Frequency distribution of thiopurine S-methyltransferase alleles in a polish population

Thiopurine S-methyltransferase (TPMT) is an enzyme that catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathioprine. TPMT activity exhibits an interindividual variability mainly a result of genetic polymorphism. Patients with intermediate or deficient TPMT activity are at risk for toxicity after receiving standard doses of thiopurine drugs. It has previously been reported that 3 variant alleles:TPMT*2, *3A, and *3C are responsible for over 95% cases of lower enzyme activity. The purpose of this study was to determine the frequency of TPMT variant alleles in a Polish population. DNA samples were obtained from 358 unrelated healthy Polish subjects of white origin, and TPMT genetic polymorphism was determined using PCR-RFLP and allele-specific PCR methods. The results showed that allelic frequencies were 0.4% for TPMT*2, 2.7% for TPMT*3A, and 0.1% for TPMT*3C, respectively. A TPMT*3B allele was not found in the studied population. The general pattern of TPMT allele disposition in the Polish population is similar to those determined for other white populations, but the frequency of total variant alleles is lower than in other European populations studied to date.     

Group III mGlu receptor agonists produce anxiolytic- and antidepressant-like effects after central administration in rats

It was well established that compounds which decrease glutamatergic transmission via blockade of NMDA or group I mGlu receptors produce anxiolytic- and antidepressant-like action in animal tests and models. Since group III metabotropic glutamate receptor (mGluR) agonists are known to reduce glutamatergic neurotransmission by the inhibition of glutamate release, we decided to investigate potential anxiolytic- and/or antidepressant-like effects of group III mGluR agonists, after central administration in rats. It was found that group III mGluR agonists, (1S,3R,4S)-1-aminocyclo-pentane-1,3,4-tricarboxylic acid (ACPT-I) and 2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid (HomoAMPA), given intrahippocampally, produced a dose-dependent anxiolytic-like effect in the conflict drinking test. The effects of ACPT-I and HomoAMPA were reversed by (RS)-alpha-cyclopropyl-4-phosphonophenyl glycine (CPPG), group III mGluR antagonist. Moreover, a dose-dependent antidepressant-like action of group III mGluR agonists, ACPT-I and (RS)-4-phosphonophenylglycine (RS-PPG), but not HomoAMPA, was found in behavioral despair test, after intracerebroventricular injections, and the effect of ACPT-I was reversed by CPPG. The results obtained indicate that group III mGluR agonists produce anxiolytic- as well as antidepressant-like effects in behavioral tests, after central administration in rats. The reduction of glutamate release by group III mGluR activation may be a possible mechanism underlying anxiolytic- and antidepressant-like properties of the tested compounds. In conclusion, the results of our studies indicate that group III mGlu receptor agonists may play a role in the therapy of both anxiety and depression.     

Identification of novel metabolites of pioglitazone in rat and dog

1. Four new metabolites of pioglitazone were identified by liquid chromatography-mass spectrometry (LC-MS/MS) as being formed by hydroxylation (M-VII and M-VIII), opening of the thiazolidinedione ring (M-X) and by desaturation of the terminal ethyl side chain or tether ethoxy moiety (M-IX), respectively. The structure of one of the hydroxylated metabolites (M-VII) was confirmed by chemical modification using the Jones reaction. 2. Oxidative cleavage of the thiazolidinedione ring is a novel pathway not previously reported for pioglitazone. 3. The hydroxylated M-VII was detected in incubations with rat, dog and human liver and kidney microsomes, and in plasma from rats and dogs dosed orally with [(3)H]pioglitazone. 4. The carboxylic acid derivative of M-VII (M-V) and its taurine conjugate were the major radioactive components in dog bile.     

Culture of human trophoblast cells in vitro as a research method--review of techniques (Part II)

In part II, management of the human trophoblast cell-cultures and methods of quality control are presented. The examples of application of this in vitro model for scientific research are also given.     

Plasminogen activator inhibitor 1 (PAI-1) levels and gene promoter polymorphisms in subjects with colorectal cancer

The high level of plasminogen activator inhibitor 1 (PAI-1) in colorectal cancer predicts poor prognosis for patients. The insertion (5G)/deletion (4G) polymorphism (the 4G/5G polymorphism) and G-->A single base substitution (the G/A polymorphism) located at promoter of PAI-1 gene may have functional significance in regulation of its expression. In the present work the level of PAI-1, distribution of genotypes and frequency of alleles of the 4G/5G and G/A polymorphisms in samples of cancer tissue and normal mucosa as well as in blood were investigated. Blood, tumor and normal tissues were obtained from 40 patients with colorectal cancer. The 4G/5G and G/A polymorphism were determined by PCR amplification using the allele specific primers. The PAI-1 level was measured by enzyme linked immunosorbent assay (ELISA). The distribution of the genotypes of both polymorphisms did not differ significantly (p > 0.05) from those predicted by the Hardy-Weinberg distribution. There were no differences in the genotype distributions and allele frequencies between blood, normal mucosa samples and cancer tissue. The 4G/5G and G/A polymorphisms were in linkage disequilibrium. The average level of PAI-1 in tumor samples was significantly (p < 0.05) higher than in normal tissue. The results obtained indicate that a higher level of PAI-1 can be associated with colorectal cancer. On the other hand, in colon cancer, the 4G/5G and G/A polymorphisms are not linked with elevated levels of PAI-1 and therefore may not be used to predict colon cancer prognosis.     

Influence of histamine on the process of human trophoblast differentiation

Inflammation Research - .     

Zonulin,inflammation and iron status in patients with early stages of chronic kidney disease

Background/aims

Zonulin is the only known regulator of intestinal permeability. It is also considered as a potential inflammatory marker in several conditions such as diabetes and inflammatory bowel syndrome. The aim of the study was to investigate zonulin levels in patients with early stages of CKD and its possible correlation with inflammation, anemia and iron status parameters.

Methods

Eighty-eight patients with early stages of CKD and 23 healthy volunteers were enrolled in the study. Zonulin, hepcidin-25, soluble transferrin receptor, interleukin-6 and high-sensitivity C-reactive protein were measured using commercially available assays.

Results

Zonulin was significantly lower among patients with CKD in comparison with healthy volunteers. There were no statistically significant differences in zonulin concentration between patients with and without inflammation. Zonulin was significantly correlated with hepcidin only in patients with inflammation. Zonulin was neither related to iron nor related to ferritin.

Conclusions

Zonulin cannot be considered as an inflammatory marker in CKD. It does not play a role in the disturbances of iron metabolism in CKD. Its physiological role remains to be elucidated.

     

Influence of 825 C>T polymorphism of G protein β3 subunit gene (GNB3) on hemodynamic response during dobutamine stress echocardiography

There is a wide interpersonal difference to dobutamine response during dobutamine stress echocardiography (DSE). The aim of this study was to determine an association between GNB3 825C>T gene polymorphism, encoding the β3-subunit of G protein, and heart rate (HR), systolic (SBP) and diastolic blood pressure (DBP) response to dobutamine during DSE. The study involved 119 patients with clinical indications for DSE. Genotyping of GNB3 825C>T (rs5443) polymorphism was based on PCR-RFLP method with BseDI restriction enzyme. Significantly higher levels of both resting SBP and DBP in 825T allele carriers vs. 825CC patients were revealed. HR of 825CC vs. CT + TT subjects was slower along the test period reaching marked difference at dobutamine level 30 μg/kg/min (109 ± 20 vs. 116 ± 18 bpm, respectively, p = 0.047). SBP and DBP were markedly lower in 825CC homozygotes compared to 825T allele carriers throughout DSE. It can be concluded that GNB3 825C>T polymorphism is associated with resting SBP and DBP in Polish Caucasian patients subjected for diagnostic DSE. The polymorphism also modulate HR, SBP and DBP response during DSE.     

Expression of genes involved in xenobiotic metabolism and transport in end-stage liver disease: up-regulation of ABCC4 and CYP1B1

BackgroundExpression of drug-metabolizing enzymes and drug transporters in liver is mainly regulated by a system of nuclear receptors. The aim of the current study was to investigate the expression of nuclear receptors, as well as these enzymes and transporters, in liver samples from patients suffering from end-stage liver disease of various etiologies (HCV infection, alcohol liver disease, and primary sclerosis cholangitis).MethodsGene expression was measured using quantitative real-time PCR with surgical specimens from livers of patients with end-stage liver disease, and non-tumoral liver tissue that served as control.ResultsOur study confirmed that the expression of most phase I enzymes is suppressed in end-stage liver disease, and is correlated with a decrease in NR1I2 and NR1I3, the main regulators of xenobiotic metabolism. While mRNA levels of phase II enzymes were generally unchanged, some ABC transporters were up-regulated. The most spectacular increases in expression were observed with ABCC4 (MRP4) – at the mRNA level, and CYP1B1 – at both the mRNA and protein levels. We also demonstrated that IL-6 can induce CYP1B1 expression independently of CYP1A1, in a human hepatocellular liver carcinoma cell line.ConclusionsAs CYP1B1 is an enzyme which converts various substrates into carcinogenous metabolites, its overexpression in liver may be one of the factors increasing the risk of hepatic cancers inpatients with liver disease. CYP1A1 and CYP1B1 are often referred to as model AHR target genes, but CYP1A1 was down-regulated in diseased liver samples. This points to the existence of differences in regulation of these two genes.     

Plasma Neutrophil Gelatinase-Associated Lipocalin (NGAL) Correlations With Cystatin C, Serum Creatinine, and Glomerular Filtration Rate in Patients After Heart and Lung Transplantation

Objective

Kidney injury represents a major clinical problem in orthotopic heart transplant (OHT) patients which seriously increases the mortality rate. The aim of this work was to evaluate the utility of a new kidney damage marker—neutrophil gelatinase-associated lipocalin (NGAL)—and its correlations with cystatin C, creatinine, and glomerular filtration rate (GFR) among patients after heart or lung transplantation.

Materials and Methods

We examined serum samples from 71 patients after heart transplantation, 7 patients after lung transplantation, and 20 healthy controls to measure serum NGAL using an enzyme-linked immunosorbent assay (ELISA) method; cystatin C using latex immunonephelometry; and creatinine using the alkaline-picrate method.

Results

Serum NGAL levels were significantly elevated among transplant patients, but did not significantly correlate with cystatin C in the transplant group (R = .13; P = .11) or in the control group (R = .26; P = .26), or GFR in the transplant group (R = −.09; P = .25) or in the control cohort (R = .22; P = .36). In the transplant group, serum NGAL positively correlated with serum creatinine (R = .27; P = .001).

Conclusions

Plasma NGAL was not a specific biomarker for monitoring chronic renal disorders. We did not exclude other pathologies that might contribute to increased serum NGAL levels.