Showing papers by "Gdańsk Medical University published in 2003"

Cap analysis gene expression for high-throughput analysis of transcriptional starting point and identification of promoter usage.

Abstract: We introduce cap analysis gene expression (CAGE), which is based on preparation and sequencing of concatamers of DNA tags deriving from the initial 20 nucleotides from 5′ end mRNAs. CAGE allows high-throughout gene expression analysis and the profiling of transcriptional start points (TSP), including promoter usage analysis. By analyzing four libraries (brain, cortex, hippocampus, and cerebellum), we redefined more accurately the TSPs of 11-27% of the analyzed transcriptional units that were hit. The frequency of CAGE tags correlates well with results from other analyses, such as serial analysis of gene expression, and furthermore maps the TSPs more accurately, including in tissue-specific cases. The high-throughput nature of this technology paves the way for understanding gene networks via correlation of promoter usage and gene transcriptional factor expression.

Sympathetic nerve activity in obstructive sleep apnoea.

Abstract: The mechanisms underlying the link between obstructive sleep apnoea (OSA) and cardiovascular disease are not completely established. However, there is increasing evidence that autonomic mechanisms are implicated. A number of studies have consistently shown that patients with OSA have high levels of sympathetic nerve traffic. During sleep, repetitive episodes of hypoxia, hypercapnia and obstructive apnoea act through chemoreceptor reflexes and other mechanisms to increase sympathetic drive. Remarkably, the high sympathetic drive is present even during daytime wakefulness when subjects are breathing normally and no evidence of hypoxia or chemoreflex activation is apparent. Several neural and humoral mechanisms may contribute to maintenance of higher sympathetic activity and blood pressure. These mechanisms include chemoreflex and baroreflex dysfunction, altered cardiovascular variability, vasoconstrictor effects of nocturnal endothelin release and endothelial dysfunction. Long-term continuous positive airway pressure treatment decreases muscle sympathetic nerve activity in OSA patients. The vast majority of OSA patients remain undiagnosed. Unrecognized OSA may contribute, in part, to the metabolic and cardiovascular derangements that are thought to be linked to obesity, and to the association between obesity and cardiovascular risk. Furthermore, acting through sympathetic neural mechanisms, OSA may contribute to or augment elevated levels of blood pressure in a large proportion of the hypertensive patient population.

Antibacterial and antifungal activity of juniper berry oil and its selected components.

Abstract: Juniper berry oil is stated to possess a wide spectrum of pharmacological activities and its monographs are included in some National Pharmacopoeias. The antibacterial and antifungal activity of the oil was reported by some authors. In our study we estimated the antibacterial and antifungal activity of three different juniper berry oils and their main components. All the micro-organisms used in this experiment were isolated from patients of Regional Hospital of Gdansk and some of them showed resistance against commonly used antibiotics. Only one of the oils (labelled A) revealed good antimicrobial properties. None of the single oil components was a stronger antibacterial and antifungal inhibitor than the oil A itself. Our data suggest that the antimicrobial activity of juniper oil A is the result of either the specific composition of the oil A (highest concentration of (-)-alpha-pinene, p-cymene and beta-pinene) or activity of a single non-identified compound. The presence of an adulterant in the oil was excluded.

Third-Generation β-Blockers Stimulate Nitric Oxide Release From Endothelial Cells Through ATP Efflux A Novel Mechanism for Antihypertensive Action

Abstract: Background— Nebivolol and carvedilol are third-generation β-adrenoreceptor antagonists, which unlike classic β-blockers, have additional endothelium-dependent vasodilating properties specifically related to microcirculation by a molecular mechanism that still remains unclear. We hypothesized that nebivolol and carvedilol stimulate NO release from microvascular endothelial cells by extracellular ATP, which is a well-established potent autocrine and paracrine signaling factor modulating a variety of cellular functions through the activation of P2-purinoceptors. Methods and Results— Contraction and relaxation of renal glomerular vasculature were measured by determination of intracapillary volume with [3H]-inulin. Biologically active NO was measured with highly sensitive porphyrinic NO microsensors in a single glomerular endothelial cell (GEC). Extracellular ATP was measured by a luciferin-luciferase assay. Enzymatic degradation of extracellular ATP by apyrase and blockade of P2Y-purinoceptors by suramin or r...

Functional activity of serotoninergic and melatoninergic systems expressed in the skin.

Abstract: We tested the expression of genes coding receptors of a cutaneous serotoninergic/melatoninergic system in whole human skin and in normal and pathologic cultured skin cells. Evaluation of serotonin (5HT), melatonin (MT), and melatonin-related receptors (MRR) showed expression of the isoforms 5HT2B, 5HT7, and MT1 genes in almost all the tested samples. Expression of other isoforms was less prevalent; 5HT2C, MRR, and MT2 were rarely detected. We also found novel isoforms for MT2, MRR, and 5HT2B and documented the process of RNA editing for 5HT2C. Testing for functional activity of these receptors with serotonin and melatonin (10(-14) to 10(-10) M) showed variable effects depending on cell type and culture conditions. Thus, serotonin stimulated proliferation of melanocytes in medium deprived of growth factors, while inhibiting cell growth in the presence of growth factors. Melatonin inhibited both apoptosis of HaCaT keratinocytes incubated in serum-free media, and proliferation of cells cultured in medium supplemented with serum. Melatonin also increased the numbers of viable fibroblasts incubated in serum free medium. N-acetylserotonin (NAS) and 5 methoxytryptamine (5MTT) were generally without effect on cell proliferation, with the exception of an inhibition of melanocyte proliferation at the higher 5MTT concentration of 10(-10) M. Thus, skin cells represent a true target for the products of the serotoninergic/melatoninergic cutaneous pathway with their actions modulating cell proliferation or viability.

Validation of cytochrome b sequence analysis as a method of species identification.

Abstract: One of the stages of dealing with biological material submitted to forensic laboratories is species identification. The aim of the present work was to validate and assess the possibility of applying sequence analysis of the region coding cytochrome b as a method of species identification in the field of forensic science. DNA originating from individuals from major phyla of vertebrates was isolated by the organic method from various specimens. Extracted DNA was subjected to PCR and direct cycle sequencing using a universal pair of primers. The validation process, performed according to TWGDAM recommendations, revealed that the technique is a very sensitive and reliable method of species identification allowing analysis of tiny amounts of material and also degraded material, and can be useful in the field of forensic genetics. The case example presented here, concerning the determination of species origin of biological evidence collected from fatal road accident, confirms that analysis can be carried out even when there is no reference sample, and the sequences obtained can be assessed through analysis of their similarity to sequences for cytochrome b present in DNA databases.

Novel properties of antimicrobial peptides.

Abstract: Endogenous peptide antibiotics are known as evolutionarily old components of innate immunity. Due to interaction with cell membrane these peptides cause permeabilization of the membrane and lysis of invading microbes. However, some studies proved that antimicrobial peptides are universal multifunctional molecules and their functions extend far beyond simple antibiotics. In this review we present an overview of the general mechanism of action of antimicrobial peptides and discuss some of their additional properties, like antitumour activity, mitogenic activity, role in signal transduction pathways and adaptive immune response.

Intraperitoneal cisplatin versus no further treatment: 8-year results of EORTC 55875, a randomized phase III study in ovarian cancer patients with a pathologically complete remission after platinum-based intravenous chemotherapy.

Abstract: First-line intravenous chemotherapy (CT) following debulking surgery is associated with prolonged survival, in particular in patients who achieve a pathological complete remission (pCR) at second-look surgery but in whom a high rate of relapses still occurs. Between 1988 and 1997, 153 patients in pCR following platinum-based intravenous CT were randomized between four courses of intraperitoneal cisplatin (P) (90 mg/m2 every 3 weeks) or observation. Overall survival (OS) was the primary endpoint, while progression-free survival (PFS) was a secondary endpoint. This intent-to-treat analysis includes 16 patients who were not eligible and 17 patients who had protocol violations. The two groups were well balanced in terms of age (median = 55 years), performance status (78% P.S. O), FIGO stage (96% stage III), histology (serous in 66%), grade (2 or 3 in 80%), and residuum before intravenous CT (>1 cm in 40%). Intraperitoneal CT was delivered mainly through intraperitoneal catheters (Port-a-Cath 61% and Tenckhoff 25%). Side effects of intraperitoneal cisplatin included vomiting [> or =grade 2 (82%)], rise in serum creatinine [> or =grade 2 (14%)], abdominal pain [grade 1-2 (38%)], and neurotoxicity [grade 2-3 (15%)]. After a median follow-up of 8 years, 80 patients (52%) have progressed with no difference in the pattern of relapse between the two groups and 75 patients (49%) have died; the respective hazard ratios for PFS and OS with 95% CI are 0.89 (0.59-1.33) and 0.82 (0.52-1.29). These results are suggestive of a treatment benefit but do not support a change in clinical practice. Other randomized clinical trials of intraperitoneal CT are reviewed and briefly discussed.

Expression level of Ubc9 protein in rat tissues

Abstract: Ubc9 is a homologue of the E2 ubiquitin conjugating enzyme and participates in the covalent linking of SUMO-1 molecule to the target protein. In this report we describe a simple and efficient method for obtaining pure human recombinant Ubc9 protein. The purified Ubc9 retained its native structure and was fully active in an in vitro sumoylation assay with the promyelocytic leukaemia (PML) peptide as a substrate. In order to better understand the physiology of Ubc9 protein we examined its levels in several rat tissues. Immunoblot analyses performed on tissue extracts revealed quantitative and qualitative differences in the expression pattern of Ubc9. The Ubc9 protein was present at a high level in spleen and lung. Moderate level of Ubc9 was detected in kidney and liver. Low amount of Ubc9 was observed in brain, whereas the 18 kDa band of Ubc9 was barely visible or absent in heart and skeletal muscle. In heart and muscle extracts the Ubc9 antibodies recognized a 38 kDa protein band. This band was not visible in extracts of other rat tissues. A comparison of the relative levels of Ubc9 mRNA and protein indicated that the overall expression level of Ubc9 was the highest in spleen and lung. In spleen, lung, kidney, brain, liver and heart there was a good correlation between the 18 kDa protein and Ubc9 mRNA levels. In skeletal muscle the Ubc9 mRNA level was unproportionally high comparing to the level of the 18 kDa protein. The presented data indicate that in the rat the expression of the Ubc9 protein appears to have some degree of tissue specificity.

Peroxisome proliferator-activated receptor alpha is downregulated in the failing human heart

Abstract: Cardiac hypertrophy in humans is associated with a decrease in myocardial fatty acid beta-oxidation (FAO) and accompanying alterations in metabolic gene expression. Flux through the cardiac FAO pathway, which is the principal source of energy production in the adult mammalian heart, is tightly controlled in accordance with energy demands. In rodents, the FAO pathway is under control of a nuclear peroxisome proliferator-activated receptor alpha (PPARalpha?. We sought to delineate the molecular regulatory events involved in the energy substrate preference switch from fatty acids to glucose during cardiac hypertrophic growth in humans. We analysed the amount of PPARalpha protein in human cardiac tissue. PPARalpha protein level was measured in homogenates prepared from left ventricular biopsies taken from five control donor hearts and compared to the amount of this transcription factor in biopsies from five patients with compensated end-stage heart failure (HF) at the time of transplantation. Using Western blot analysis with a monoclonal antibody against human PPARalpha, we observed a significant decrease (54%) in the mean amount of PPARalpha in the group of HF patients compared to that in the donor tissue. This study indicates that the decrease in cardiac PPARalpha transcription factor gene expression observed in the failing human heart could play an important role in a reduction in fatty acid utilisation by the adult heart during cardiac hypertrophy.

Increased activity of glycerol 3-phosphate dehydrogenase and other lipogenic enzymes in human bladder cancer.

Abstract: Common molecular changes in cancer cells are high carbon flux through the glycolytic pathway and overexpression of fatty acid synthase, a key lipogenic enzyme. Since glycerol 3-phosphate dehydrogenase creates a link between carbohydrates and the lipid metabolism, we have investigated the activity of glycerol 3-phosphate dehydrogenase and various lipogenic enzymes in human bladder cancer. The data presented in this paper indicate that glycerol 3-phosphate dehydrogenase activity in human bladder cancer is significantly higher compared to adjacent non-neoplastic tissue, serving as normal control bladder tissue. Increased glycerol 3-phosphate dehydrogenase activity is accompanied by increased enzyme activity, either directly (fatty acid synthase) or indirectly (through ATP-citrate lyase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and citrate synthase) involved in fatty acid synthesis. Coordinated upregulation of glycerol 3-phosphate dehydrogenase and lipogenic enzymes activities in human bladder cancer suggests that glycerol 3-phosphate dehydrogenase supplies glycerol 3-phosphate for lipid biosynthesis.

Speciation of Mg, Mn and Zn in extracts of medicinal plants

Abstract: Aqueous extracts of birch leaves ( folium Betulae), peppermint leaves ( folium Menthae), sage leaves ( folium Salviae), valerian roots ( radix Valerianae), and dandelion roots ( radix Taraxaci) are analysed for the three essential elements magnesium, manganese and zinc Ultrafiltration reveals that 60–100% of these metals are present as low molecular weight species (<5000 Da) Further characterisation of the low molecular weight fraction is done by using size exclusion chromatography and different detectors, namely element specific AAS detection, diode array UV–VIS detection and electrochemical detection The similarities and differences of the plant extracts are well reflected by the respective chromatograms, and typical plant constituents can be identified by their spectral and electrochemical properties (eg flavonoids in Betula) Mg and Mn species are selectively detected by AAS in closely neighbouring chromatographic regions for all five samples However, there are significant differences between the samples investigated In all cases a good correlation exists between detection of these metals (Mg, Mn) and pulsed amperometric detection (PAD), which is selective for carbohydrates The respective molecular weight of carbohydrate species is in the range of approximately 300–600 Da The distribution of zinc species (detectable only in the Betula extract) is totally different from that of Mg and Mn species For zinc, many more species are detected, and there is no correlation to carbohydrates; instead (poly)phenols are involved in zinc complexation

Enhanced glycerol 3-phosphate dehydrogenase activity in adipose tissue of obese humans.

Abstract: The primary purpose of this investigation was to determine whether adipose tissue glycerol 3-phosphate dehydrogenase activity is associated with human obesity. The data presented in this paper indicate that the glycerol 3-phosphate dehydrogenase activity in adipose tissue from morbidly obese subjects is approximately 2-fold higher than from lean individuals. Moreover, positive correlation between adipose tissue glycerol 3-phosphate dehydrogenase activity and body mass index (BMI) (r = 0.5; p < 0.01) was found. In contrast, the adipose tissue fatty acid synthase (FAS) and ATP-citrate lyase (ACL) activities in morbidly obese patients are significantly lower than in lean subjects. Furthermore, negative correlation between adipose tissue FAS activity and BMI (r = -0.3; p < 0.05) as well as between ACL activity and BMI (r = -0.3; p < 0.05) was found. These data indicate that elevated glycerol 3-phosphate dehydrogenase might contribute to the increase of triacylglycerol (TAG) synthesis in obese subjects, however, fatty acids necessary for glycerol 3-phosphate esterification must be derived (because of lower FAS and ACL activities) mainly from TAG in circulating lipoproteins formed in liver (VLDL), and/or from the intake with food (chylomicrons). The conclusion is, that the enhanced activity of glycerol 3-phosphate dehydrogenase, and hence the generation of more glycerol 3-phosphate in adipose tissue offers a novel explanation for increased TAG production in adipose tissue of obese subjects.

Prediction of gradient retention from the linear solvent strength (LSS) model, quantitative structure-retention relationships (QSRR), and artificial neural networks (ANN)

Abstract: The linear solvent strength (LSS) model combined with quantitative structure-retention relationships (QSRR) and artificial neural network (ANN) analysis has been shown to permit approximate prediction of the gradient high-performance liquid chromatography (HPLC) retention time for any analyte on a once-characterized column. The approach applies well to the reversed-phase HPLC mode with a methanol-water (buffer) eluent of linearly changing composition. Its suitability was tested for a representative series of structurally diverse analytes. In this approach the determination of retention times, t R , in two gradient runs for a predesigned model series of 15 analytes is first needed. Next, model QSRR equations describing t R in terms of analyte structure are derived to characterize the HPLC systems of interest. To quantitatively characterize the structure of the analytes the following three structural descriptors from molecular modeling are employed: total dipole moment; electron excess charge of the most negatively charged atom; and water-accessible molecular surface area. Using these data a general QSRR equation is derived which is valid for a given column/eluent system. Next, having the structural descriptors for any analyte to be chromatographed in such a characterized HPLC system, one employs the previously derived general QSRR equation to calculate the analyte's retention time. The expected gradient retention time for any gradient conditions can be calculated by means of appropriate LSS equations. Independent of the standard QSRR calculation procedure based on multiple regression analysis (MRA), predictions of gradient retention times were performed by means of artificial neural networks (ANN). It has been found that the predictive power of ANN is similar to that of MRA. The combined LSS/QSRR approach has been demonstrated to provide approximate, yet otherwise unattainable, a priori predictions of gradient retention of analytes based solely on their chemical formulae. That way a rational chemometric basis for a systematic optimization of chromatographic separations has been elaborated as an alternative to the trial-and-error method normally applied at present.

MR urography of obstructive uropathy: diagnostic value of the method in selected clinical groups.

Abstract: The aim of the study was to evaluate the role of MR urography (MRU) in the diagnosis of obstructive uropathy in selected groups of patients The groups involved following pathologies: calculi; strictures of ureteropelvic junction (UPJ); benign and malignancy-induced ureterostenosis Sixty patients with clinical diagnosis of obstructive uropathy were subjected to static fluid MRU (sMRU) with the use of 3D turbo spin echo (TSE) sequence in a 05-T magnet The examination was completed with conventional MR sequences and in 12 cases additionally with sequences after the administration of Gd-DTPA and excretory MRU The results were compared with intravenous urography (IVU), CT, US, clinical and histopathological data The degree of the urinary tract dilatation as well as the level and type of obstruction were estimated In patients with urolithiasis sMRU correctly depicted the degree of ureterohydronephrosis in 85%, in cases of UPJ stenosis and malignancy-induced ureterostenosis in 100% and in the group of benign ureterostenosis in 91% of patients Determination of obstruction level in patients with stones was adequate in 92% and in cases of non-calculous ureteral strictures in 100% of patients The sMRU sequence alone could not specify the nature of obstruction except 1 case of bladder carcinoma Filling defects in ureters visible on MR urograms were verified with IVU or CT to exclude intrinsic tumours Completed with conventional MR sequences sMRU enabled the depiction of solid mass or infiltration in 83% cases of malignancy-induced ureterostenosis, and in the remaining groups of patients neoplastic process was excluded in 91% In conjunction with excretory MRU and conventional MR images sMRU appears to be a highly useful technique in assessment of obstructive uropathy, especially that of non-calculous origin Among different clinical applications MRU is superior in the evaluation of dilated urinary tract in altered anatomical conditions (eg in patients with ileal neobladder)

In vivo alanine/EPR dosimetry in daily clinical practice: a feasibility study

Abstract: Purpose: The objective of this study was evaluation of accuracy of in vivo dosimetry using electron paramagnetic resonance (EPR) in alanine. Additionally, we aimed to identify sources of uncertainty in dose determination and quantitative assessment of physical factors that may result in discrepancies between the measured and planned single-fraction doses. Methods and materials The measurements were performed using detectors in a form of 1.6 cm × 1.6 cm polyethylene sachets filled with powdered L -alanine. The detectors were taped to the patient’s skin and measured the entrance doses for 60Co and electron beams. Some detectors were covered with buildup material, and some measured the “skin dose.” The EPR measurements were performed with a Varian E-4 spectrometer. Results The calculated uncertainty of EPR measured doses was dependent on measured doses and varied from 6.6% for 0.5 Gy to 3.2% for 2 Gy. The calculated uncertainty was in concordance with experimentally determined reproducibility of EPR signals. However, the deviations between measured and planned doses exceeded the uncertainty range of EPR measurements, which can be attributed to uncertainty in determination of actually delivered doses to the detectors, on the basis of treatment planning data. Conclusion The accuracy of dose determination by EPR measurements was shown to be achievable within the 5% limit recommended by the ICRU for doses above 0.7 Gy. The accuracy of in vivo verification of radiotherapy doses by in vivo EPR dosimetry can be improved by meticulous selection of measurement conditions, i.e., radiation fields and detector positions, ensuring accurate calculation of doses delivered to the dosimeters.

Antifungal activity of biflavones from Taxus baccata and Ginkgo biloba.

Abstract: Bilobetin and 4'''-O-methylamentoflavone were isolated and identified in the needles of Taxus baccata, for the first time in this species. The antifungal activity of biflavones from T. baccata and Ginkgo biloba, namely amentoflavone, 7-O-methylamentoflavone, bilobetin, ginkgetin, sciadopitysin and 2,3-dihydrosciadopitysin towards the fungi Alternaria alternata, Fusarium culmorum, Cladosporium oxysporum was determined employing computer-aided image analysis couplet to a microscope. Bilobetin exhibited a significant antifungal activity with values of ED50 14, 11 and 17 microM respectively. This compound completely inhibited the growth of germinating tubes of Cladosporium oxysporum and Fusarium culmorum at a concentration 100 microM. Activity of ginkgetin and 7-O-methylamentoflavone towards Alternaria alternata was stronger than that of bilobetin. Moreover, slight structural changes in the cell wall of Alternaria alternata exposed to ginkgetin at concentration of 200 microM were observed.

Immune response to influenza vaccination in an elderly population.

Abstract: The purpose of this study was to assess the serum antibody responses to both the hemagglutinin and the neuraminidase antigens of inactivated influenza vaccine in 45 elderly and 28 younger adults. After vaccination, antihemagglutinin antibody levels increased significantly and mean fold increases ranged from 2.8 to 22.0. Seroprotection rates were between 42.2 and 91.1% 1 month after vaccination and 15.6 and 84.4% 5 months afterward. Seroresponse rates ranged from 42.2 to 91.1% 1 month after vaccination and 15.6 to 82.2% 5 months afterward. After vaccination antineuraminidase antibody levels increased significantly and mean fold increases ranged from 3.6 to 12.3. Significantly higher antibody responses to both hemagglutinin and neuraminidase were observed for antigen A(H3N2) than for antigens A(H1N1) and B. In most instances there were no statistically significant differences between the elderly and the control subjects. Influenza vaccine was immunogenic in the institutionalized elderly, who developed good antibody responses to influenza hemagglutinin and neuraminidase antigens.

The effect of insulin on expression level of nucleoside transporters in diabetic rats.

Abstract: Evidence that the time course of insulin-induced changes in adenosine level in diabetic rats is different from that observed for expression of adenosine kinase prompted us to study the insulin effect on expression of nucleoside transporters in tissues of diabetic rats. RNase protection assay demonstrated that mRNA levels of equilibrative (rENT) and Na+-dependent nucleoside transporters (rCNT) were altered in diabetic tissues. The rENT1 mRNA level with respect to values obtained in age- and sex-matched nondiabetic rats was decreased by 45, 32, and 10% in diabetic heart, liver, and kidney, respectively. The level of rENT2 mRNA was lowered by 40% in diabetic kidney and heart, and by 24% in diabetic liver. Changes in the expression pattern of rCNT1 and rCNT2 in diabetic tissues differed significantly from that observed for rENT. The levels of rCNT1 and rCNT2 mRNA did not change significantly in diabetic kidney. In diabetic heart, the mRNA levels of rCNT1 and rCNT2 increased 1.7- and 2-fold, respectively. Changes in expression of nucleoside transporters were accompanied by alterations in adenosine content. Administration of insulin to diabetic rats resulted in a drop in adenosine concentration in examined tissues and return of the rCNT1, rCNT2, and rENT2 but not rENT1 mRNA levels to values observed in nondiabetic rats. In summary, these data demonstrate that insulin affects expression of nucleoside transporters in a cell-specific manner. We conclude that change in the expression level of the nucleoside transporters occurring in tissues of diabetic rat is an important factor influencing adenosine levels in the cell.