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The oviductal epithelium is crucial for the integrity of the female organ. Previously we got evidence that the surface proteome of oviductal epithelial cells (Oecs) is promptly altered in response to insemination and thus suggested that this early phase plays a notable regulatory role in maintaining cellular function. This study further aimed to assess the effect of semen on the cellular and molecular mechanisms in rabbit Oecs. A quantitative gel-based proteomic approach was applied to analyze changes at three time points (0h, 1h, 2h) after intrauterine insemination (IUI) compared to time matched controls. Within two hours the abundance of 22 protein species was evidently altered in the intracellular fraction. Functional analysis revealed that the proteins were primarily involved in proteostasis as well as metabolic processes. The analysis of phosphoproteins specified a role of mitogen-activated protein kinase (MAPK) signaling molecules. Concurrently, semen increased oviduct-specific glycoprotein (OVGP1) secretion. A correlation between OVGP1 abundance and microtubule-associated proteins 1A/1B-light chain 3 lipidation was observed. The localization and changes in abundance of selected proteins were corroborated by antibody-based methods. These results clearly show that the early phase of interaction acts as a trigger for cellular adaptation to meet an altered demand in the female organ., Significance: The oviductal epithelium and its secreted proteins exert a pivotal role in reproductive processes, including the final maturation of male gametes. Thereby, the regulation and subsequently the functionality of the oviductal epithelial cell layer are important factors for the establishment of the appropriate milieu in the female reproductive tract. Notably, male gametes themselves have been shown to be an extrinsic modulatory factor of the oviductal epithelium. Accordingly a comprehensive knowledge about the underlying cellular and molecular mechanisms in the epithelial cells is of interest, also with regard to in vitro purposes. So far, the role of the early phase of interaction in the female organ has not been considered in detail. To get a further insight into the underlying cellular and molecular mechanisms, herein we analyzed the effect of semen on oviductal epithelial cells (Oecs) on the intracellular proteome level within the first two hours after insemination. The present study revealed a directed response of Oecs in vivo and disclosed intracellular pathways that are affected by the interplay between semen and the female reproductive tract. The prompt adaptation of the secretory activity and remodeling of the oviductal epithelium was accompanied by the concerted alterations of protein species that are primarily involved in the maintenance of cellular homeostasis. Besides emphasizing the importance of the early interaction phase for subsequent reproductive processes, the gained knowledge might further be implemented for in vitro applications as well., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Retraction of mesenchymal stromal cells supports the invasion of colorectal cancer cells (CRC) into the adjacent compartment. CRC-secreted 12(S)-HETE enhances the retraction of cancer-associated fibroblasts (CAFs) and therefore, 12(S)-HETE may enforce invasivity of CRC. Understanding the mechanisms of metastatic CRC is crucial for successful intervention. Therefore, we studied pro-invasive contributions of stromal cells in physiologically relevant three-dimensional in vitro assays consisting of CRC spheroids, CAFs, extracellular matrix and endothelial cells, as well as in reductionist models. In order to elucidate how CAFs support CRC invasion, tumour spheroid-induced CAF retraction and free intracellular Ca 2+ levels were measured and pharmacological- or siRNA-based inhibition of selected signalling cascades was performed. CRC spheroids caused the retraction of CAFs, generating entry gates in the adjacent surrogate stroma. The responsible trigger factor 12(S)-HETE provoked a signal, which was transduced by PLC, IP3, free intracellular Ca 2+ , Ca 2+ -calmodulin-kinase-II, RHO/ROCK and MYLK which led to the activation of myosin light chain 2, and subsequent CAF mobility. RHO activity was observed downstream as well as upstream of Ca 2+ release. Thus, Ca 2+ signalling served as central signal amplifier. Treatment with the FDA-approved drugs carbamazepine, cinnarizine, nifedipine and bepridil HCl, which reportedly interfere with cellular calcium availability, inhibited CAF-retraction. The elucidation of signalling pathways and identification of approved inhibitory drugs warrant development of intervention strategies targeting tumour-stroma interaction.

Lymph node metastasis of breast cancer is a clinical marker of poor prognosis. Yet, there exist no therapies targeting mechanisms of intravasation into lymphatics. Herein we report on an effect of the antidyslipidemic drug fenofibrate with vasoprotective activity, which attenuates breast cancer intravasation in vitro, and describe the potential mechanisms. To measure intravasation in a 3-dimensional co-culture model MDA-MB231 and MCF-7 breast cancer spheroids were placed on immortalised lymphendothelial cell (LEC) monolayers. This provokes the formation of circular chemorepellent induced defects (CCIDs) in the LEC barrier resembling entry ports for the intravasating tumour. Furthermore, the expression of adhesion molecules ICAM-1, CD31 and FAK was investigated in LECs by western blotting as well as cell-cell adhesion and NF-κB activity by respective assays. In MDA-MB231 cells the activity of CYP1A1 was measured by EROD assay. Fenofibrate inhibited CCID formation in the MDA-MB231/LEC- and MCF-7/LEC models and the activity of NF-κB, which in turn downregulated ICAM-1 in LECs and the adhesion of cancer cells to LECs. Furthermore, CD31 and the activity of FAK were inhibited. In MDA-MB231 cells, fenofibrate attenuated CYP1A1 activity. Combinations with other FDA-approved drugs, which reportedly inhibit different ion channels, attenuated CCID formation additively or synergistically. In summary, fenofibrate inhibited NF-κB and ICAM-1, and inactivated FAK, thereby attenuating tumour intravasation in vitro. A combination with other FDA-approved drugs further improved this effect. Our new concept may lead to a novel therapy for cancer patients.

Phase separation in the cytoplasm is emerging as a major principle in intracellular organization. In this process, sets of macromolecules assemble themselves into liquid compartments that are distinct from the surrounding medium but are not delimited by membrane boundaries. Here, we discuss how phase separation, in which a component of one of the two phases is vesicles rather than macromolecules, could underlie the formation of synaptic vesicle (SV) clusters in proximity to presynaptic sites. The organization of SVs into a liquid phase could explain how SVs remain tightly clustered without being stably bound to a scaffold so that they can be efficiently recruited to release site by active zone components., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objectives: The aim of this study was the estimation of effects induced by chronic administration of supraphysiological dose of TE and prolonged exercise in male rats on anxiety levels (alterations in exploratory activity patterns in EPM)., Material and Methods: Two sedentary (control - C and testosterone-enanthate - T) and two exercise (exercise - E and testosterone-enanthate plus exercise - T+E) groups (n=32) underwent adequate protocols - the swimming protocol (1 h/day) and TE (20 mg/kg/w, s.c.) for six weeks. Testing was performed in EPM., Results: Anxiolytic effects of exercise were manifested as increased exploratory activity in EPM - increase in cumulative duration in open arms, the number of rearings and head-dippings, and TEA. Supraphysiological dose of TE decreased the number of rearings and head-dippings, cumulative duration in open arms and TEA compared to the control group, while this effect of TE was more pronounced compared to the exercise group. The applied dose of TE attenuated beneficial effects of exercise by means of all estimated parameters., Conslusions: Our results confirmed the beneficial effect of exercise on anxiety levels observed in EPM by means of parameters considering the alterations in exploratory activity. Supraphysiological dose of TE resulted in anxiogenic-like behavior in EPM. The effect of TE was so pronounced that the beneficial effect of exercise was reversed to the control values (or even below them). Based on the results of this trial, we propose that use of the TEA (a new parameter for overall exploratory activity) can improve the evaluation of EPM test results.

Secretion of 12(S)-HETE by breast cancer emboli provokes "circular chemorepellent induced defects" (CCIDs) in the adjacent lymphatic vasculature facilitating their intravasation and lymph node metastasis which determines prognosis. Therefore, elucidating the mechanism of lymph endothelial cell (LEC) wall disintegration may provide cues for anti-metastatic intervention. The role of intracellular free Ca(2+) for CCID formation was investigated in LECs using MCF-7 or MDA-MB231 breast cancer cell spheroids in a three-dimensional cell co-culture model. 12(S)-HETE elevated the Ca(2+) level in LEC by activating PLC/IP3. Downstream, the Ca(2+)-calmodulin kinase MYLK contributed to the phosphorylation of Ser19-MLC2, LEC contraction and CCID formation. Approved clinical drugs, lidoflazine, ketotifen, epiandrosterone and cyclosporine, which reportedly disturb cellular calcium supply, inhibited 12(S)-HETE-induced Ca(2+) increase, Ser19-MLC2 phosphorylation and CCID formation. This treatment strategy may reduce spreading of breast cancer through lymphatics., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minor component of total plasma membrane lipids, but it has a substantial role in the regulation of many cellular functions, including exo- and endocytosis. Recently, it was shown that PI(4,5)P2and syntaxin 1, a SNARE protein that catalyzes regulated exocytosis, form domains in the plasma membrane that constitute recognition sites for vesicle docking. Also, calcium was shown to promote syntaxin 1 clustering in the plasma membrane, but the molecular mechanism was unknown. Here, using a combination of superresolution stimulated emission depletion microscopy, FRET, and atomic force microscopy, we show that Ca(2+)acts as a charge bridge that specifically and reversibly connects multiple syntaxin 1/PI(4,5)P2complexes into larger mesoscale domains. This transient reorganization of the plasma membrane by physiological Ca(2+)concentrations is likely to be important for Ca(2+)-regulated secretion., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Purpose: The purpose of this study is to investigate the effect of two of the most important functional CYP1A2 variations -3860G > A and -163C > A on carbamazepine pharmacokinetics in Serbian pediatric epileptic patients., Methods: The study involved 40 Serbian pediatric epileptic patients on steady-state carbamazepine treatment. Genotyping for -3860G > A and -163C > A was carried out using PCR-RFLP method, and carbamazepine plasma concentrations were determined by high pressure liquid chromatography (HPLC) method. For pharmacokinetic analysis, NONMEM software with implementation of ADVAN 1 subroutine was used., Results: CYP1A2 polymorphism -163C > A was found at the frequency of 65.0 %, while -3860G > A was not detected. The correlation between weight-adjusted carbamazepine dose and carbamazepine concentration after dose adjustment was significant only in carriers of -163C/C and C/A genotypes (r = 0.68, p = 0.0004). The equation that described population clearance (CL) was CL (l/h) = 0.176 + 0.0484 * SEX + 0.019 * CYP1A2 + 0.000156 * DD, where SEX has a value of 1 if male and 0 if female, CYP1A2 has a value of 1 if -163A/A and 0 if -163C/C or C/A, and DD is the total carbamazepine daily dose (mg/day)., Conclusions: CYP1A2 -163A/A genotype influence carbamazepine pharmacokinetics. In addition to sex and total carbamazepine daily dose, -163C > A CYP1A2 polymorphism should be considered as a predictor of carbamazepine clearance.

Artificial SNARE analogues derived from SNARE proteins, which mediate synaptic membrane fusion, are of interest. They mimic the tetrameric α-helix bundle of the SNARE motif with various bio-oligomer recognition units. Interaction between complementary oligomers linked to the respective membrane by lipid or peptide anchors leads to proximity of vesicles and to fusion of lipid bilayers. β-Peptide nucleic acids were introduced as hybrid oligomers with the native SNARE protein transmembrane/linker sequence, in order to evaluate a fusion system that allows distance tuning of approaching membranes. Formation of a four-base pair β-PNA double strand with 20 Å length is sufficient for vesicle membrane fusion. Elongation of the recognition β-PNA duplex in the linker region yielded a 40 Å β-peptide duplex and provided a vesicle-vesicle distance that only supported hemifusion of vesicle membranes., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

RELA, RELB, CREL, NFKB1 and NFKB2, and the upstream regulators NEMO and NIK were knocked-down in lymph endothelial cells (LECs) and in MDA-MB231 breast cancer spheroids to study the contribution of NF-κB in vascular barrier breaching. Suppression of RELA, NFKB1 and NEMO inhibited "circular chemo-repellent induced defects" (CCIDs), which form when cancer cells cross the lymphatic vasculature, by ~20-30%. Suppression of RELB, NFKB2 and NIK inhibited CCIDs by only ~10-15%. In MDA-MB231 cells RELA and NFKB1 constituted MMP1 expression, which caused the activation of PAR1 in adjacent LECs. The knock-down of MMP1 in MDA-MB231 spheroids and pharmacological inhibition of PAR1 in LECs inhibited CCID formation by ~30%. Intracellular Ca(2+) release in LECs, which was induced by recombinant MMP1, was suppressed by the PAR1 inhibitor SCH79797, thereby confirming a functional intercellular axis: RELA/NFKB1 - MMP1 (MDA-MB231) - PAR1 (LEC). Recombinant MMP1 induced PAR1-dependent phosphorylation of MLC2 and FAK in LECs, which is indicative for their activity and for directional cell migration such as observed during CCID formation. The combined knock-down of the NF-κB pathways in LECs and MDA-MB231 spheroids inhibited CCIDs significantly stronger than knock-down in either cell type alone. Also the knock-down of ICAM-1 in LECs (a NF-κB endpoint with relevance for CCID formation) and knock-down of MMP1 in MDA-MB231 augmented CCID inhibition. This evidences that in both cell types NF-κB significantly and independently contributes to tumour-mediated breaching of the lymphatic barrier. Hence, inflamed tumour tissue and/or vasculature pose an additional threat to cancer progression.

Even though a number of different in vitro fusion assays have been developed to analyze protein mediated fusion, they still only partially capture the essential features of the in vivo situation. Here we established an in vitro fusion assay that mimics the fluidity and planar geometry of the cellular plasma membrane to be able to monitor fusion of single protein-containing vesicles. As a proof of concept, planar pore-spanning membranes harboring SNARE-proteins were generated on highly ordered functionalized 1.2 μm-sized pore arrays in Si3N4. Full mobility of the membrane components was demonstrated by fluorescence correlation spectroscopy. Fusion was analyzed by two color confocal laser scanning fluorescence microscopy in a time resolved manner allowing to readily distinguish between vesicle docking, intermediate states such as hemifusion and full fusion. The importance of the membrane geometry on the fusion process was highlighted by comparing SNARE-mediated fusion with that of a minimal SNARE fusion mimetic.

This study considers the use of dry Jerusalem artichoke (JA) as a functional nutrient in developing food products with enhanced nutritional characteristics and low glycaemic index (GI). Three different formulations based on buckwheat and JA were developed and processed using extrusion technology. Nutritional properties including the levels of total dietary fibre (TDF), protein, inulin, total carbohydrates and lipids were analysed. A clinical study was performed on ten healthy volunteers (aged between 21 and 56) to determine the level of GI and glycaemic load (GL). The results revealed that JA significantly (P<0.05) increased the levels of TDF and inulin whilst decreasing carbohydrates, lipids and proteins. The resulting products had a significant (P<0.05) effect on IAUC between reference food and extruded products, GI and GL. Samples containing 80% of Jerusalem artichoke were considered as a low GI food whilst samples containing 30% and 60% of Jerusalem artichoke as a medium GI food. A similar trend was seen in terms of GL., (Copyright © 2015. Published by Elsevier Ltd.)

Objective: Adrenal crisis (AC) is a life-threatening complication of adrenal insufficiency (AI), which according to retrospective data represents a significant clinical complication. Here we aimed to prospectively assess incidence of AC and mortality associated with AC in patients with chronic AI., Methods: A total of 423 patients with AI (primary AI, n = 221; secondary AI, n = 202) were prospectively followed up for 2 years. Baseline assessment included a general questionnaire and detailed written instructions on glucocorticoid dose adaptation during stress. Patients received follow-up questionnaires every 6 months and were contacted by phone in case of reported adrenal crisis., Results: A total of 423 data sets were available for baseline analysis, and 364 patients (86%) completed the whole study. Sixy-four AC in 767.5 patient-years were documented (8.3 crises per 100 patient-years). Precipitating causes were mainly gastrointestinal infection, fever, and emotional stress (20%, respectively) but also other stressful events (eg, major pain, surgery, strenuous physical activity, heat, pregnancy) or unexplained sudden onset of AC (7%) were documented. Patients with a previous AC were at higher risk of crisis (odds ratio 2.85, 95% confidence interval 1.5-5.5, P < .01). However, no further risk factors could be identified. Ten patients died during follow-up; in four cases death was associated with AC (0.5 AC related deaths per 100 patient-years)., Conclusion: Even in educated patients with chronic adrenal insufficiency, AC occurs in a substantial proportion of cases. Furthermore, we identified AC-associated mortality in approximately 6% of AC. Our findings further emphasize the need for improved management of AC in patients with chronic AI.

The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein-protein interactions. Here using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains (TMDs) and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the TMDs differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes.

Objectives: The aim of this study was to investigate the role of the lactic acidosis, as an early predictor of significant consequences and/or a fatal outcome in term neonates after a perinatal asphyxia., Background: Severe perinatal asphyxia can generate multiple organ dysfunction and neonatal mortality., Methods: In routine clinical practice, after an admission to the Intensive Care Unit, lactate concentration was determined in capillary blood samples during the first one to six hours after birth in 55 term newborns with the post-asphyxial hypoxic-ischemic encephalopathy. The control group consisted of 36 healthy term neonates randomly selected in the maternity ward at the Gynecology and Obstetrics Clinic., Results: Significantly higher concentrations of lactate (p 8.7 mmol/L with 80 % sensitivity and 82% specificity indicated the development of the hypoxic-ischemic encephalopathy stage II/III, while the lactate level>9.95 mmol/L was a predictor of death, with 75% sensitivity and 74.4% specificity., Conclusion: Determination of lactate concentrations in serum of term newborns associated with risk factors for the perinatal asphyxia is a useful tool in diagnosing metabolic disorders and ischemic damage, particularly severe clinical forms (Tab. 2, Fig. 3, Ref. 34).

Although various strength tests and their outcome measures have been proposed for anterior cruciate ligament (ACL) reconstruction (ACLR), their measurement properties still remain relatively underexplored. The aim of this study was to investigate the longitudinal construct validity of the standard isokinetic (IKT) and isometric test (IMT), and of the IMT of alternating consecutive maximal contractions (ACMC). In addition, the concurrent validity of ACMC was assessed and compared with the validity of IMT. The strength of quadriceps and hamstrings in 20 male athletes with an anterior cruciate ligament (ACL) injury were assessed before ACLR, 4 and 6 months after ACLR, by means of IMT, ACMC, and IKT performed at 60 and 180° · s(-1). Significant between-session differences in muscle strength variables were found in the involved quadriceps (F > 6.5; p ≤ 0.05), but not in the uninvolved leg (F < 2.5; p > 0.05). Coefficients of variations in the uninvolved leg (all below 13.5%) were lower than the involved leg (11.7-22.1%). Intraclass correlation coefficients were moderate-to-high for the uninvolved leg and low-to-high for quadriceps of the involved leg. The concurrent validity of ACMC with respect to the IKT (r = 0.57-0.92; p ≤ 0.05) was comparable with the validity of IMT (r = 0.52-0.87; p ≤ 0.05). We conclude that the explored longitudinal construct validity of most of the evaluated variables could be sufficiently sensitive to detect the effects of the applied rehabilitation procedures. In addition, the obtained sensitivity and concurrent validity and the potential advantages of ACMC over IMT, all suggest that ACMC could be a particularly promising method for routine testing of neuromuscular function after ACLR.

Objective: Nandrolone decanoate (ND) is frequently used anabolic androgenic steroid (AAS) among the athletes. Despite the health risks, there is significant increase in prevalence of AAS abuse., Design: The aim of this study was to investigate the effects of chronic exposure to ND at supraphysiological dose (to mimic the doses for human AAS abusers) on anxiety levels in adult rats., Settings: We performed several behavioral tests (open field test, elevated plus maze test, beam-walking test, evoked beam-walking test and tail suspension test) for estimation of anxiety in rats. Adult rats received 20 mg/kg intraperitoneal injection of ND weekly for four weeks. Behavioral test were performed on the seventh day after the last dose of ND., Results: Anxiogenic-like pattern of behavior was clearly observed in several behavioral tests, such as open field test (decrease of total distance moved and cumulative duration of moving, decrease of an average velocity of the animals, decrease of frequency and total time in centre zone); elevated plus maze (decreased total time spent in open arms and the number of entries in open arms of the elevated plus maze); evoked beam-walking test (decreased time to cross the beam) and tail suspension test (increased latency to first immobility and decreased total duration of immobility)., Main Findings: Results of this study show that four-week treatment with the supraphysiological dose of ND produced anxiogenic effects in sedentary male rats., Conclusion: Our results show that rats after chronic treatment with a supraphysiological dose of ND exhibited anxiety-like behavior.

There are contradictory opinions if late-onset systemic lupus erythematosus (SLE) is associated with a different, more benign disease course and better prognosis than early-onset SLE. The objective of this study was to evaluate the clinical manifestations, course, treatment, and prognosis of late-onset SLE. Patients who developed SLE after/or at the age of 50 years were considered late-onset SLE and compared to a group of randomly selected patients aged younger than 50 years at the diagnosis, matched for disease duration. Lower frequency of cutaneous manifestations (p = 0.01) and higher frequency of cytopenias (p = 0.02) were registrated at the SLE onset in the late-onset group. Atypical clinical presentation of SLE contributed to a longer delay of diagnosis in late-onset SLE patients (p = 0.005), who fullfiled less American College of Rheumatology criteria at the diagnosis (p = 0.022). Cumulative incidence of clinical manifestations showed lower frequency of cutaneous (p = 0.017), neuropsychiatric manifestations (p = 0.021), lupus nephritis (p = 0.006), and higher frequency of Sjogren's syndrome (p = 0.025) in the late-onset group. Late-onset SLE patients received lower doses of corticosteroid (p = 0.006) and cyclophosphamide (p = 0.001) and had more cyclophosphamide-induced complications (p = 0.005). Higher prevalence of comorbid conditions in the late-onset group (p = 0.025), and higher Systemic Lupus International Collaborating Clinics/American College of Rheumatology damage index was noticed (p = 0.018). Despite the less major organ involvement and more benign course of disease, late-onset SLE has poorer prognosis, because of the higher frequency of comorbid conditions and higher organ damage, due to the aging and longer exposition to a classical vascular risk factors.

Synaptic-vesicle exocytosis is mediated by the vesicular Ca(2+) sensor synaptotagmin-1. Synaptotagmin-1 interacts with the SNARE protein syntaxin-1A and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2). However, it is unclear how these interactions contribute to triggering membrane fusion. Using PC12 cells from Rattus norvegicus and artificial supported bilayers, we show that synaptotagmin-1 interacts with the polybasic linker region of syntaxin-1A independent of Ca(2+) through PIP2. This interaction allows both Ca(2+)-binding sites of synaptotagmin-1 to bind to phosphatidylserine in the vesicle membrane upon Ca(2+) triggering. We determined the crystal structure of the C2B domain of synaptotagmin-1 bound to phosphoserine, allowing development of a high-resolution model of synaptotagmin bridging two different membranes. Our results suggest that PIP2 clusters organized by syntaxin-1 act as molecular beacons for vesicle docking, with the subsequent Ca(2+) influx bringing the vesicle membrane close enough for membrane fusion.

The novel test based on isometric alternating consecutive maximal contractions performed by two antagonistic muscles has been recently proposed as a test of muscle function in healthy subjects. The aim of this study was to evaluate reliability and sensitivity of a novel test as a test of knee muscles function in athletes recovering from anterior cruciate ligament reconstruction. Fifteen male athletes with recent ligament reconstruction (4.0 ± 0.1 months following the surgery) and 15 sport and physical education students participated in the study. Peak torques of the quadriceps and hamstring muscles assessed both through the alternating consecutive maximal contractions and standard isokinetic test performed at 60 º/s and 180 º/s served for calculation of the hamstrings-to-quadriceps ratio and the bilateral difference in strength. When applied on individuals recovering from anterior cruciate ligament reconstruction, the novel test revealed a high within-day reliability and sensitivity for detecting imbalances both between antagonistic and between contralateral muscles. The present findings suggest that alternating consecutive maximal contractions could be used as a test of muscle function that is either complementary or alternative to the isokinetic test, particularly in the laboratories where the isokinetic devices are not available. Potential advantages of the novel test could be both a brief testing procedure and a possibility to conduct it using relatively inexpensive devices such as custom made kits containing a single one-axis force transducer.

Long-range correlated motions in proteins are candidate mechanisms for processes that require information transfer across protein structures, such as allostery and signal transduction. However, the observation of backbone correlations between distant residues has remained elusive, and only local correlations have been revealed using residual dipolar couplings measured by NMR spectroscopy. In this work, we experimentally identified and characterized collective motions spanning four β-strands separated by up to 15 Å in ubiquitin. The observed correlations link molecular recognition sites and result from concerted conformational changes that are in part mediated by the hydrogen-bonding network.

Objective: Adrenal crisis (AC) is a life-threatening complication of adrenal insufficiency (AI). Here, we evaluated frequency, causes and risk factors of AC in patients with chronic AI., Methods: In a cross-sectional study, 883 patients with AI were contacted by mail. Five-hundred and twenty-six patients agreed to participate and received a disease-specific questionnaire., Results: Four-hundred and forty-four datasets were available for analysis (primary AI (PAI), n=254; secondary AI (SAI), n=190). Forty-two percent (PAI 47% and SAI 35%) reported at least one crisis. Three hundred and eighty-four AC in 6092 patient years were documented (frequency of 6.3 crises/100 patient years). Precipitating causes were mainly gastrointestinal infection and fever (45%) but also other stressful events (e.g. major pain, surgery, psychic distress, heat and pregnancy). Sudden onset of apparently unexplained AC was also reported (PAI 6.6% and SAI 12.7%). Patients with PAI reported more frequent emergency glucocorticoid administration (42.5 vs 28.4%, P=0.003). Crisis incidence was not influenced by educational status, body mass index, glucocorticoid dose, DHEA treatment, age at diagnosis, hypogonadism, hypothyroidism or GH deficiency. In PAI, patients with concomitant non-endocrine disease were at higher risk of crisis (odds ratio (OR)=2.02, 95% confidence interval (CI) 1.05-3.89, P=0.036). In SAI, female sex (OR=2.18, 95% CI 1.06-4.5, P=0.035) and diabetes insipidus (OR=2.71, 95% CI 1.22-5.99, P=0.014) were associated with higher crisis incidence., Conclusion: AC occurs in a substantial proportion of patients with chronic AI, mainly triggered by infectious disease. Only a limited number of risk factors suitable for targeting prevention of AC were identified. These findings indicate the need for new concepts of crisis prevention in patients with AI.

A combined test performed at the 12th week of gestation enables us to classify the pregnancy as high risk (risk higher than 1:300) or low risk (risk lower than 1:300) for congenital foetal anomalies, with great accuracy of 85 - 90%. According to the available data, the frequency of false positive results is estimated at around 5%. The objective of the study was to examine possible correlation between the serum marker values and amniocentesis results in prenatal diagnostics of congenital foetal anomalies. The study included 745 pregnant women monitored by the Genetic Counselling Service of the Clinic of Gynaecology and Obstetrics of the Clinics Centre Kragujevac. The subjects were included in the study under condition that CRL (embryonic crown-rump length) was from 45 to 84 mm and that the gestational age was at 11-13+6 weeks. Free beta HCG and PAPP-A were determined from venous blood using commercial DPS-USA tests. Tests were based on the analytic principle of the immuno-chemiluminescence technique and were performed by application of the automatic Immulite 2000 analyzer by DPC-USA. The foetal nuchal translucency thickness (NT) and CRL were measured by Colour Doppler. The chromosome identification was performed after a certain number of cell divisions by stopping the cell division in metaphase of mitosis when the chromosomes were the most distinguishable. The foetal karyotype was prepared using G bands. In the total sample of pregnant women (n=745), there were six cases of pathological foetal karyotype. A statistical paradox in the frequency of congenital foetal anomalies in favour of younger population was noticed. A high coefficient of Spearman's rank correlation suggests great importance of the combined test in the detection of congenital foetal anomalies (p<0,05). A high consistency was also proved for components of biochemical screening and ultrasonographic markers. The combined test, as a method of prenatal screening in the first trimester of pregnancy, if used at 11 - 13+6 weeks' gestation and for CRL of 45-84 mm, has a great importance in the detection of congenital foetal anomalies.

Objectives: While the neurotransmitter role of glutamate in the gastrointestinal tract has been shown, its effects on smooth muscle of the human ureter have not previously been investigated. In our study we have investigated the effects of exogenous glutamate on the spontaneous activity of isolated human ureter, taken from 14 adult patients after nephrectomy., Methods: The segment of ureter, excised 3 cm distal from the pyeloureteral junction, was isolated in an organ bath. Both longitudinal tension and intraluminal pressure of the segment were recorded simultaneously., Results: Glutamate administered in the lumen of the isolated ureteral segments (7.8 x 10(-7) M/L-3.5 x 10(-2) M/L) was ineffective. When added to the isolated organ bath from the serous side of the ureteral segment, glutamate (7.9 x 10(-6) M/L-10.6 x 10(-3) M/L) and N-Methyl-D-aspartic acid (NMDA) (9.1 x 10(-8) M/L-3.1 x 10(-5) M/L) produced a concentration-dependent increase in spontaneous activity of the isolated preparations, while kainic acid (6.3 x 10(-8) M/L-10.5 x 10(-5) M/L) and (+/-)-trans-1-Aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD) (7.7 x 10(-8) M/L -6.5 x 10(-5) M/L) were ineffective., Conclusions: The results of our study suggest that an excitatory neurotransmitter glutamate stimulates spontaneous activity of the human ureter through activation of NMDA ionotropic receptors, located on smooth muscle cells or intramural nerve fibers.

Although the contractile effects of glutamate and related excitatory amino acids on gut smooth muscle strips have been demonstrated, the mechanisms, and particularly the physiological importance of that action, remain unknown. In this study, glutamate, aspartate, AMPA, quisqualate, cis-ACPD and (2R,4R)-APDC evoked concentration-dependent contraction of isolated adult rat gastric fundus, with EC50 values of 210 microM, 150 microM, 20 microM, 33 microM, and 2.7 microM and 7.9 microM, respectively. L-SOP (0.1 microM-1.9 mM) did not change the basal tone of the preparations. The maximal contractions evoked by glutamate (20 mM) were 38.83% compared with those elicited by acetylcholine (20 microM). The glutamate-evoked contractions were not affected by atropine, verapamil and nicardipine, blocked by CNQX (0.01 microM), or potentiated by Mg2+ (0.01-100 microM), ketamine (0.01-100 microM) and DL-AP5 (0.1-100 microM), as well as L-trans-2,4-PDC (1-100 microM). Analysis of glutamate's action on rat rectum (EC50 = 44 microM) could only be carried out at the early stages, as half of the preparations were not affected by glutamate. Only 5 out of 26 human longitudinal and circular smooth muscle preparations taken from the stomach and three segments of the large intestine were very slightly contracted by glutamate, excluding further analysis. The contractile effects of glutamate on rat gut smooth muscles were mediated by multiple GluR (non-NMDA > NMDA > group I/II mGluRs) located primarily on smooth muscle cells but functional GluRs on neurons and/or nerve fibers of myenteric nervous plexuses could not be excluded. To fully understand the physiological significance of glutamate-evoked contractions in the gut, more research is required, most likely using many different methodological approaches.

Many diagnostic and therapeutic procedures depend on medical images. In order to overcome imperfections of the obtained images, which are due to the acquisition process, and to extract new information from the available images, many techniques have been developed. In this study, a new method of image segmentation and 3D reconstruction based on active contour models ('snakes') was applied in analyzing computed tomography (CT) images in patients with acute head trauma. Using this method, lesion to brain (LBR) and ventricle to brain ratio (VBR) parameters, as well as 3D reconstruction of traumatic lesion, was obtained accurately. In our study group, 215 patients (mean age 42.4+/-23.5 years, 138/215 (64.2%) males) were included. Among them, 72 (33.5%) did not survive during hospitalisation in the Emergency Department. LBR correlated with the Glasgow Coma Score and the intrahospital outcome (r=-0.457 and r=0.515, respectively). Besides, non-survivors had greater LTB values (0.042+/-0.034) than survivors (0.005+/-0.011). However, VBR did not correlate with these clinical parameters. In addition, LBR was significantly higher in the patients with other pathologic CT findings. The proposed methodology, based on extracting maximum information from available CT scans, could be a basis for further medical decision making in patients with acute head trauma.

RNA splicing is a tightly regulated process. It is essential for gene expression and, therefore, intervenes in every biological phenomenon in mammals. RNA splicing regulation is cell type-specific in such a way that a cellular situation can be characterised by its repertoire of spliced events, the spliceome. Comparison of the splicing repertoire of two situations identifies alternative exons and introns. This regulation involves cis-acting sequences and transacting factors. Mutations, as well as modifications of signalling pathways, can alter the accuracy of splicing. Since deletion of exons or retention of introns within coding sequences modifies the corresponding proteins and functional domains of proteins are encoded by contiguous exons, identifying changes in the spliceome pinpoints functional domains, which are specifically regulated at the level of RNA splicing. We have developed a new method of gene profiling, qualitative gene profiling, that allows the comparative study of the repertoires of spliced events that characterise distinct physiopathological situations. We present in this review the different uses of this new genomic technique that can help each step of the R&D process in the pharmaceutical industry, and that represents a short cut towards functional genomics and pharmacogenomics.

Many diagnostic and therapeutic procedures depend on medical images. In order to overcome imperfections of obtained images which are due to acquisition process and to obtain new information from available images, many techniques have been developed. In this study relatively new method of image segmentation, active contour model--"snakes" was applied in analyzing computed tomography (CT) images in patients with acute head trauma. Using this method, lesion to brain (LBR) and ventricle to brain ratio (VBR) were obtained accurately. Quantitative variable LBR, is significantly higher in patients with other pathologic CT findings and who do not survive during hospitalization. Thus, by applying segmentation "snakes" model it is possible to extract maximum information from available CT scans. These variables could be basis for medical decision making in patients with acute head trauma.

This review summarizes some of the structural information that has been obtained on the gastric H,K ATPase. Methods such as tryptic digestion, site specific labeling and in vitro translation combine to provide a ten membrane segment model with however reservations as to the full transmembrane nature of M5 or M6. Labeling this region with the thiophilic luminal face reagent omeprazole provided cogent evidence that cys 813 but not cys 822 was labeled. On the other hand, cysteine mutagenesis provided evidence that removal of cys 813 did not affect inhibition of Rb transport by omeprazole whereas removal of cys 822 although not affecting ATPase activity abolished omeprazole inhibition of transport. A model to reconcile these data is presented where M5 and M6 although intramembranal are not transmembrane hairpin structures. Analysis of the region of alpha beta interaction by tryptic digestion and WGA chromatography to define those fragments of alpha that remain beta associated shows that leu 853 to arg 922 in the TM7-loop are a major region of association with the beta subunit. Yeast two hybrid analysis, when combined with these data and those from a chimeric construct, indicates that the sequence Q 907 to R 922 is the important element of interaction in the alpha subunit and no other extracytoplasmic domain was found to interact. Two regions of the beta subunit interact with this region of the alpha subunit between Q64 and N130 as well as A156 and R188. Apparently the beta subunit is folded around a small region of the large extracytoplasmic loop between TM7 and TM8, closer to TM8.

To define the pharmacological properties of a newly developed transdermal nicotine system (TNS) designed to facilitate abstention from smoking, three human pharmacology studies were performed in healthy, nicotine-dependent smokers. The first study, in which cigarette smoking served as the standard mode of nicotine intake, was carried out to evaluate the most suitable non-invasive methods for detecting the pharmacodynamic effects of nicotine and to provide a baseline for comparisons with the results of the subsequent studies to assess the effects of single and repeated applications of the TNS. The pharmacodynamic changes induced by smoking were generally most pronounced after the first cigarette following 10 hours' abstinence. The most sensitive parameters were heart rate, which increased, stroke volume measured by impedance cardiography, which decreased, cutaneous blood flow measured by Laser Doppler flowmetry and skin temperature, which diminished to a statistically significant extent after each cigarette. Increases in blood pressure were not very pronounced. Plasma catecholamines were consistently elevated after each cigarette, but the changes were not statistically significant. Compared with those induced by cigarette smoking, the cardiovascular effect seen after either a single application of the TNS (10, 20 and 40 mg/24 h) or repeated application of a TNS delivering 14 mg nicotine/24 h were minor. A slight increase in blood pressure was detectable only on the first day of application and had disappeared after 10 days' repetitive application, suggesting the development of partial tolerance. Heart rate was slightly increased by 3-7% and stroke volume decreased by 5-12% on the tenth day of TNS application.(ABSTRACT TRUNCATED AT 250 WORDS)