Oxygen-enriched air reduces breathing gas consumption over air.

Owing to the unfamiliar environment, recreational and professional diving is confronted with several challenges. Usage of self-contained under-water breathing apparatuses during the dive provides the indispensable breathing gas supply for the diver. Instead of air, oxygen-enriched breathing gases (EANx or nitrox) are used with increasing frequency. Unfortunately, their usage implies negative effects because the elevated oxygen partial pressure (pO2) increases oxidative stress. As a result, the increased formation of reactive oxygen species exerts negative effects on the central nervous system, lungs, vasculature and eyes. However, these disadvantages can be avoided if appropriate rules are followed, e.g. a pO2<1.4 bar. EANx breathing gases have, on the other hand, major advantages as they help reducing narcotic nitrogen effects and bubble formation. Several land-based studies had proven a reduced ventilation of exercising subjects if EANx was used instead of air. As breathing gas is the most valuable under-water good, we wanted to translate the on-land results into under-water results. Appropriate studies now demonstrate a novel EANx property as under-water ventilation is also reduced with EANx. In this short communication, we present this additional advantage of EANx-breathing. This benefit seems to be of particular importance as it delays unforeseen running-out-of-gas and thus, contributes to further improving diving safety.

Mental health among currently enrolled medical students in Germany.

OBJECTIVES: The study identifies the prevalence of common mental disorders according to the patient health questionnaire (PHQ) and the use of psychotropic substances in a sample of currently enrolled medical students. STUDY DESIGN: A cross-sectional survey with a self-administrated questionnaire. METHODS: All newly enrolled medical students at the University of Dusseldorf, with study beginning either in 2012 or 2013, respectively, were invited to participate. The evaluation was based on 590 completed questionnaires. Mental health outcomes were measured by the PHQ, including major depression, other depressive symptoms (subthreshold depression), anxiety, panic disorders and psychosomatic complaints. Moreover, information about psychotropic substances use (including medication) was obtained. Multiple logistic regression analysis was used to estimate associations between sociodemographic and socio-economic factors and mental health outcomes. RESULTS: The prevalence rates, measured by the PHQ, were 4.7% for major depression, 5.8% for other depressive symptoms, 4.4% for anxiety, 1.9% for panic disorders, and 15.7% for psychosomatic complaints. These prevalence rates were higher than those reported in the general population, but lower than in medical students in the course of medical training. In all, 10.7% of the students reported regular psychotropic substance use: 5.1% of students used medication 'to calm down,' 4.6% 'to improve their sleep,' 4.4% 'to elevate mood,' and 3.1% 'to improve cognitive performance.' In the fully adjusted model, expected financial difficulties were significantly associated with poor mental health (odds ratio [OR]: 2.14; 95% confidence interval [CI]: 1.31-3.48), psychosomatic symptoms (OR:1.85; 95% CI: 1.11-3.09) and psychotropic substances use (OR: 2.68; 95% CI: 1.51-4.75). CONCLUSION: The high rates of mental disorders among currently enrolled medical students call for the promotion of mental health, with a special emphasis on vulnerable groups.

[Needlestick Injuries to Medical Students]. / Nadelstichverletzungen bei Studierenden der Humanmedizin.

The study aimsed at surveying and analysing the prevailing risks for medical students due to so-called needlestick injuries, I. e., injuries to the skin by handling sharp objects by which blood of patients can be transmitted to the health professional. After introducing preventive measures in a typical German university hospital, a total of 1 903 students of human medicine in their clinical period from 2009 to 2012 (from a total of 2 024 subjects - a rate of 94.0%) were questioned in detail about potential needlestick or other injuries related to their work. The results show that such injuries happen particularly during the clinical period of the medical studies: While only 20.6% of the students indicated a needlestick injury at the beginning of this period, half of the students (50.9%) had experienced at least one injury at the end of the clinical period. The activities mentioned most frequently were taking of blood samples and injections. Needlestick injuries happened most frequently in surgical units, in internal medicine, and in gynaecology. Accidents happened mostly during secondary employment, medical traineeship, or in the context of practical nursing. In consequence, measures for improvement of the primary prevention should start with training on the one hand: Only briefing seems to be insufficient - intensive exercises in using stick-proof instruments seems to be more promising. On the other hand, the comprehensive introduction of stick-proof instruments has to be supported.

Sample prefractionation with liquid isoelectric focusing enables in depth microbial metaproteome analysis of mesophilic and thermophilic biogas plants.

Biogas production from energy crops and biodegradable waste is one of the major sources for renewable energies in Germany. Within a biogas plant (BGP) a complex microbial community converts biomass to biogas. Unfortunately, disturbances of the biogas process occur occasionally and cause economic losses of varying extent. Besides technical failures the microbial community itself is commonly assumed as a reason for process instability. To improve the performance and efficiency of BGP, a deeper knowledge of the composition and the metabolic state of the microbial community is required and biomarkers for monitoring of process deviations or even the prediction of process failures have to be identified. Previous work based on 2D-electrophoresis demonstrated that the analysis of the metaproteome is well suited to provide insights into the apparent metabolism of the microbial communities. Using SDS-PAGE with subsequent mass spectrometry, stable protein patterns were evaluated for a number of anaerobic digesters. Furthermore, it was shown that severe changes in process parameters such as acidification resulted in significant modifications of the metaproteome. Monitoring of changing protein patterns derived from anaerobic digesters, however, is still a challenge due to the high complexity of the metaproteome. In this study, different combinations of separation techniques to reduce the complexity of proteomic BGP samples were compared with respect to the subsequent identification of proteins by tandem mass spectrometry (MS/MS): (i) 1D: proteins were tryptically digested and the resulting peptides were separated by reversed phase chromatography prior to MS/MS. (ii) 2D: proteins were separated by GeLC-MS/MS according to proteins molecular weights before tryptic digestion, (iii) 3D: proteins were separated by gel-free fractionation using isoelectric focusing (IEF) conducted before GeLC-MS/MS. For this study, a comparison of two anaerobic digesters operated at mesophilic and at thermophilic conditions was conducted. The addition of further separation dimensions before protein identification increased the number of identified proteins. On the other hand additional fractionation steps increased the experimental work load and the time required for LC-MS/MS measurement. The high resolution of the 3D-approach enabled the detection of approximately 750 to 1650 proteins covering the main pathways of hydrolysis, acidogenesis, acetogenesis and methanogenesis. Methanosarcinales dominated in the mesophilic BGP, whereas Methanomicrobiales were highly abundant in the thermophilic BGP. Pathway analysis confirmed the taxonomic results and revealed that the acetoclastic methanogenesis occurred preferentially at mesophilic conditions, whereas exclusively hydrogenotrophic methanogenesis was detected in thermophilic BGP. However, for the identification of process biomarkers by comprehensive screening of BGP it will be indispensable to find a balance between the experimental efforts and analytical resolution.

[Comparison of pulmonary compliance and the work of breathing with pulmonary function parameters in men]. / Vergleichende Messungen der Lungen-Compliance und der Atemarbeit mit anderen Lungenfunktionsparametern bei männlichen Probanden.

BACKGROUND: Dynamic and static pulmonary compliance as well as the work of breathing are essential parameters for describing respiratory mechanics. However, current reference values or correlations with other pulmonary function variables have been established only in studies with small numbers of participants. Therefore, the aim of the present study was to compare pulmonary compliance and the work of breathing with different pulmonary function parameters in a large number of men. METHODS: In 208 healthy men, aged 20 to 70 years, spirometry, bodyplethysmography and carbon monoxide transfer factor measurements were conducted. The esophageal pressure method was used to measure dynamic (C (dyn)) and static (C (stat)) compliance. RESULTS: C (dyn) and C (stat) were significantly correlated with total lung capacity (TLC), vital capacity (VC), forced expiratory volume in the first second (FEV (1)) and carbon monoxide transfer factor (T (LCO)). C (dyn) decreased with increasing age, while C (stat) was correlated with height. The elastic and non-elastic work of breathing were significantly correlated with airway resistance, but not with aging. CONCLUSIONS: Lung volumes are important for the interpretation of pulmonary compliance and the work of breathing and therefore have to be considered in the assessment of respiratory mechanics. In contrast, the work of breathing is essentially influenced by the airway resistance. With increasing age there is only a small decrease in pulmonary compliance and the work of breathing.

Reference values for dynamic and static pulmonary compliance in men.

The aim of the present study was to determine new reference values and predictive variables for dynamic and static pulmonary compliance in men. The investigation was conducted as a prospective study in healthy, non-smoking men with normal pulmonary function parameters including spirometry, bodyplethysmography and CO diffusing capacity. The esophageal pressure method was used to measure dynamic compliance (Cdyn), specific dynamic compliance (Cdyn/ITGV), static compliance (Cstat) and specific static compliance (Cstat/ITGV). Lung recoil pressures were recorded at different levels of total lung capacity (TLC). A total of 208 men aged 20-69 years were included in the study. The mean values for the compliance parameters were: Cdyn: 2.91+/-1.08 L/kPa; Cdyn/ITGV: 0.71 +/- 0.30 kPa (-1); Cstat: 3.34 +/- 1.04 L/kPa; Cstat/ITGV: 0.82 +/- 0.31 kPa (-1). Cdyn, Cdyn/ITGV and Cstat/ITGV were significantly correlated with age and Cstat was related to height, but in multiple regression analyses the predictability for compliance parameters was very low. Lung recoil pressures at all TLC levels significantly decreased with ageing. In conclusion, we demonstrated that the contribution of anthropometric variables to the regression equations of pulmonary compliance was low. With ageing the static pressure-volume curve of the lung shifted to the left without substantial alteration of the slope.

A membrane-embedded glutamate is required for ligand binding to the multidrug transporter EmrE.

EmrE is an Escherichia coli multidrug transporter that confers resistance to a variety of toxins by removing them in exchange for hydrogen ions. The detergent-solubilized protein binds tetraphenylphosphonium (TPP(+)) with a K(D) of 10 nM. One mole of ligand is bound per approximately 3 mol of EmrE, suggesting that there is one binding site per trimer. The steep pH dependence of binding suggests that one or more residues, with an apparent pK of approximately 7.5, release protons prior to ligand binding. A conservative Asp replacement (E14D) at position 14 of the only membrane-embedded charged residue shows little transport activity, but binds TPP(+) at levels similar to those of the wild-type protein. The apparent pK of the Asp shifts to <5.0. The data are consistent with a mechanism requiring Glu14 for both substrate and proton recognition. We propose a model in which two of the three Glu14s in the postulated trimeric EmrE homooligomer deprotonate upon ligand binding. The ligand is released on the other face of the membrane after binding of protons to Glu14.

Sorting of P-type ATPases in polarized epithelial cells.

The Na,K-ATPase and the H,K-ATPase are highly homologous members of the P-type family of ion transporting ATPase. Despite their structural similarity, these two pumps are sorted to different destinations in polarized epithelial cells. While the Na,K-ATPase is restricted to the basolateral surfaces of most epithelial cells types, the H,K-ATPase is concentrated at the apical plasmalemma and in a pre-apical vesicular storage compartment in the parietal cells of the stomach. We have generated molecular chimeras composed of complementary portions of these two pumps' alpha-subunits. By expressing these pump constructs in polarized epithelial cells in culture, we have been able to identify sequence domains which participate in the targetting of the holoenzyme. We find that information embedded within the sequence of the fourth transmembrane domain of the H,K-ATPase is sufficient to account for this protein's apical localization. Stimulation of gastric acid secretion results in insertion of the intracellular H,K-ATPase pool into the apical plasma membrane and inactivation of acid secretion is accompanied by the re-internalization of these pumps. We have identified a tyrosine-based signal in the cytoplasmic tail of the H,K-ATPase beta-subunit which appears to be required for this endocytosis. We have mutated the critical tyrosine residue to alanine and expressed the altered protein in transgenic mice. The H,K-ATPase remains continuously at the apical cell surface in parietal cells from these animals, and they constitutively hypersecrete gastric acid. These results demonstrate that the beta-subunit sequence mediates the internalization of the H,K-ATPase and is required for the cessation of gastric acid secretion. Thus, at least two sorting signals are required to ensure the proper targetting and regulation of the gastric H,K pump.

Identification of sorting determinants in the C-terminal cytoplasmic tails of the gamma-aminobutyric acid transporters GAT-2 and GAT-3.

In order to perform their physiologic functions, polarized epithelial cells must target ion transport proteins to the appropriate domains of their plasma membranes. Molecular signals responsible for polarized sorting have been identified for several membrane proteins which span the bilayer once. Most ion transport proteins are polytopic, however, and little is known of the signals responsible for the targeting of this class of polypeptides. Members of the gamma-aminobutyric acid (GABA) transporter family are polytopic membrane proteins found endogenously in both epithelial cells and neurons. We have identified narrowly defined sequences which are required for the proper accumulation of two members of this transporter family in Madin-Darby canine kidney cells. The highly homologous GABA transporter isoforms, GAT-2 and GAT-3, localize to the basolateral and apical surfaces, respectively, when expressed stably in Madin-Darby canine kidney cells. We have generated deletion constructs and chimeric transporters composed of complimentary portions of GAT-2 and GAT-3. We find that information which directs their differential sorting is present in the C-terminal cytoplasmic tails of these two polypeptides. A sequence of 22 amino acids at the C terminus of GAT-2 is required for the transporter's basolateral distribution and is capable of directing GAT-3 to the basolateral surface when appended to the C terminus of this normally apical polypeptide. The deletion of 32 amino acids from the C terminus of GAT-3 causes this transporter to become mislocalized to both surfaces. Moreover, removal of the final three amino acids of GAT-3 (THF) similarly disrupts its apical sorting. The GAT-3 C-terminal sequence resembles motifs which interact with PDZ domains, raising the possibility that the steady state distribution of GAT-3 at the apical plasmalemmal surface requires a protein-protein interaction mediated by its extreme C-terminal cytoplasmic tail. These data provide the first characterization of a protein-based signal required for the apical distribution of a membrane protein.

A basolateral sorting signal is encoded in the alpha-subunit of Na-K-ATPase.

Na-K-ATPase and H-K-ATPase are highly homologous ion pumps that exhibit distinct plasma membrane distributions in epithelial cells. We have studied the alpha-subunits of these heterodimeric pumps to identify the protein domains responsible for their polarized sorting. A chimeric alpha-subunit construct (N519H) was generated in which the first 519 amino acid residues correspond to the Na-K-ATPase sequence and the remaining 500 amino acids are derived from the H-K-ATPase sequence. In stably transfected LLC-PK1 cell lines, we found that the N519H chimera is restricted to the basolateral surface under steady-state conditions, suggesting that residues within the NH2-terminal 519 amino acids of the Na-K-ATPase alpha-subunit contain a basolateral sorting signal. H-K-ATPase beta-subunit expressed alone in LLC-PK1 cells accumulates at the apical surface. When coexpressed with N519H, the H-K-ATPase beta-subunit assembles with this chimera and accompanies it to the basolateral surface. Thus the NH2-terminal basolateral signal in the Na-K-ATPase alpha-subunit masks or is dominant over any apical sorting information present in the beta-polypeptide. In gastric parietal cells, the H-K-ATPase beta-subunit targets the H-K-ATPase to an intracellular vesicular compartment which fuses with the plasma membrane in response to secretagogue stimulation. To test whether the chimera-H-K-ATPase beta-subunit complex is directed to a similar compartment in LLC-PK1 cells, we treated transfected cells with drugs that raise intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels. Elevation of cytosolic cAMP increased the surface expression of both the N519H chimera and the H-K-ATPase beta-subunit. This increase in surface expression, however, appears to be the result of transcriptional upregulation and not recruitment of chimera to the surface from a cAMP-inducible compartment.

Sorting and trafficking of ion transport proteins in polarized epithelial cells.

Transport proteins are targeted to specific plasmalemmal domains in polarized epithelial cells. The molecular signals that govern these sorting events are just beginning to be elucidated. In many cases, the cell surface delivery of transport proteins is subjected to tight regulation. Several different mechanisms appear to participate in these trafficking processes.

Localization of the effector-specifying regions of Gi2alpha and Gqalpha.

Heterotrimeric G proteins transmit hormonal and sensory signals received by cell surface receptors to effector proteins that regulate cellular processes. Members of the highly conserved family of alpha subunits specifically modulate the activities of a diverse array of effector proteins. To investigate the determinants of alpha subunit-effector specificity, we localized the effector-specifying regions of alphai2, which inhibits adenylyl cyclase, and alphaq, which stimulates phosphoinositide phospholipase C using chimeric alpha subunits. The chimeras were generated using an in vivo recombination method in Escherichia coli. The effector-specifying regions of both alphai2 and alphaq were localized within the GTPase domain. An alphaq/alphai2/alphaq chimera containing only 78 alphai2 residues within the GTPase domain robustly inhibited adenylyl cyclase. This alphai2 segment includes regions corresponding to two of the three regions of alphas that activate adenylyl cyclase, but does not include any of the alpha subunit regions that switch conformation upon binding GTP. Replacement of the alphaq residues that comprise the helical domain with the homologous alphai2 residues resulted in a chimeric alpha subunit that activated phospholipase C. Combined with previous studies of the effector-specifying residues of alphas and alphat, our results suggest that the effector specificity of alpha subunits is generally determined by the GTPase and not the helical domain.

Polarized expression of GABA transporters in Madin-Darby canine kidney cells and cultured hippocampal neurons.

At least three high affinity Na+- and Cl--dependent gamma-aminobutyric acid (GABA) transporters are known to exist in the rat and mouse brain. These transporters share 50-65% amino acid sequence identity with the kidney betaine transporter which also transports GABA but with lower affinity. The betaine transporter (BGT) is expressed on the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. Recent evidence suggests that the signals and mechanisms involved in membrane protein sorting share many functional characteristics in polarized neurons and epithelial cells. It was previously shown that the rat GABA transporter GAT-1 is located in the presynaptic membrane of axons where it plays a role in terminating GABAergic neurotransmission. When expressed in MDCK cells by transfection, GAT-1 was sorted to the apical membrane. In this report, we have localized the other two GABA transporters, GAT-2 and GAT-3, in transfected MDCK cells by GABA uptake, immunofluorescence, and cell surface biotinylation. GAT-3, like GAT-1, localized to the apical membrane of MDCK cells while GAT-2, like BGT, localized to the basolateral membrane. We have also expressed BGT in low density cultures of hippocampal neurons by microinjection and immunolocalized it to the dendrites. The distribution of GAT-3 in these neurons after transfection was axonal as well as somatodendritic. These results indicate that highly homologous subtypes of GABA transporters are sorted differently when expressed in epithelial cells or neurons and suggest that these two cell types share the capacity to distinguish among these isoforms and target them to distinct destinations.

Interactions of VirB9, -10, and -11 with the membrane fraction of Agrobacterium tumefaciens: solubility studies provide evidence for tight associations.

The eleven predicted gene products of the Agrobacterium tumefaciens virB operon are believed to form a transmembrane pore complex through which T-DNA export occurs. The VirB10 protein is required for virulence and is a component of an aggregate associated with the membrane fraction of A. tumefaciens. Removal of the putative membrane-spanning domain (amino acids 22 through 55) disrupts the membrane topology of VirB10 (J. E. Ward, E. M. Dale, E. W. Nester, and A. N. Binns, J. Bacteriol. 172:5200-5210, 1990). Deletion of the sequences encoding amino acids 22 to 55 abolishes the ability of plasmid-borne virB10 to complement a null mutation in the virB10 gene, suggesting that the proper topology of VirB10 in the membrane may indeed play a crucial role in T-DNA transfer to the plant cell. Western blot (immunoblot) analysis indicated that the observed loss of virulence could not be attributed to a decrease in the steady-state levels of the mutant VirB10 protein. Although the deletion of the single transmembrane domain would be expected to perturb membrane association, VirB10 delta 22-55 was found exclusively in the membrane fraction. Urea extraction studies suggested that this membrane localization might be the result of a peripheral membrane association; however, the mutant protein was found in both inner and outer membrane fractions separated by sucrose density gradient centrifugation. Both wild-type VirB10 and wild-type VirB9 were only partially removed from the membranes by extraction with 1% Triton X-100, while VirB5 and VirB8 were Triton X-100 soluble. VirB11 was stripped from the membranes by 6 M urea but not by a more mild salt extraction. The fractionation patterns of VirB9, VirB10, and VirB11 were not dependent on each other or on VirB8 or VirD4. The observed tight association of VirB9, VirB10, and VirB11 with the membrane fraction support the notion that these proteins may exist as components of multiprotein pore complexes, perhaps spanning both the inner and outer membranes of Agrobacterium cells.

Effect of an acute beta-adrenergic blockade on the relationship between ventilatory and plasma lactate threshold.

The present study assesses interobserver variability and day-to-day reproducibility of ventilatory threshold T(ven) and lactate threshold T(lac) in healthy young men. The data were obtained before and after acute beta-adrenergic blockade with metoprolol (100 mg/d p.o.). Fifteen healthy young men underwent progressively increased treadmill ergometry tests on two separate days (day 1, 2). Ten out of fifteen subjects participated in a third exercise test under an acute beta-adrenergic blockade (day 3). Interobserver variability of oxygen uptake at T(ven) (8%; +/- 0.136 l/min) and T(lac) (5%; +/- 0.984 l/min) was small and day-to-day reproducibility of T(ven) (7%; +/- 0.131 l/min) and T(lac) (7%; +/- 0.133 l/min) high. Under acute beta-adrenergic blockade T(ven) occurred at a significantly lower work load (360 +/- 117 s) as compared with T(ven) of days 1 and 2 (477 +/- 153 s; p < 0.05), and T(lac) of day 3 after beta-adrenergic blockade (456 +/- 76 s; p < 0.05). Therefore, a significantly lower oxygen uptake (1.409 +/- 0.29 l/min) could be observed at T(ven) of day 3 as compared with T(ven) of days 1 and 2 (1.852 +/- 0.30; p < 0.001), and T(lac) of days 1 and 2 (1.724 +/- 0.22; p < 0.001). There was a significant linear correlation between oxygen uptake at T(ven) and T(lac) before beta-adrenergic blockade (r = 0.86; p < 0.001). This correlation, however, was lost following an acute beta-adrenergic blockade (r = 0.56; n.s.).(ABSTRACT TRUNCATED AT 250 WORDS)

Greater diagnostic sensitivity of treadmill versus cycle exercise testing of asymptomatic men with coronary artery disease.

Maximal hemodynamic and ventilatory responses using cycle and treadmill ergometer were compared in 52 asymptomatic patients with angiographically proved coronary artery disease. Moreover, test sensitivity with respect to ST-segment depression and typical angina pectoris were compared between exercise modes used. Exercise tests were performed on different days in randomized order. In 42 patients, exercise-induced myocardial ischemia, expressed as a fraction of left ventricular circumference, was assessed by thallium-201 scintigraphy. The main finding of this study was a significantly higher maximal oxygen uptake (1.87 +/- 0.4 vs 2.2 +/- 0.5 liters/min; p less than 0.001), heart rate (148 +/- 19 vs 158 +/- 18 beats/min; p less than 0.001) and rate-pressure product (28.3 +/- 5 x 10(3) vs 30.7 +/- 5 x 10(3); p less than 0.001) during treadmill walking than during cycling. Therefore, stress-induced myocardial ischemia was significantly more extensive after treadmill walking (31 +/- 37 degrees vs 45 +/- 40 degrees; p less than 0.001). Moreover, there were significantly more patients with signs of myocardial ischemia (ST-segment depression or typical angina pectoris, or both) during treadmill than during cycle ergometry (35 vs 25 patients; p less than 0.05). However, lactate levels measured at peak exercise (4.07 +/- 2.0 vs 4.38 +/- 1.9 mmol/liter) and 3 minutes into the recovery period (5.60 +/- 2.2 vs 5.80 +/- 2.2 mmol/liter) were comparable between both methods, indicating no significant difference in anaerobic energy production. These findings suggest that walking on a treadmill represents an exercise method with a greater ability than cycling to detect coronary artery disease.