thought to be the major enzyme responsible for IMTG hydrolysis in skeletal muscle. In adipocytes, regulation of HSL by intracellular redistribution has been demonstrated. The existence of such regulatory mechanisms in skeletal muscle has long been hypothesized but has never been demonstrated. The aim of this study was to characterize the PAT family proteins associated with IMTG and to investigate the effect of epinephrine stimulation or muscle contraction on skeletal muscle TG content and HSL intracellular distribution. Rat soleus muscles were either incubated with epinephrine or electrically stimulated for 15 min. Single muscle fibers were used for morphological analysis by confocal and transmission electron microscopy. We show a decrease in IMTG in response to both lipolytic stimuli. Furthermore, we identify two PAT family proteins, ADRP and TIP47, associated with IMTG. Finally, we demonstrate HSL translocation to IMTG and ADRP after stimulation with epinephrine or contraction.     

Interactions of chromogranin A-derived vasostatins and monolayers of phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine

Vasostatin-I (CgA1-76) is a naturally occurring and biologically active N-terminal peptide derived from chromogranin A (CgA), produced and secreted at high concentrations by neuroendocrine tissues and also from a range of neuroendocrine tumors. This study aims to examine the hypothesis that in the absence of classical protein receptors CgA1-76 may, like its two derived peptides CgA1-40 and CgA47-66, perturb the lipid microenvironment of other membrane receptors, as a basis for the largely inhibitory activities of these CgA peptides. The nature of the interactions between phospholipids and vasostatin-derived fragments was studied in the Langmuir film balance apparatus at 37 degrees C. The synthetic peptides CgA1-40 and CgA47-66 and a recombinant fragment (VS-I) containing vasostatin-I (Ser-Thr-Ala-CgA1-78) were compared for their effects on monolayers of phosphatidylcholine and phosphatidylethanolamine from pig brain and defined species of phosphatidylserine. Marked differences in surface pressure-area isotherms and phase-transition plateaus were apparent with the three classes of phospholipids on VS-I, CgA1-40 and CgA47-66 in physiological buffer or pure water. The results indicate that VS-I and CgA47-66 at 5-10 nM concentrations may engage in electrostatic as well as hydrophobic interactions with membrane-relevant phospholipids at physiological conditions, VS-I in particular enhancing the fluidity of saturated species of phosphatidylserine.     

Impaired mucus-bicarbonate barrier in Helicobacter pylori-infected mice

To resist the harsh intrinsic milieu, several lines of defense exist in the stomach. The aim of this study was to investigate the effect of the gastric pathogen Helicobacter pylori on these mechanisms in vivo. We used FVB/N mice expressing human alpha-1,3/4-fucosyl transferase (producing Lewis b epitopes) and inoculated with H. pylori 1. Mice were anesthetized with isoflurane or Hypnorm-midazolam, the stomach was exteriorized, and the surface of the corpus mucosa was exposed. Mucus thickness was measured with micropipettes, juxtamucosal pH (pH(jm)) was measured with pH-sensitive microelectrodes, blood flow was measured with laser-Doppler flowmetry, and mRNA levels of the bicarbonate transporter SLC26A9 were quantified with real-time PCR. The increase in mucosal blood flow seen in response to luminal acid (pH 1.5) in control animals (140 +/- 9% of control) was abolished in infected mice. The firmly adherent mucus layer was significantly thinner in infected mice (31 +/- 2 microm) than in control mice (46 +/- 5 microm), and no mucus accumulation occurred in infected mice. pH(jm) decreased significantly more on exposure to luminal acid in infected mice (luminal pH 1.5, pH(jm) 2.4 +/- 0.7) than in control mice (pH(jm) 6.4 +/- 0.5). Despite reduced pH(jm), SLC26A9 mRNA expression was significantly, by increased 1.9-fold, in infected mice. The reduction in pH(jm) by infection with H. pylori might be due to a reduced firmly adherent mucus layer, increased mucus permeability to H(+), and/or inhibition of bicarbonate transport. The upregulation of SLC26A9 in H. pylori-infected epithelium might be a result of continuous inhibition of the transporter, e.g., by ammonium, a H. pylori product, which has been previously shown to inhibit SLC26A9.     

Induction of a midbrain dopaminergic phenotype in Nurr1-overexpressing neural stem cells by type 1 astrocytes.

The implementation of neural stem cell lines as a source material for brain tissue transplants is currently limited by the ability to induce specific neurochemical phenotypes in these cells. Here, we show that coordinated induction of a ventral mesencephalic dopaminergic phenotype in an immortalized multipotent neural stem cell line can be achieved in vitro. This process requires both the overexpression of the nuclear receptor Nurr1 and factors derived from local type 1 astrocytes. Over 80% of cells obtained by this method demonstrate a phenotype indistinguishable from that of endogenous dopaminergic neurons. Moreover, this procedure yields an unlimited number of cells that can engraft in vivo and that may constitute a useful source material for neuronal replacement in Parkinson's disease.     

Mortality among twins after age 6: fetal origins hypothesis versus twin method.

OBJECTIVE--To test the validity of the fetal origins hypothesis and the classic twin method. DESIGN--Follow up study of pairs of same sex twins in which both twins survived to age 6. SETTING--Denmark. SUBJECTS--8495 twin individuals born 1870-1900, followed through to 31 December 1991. MAIN OUTCOME MEASURES--Mortality calculated on a cohort basis. RESULTS--Mortality among twins and the general population was not significantly different except among females aged 60-89, in whom mortality among twins was 1.14 times (SE 0.03) higher than in the general population. Mortality among female dizygotic twins was 1.77 times (0.18) higher than among monozygotic twins at age 30-59. Otherwise, mortality for monozygotic and dizygotic twins did not consistently differ after age 6. CONCLUSION--According to the fetal origins hypothesis the risk of adult morbidity and mortality is heightened by retardation in intrauterine growth. Twins, and in particular monozygotic twins, experience growth retardation in utero. The findings in the present study suggest that the fetal origins hypothesis is not true for the retardation in intrauterine growth experienced by twins. Furthermore, the data are inconsistent with the underlying assumption of a recent claim that the classic twin method is invalid for studies of adult diseases. The present study is, however, based on the one third of all pairs of twins in which both twins survived to age 6. The possible impact of this selection can be evaluated in future studies of cohorts of younger twins with lower perinatal and infant mortality.