Nocistatin and nociceptin given centrally induce opioid-mediated gastric mucosal protection

Nociceptin (N/OFQ) and nocistatin (NST) are two endogenous neuropeptides derived from the same precursor protein, preproN/OFQ. The aim of the present work was to study the effect of NST on the ethanol-induced mucosal damage compared with that of N/OFQ following intracerebroventricular (i.c.v.) administration in the rat and to analyze the mechanism of the gastroprotective action. It was found that both NST and N/OFQ reduced the mucosal lesions in the same dose range (0.2–1 nmol i.c.v.), but in higher doses (2–5 nmol i.c.v.) the gastroprotective effect of both peptides was highly diminished. The gastroprotective effect of N/OFQ (1 nmol), but not that of NST (1 nmol), was reduced by the selective nociceptin receptor antagonist J-113397 (69 nmol i.c.v.). Similarly, decrease of the gastroprotective effect was observed after the combination of NST (1 nmol) with N/OFQ (0.6 or 1 nmol). However, addition of the gastroprotective effects was observed, when lower dose (0.2 nmol) of NST was given prior to N/OFQ (0.6 nmol). The gastroprotective effect of both N/OFQ and NST was antagonized by naloxone (27 nmol), β-funaltrexamine (20 nmol), naltrindole (5 nmol) and norbinaltorphimine (14 nmol), the μ-, δ- and κ-opioid receptor antagonists, respectively, given i.c.v. The mucosal protection was significantly decreased after bilateral cervical vagotomy. The present findings suggest that NST similar to N/OFQ, may also induce gastric mucosal protective action initiated centrally in a vagal-dependent mechanism. Opioid component is likely to be involved in the gastroprotective effect of both NST and N/OFQ.     

Expression and Purification of Biologically Active Rat Bone Morphogenetic Protein-4 produced as Inclusion Bodies in Recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Rat bone morphogenetic protein-4 (rBMP-4) cDNA was cloned from rat osteoblasts by RT-PCR and expressed in E. coli. Monomeric, dimeric and polymeric forms of recombinant rat BMP-4 (rrBMP-4) were obtained from inclusion bodies after solubilization with urea. The dimer was separated from the remaining polymer and host cell contaminants using size exclusion chromatography. Furthermore, purified rrBMP-4 was stabilized at low urea concentration (40 mm) and at pH 8.5 through the addition of bovine serum albumin. Both, rrBMP-4 dimer and polymer were biologically active as tested by the induction of alkaline phosphatase activity in MC3T3-E1 cells.     

Organic fertilization changes the response of mycelium of arbuscular mycorrhizal fungi and their sporulation to mineral NPK supply

The synergetic effect of organic (cow manure) and mineral fertilization on the development arbuscular mycorrhizal (AM) fungi was demonstateded. The length of AM mycelium and sporulation were used as sensitive markers of the physiological state of soil AM fungal population. In manured treatments, both parameters increased in proportion with increasing mineral fertilization. In unmanured soil, the opposite trend was observed for the length of AM hyphae, which decreased with increasing mineral fertilization. Correlation analysis showed the dependence of length of AM hyphae and sporulation on soil available phosphorus. The correlation was negative in soil with no mineral fertilization and positive in soil supplied with luxury doses of mineral fertilizer.     

Sra-1 interacts with Kette and Wasp and is required for neuronal and bristle development in Drosophila

Regulation of growth cone and cell motility involves the coordinated control of F-actin dynamics. An important regulator of F-actin formation is the Arp2/3 complex, which in turn is activated by Wasp and Wave. A complex comprising Kette/Nap1, Sra-1/Pir121/CYFIP, Abi and HSPC300 modulates the activity of Wave and Wasp. We present the characterization of Drosophila Sra-1 (specifically Rac1-associated protein 1). sra-1 and kette are spatially and temporally co-expressed, and both encoded proteins interact in vivo. During late embryonic and larval development, the Sra-1 protein is found in the neuropile. Outgrowing photoreceptor neurons express high levels of Sra-1 also in growth cones. Expression of double stranded sra-1 RNA in photoreceptor neurons leads to a stalling of axonal growth. Following knockdown of sra-1 function in motoneurons, we noted abnormal neuromuscular junctions similar to what we determined for hypomorphic kette mutations. Similar mutant phenotypes were induced after expression of membrane-bound Sra-1 that lacks the Kette-binding domain, suggesting that sra-1 function is mediated through kette. Furthermore, we could show that both proteins stabilize each other and directly control the regulation of the F-actin cytoskeleton in a Wasp-dependent manner.     

The Drosophila ARF6-GEF Schizo controls commissure formation by regulating Slit

The CNS of bilateral symmetric organisms is characterized by intensive contralateral axonal connections. Genetic screens in Drosophila have identified only a few genes required for guiding commissural growth cones toward and across the midline. Two evolutionarily conserved signaling molecules, Netrin and Slit, are expressed in the CNS midline cells. Netrin acts primarily as an attractive signaling cue, whereas Slit mediates repulsive functions. Here, we describe a detailed analysis of the Drosophila gene schizo, which is required for commissure formation. schizo leads to a commissural phenotype reminiscent of netrin mutant embryos. Double-mutant analyses indicate that Netrin and Schizo act independently. The schizo mutant phenotype can be suppressed by either expressing netrin in the CNS midline cells or by a reduction of the slit gene dose, indicating that the balance of attractive and repulsive signaling is impaired in schizo mutants. Overexpression of the schizo RNA in the CNS midline using the GAL4/UAS system leads to a slit phenocopy, suggesting that schizo primarily antagonizes Slit signaling. This is further supported by cell type-specific rescue experiments. The schizo gene generates at least two proteins containing a conserved Sec7 and a pleckstrin homology domain (PH) characteristic for guanine nucleotide exchange factors (GEF) acting on ARF GTPases, which are known to regulate endocytosis. In support of the notion that schizo regulates Slit expression via endocytosis, we found that block of endocytosis leads to a schizo-like phenotype. We thus propose that the balance of the two signaling cues Netrin and Slit can be regulated, controlling membrane dynamics.     

Therapy-induced antibodies against the antiviral and antiproliferative effects of interferons in patients with chronic hepatitis C virus infection

Sera from 86 patients with chronic hepatitis C virus (HCV) infection treated with recombinant interferons-alpha (rIFN-alpha) were screened for IFN-binding and antiviral effect-neutralizing antibodies. Out of the 61 patients treated with rIFN-alpha2b, 46% had binding and 28% had neutralizing antibodies. 44% of the 25 patients treated with rIFN-alpha2a developed binding antibodies and 24% had neutralizing antibodies. Contradictory data were observed concerning the appearance of anti-IFN antibodies and the outcome of IFN therapy. A significantly higher number of the patients with a sustained response to rIFN-alpha2b therapy formed antibodies than the number among the non-responder patients. At the same time, in the patients treated with rIFN-alpha2a, opposite data were found. The activity of the antibodies in some sera was studied against the antiproliferative effect of IFNs on Daudi cells by measuring the [3H]thymidine incorporation. The binding antibodies without neutralization of the antiviral effect of the IFNs inhibited the antiproliferative activity of the rIFNs, similarly to antibodies having both IFN-binding and antiviral effect-neutralizing capacities. At the same time, the antiproliferative effect of the natural IFN was less affected. It is suggested that the antiproliferative assay is more sensitive than the antiviral method for demonstration of the presence of antibodies exerting an inhibitory effect on the biological activities of IFN.     

The space-use strategy of plants with different growth forms,in a field experiment with manipulated nutrients and light

The biomass allocation pattern in plants is known to depend on the below and above-ground resource availabilities. In a herbaceous multi-species stand, it can be expected that the effects of nutrient and light availability on plants’ general space-use strategy are fundamentally different. We hypothesized that nutrient status alters the amount of biomass produced per unit canopy volume (biomass density), but not so much the biomass vertical distribution pattern. Changes in light availability, in contrast, should affect the vertical distribution pattern of biomass but not biomass density. We were also interested in whether the effect of resource manipulation on a plant’s space-use strategy depends on its basic morphological characteristics (growth form). The results from a four-year permanent plot experiment in a species-rich grassland, with fertilization and additional illumination from mirrors applied to 40 × 40 cm plots, showed that our main hypothesis was correct. Fertilization significantly affected biomass density above as well as below-ground, while additional illumination generally did not. Light addition altered the vertical distribution pattern of above-ground biomass, which remained unaffected by the fertilizer treatment.     

Rapid determination of tamsulosin in human plasma by high-performance liquid chromatography using extraction with butyl acetate

A high-performance liquid chromatographic method with fluorescence detection for the determination of tamsulosin in human plasma is reported. The sample preparation involved liquid-liquid extraction of tamsulosin from alkalised plasma with butyl acetate and back-extraction of the drug to the phosphate buffer (pH 2). Butyl acetate is preferable to more commonly used ethyl acetate because of its much lower solubility in water. Liquid chromatography was performed on an octadecylsilica column (55 mm x 4 mm, 3 microm particles), the mobile phase consisted of acetonitrile-30 mM dihydrogenpotassium phosphate (25:75 v/v). The run time was 3.5 min. The fluorimetric detector was operated at 228/326 nm (excitation/emission wavelength). An analogue of tamsulosin, (R)-5-[2-[(3-(2-ethoxyphenoxy)propyl)amino]-2-methylethyl]-2-methoxybenzensulfonamide was used as the internal standard. The limit of quantitation was 0.4 ng/ml using 1 ml of plasma. Within-day and between-day precision expressed by relative standard deviation was less than 10% and inaccuracy did not exceed 5%. The assay was applied to the analysis of samples from several pharmacokinetic studies.