Restraint stress affects circulating NUCB2/nesfatin-1 and phoenixin levels in male rats.

The two peptides phoenixin and nesfatin-1 are colocalized in hypothalamic nuclei involved in the mediation of food intake and behavior. Phoenixin stimulates food intake and is anxiolytic, while nesfatin-1 is an anorexigenic peptide shown to increase anxiety and anhedonia. Interestingly, central activation of both peptides can be stimulated by restraint stress giving rise to a role in the mediation of stress. Thus, the aim of the study was to test whether also peripheral circulating levels of NUCB2/nesfatin-1 and phoenixin are altered by restraint stress. Male ad libitum fed Sprague Dawley rats equipped with a chronic intravenous catheter were subjected to restraint stress and plasma levels of NUCB2/nesfatin-1, phoenixin and cortisol were measured over a period of 240 min and compared to levels of freely moving rats. Peripheral cortisol levels were significantly increased in restrained rats at 30, 60, 120 and 240 min compared to controls (p < 0.05). In contrast, restraint stress decreased plasma phoenixin levels at 15 min compared to unstressed conditions (0.8-fold, p < 0.05). Circulating NUCB2/nesfatin-1 levels were increased only at 240 min in restrained rats compared to those in unstressed controls (1.3-fold, p < 0.05). In addition, circulating NUCB2/nesfatin-1 levels correlated positively with phoenixin levels (r = 0.378, p < 0.001), while neither phoenixin nor nesfatin-1 were associated with cortisol levels (r = 0.0275, and r=-0.143, p> 0.05). These data suggest that both peptides, NUCB2/nesfatin-1 and phoenixin, are affected by restraint stress, although less pronounced than circulating cortisol.

Central blockage of nesfatin-1 has anxiolytic effects but does not prevent corticotropin-releasing factor-induced anxiety in male rats.

Nesfatin-1, a pleotropic peptide, was recently implicated in the regulation of anxiety and depression-like behavior in rats. However, the underlying mechanisms remain unclear so far. Thus, this study aimed to investigate the role of endogenous nesfatin-1 in the mediation of anxiety and depression-like behavior induced by corticotropin-releasing factor (CRF). Therefore, normal weight male intracerebroventricularly (icv) cannulated Sprague Dawley rats received two consecutive icv injections of anti-nesfatin-1 antibody or IgG control antibody followed by CRF or saline, before being exposed to a behavioral test. In the elevated zero maze test, assessing anxiety and explorative behavior, blockade of nesfatin-1 using an anti-nesfatin-1 antibody under basal conditions increased the number of entries into the open arms compared to control antibody/vehicle (1.6-fold, p < 0.05) and the time in open arms compared to the other groups (p < 0.05). Control antibody/CRF-treated animals tended to spend less time in the open arms compared to control antibody/vehicle (0.7-fold, p = 0.17), an effect not altered by the nesfatin-1 antibody (control antibody/CRF-treated animals vs. nesfatin-1 antibody/CRF group, p = 1.00). In the novelty-induced hypophagia test, assessing anhedonia as part of depression-like behavior, no significant differences were observed between the four groups for the latency to the first bout, number of bouts and the amount of palatable snack eaten (p > 0.05). In summary, CRF tended to increase anxiety and explorative behavior an effect not altered by blockade of nesfatin-1, whereas no significant effect of CRF on anhedonia was observed. Blockade of endogenous nesfatin-1 significantly decreased anxiety-like behavior giving rise to a physiological role of brain nesfatin-1 in the mediation of anxiety.

Intracerebroventricular injection of phoenixin alters feeding behavior and activates nesfatin-1 immunoreactive neurons in rats.

Phoenixin is a novel neuropeptide initially associated with reproductive functions, but subsequently also with feeding behavior. Nesfatin-1 is also involved in the regulation of food intake and has been shown to largely colocalize with phoenixin in the rat brain; however, a functional link is missing so far. The current study investigated whether phoenixin activates nesfatin-1 immunoreactive nuclei in the rat brain. Male Sprague Dawley rats chronically equipped with an intracerebroventricular cannula were injected with vehicle (5 µl ddH2O) or phoenixin (1.7 nmol in 5 µl ddH2O, n = 5-6 group). Behavior was assessed manually and c-Fos as well as nesfatin-1 immunoreactivity using immunohistochemistry. Phoenixin significantly increased feeding and drinking behavior as well as locomotor activity compared to vehicle (p < 0.01). Moreover, phoenixin injected intracerebroventricularly (icv) activated several nuclei throughout the rat brain as assessed using c-Fos; the number of c-Fos/nesfatin-1 immunoreactive neurons was increased in the lateral septal nucleus (4-fold), supraoptic nucleus (107-fold), paraventricular nucleus (6-fold) and the nucleus of the solitary tract (18-fold) compared to vehicle (p < 0.05). In summary, phoenixin activates several nesfatin-1 immunoreactive nuclei in the rat brain. This activation may play a role in the modulation of food intake.

Alteration of peptidergic gut-brain signaling under conditions of obesity.

The prevalence of obesity is rising worldwide; therefore, the World Health Organization introduced the term 'globesity'. This rise also causes an increase of associated diseases such as cardiovascular diseases, type 2 diabetes, several malignomas as well as psychiatric disorders. In order to face this medical challenge, a better understanding of the pathophysiological alterations under conditions of obesity is necessary. Hunger and satiety are largely regulated by peptidergic hormones predominantly produced in the gastrointestinal tract and signaling to the brain via the gut-brain axis. While several hormones are known to decrease food intake such as nesfatin-1, cholecystokinin (CKK), glucagon-like peptide 1 (GLP-1), pancreatic polypeptide (PP) and peptide YY (PYY), only one peripherally produced and centrally acting hormone - ghrelin - is known so far that stimulates food intake. Several alterations of the signaling of these hormones have been described in the past years e.g. an attenuated postprandial response of CCK, GLP-1 and PYY as well as a reduced postprandial suppression of ghrelin that might contribute to the development and/or maintenance of obesity and will be discussed in the present review. Lastly, gaps in knowledge will be highlighted.

A new photobioreactor concept enabling the production of desiccation induced biotechnological products using terrestrial cyanobacteria.

Cyanobacteria offer great potential for the production of biotechnological products for pharmaceutical applications. However, these organisms can only be cultivated efficiently using photobioreactors (PBR). Under submerged conditions though, terrestrial cyanobacteria mostly grow in a suboptimal way, which makes this cultivation-technique uneconomic and thus terrestrial cyanobacteria unattractive. Therefore, a novel emersed photobioreactor (ePBR) has been developed, which can provide the natural conditions for these organisms. Proof of concept as well as first efficiency tests are conducted using the terrestrial cyanobacteria Trichocoleus sociatus as a model organism. The initial maximum growth rate of T. sociatus (0.014±0.001h(-1)) in submerged systems could be increased by 35%. Furthermore, it is now possible to control desiccation-correlated product formation and related metabolic processes. This is shown for the production of extracellular polymeric substances (EPS). In this case the yield of 0.068±0.006g of EPS/g DW could be increased by more than seven times.

Application of biofilm bioreactors in white biotechnology.

The production of valuable compounds in industrial biotechnology is commonly done by cultivation of suspended cells or use of (immobilized) enzymes rather than using microorganisms in an immobilized state. Within the field of wastewater as well as odor treatment the application of immobilized cells is a proven technique. The cells are entrapped in a matrix of extracellular polymeric compounds produced by themselves. The surface-associated agglomerate of encapsulated cells is termed biofilm. In comparison to common immobilization techniques, toxic effects of compounds used for cell entrapment may be neglected. Although the economic impact of biofilm processes used for the production of valuable compounds is negligible, many prospective approaches were examined in the laboratory and on a pilot scale. This review gives an overview of biofilm reactors applied to the production of valuable compounds. Moreover, the characteristics of the utilized materials are discussed with respect to support of surface-attached microbial growth.

A structural chemogenomics analysis of aminergic GPCRs: lessons for histamine receptor ligand design.

BACKGROUND AND PURPOSE: Chemogenomics focuses on the discovery of new connections between chemical and biological space leading to the discovery of new protein targets and biologically active molecules. G-protein coupled receptors (GPCRs) are a particularly interesting protein family for chemogenomics studies because there is an overwhelming amount of ligand binding affinity data available. The increasing number of aminergic GPCR crystal structures now for the first time allows the integration of chemogenomics studies with high-resolution structural analyses of GPCR-ligand complexes. EXPERIMENTAL APPROACH: In this study, we have combined ligand affinity data, receptor mutagenesis studies, and amino acid sequence analyses to high-resolution structural analyses of (hist)aminergic GPCR-ligand interactions. This integrated structural chemogenomics analysis is used to more accurately describe the molecular and structural determinants of ligand affinity and selectivity in different key binding regions of the crystallized aminergic GPCRs, and histamine receptors in particular. KEY RESULTS: Our investigations highlight interesting correlations and differences between ligand similarity and ligand binding site similarity of different aminergic receptors. Apparent discrepancies can be explained by combining detailed analysis of crystallized or predicted protein-ligand binding modes, receptor mutation studies, and ligand structure-selectivity relationships that identify local differences in essential pharmacophore features in the ligand binding sites of different receptors. CONCLUSIONS AND IMPLICATIONS: We have performed structural chemogenomics studies that identify links between (hist)aminergic receptor ligands and their binding sites and binding modes. This knowledge can be used to identify structure-selectivity relationships that increase our understanding of ligand binding to (hist)aminergic receptors and hence can be used in future GPCR ligand discovery and design.

Quality of life and employment status are correlated with antiepileptic monotherapy versus polytherapy and not with use of "newer" versus "classic" drugs: results of the "Compliant 2006" survey in 907 patients.

"Classic" and "newer" antiepileptic drugs (AEDs) were compared in an epidemiological survey regarding patient's acceptance of AEDs, quality of life (QoL), and employment. Data from 907 outpatients, 45.9% female (mean age: 44.8 ± 17.9 years), were evaluated by 90 neurologists in private practices, who were also involved in a non-interventional study by Sanofi-Aventis Deutschland GmbH, regarding medication, seizure type, illness duration, employment, patients' acceptance of AEDs (4-point scale where 1=very good), and QoL (6-point scale where 1=very good). Among the patients, 69.7% were on monotherapy, 25.4% were taking two AEDs, and 4.9% were taking more than two AEDs. Patient's acceptance of AEDs (mean ± SD=1.65 ± 0.62) and QoL (2.34 ± 0.89) were "good." Among patients aged 18-65 years, 68.6% were employed. QoL and acceptance were lower with polytherapy. Older age and polytherapy were associated with lower probability of employment. No differences emerged between "classic" and "newer" AED monotherapy. Polytherapy-associated lower QoL could be due to severity of illness or adverse effects of treatment.

Detection of bovine viral diarrhoea virus infected cattle--testing tissue samples derived from ear tagging using an Erns capture ELISA.

A new diagnostic approach testing tissue samples derived from cattle ear tagging for bovine viral diarrhoea virus (BVDV) antigen in a commercially available antigen capture enzyme-linked immunosorbent assay (ACE) was developed. To validate this method, 99 positive and 469 negative samples were tested. With those samples the assay yielded a sensitivity of 100% and specificity of >or=99.6%. Serum and ear tissue samples from 11 persistently infected (PI) BVDV calves were tested. While serum samples were negative after intake of colostrum, the ear tissue samples could be detected positive for BVDV all the time. Testing multiple samples derived from the same ear from PI cattle yielded positive results and low variation. Using cattle ear tags combining the ear tag application with sampling of a small ear tissue plug and testing those tissue samples with an ACE could be a reliable and economic way of BVDV testing.

Mechanism of terfenadine block of ATP-sensitive K(+) channels.

The ATP-sensitive K(+) (K(ATP)) channel is a complex of a pore-forming inwardly rectifying K(+) channel (Kir6.2) and a sulphonylurea receptor (SUR). The aim of the present study was to gain further insight into the mechanism of block of K(ATP) channels by terfenadine. Channel activity was recorded both from native K(ATP) channels from the clonal insulinoma cell line RINm5F and from a C-terminal truncated form of Kir6.2 (Kir6.2Delta26), which - in contrast to Kir6.2 - expresses independently of SUR. Kir6.2Delta26 channels were expressed in COS-7 cells, and enhanced green fluorescent protein (EGFP) cDNA was used as a reporter gene. EGFP fluorescence was visualized by a laser scanning confocal microscope. Terfenadine applied to the cytoplasmic side of inside-out membrane patches concentration-dependently blocked both native K(ATP) channel and Kir6.2Delta26 channel activity, and the following values were calculated for IC(50) (the terfenadine concentration causing half-maximal inhibition) and n (the Hill coefficient): 1.2 microM and 0.7 for native K(ATP) channels, 3.0 microM and 1.0 for Kir6. 2Delta26 channels. Terfenadine had no effect on slope conductance of either native K(ATP) channels or Kir6.2Delta26 channels. Intraburst kinetics of Kir6.2Delta26 channels were not markedly affected by terfenadine and, therefore, terfenadine acts as a slow channel blocker on Kir6.2Delta26 channels. Terfenadine-induced block of Kir6. 2Delta26 channels demonstrated no marked voltage dependence, and lowering the intracellular pH to 6.5 potentiated the inhibition of Kir6.2Delta26 channels by terfenadine. These observations indicate that terfenadine blocks pancreatic B-cell K(ATP) channels via binding to the cytoplasmic side of the pore-forming subunit. The presence of the pancreatic SUR1 has a small, but significant enhancing effect on the potency of terfenadine.

Growth performance, metabolic and endocrine traits, and absorptive capacity in neonatal calves fed either colostrum or milk replacer at two levels.

Colostrum (CO) contains high amounts, whereas whole milk and milk replacer (MR) contain small amounts, of bioactive and growth-promoting substances, such as IGF-I. An experiment was designed to study the effects of feeding CO or MR on the first 3 d to neonatal calves, followed by whole milk up to d 7, at low and high density. Intestinal absorptive capacity, plasma metabolite and hormone concentrations, and growth performance were measured during the 1st wk of life. Body weight increased (P < .05) similarly in calves fed low or high amounts of CO but did not rise in MR-fed calves. Loose feces were more frequent (P < .05) and absorption of xylose on d 5 was lower (P < .01) in MR- than in CO-fed calves, but there were no effects of feeding density within CO-fed or within MR-fed groups. However, high feeding density within CO-fed groups enhanced (P < .05) total protein, globulin, triglyceride, cholesterol, and insulin concentrations, whereas in the initially high and low MR-fed groups only plasma glucose and insulin after the first meal and plasma NEFA on d 2 were modified (P < .05) by different feeding density. Thus, feeding different amounts of CO partly influenced protein and fat metabolism in calves during the 1st wk of life, but it did not measurably affect intestinal function. However, feeding different amounts of MR, in the absence of CO, barely affected metabolic and endocrine traits and absorptive capacity. Thus, high density CO feeding, and therefore a high supply of nutrients, together with greater amounts of bioactive and growth-promoting substances influenced neonatal metabolism and growth more than a high density of MR feeding containing only small amounts of bioactive and growth-promoting substances. Factors in addition to nutrient density seem to be important for the development of neonatal calves.

Disopyramide block of K(ATP) channels is mediated by the pore-forming subunit.

The class Ia antiarrhythmic agent disopyramide blocks native ATP-sensitive K+ (K(ATP)) channels at micromolar concentrations. The K(ATP) channel is a complex of a pore-forming inwardly rectifying K+ channel (Kir6.2) and a sulfonylurea receptor (SUR). The aim of the present study was to further localize the site of action of disopyramide. We have used a C-terminal truncated form of Kir6.2 (Kir6.2delta26), which--in contrast to Kir6.2--expresses independently of SUR. Kir6.2delta26 channels were expressed in African green monkey kidney COS-7 cells, and enhanced green fluorescent protein (EGFP) cDNA was used as a reporter gene. EGFP fluorescence was visualized by a laser scanning confocal microscope. Disopyramide applied to the cytoplasmic membrane surface of inside-out patches inhibited Kir6.2delta26 channels half-maximally at 7.1 microM (at pH 7.15). Lowering the intracellular pH to 6.5 potentiated the inhibition of Kir6.2delta26 channels by disopyramide. These observations suggest that disopyramide directly blocks the pore-forming Kir6.2 subunit, in particular at reduced intracellular pH values that occur under cardiac ischaemia.

Effects of P2 purinoceptor agonists on membrane potential and intracellular Ca2+ of human cardiac endothelial cells.

Vasoactive agonists like adenosine-5'-triphosphate (ATP) increase intracellular Ca2+ ([Ca2+]i) in vascular endothelial cells with an initial peak due to inositol 1,4,5-triphosphate-mediated Ca2+ release from intracellular stores followed by a sustained plateau that is dependent on the presence of extracellular Ca2+, thus leading to an increased synthesis and release of prostacyclin and nitric oxide. We studied the effects of nucleotides on membrane potential and [Ca2+]i in confluent human microvascular cardiac endothelial cells obtained from patients with dilated cardiomyopathy. The whole-cell configuration of the patch-clamp technique and a confocal laser scanning microscope employing fluo-3 as a Ca2+ indicator were used. Both uridine-5'-triphosphate (UTP) and 2-methylthioadenosine-5'-triphosphate (2MeSATP) induced depolarizations in human microvascular cardiac endothelial cells and increased [Ca2+]i with a rank order of potency 2MeSATP>ATP=UTP (EC50 values (in microM) were 0.084 2MeSATP, 0.67 ATP and 1.1 UTP). This suggests that both P2u and P2y purinoceptors are present on human microvascular cardiac endothelial cells. Maximal [Ca2+]i responses of confluent human microvascular cardiac endothelial cell monolayers to UTP were lower when compared to 2MeSATP. Nucleotide-induced increases in [Ca2+]i consisted of a transient peak, which was also observed in the absence of extracellular Ca2+, and a sustained [Ca2+]i plateau. This plateau, which was not observed in all monolayers studied, was not markedly influenced by increasing extracellular [K+]. Previous incubation with thapsigargin abolished ATP-induced increases of [Ca2+]i. It is concluded that human microvascular cardiac endothelial cells express both P2y and P2u purinoceptors. P2 purinoceptor agonists release Ca2+ from intracellular thapsigargin-sensitive stores and stimulate capacitative Ca2+ influx pathways. K+ efflux through Ca2+-dependent K+ (K(Ca)) channels does not play a major role in the regulation of nucleotide-induced Ca2+ influx in human microvascular cardiac endothelial cells, which might be related to an impaired function of the cells.

Persistent high serum levels of cartilage oligomeric matrix protein in a subgroup of patients with traumatic knee injury.

The objective was to assess whether changes of cartilage oligomeric matrix protein (COMP) serum levels can predict the development of osteoarthritis following traumatic knee injury. Sera and synovial fluids were acquired at surgery (T0) and postoperatively during the first (T1) and second (T2) year from 30 knee-injured patients. COMP levels and anti-COMP autoantibodies were quantified by ELISA. Radiographs and patient questionnaires were used to assess outcomes. At T0, compared with controls (1.6 +/- 1.6 micrograms/ml), the serum COMP concentration was significantly elevated (6.5 +/- 2.8 micrograms/ml) with a tendency to further increase (T0 vs. T1, P = 0.076) and subsequently decrease (T1 vs. T2, P = 0.074). However, individual variations are observed, e.g. persistently high (8/30) or increasing (T0 to T2, 8/30) serum COMP. Ten of these patients have elevated COMP at T2 that increased from T0. COMP levels in serum and synovial fluid correlated significantly (P = 0.012). Interestingly, some patients who revealed increasing serum levels of COMP from T0 to T2 displayed anti-COMP autoantibodies. These data suggest that local immune response could contribute to further joint damage. The subgroup of 10 patients (33%) with elevated and increasing serum COMP levels and in particular the patients with antibodies against cartilage matrix molecules appear at increased risk for developing posttraumatic osteoarthritis.

[The hematocrit values and hemoglobin content of blood and the level of total protein, free fatty acids, glucose, lactate, Ca, Mg, Na, K, Pa, Fe, Fe-binding capacity, Cu and Zn in the blood plasma of newborn calves and their mothers immediately after birth]. / Untersuchungen über den Hämatokritwert und den Hämoglobingehalt des Blutes sowie den Gehalt an Gesamtprotein, freien Fettsäuren, Glucose, Lactat, Ca, Mg, Na, K, Pa, Fe, Fe-Bindungskapazität, Cu und Zn im Blutplasma von neugeborenen Kälbern sowei bei den Muttertieren unmittelbar nach der Geburt.

Blood samples were collected from 22 calves in three weight classes (A: 29.3 +/- 0.5 kg, B: 36.0 +/- 1.3 kg, C: 42.7 +/- 3.7 kg), 1-3 minutes after parturition and prior to uptake of foremilk as well as 24-26 hours after parturition. Other blood samples were collected from 45 calves, 5-60 minutes after parturition, and from their mothers, 3-5 or 5-60 minutes from calving. Haematocrit and haemoglobin in the blood of the calves, immediately after parturition, were higher the values recorded from adult cattle. Major differences were found to exist between individual calves regarding total protein of blood plasma. Protein levels in 14 calves were below 50 g/l, within 24-26 hours from parturition. Free fatty acids in blood plasma of calves were lower than those in cattle, 1-3 minutes from parturition, and were higher, 5-60 minutes from parturition. Glucose levels in Group C were higher than those in A and B, 1-3 minutes postpartum. Lactate in Group C was higher than that in B. An account is also given of blood plasma levels of Ca, Pa, Mg, Na, K, Cu, and Zn.