Changes in urine autofluorescence in ovarian cancer patients.

Ovarian cancer is the type of cancer with the highest mortality rate among gynaecologic malignancies. Due to lack of screening tools, this disease is mainly diagnosed at a progressed stage, when it is too late to adequate therapy. Despite many attempts, enough sensitive and specific biomarker was not still uncovered. Fluorescence spectroscopy has proven to be a useful diagnostic tool with high efficiency. Fluorescence detection has three major advantages over other light-based investigation methods: high sensitivity, high speed, and reliability. Biological materials consist of a number of intrinsic fluorescent compounds -autofluorophores, which are associated with cardinal metabolic pathways. It is well known, that cancerous tissue metabolism is altered compared to healthy one, what influence also intrinsic fluorophores composition of bodily fluids. Urine is one of the biological fluids that could be obtained most easily and displays a blue - green fluorescence that can change in case of pathological process. Analysis of urine autofluorescence is non invasive and simple technique. Using fluorescent spectroscopy, ovarian cancer patients and healthy control group were discerned with high significance, so we predict that fluorescence analysis of urine could be a potential means of ovarian cancer screening.

Autonomic recovery following sprint interval exercise.

The autonomic nervous activity was assessed following supramaximal exercise through heart rate (HR) and blood pressure (BP) variability (HRV and BPV) and baroreflex sensitivity (BRS). The beat-to-beat HR and BP were recorded during the supine and standing states before (PRE) and at 60 (R60) and 120 min (R120) following single (one Wingate, 1W) and multiple sprint intervals (four Wingates interspersed with 4 min of light cycling, 4W). The supine low frequency (LF) component was increased (P<0.001) and the high frequency (HF) was reduced (P<0.01) at R60 (LF, 178.1 ± 11.0; HF, 74.8 ± 10.5) compared with PRE (LF, 140.2 ± 7.4; HF, 110.4 ± 7.2) after both exercises. Supine systolic BPV LF:HF was higher at R60 (4.6 ± 1.4) compared with PRE (6.8 ± 2.4) only after 4W (P=0.035). Supine BRS was lower (P<0.001) at R60 (6.8 ± 1.1) than at PRE (15.3 ± 1.8) and R120 (11.3 ± 1.3). BRS at R120 remained lower after 4W (P=0.02). Standing BRS was less (P<0.001) at R60 (2.3 ± 0.5) than at PRE (5.6 ± 0.8) or R120 (3.7 ± 0.6) and returned to PRE values only after 1W. We concluded that (a) autonomic balance is shifted to a greater sympathetic and less parasympathetic activation following both types of exercise, (b) it takes longer than 1 h to recover following supramaximal exercise and (c) the recovery is longer after 4W than 1W.

Effects of exercise stress on the endocannabinoid system in humans under field conditions.

The effects of physical exercise stress on the endocannabinoid system in humans are almost unexplored. In this prospective study, we investigated in a crossover design and under field conditions at different altitudes the effects of physical exercise on the endocannabinoid system (ECS) in 12 trained healthy volunteers. For determination of alterations on the ECS three different protocols were analyzed: Protocol A (physical exercise at lower altitude) involved strenuous hiking below 2,100 m, whereas Protocol B (physical exercise by active ascent to high altitude) involved hiking up to 3,196 m, an accommodation at the cottage and a descent the next day. Protocol C (passive ascent) included a helicopter ascent to 3,196 m, an overnight stay at this altitude and a flight back to the base camp the following day. The cumulative hiked altitude in Protocol A and B was comparable (~1,650 m). The blood EC concentrations of anandamide increased significantly in Protocol A/B from baseline (T0) 0.12 ± 0.01/0.16 ± 0.02 (mean ± SEM) to 0.27 ± 0.02/0.42 ± 0.02 after exercise (T1) (p < 0.05). Anandamide levels in Protocol C remained stable at 0.20 ± 0.02. We conclude that the ECS is activated upon strenuous exercise whereas the combination with hypoxic stress further increases its activity. The reduced partial pressure of oxygen at high altitude alone did not affect this system. In summary, physical exercise activates the endocannabinoid system, whereas the combination with high altitude enhances this activation. This discloses new perspectives to adaptation mechanisms to physical exercise.

Differential Urinary Proteomic Analysis of Endometrial Cancer.

Endometrial cancer is one of the most frequent gynecological malignancies present in more than 95 % of all uterine cancers. In spite of that, screening of such disease is not commonly performed in clinical practice due to enormous costs and relatively low sensitivity. Therefore, developing an effective screening test to diagnose endometrial cancer at early stages is of great importance for the clinical area of investigation. In this work, we applied urinary proteomics (i.e., bottom-up proteomic approach followed by nano HPLC-ESI-MS/MS) in patients with endometrial cancer, with respect to find proteins aimed for the early diagnostics and screening. According to the results, the significant semi-quantitative changes were observed in urinary proteome of treated patients. The proteins that may be pivotal in pathogenesis of endometrial cancer, like cadherin-1 (CDH1), vitronectin (VTN) and basement membrane specific-heparan sulphate proteoglycan core protein (HSPG2) were down-regulated, when compared to the control group. Ultimately, it can be stated that urinary proteomics has a potential for the searching of cancer protein biomarkers based on their altered concentration.

Combining parallel pattern generation of electrohydrodynamic lithography with serial addressing.

Electrohydrodynamic lithography (EHDL) is a parallel patterning process which typically makes use of topographically structured electrodes to guide pattern formation along areas of higher electrical field strength. The main driving force for pattern formation is an electrostatic pressure acting on a thin film polymer surface caused by a voltage applied between a top and bottom electrode. We here demonstrate that the principle can be applied using an addressable electrode composed of interdigitated fingers. Depending on the applied voltages, line patterns with different periodicities were fabricated. Our proof-of-concept experiments pave the way for a parallel pattern replication process where a serially addressed master is used. We complement the experiments by modelling the potentials across the electrodes and electrostatic forces acting on the polymer surface using different addressing schemes. Numerical simulations of the experimental setup pointed to some critical issues we experienced during the design of the experiments.

Mesencephalic dopamine neuron number and tyrosine hydroxylase content: Genetic control and candidate genes.

The mesotelencephalic dopamine system shows substantial genetic variation which fundamentally affects normal and pathological behaviors related to motor function, motivation, and learning. Our earlier radioenzyme assay studies demonstrated significantly higher activity of tyrosine hydroxylase (TH), the first and rate limiting enzyme in the biosynthesis of catecholamine neurotransmitters, in the substantia nigra-ventral tegmental area of BALB/cJ mice in comparison with that of C57BL/6ByJ mice. Here, using quantitative immunoblotting and immunocytochemistry, we tested the hypothesis that mesencephalic TH protein content and number of nigral TH-positive neurons show strain-dependent differences in C57BL/6ByJ and BALB/cJ parallel to those observed in the TH activity studies. Immunoblotting experiments detected significantly higher mesencephalic TH protein content in BALB/cJ in comparison to C57BL/6ByJ (P<0.05). Immunocytochemical studies demonstrated that the number of TH-positive cells in substantia nigra was 31.3% higher in BALB/cJ than that in C57BL/6ByJ (P<0.01), while the average dopamine neuron volume was not significantly different. In a search for candidate genes that modulate TH content and the size of mesencephalic dopamine neuron populations we also studied near-isogenic mouse sublines derived from the C57BL/6ByJ and BALB/cJ progenitor strains. A whole-genome scan with 768 single nucleotide polymorphism markers indicated that two sublines, C4A6/N and C4A6/B, were genetically very similar (98.3%). We found significantly higher mesencephalic TH protein content in C4A6/B in comparison to C4A6/N (P=0.01), and a tendency for higher number of dopamine neurons in the substantia nigra in C4A6/B in comparison to C4A6/N, which, however, did not reach statistical significance. To identify the genetic source of the TH content difference we analyzed the single nucleotide polymorphism (SNP) genotype data of the whole-genome scan, and detected two small differential chromosome segments on chr. 13 and chr. 14. Microarray gene expression studies and bioinformatic analysis of the two differential regions implicated two cis-regulated genes (Spock1 and Cxcl14, chr. 13), and two growth factor genes [bone morphogenetic protein 6 (Bmp6) (chr. 13), and fibroblast growth factor 14 (Fgf14) (chr. 14)]. Taken together, the results suggest that (1) nigral dopamine neuron number and TH protein content may be genetically associated but further studies are needed to establish unequivocally this linkage, and (2) Spock1, Cxcl14, Bmp6, and Fgf14 are novel candidates for modulating the expression and maintenance of TH content in mesencephalic dopamine neurons in vivo.

Genome-wide screen for differentially methylated long noncoding RNAs identifies Esrp2 and lncRNA Esrp2-as regulated by enhancer DNA methylation with prognostic relevance for human breast cancer.

The majority of long noncoding RNAs (lncRNAs) is still poorly characterized with respect to function, interactions with protein-coding genes, and mechanisms that regulate their expression. As for protein-coding RNAs, epigenetic deregulation of lncRNA expression by alterations in DNA methylation might contribute to carcinogenesis. To provide genome-wide information on lncRNAs aberrantly methylated in breast cancer we profiled tumors of the C3(1) SV40TAg mouse model by MCIp-seq (Methylated CpG Immunoprecipitation followed by sequencing). This approach detected 69 lncRNAs differentially methylated between tumor tissue and normal mammary glands, with 26 located in antisense orientation of a protein-coding gene. One of the hypomethylated lncRNAs, 1810019D21Rik (now called Esrp2-antisense (as)) was identified in proximity to the epithelial splicing regulatory protein 2 (Esrp2) that is significantly elevated in C3(1) tumors. ESRPs were shown previously to have a dual role in carcinogenesis. Both gain and loss have been associated with poor prognosis in human cancers, but the mechanisms regulating expression are not known. In-depth analyses indicate that coordinate overexpression of Esrp2 and Esrp2-as inversely correlates with DNA methylation. Luciferase reporter gene assays support co-expression of Esrp2 and the major short Esrp2-as variant from a bidirectional promoter, and transcriptional regulation by methylation of a proximal enhancer. Ultimately, this enhancer-based regulatory mechanism provides a novel explanation for tissue-specific expression differences and upregulation of Esrp2 during carcinogenesis. Knockdown of Esrp2-as reduced Esrp2 protein levels without affecting mRNA expression and resulted in an altered transcriptional profile associated with extracellular matrix (ECM), cell motility and reduced proliferation, whereas overexpression enhanced proliferation. Our findings not only hold true for the murine tumor model, but led to the identification of an unannotated human homolog of Esrp2-as which is significantly upregulated in human breast cancer and associated with poor prognosis.

Oriented immobilisation of engineered single-chain antibodies to develop biosensors for virus detection.

Single chain variable fragment (scFv) molecules were selected from a synthetic phage display library then cloned into a generic vector for expression of the scFv fused to the light chain constant domain of human immunoglobulin with a C-terminal cysteine residue (scFvC(L)cys). A heterobifunctional maleimide linker was synthesised and a strategy for functionalization of gold with the scFvC(L)cys fusion proteins elaborated. Successful covalent attachment of functional scFvC(L)cys was demonstrated using a surface plasmon resonance-based sensor. The results showed that the immobilised scFvC(L)cys molecules were functional and specific binding curves (with response relative to the concentration of virus antigen) were obtained over more than 25 cycles of binding and dissociation. ScFv molecules lacking the C-terminal cysteine performed poorly in similar experiments. The work demonstrates the feasibility of using simple scFv selection and cloning procedures combined with oriented immobilisation of scFvC(L)cys fusion proteins for robust antigen sensing surfaces in immunosensor or other biotechnological applications.

A novel strategy for the expression of foreign genes from plant virus vectors.

Potato virus X (PVX)-based vector constructs were generated to investigate the use of an internal ribosome entry site (IRES) sequence to direct translation of a viral gene. The 148-nucleotide IREScp sequence from a crucifer-infecting strain of tobacco mosaic virus was used to direct expression of the PVX coat protein (CP). The IRES was inserted downstream of the gene encoding green fluorescent protein (GFP) and upstream of the PVX CP, in either sense or antisense orientation, such that CP expression depended on ribosome recruitment to the IRES. Stem-loop structures were inserted at either the 3'- or 5'-end of the IRES sequence to investigate its mode of action. In vitro RNA transcripts were inoculated to Nicotiana benthamiana plants and protoplasts: levels of GFP and CP expression were analysed by enzyme-linked immunosorbent assay and the rate of virus cell-to-cell movement was determined by confocal laser scanning microscope imaging of GFP expression. PVX CP was expressed, allowing cell-to-cell movement of virus, from constructs containing the IRES sequence in either orientation, and from the construct containing a stem-loop structure at the 5'-end of the IRES sequence. No CP was expressed from a construct containing a stem-loop at the 3'-end of the IRES sequence. Our results suggest that the IRES sequence is acting in vivo to direct expression of the 3'-proximal open reading frame in a bicistronic mRNA thereby demonstrating the potential of employing IRES sequences for the expression of foreign proteins from plant virus-based vectors.

Differential expression of tissue transglutaminase during in vivo apoptosis of thymocytes induced via distinct signalling pathways.

A significant increase in the expression and activity of tissue transglutaminase (tTG), one of the effector elements of apoptosis, was observed during involution of thymus elicited by treatment with either anti-CD3 antibody or dexamethasone or by irradiation. The blood plasma concentration of epsilon(gamma-glutamyl)lysine isodipeptide, the end-product of the digestion of transglutaminase cross-linked proteins, was also elevated in each of these cases. tTG was localized in cells of the cortical layer of the thymus and immunofluorescence double staining revealed that the enzyme appeared in the apoptotic cells. None of these observations could be made when apoptosis was induced by fas-receptor stimulation. The lack of tTG activity in fas-stimulated cells was accompanied with a less organized apoptotic morphology. Our data suggest that distinct signalling pathways, which induce apoptosis within the same cell type, can differentially regulate the expression of tTG, and this enzyme may be involved in structural stabilization of the apoptotic cells.

Organ-specificity of the extravasation process: an ultrastructural study.

UNLABELLED: The process of extravasation of the high metastatic Lewis lung carcinoma line was examined in different organs. Four of the five organs (liver, lungs, brain and adrenals) represent the most frequent metastatic sites in humans. In the case of each organ 150-350 tumor cells were analysed. The interaction of tumor cells with endothelial cells and the basement membrane showed significant differences between the organs. In the liver and lungs, endothelial cells were found to migrate onto the surface of the tumor cells, resulting in the removal of tumor cells from the circulation. The process was initiated by development of cytoplasmic projections on the luminal surface of the endothelial cells. In the liver only half of the tumor cells showed basement membrane degradation even after 24 h, although 6 h after injection 40% of the tumor cells were sequestered from the circulation. In the adrenals and brain, tumor cells were not covered by endothelial cells instead, limited retraction of endothelial cells was followed by penetration of the basement membrane. In the kidney both types of tumor cell-endothelial cell interactions were observed, but the process of extravasation was not completed, stopping as the tumor cells reached the basement membrane or the mesangial matrix. The time course of tumor cell extravasation also showed significant differences between the organs. The process was most rapid in case of the liver and adrenals. By 6 h 40-50% of the tumor cells were in the process of extravasation or were in an extracapillary position. These organs are preferential metastatic sites of this tumor line. The time of extravasation was much longer in the other organs (lungs 16 h, brain 48 h), for which this tumor line shows no preference. CONCLUSIONS: (1) Type and duration of tumor cell extravasation differ between the organs. (2) The time needed to reach extraluminal position, but not the type of extravasation correlates with the organ preference. (3) Endothelial cells of the lungs and liver can play a much more active role in the process of extravasation than previously suggested. (4) Tumor cells can complete the metastatic process without reaching a complete extracapillary position; contact with the basement membrane or extracellular matrix seems to be sufficient.

Cell death in HIV pathogenesis and its modulation by retinoids.

Patients infected with the human immunodeficiency virus exhibit a progressive decline in the CD4 T-cell number, resulting in immunodeficiency and increased susceptibility to opportunistic infections and malignancies. Although CD4 T cell production is impaired in patients infected with HIV, there is now increasing evidence that the primary basis of T cell depletion is accelerated apoptosis of CD4 and CD8 T cells. The rate of lymphocyte apoptosis in HIV infection correlates inversely with the progression of the disease: it is low in long-term progressors and in patients undergoing highly active antiretroviral therapy. Interestingly, only a minor fraction of apoptotic lymphocytes are infected by HIV, indicating that the enhanced apoptosis does not necessarily always serve to remove the HIV+ cells and results from mechanisms other than direct infection. Thus, understanding and influencing the mechanisms of HIV-associated lymphocyte apoptosis may lead to new therapies for HIV disease. In this paper the potential effects of retinoids on CD4 T cell apoptosis is discussed.

Effects of dopamine and dopamine-active compounds on oxytocin and vasopressin production in rat neurohypophyseal tissue cultures.

The effects of dopamine (DA) or DA-active drugs on the synthesis of neurohypophyseal (NH) hormones were studied in 13-14 day cultures of isolated NH tissue from rats. The following DA-active compounds were used (10(-6) M in each medium): DA, apomorphine (APM), Pro-Lys-Gly (PLG), butaclamol (B), haloperidol (HP), chlorpromazine (CPZ) and sulpiride (SP). The oxytocin (OT) and vasopressin (VP) contents of the condensed media were determined by RIA after a 1 or 2 h incubation. Significantly increased contents of OT and VP were detected in the tissue culture media following DA, APM or PLG administration. This elevation of NH hormone production could be blocked by previous administration of B or the DA receptor antagonists HP, CPZ or SP. The application of B after DA agonists proved ineffective. The results indicate that NH hormone production can be directly influenced by the DA-ergic system. The DA-ergic control of NH hormone secretion in rats can occur independently of the hypothalamus, at the level of the posterior pituitary.

Inhibitory effect of galanin on dopamine induced increased oxytocin secretion in rat neurohypophyseal tissue cultures.

The regulation of oxytocin (OT) release by galanin (GAL) at the neurohypophyseal (NH) nerve terminal is not adequately understood. The effect of GAL on the secretion of OT was studied in 13- to 14-day cultures of isolated rat NH tissue. By this time, the hormone content of the medium had become constant. The OT content of the supernatant medium was determined by RIA after a 1- or 2-h incubation. A significantly decreased content of OT was found following incubation with 10(-6)-10(-8) M doses of GAL. Dopamine (DA) and the DA-active drugs apomorphine (APM) and Pro-Lys-Gly (PLG) (10(-6) M in each medium) increased the OT synthesis of NH tissue cultures. This elevation of OT secretion could be blocked by the administration of GAL together with DA, APM or PLG. The DA-blocking effect of GAL was prevented by previous treatment with the GAL receptor antagonist galantid (M15). The results indicate that OT release from the NH is directly influenced by the GAL-ergic system. The GAL-ergic control of OT secretion from NH tissue in rats can occur at the level of the posterior pituitary.

Inhibitory effect of galanin on dopamine-induced enhanced vasopressin secretion in rat neurohypophyseal tissue cultures.

The effect of galanin (GAL) on vasopressin (VP) secretion was studied in 13-14-day cultures of isolated rat neurohypophyseal (NH) tissue. The VP content of the supernatant was determined by radioimmunoassay (RIA) after a 1- or 2-h incubation. A significantly decreased content of VP was detected following the administration of 10(-6)-10(-9) M doses of GAL. Dopamine (DA) and the DA-active drugs apomorphine (APM) and Pro-Lys-Gly (PLG) (10(-6) M in each medium) increased the VP level of NH tissue cultures. This VP concentration elevation could be blocked by the administration of GAL together with DA, APM or PLG. The DA-blocking effect of GAL was prevented by previous treatment with the GAL receptor antagonist galantid (M15). The results indicate that VP release is directly influenced by the GAL-ergic system. The GAL-ergic control of VP secretion from NH tissue in rats can occur independently of the hypothalamus, at the level of the posterior pituitary.

Properties of a panel of single chain variable fragments against Potato leafroll virus obtained from two phage display libraries.

Twelve single chain variable fragment (scFv) antibodies that bind to particles of Potato leafroll virus (PLRV) were obtained from two naive phage display libraries. Phages were selected against PLRV particles or dissociated PLRV particles immobilised onto tubes. Individual PLRV-binding scFv were identified by ELISA, after their expression either fused to the surface of phage particles, or as soluble scFv (scFv-c-myc), or as scFv-alkaline phosphatase fusion proteins (scFv-AP), obtained by subcloning into pSKAP/S. These procedures resulted in the isolation of scFv with different properties. For example, some of the scFv reacted strongly with virus particles but not with dissociated capsid protein, which suggests that they had reacted with discontinuous epitopes. Others reacted with dissociated capsid proteins and SDS-denatured protein, which suggests that they had reacted with continuous epitopes. ScFv were also subcloned into pC(L) for expression as fusion proteins with human kappa constant region (scFv-C(L)). Expression of these constructs in Escherichia coli yielded 0.2-1 mg protein per litre of bacterial culture. The different scFv fusion proteins were evaluated in ELISA to detect PLRV in leaf extracts of Physalis floridana. Absorbance values obtained with the fusion proteins were greater than those obtained with the scFv-c-myc, and were similar to those obtained in assays done using monoclonal or polyclonal antibodies.

In situ derivatisation using pressurized liquid extraction to determine phenols, sterols and carboxylic acids in environmental samples and microbial biomasses.

Pressurized liquid extraction was combined with in-situ derivatisation to extract polar analytes such as phenols (including chlorophenols) sterols and carboxylic acids from environmental and microbial samples. This one-step protocol uses acetic anhydride as an acetylation agent, N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) as an silylation agent, and boron trifluoride-methanol, phenyltrimethyl ammoniumhydroxide and trimethyl sulfoniumhydroxide as methylation agents. It results in faster extraction rates and better or comparable extraction efficiencies when compared to classical approaches. The addition of a silylation agent also facilitates the extraction kinetics of analytes not accessible to silylation (e.g. polycyclic aromatic hydrocarbons or alkylbenzenes). This may be attributed to a dissociative action of the agent to weaken analyte-matrix interactions.

Cr(III) and Cr(VI) on-line preconcentration and determination with high performance flow flame emission spectrometry in natural samples.

Methods for the on-line chromatographic preconcentration of Cr(III) and Cr(VI) have been developed. Cr(VI) has been preconcentrated on an RP C18 silica based column with tetrabutylammonium-bromide (TBABr) as ion-pairing agent. Specially for Cr(III) a new and effective preconcentration technique based on the sorption of Cr(III)-ions in a C18 column in presence of KH-phthalate has been developed. The efficiency of sample introduction into the atomic emission spectrometer could be improved by hydraulic high pressure nebulization. For the detection of chromium the acetylene/N(2)O flame has been used as a powerful emission spectrometric source. Applying these steps the detection limit (3sigma) could be improved to 25 pg/mL for Cr(III) and to 20 pg/mL for Cr(VI). The method has been applied for the chromium speciation in natural water samples.

Expression of the E2 envelope glycoprotein of bovine viral diarrhoea virus (BVDV) elicits virus-type specific neutralising antibodies.

A region of genome from the NADL strain of BVDV corresponding to the coding sequence for the E2 glycoprotein has been molecularly cloned using RT-PCR. The viral cDNA sequence was used to construct vaccinia virus recombinants that expressed either the entire E2 coding sequence or fragments of it. These recombinants were used to immunise mice of three H-2 haplotypes to investigate their ability to elicit a neutralising antibody response against BVDV. Sera from mice immunised with the recombinant expressing full length E2 contained high levels of virus neutralising antibodies that in addition to giving neutralisation of the homologous NADL strain were also able to neutralise the Oregon C24V reference strain. These sera failed to give any neutralisation of the Osloss reference strain providing evidence for the division of BVDV isolates into at least two distinct E2 serotypes. These results were confirmed in gnotobiotic lambs. Expression of E2 fragments revealed the presence of at least two distinct neutralising epitopes, one of which was localised within the carboxy terminal 90 amino acids of the protein.

Total and free plasma neuroleptic levels in schizophrenic patients.

Nineteen male patients, under 35 years of age, newly admitted with a diagnosis of schizophrenia, were treated with either chlorpromazine or haloperidol at a fixed dosage for 25 days. Both total and free plasma neuroleptic levels were measured using a radioreceptor assay. Clinical response was measured by the Brief Psychiatric Rating Scale (BPRS). On day 25, nonresponders (those with a decrease of less than 8 points on the BPRS) had both total and free plasma neuroleptic levels within the range observed in responders. It is therefore concluded that lack of therapeutic response is generally not due to inadequate plasma drug levels.