TaqMan REAL-Time PCR-based approach for differentiation between <Emphasis Type="Italic">Globodera rostochiensis</Emphasis> (golden nematode) and <Emphasis Type="Italic">Globodera artemisiae</Emphasis> species

Cyst nematodes from the genus Globodera are common, and widely distributed parasites of Solaraceae. Intact cysts persist in soil even up to 10 years without detriment. Out of two Globodera species occurring in Poland, Globodera rostochiensis is considered by European and Mediterranean Plant Protection Organization (EPPO) as a quarantine pest, while Globodera artemisiae is not. Therefore, the distinction between these two species is crucial. Classic methods of detection and differentiation are laborious and time-consuming. Instead, application of molecular biology techniques allows obtaining of rapid and reliable results. The aim of this study was to establish detection and differentiation method of two species, G. rostochiensis and G. artemisiae, based upon real-time polymerase chain reaction with the use of TaqMan probes. In reaction with primers and probes specific for the nematodes' ribosomal DNA (rDNA), the samples used were DNAs isolated from the two species, alone or in mixture, as well as crushed single cysts. Applied probes enable not only to identify the species in DNA mixtures but also in a single cyst. The use of a crushed cyst eliminates long-lasting procedure of DNA isolation and reduces costs of analysis.     

Ceruloplasmin and antioxidative enzymes in pre-eclampsia

Objective: To evaluate diagnostic value of ceruloplasmin together with other enzymatic and nonenzymatic antioxidants (superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and uric acid) and to evaluate the level of oxidative stress in patients with pre-eclampsia (PE) and compare it with normal pregnancy.

Methods: In this prospective study, antioxidative markers were investigated in two groups of pregnant women: patients with pre-eclampsia (n?=?32) and the healthy pregnant women (n?=?60). The following antioxidative markers and enzymes were evaluated: serum ceruloplasmin levels, uric acid, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px).

Results: Serum levels of ceruloplasmin, uric acid and SOD were significantly higher in the PE group compared to the control group. Serum levels of GSH-Px were not significantly higher in the PE group compared to the control group. Serum ceruloplasmin and serum uric acid have the best diagnostic accuracy for oxidative stress in PE and are more accurate compared to antioxidative enzymes -SOD and specially more accurate than GSH-Px.

Conclusions: Serum ceruloplasmin level may have significant role as the markers of oxidative stress in pre-eclampsia especially when used in combination with uric acid levels.     

Billateral Polycystic Kidneys in a Girl with WAGR Syndrome

The WAGR contiguous gene deletion syndrome is a combination of Wilms tumor, aniridia, genito-urinary abnormalities, and mental retardation. An 8.5-year-old girl was initially investigated at the age of 18 months for congenital bilateral aniridia, cataracts, glaucoma and epicantus. The ultrasound (US) scan showed polycystic kidney disease. FISH study revealed deletion of the WT1 and PAX6 gene in the 11p13 WAGR region. Forty days after the first kidney US, the second US revealed a 3 cm tumor in the right kidney: a Wilms tumour, treated successfully with the Wilm’s tumor protocol. The authors conclude that the identification of the deletions in the WAGR region in patients with aniridia should definitely be done. In addition, Wilms tumor can have a very rapid growth, which, per se requires frequent and careful ultrasound kidney controls. Polycystic kidneys can be part of the WAGR presentation.     

Cocaine- and amphetamine-regulated transcript (CART) peptide activates the extracellular signal-regulated kinase (ERK) pathway in AtT20 cells via putative G-protein coupled receptors

CART peptides are important neurotransmitters, but little is known about their receptors or signaling pathways in cells. In this study we describe the effects of CART 55-102 on the stimulation of extracellular signal-related kinase (ERK) in a pituitary-derived cell line. CART 55-102 treatment resulted in markedly enhanced ERK phosphorylation in AtT20 and GH3 cells, but had no significant effect on ERK phosphorylation levels in a variety of other cell types that were examined. The peptide activated ERK1 and 2 in AtT20 cells in a dose- and time-dependent manner, but an inactive peptide, CART 1-27, had no effect. U0126, an inhibitor of the MEK kinases, blocked the CART-stimulated activation of ERKs. ERK activation was also attenuated by pertussis toxin pre-treatment, but not by genistein, suggesting a Gi/o-dependent mechanism. Overall, these data strongly support the existence of a specific receptor for CART peptide that is a G-protein coupled receptor utilizing a Gi/o mechanism involving MEK1 and 2.     

Distribution of HBV genotypes and mutants among hepatitis B infected patients from northern Poland

Hepatitis B virus (HBV) infection is one of the major global epidemiological problems. The aim of our study was to determine the distribution of HBV genotypes in Poland since the data concerning the spread of HBV viruses in the central-eastern region of Europe is still very limited. HBV DNA was extracted from 58 serum samples. To quantify the level of HBV DNA the Roche Amplicor HBV Monitor Assay was used. To genotype and assign HBV subtypes DNA sequencing methods were performed. The HBV virus from 43 serum samples from hepatitis B infected patients was genotype A (74.1%), 12 cases had genotype D (20.7%), and 3 had the rare in Europe genotype F (5.2%). Prediction of HBV serological subtypes based on HBsAg sequencing showed almost 100% occurrence of subtype adw2 in the group of genotype A samples, three different subtypes in genotype D (ayw2, ayw3, and ayw4), and equal distribution of subtype adw4q- in all 3 cases of genotype F, also the most prevalent subtype in the Amerindians. Our results coincide with the general European HBV prevalence. However, HBV genotype F, which is not a common genotype in European countries, was detected and so was relatively high occurrence of genotype D, which may reflect historical and ethnical migration events in Poland in the past.     

Hemodialysis Affects Phenotype and Proliferation of CD4-Positive T Lymphocytes

CD4⁺ T lymphocytes of patients with chronic kidney disease (CKD) are characterized by reduced levels of crucial surface antigens and changes in the cell cycle parameters. Recombinant human erythropoietin (rhEPO) normalizes their altered phenotype and proliferative capacity. Mechanisms leading to the deficient responses of T lymphocytes are still not clear but it is postulated that immunological changes are deepened by hemodialysis (HD). Study of activation parameters of CD4⁺ T lymphocytes in hemodialyzed and predialysis CKD patients could bring insight into this problem. Two groups of patients, treated conservatively (predialysis, PD) and hemodialyzed (HD), as well as healthy controls, were included into the study; neither had received rhEPO. Proportions of main CD4⁺CD28⁺, CD4⁺CD25⁺, CD4⁺CD69⁺, CD4⁺CD95⁺, and CD4⁺HLA-DR⁺ lymphocyte subpopulations and proliferation kinetic parameters were measured with flow cytometry, both ex vivo and in vitro. No differences were seen in the proportions of main CD4⁺ lymphocyte subpopulations (CD4⁺CD28⁺, CD4⁺CD25⁺, CD4⁺HLA-DR⁺, CD4⁺CD69⁺, CD4⁺CD95⁺) between all examined groups ex vivo. CD4⁺ T lymphocytes of HD patients exhibited significantly decreased expression of co-stimulatory molecule CD28 and activation markers CD25 and CD69 after stimulation in vitro when compared with PD patients and healthy controls. HD patients showed also decreased percentage of CD4⁺CD28⁺ lymphocytes proliferating in vitro; these cells presented decreased numbers of finished divisions after 72 h of stimulation in vitro and had longer G0→G1 time when compared to healthy controls. CD4⁺ T lymphocytes of PD patients and healthy controls were characterized by similar cell cycle parameters. Our study shows that repeated hemodialysis procedure influences phenotype and proliferation parameters of CD4⁺ T lymphocytes.     

Cdc42 is crucial for the establishment of epithelial polarity during early mammalian development.

To study the role of Cdc42 in the establishment of epithelial polarity during mammalian development, we generated murine Cdc42-null embryonic stem cells and analyzed peri-implantation development using embryoid bodies (EBs). Mutant EBs developed endoderm and underlying basement membrane, but exhibited defects of cell polarity, cell-cell junctions, survival, and cavitation. These defects corresponded to a decreased phosphorylation and membrane localization of aPKC, a reduced phosphorylation of GSK3beta, and a diminished activity of Rac1. However, neither Rac1 nor the kinase function of GSK3beta seem to contribute to cell polarization and cell-cell contacts. In contrast, EBs expressing dominant-negative (dn) PKCzeta mimicked well the phenotype of Cdc42-null EBs, suggesting a major role of aPKC in mediating cell polarization downstream of Cdc42. Finally, aggregation experiments with endodermal cell lines suggested that Cdc42 might affect formation of adherens and tight junctions by PKCzeta-dependent regulation of the protein levels of p120 catenin and E-cadherin.     

The nucleotide sequence of a Polish isolate of Tomato torrado virus

A new virus was isolated from greenhouse tomato plants showing symptoms of leaf and apex necrosis in Wielkopolska province in Poland in 2003. The observed symptoms and the virus morphology resembled viruses previously reported in Spain called Tomato torrado virus (ToTV) and that in Mexico called Tomato marchitez virus (ToMarV). The complete genome of a Polish isolate Wal’03 was determined using RT-PCR amplification using oligonucleotide primers developed against the ToTV sequences deposited in Genbank, followed by cloning, sequencing, and comparison with the sequence of the type isolate. Phylogenetic analyses, performed on the basis of fragments of polyproteins sequences, established the relationship of Polish isolate Wal’03 with Spanish ToTV and Mexican ToMarV, as well as with other viruses from Sequivirus, Sadwavirus, and Cheravirus genera, reported to be the most similar to the new tomato viruses. Wal’03 genome strands has the same organization and very high homology with the ToTV type isolate, showing only some nucleotide and deduced amino acid changes, in contrast to ToMarV, which was significantly different. The phylogenetic tree clustered aforementioned viruses to the same group, indicating that they have a common origin. The nucleotide sequence of the Wal’03 isolate reported in this article has been submitted to the GenBank and is available under accession numbers: EU563948 (RNA1) and EU563947 (RNA2).     

Does simultaneous determination of LDL and HDL particle size improve prediction of coronary artery disease risk?

BACKGROUND: Alterations in plasma lipoprotein subclass distribution affect the risk for coronary artery disease (CAD). However, it is unclear whether the determination of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) phenotypes may or may not improve the ability to predict CAD development. METHODS: Polyacrylamide gradient (3-31%) gel electrophoresis was used to simultaneously determine size and distribution of lipoprotein subclasses in 181 CAD patients and 178 controls. RESULTS: Mean LDL and HDL subclass sizes were significantly smaller in patients than in controls (p < 0.001). Multivariate logistic regression analysis showed that small dense LDL particles were independent CAD risk predictors (OR = 2.867, p < 0.01), even when adjusted for other traditional risk factors, while small HDL particles lost their significance after adjustment (OR = 2.071, p = 0.054). The area under the ROC curve for LDL (0.671) and HDL (0.643) particle size measurement demonstrated low clinical accuracy when compared to the combination of traditional lipid risk factor measurements. CONCLUSIONS: CAD is associated with the predominance of smaller LDL and HDL particles. However, simultaneous determination of these two lipoprotein phenotypes provides no additional power in discriminating CAD and non-CAD subjects, beyond that obtained by the traditional risk factors.     

Serum activity of DPPIV and its expression on lymphocytes in patients with melanoma and in people with vitiligo

Background

Evidence is accumulating that chronic inflammation may have an important role in prostate cancer (PCa). The COX-2 polymorphism rs2745557 (+202 C/T) has been extensively investigated as a potential risk factor for PCa, but the results have thus far been inconclusive. This meta-analysis was performed to derive a more precise estimation of the association.

Methods

A comprehensive search was conducted to identify all case-control studies of COX-2 rs2745557 polymorphism and PCa risk. We used odds ratios (ORs) to assess the strength of the association, and 95% confidence intervals (CIs) give a sense of the precision of the estimate. Statistical analyses were performed by Review Manage, version 5.0 and Stata 10.0.

Results

A total of 8 available studies were considered in the present meta-analysis, with 11356 patients and 11641 controls for rs2745557. When all groups were pooled, there was no evidence that rs2745557 had significant association with PCa under co-dominant, recessive, over-dominant, and allelic models. However, our analysis suggested that rs2745557 was associated with a lower PCa risk under dominant model in overall population (OR = 0.85, 95%CI = 0.74-0.97, P = 0.02). When stratifying for race, there was a significant association between rs2745557 polymorphism and lower PCa risk in dominant model comparison in the subgroup of Caucasians (OR = 0.86, 95%CI = 0.75-0.99, P = 0.04), but not in co-dominant, recessive, over-dominant and allelic comparisons.

Conclusion

Based on our meta-analysis, COX-2 rs2745557 was associated with a lower PCa risk under dominant model in Caucasians.