The signalling profile of recombinant human orexin-2 receptor

Orexin-A and orexin-B orchestrate their diverse central and peripheral effects via two G-protein coupled receptors, OX1R and OX2R, which activate multiple G-proteins. In many tissues, orexins activate extracellular signal-regulated kinase (ERK(1/2)) and p38 mitogen-activated protein kinase (MAPK); however, the mechanism by which OX2R alone mediates MAPK activation is not understood. This study describes the intracellular signalling pathways involved in OX2R-mediated ERK(1/2) and p38 MAPK activation. In HEK-293 cells stably over-expressing recombinant human OX2R, orexin-A/B resulted in a rapid, dose and time dependent increase in activation of ERK(1/2) and p38 MAPK, with maximal activation at 10 min for ERK(1/2) and 30 min for p38 MAPK. Using dominant-negative G-proteins and selective inhibitors of intracellular signalling cascades, we determined that orexin-A and orexin-B induced ERK(1/2) and p38 MAPK activation through multiple G-proteins and different intracellular signalling pathways. ERK(1/2) activation involves Gq/phospholipase C (PLC)/protein kinase C (PKC), Gs/adenylyl cyclase (AC)/cAMP/protein kinase A (PKA) and Gi cascades; however, the Gq/PLC/PKC pathway, as well as PKA is not required for OX2R-mediated p38 MAPK activation. Interestingly, orexin-B-induced ERK(1/2) activation is predominantly mediated through the Gq/PLC/PKC pathway. In conclusion, this is the first comprehensive signalling study of the human OX2R recombinant receptor, showing ERK(1/2) and p38 MAPK activation are regulated by differential signalling pathways in HEK-293 cells, and that the ERK(1/2) activation is severely affected by naturally occurring mutants associated with narcolepsy. Moreover, it is evident that the human OX2R has ligand specific effects, with orexin-B being more potent in this transfected system and this distinct modulation of the MAPKs through OX2R, may translate to the regulation of diverse biological actions of orexins.     

Comparative transcriptome analysis of Methylibium petroleiphilum PM1 exposed to the fuel oxygenates methyl tert-butyl ether and ethanol

High-density whole-genome cDNA microarrays were used to investigate substrate-dependent gene expression of Methylibium petroleiphilum PM1, one of the best-characterized aerobic methyl tert-butyl ether (MTBE)-degrading bacteria. Differential gene expression profiling was conducted with PM1 grown on MTBE and ethanol as sole carbon sources. Based on microarray high scores and protein similarity analysis, an MTBE regulon located on the megaplasmid was identified for further investigation. Putative functions for enzymes encoded in this regulon are described with relevance to the predicted MTBE degradation pathway. A new unique dioxygenase enzyme system that carries out the hydroxylation of tert-butyl alcohol to 2-methyl-2-hydroxy-1-propanol in M. petroleiphilum PM1 was discovered. Hypotheses regarding the acquisition and evolution of MTBE genes as well as the involvement of IS elements in these complex processes were formulated. The pathways for toluene, phenol, and alkane oxidation via toluene monooxygenase, phenol hydroxylase, and propane monooxygenase, respectively, were upregulated in MTBE-grown cells compared to ethanol-grown cells. Four out of nine putative cyclohexanone monooxygenases were also upregulated in MTBE-grown cells. The expression data allowed prediction of several hitherto-unknown enzymes of the upper MTBE degradation pathway in M. petroleiphilum PM1 and aided our understanding of the regulation of metabolic processes that may occur in response to pollutant mixtures and perturbations in the environment.     

Root growth dynamics linked to above-ground growth in walnut (Juglans regia)

Background and Aims Examination of plant growth below ground is relatively scant compared with that above ground, and is needed to understand whole-plant responses to the environment. This study examines whether the seasonal timing of fine root growth and the spatial distribution of this growth through the soil profile varies in response to canopy manipulation and soil temperature.Methods Plasticity in the seasonal timing and vertical distribution of root production in response to canopy and soil water manipulation was analysed in field-grown walnut (Juglans regia ‘Chandler’) using minirhizotron techniques.Key Results Root production in walnuts followed a unimodal curve, with one marked flush of root growth starting in mid-May, with a peak in mid-June. Root production declined later in the season, corresponding to increased soil temperature, as well as to the period of major carbohydrate allocation to reproduction. Canopy and soil moisture manipulation did not influence the timing of root production, but did influence the vertical distribution of roots through the soil profile. Water deficit appeared to promote root production in deeper soil layers for mining soil water. Canopy removal appeared to promote shallow root production.Conclusions The findings of this study add to growing evidence that root growth in many ecosystems follows a unimodal curve with one marked flush of root growth in coordination with the initial leaf flush of the season. Root vertical distribution appeared to have greater plasticity than timing of root production in this system, with temperature and/or carbohydrate competition constraining the timing of root growth. Effects on root distribution can have serious impacts on trees, with shallow rooting having negative impacts in years with limited soil water or positive impacts in years with wet springs, and deep rooting having positive impacts on soil water mining from deeper soil layers but negative impacts in years with wet springs.     

Prevalence of diarrhoeal pathogens among children under five years of age with and without diarrhoea in Guinea-Bissau

BackgroundChildhood diarrhoea, a major cause of morbidity and mortality in low-income regions, remains scarcely studied in many countries, such as Guinea-Bissau. Stool sample drying enables later qPCR analyses of pathogens without concern about electricity shortages.MethodsDried stool samples of children under five years treated at the Bandim Health Centre in Bissau, Guinea-Bissau were screened by qPCR for nine enteric bacteria, five viruses, and four parasites. The findings of children having and not having diarrhoea were compared in age groups 0–11 and 12–59 months.ResultsOf the 429 children– 228 with and 201 without diarrhoea– 96.9% and 93.5% had bacterial, 62.7% and 44.3% viral, and 52.6% and 48.3% parasitic pathogen findings, respectively. Enteroaggregarive Escherichia coli (EAEC; 60.5% versus 66.7%), enteropathogenic E. coli (EPEC; 61.4% versus 62.7%), Campylobacter (53.2% versus 51.8%), and enterotoxigenic E. coli (ETEC; 54.4% versus 44.3%) were the most common bacterial pathogens. Diarrhoea was associated with enteroinvasive E. coli (EIEC)/Shigella (63.3%), ETEC (54.4%), astrovirus (75.0%), norovirus GII (72.6%) and Cryptosporidium (71.2%). The only pathogen associated with severe diarrhoea was EIEC/Shigella (p<0.001). EAEC was found more frequent among the infants, and EIEC/Shigella, Giardia duodenalis and Dientamoeba fragilis among the older children.ConclusionsStool pathogens proved common among all the children regardless of them having diarrhoea or not.     

Antidepressant effect of taurine in diabetic rats

Clinical and preclinical studies have shown that diabetic individuals present more depressive behaviors than non-diabetic individuals. Taurine, one of the most abundant free amino acids in the central nervous system, modulates a variety of biological functions and acts as an agonist at GABA_A receptors. Our objective was to assess the antidepressant effect of taurine in diabetic rats. Additionally, we studied the effect of taurine on weight gain, water and food intake, and blood glucose levels in diabetic and non-diabetic rats. Male Wistar rats were divided into control (CTR) and streptozotocin-induced diabetic (STZ) groups and were administered daily 0, 25, 50 or 100?mg/kg of taurine (n?=?10 per subgroup) intraperitoneally. After 28?days of treatment, the animals were exposed to the forced swimming test, and their behaviors were recorded. Weight gain, water and food intake, and blood glucose levels were measured weekly. Our results showed that STZ rats had a higher immobility duration than CTR rats, and taurine decreased this depressive-like behavior in STZ rats at doses of 25 and 100?mg/kg. Both of these doses of taurine also decreased water intake and improved weight gain in STZ rats. All doses of taurine decreased the water intake in CTR rats. Taurine, at a dose of 100?mg/kg, decreased food intake and blood glucose levels in STZ rats. Because taurine is a GABA agonist and both amino acids are lower in the plasma of diabetic and depressive individuals, we hypothesize that taurine may represent a new adjuvant drug for the treatment of depression in diabetic individuals.     

Synthesis and cytotoxic activity of heterocyclic ring-substituted betulinic acid derivatives

A new series of betulinic acid derivatives have been synthesized by introducing heterocyclic ring between C-2 and C-3 positions of betulinic acid. Further modifications were also carried out by reduction of C-20(29) unsaturated bond and substitution of C-28 carboxyl group by ester and amide linkage to enhance the selectivity. Compound 11 resulted in IC(50) of 2.44, 2.5, and 2.7 microg/ml on MIAPaCa, PA-1, and SW620 cancer cell lines, respectively. Compound 38 resulted in IC(50) of 0.67 microg/ml on MIAPaCa cell line.     

Early Trichinella spiralis and Trichinella nativa infections induce similar gene expression profiles in rat jejunal mucosa

Trichinella spiralis causes a significantly higher parasite burden in rat muscle than Trichinella nativa. To assess whether the difference in infectivity is due to the early intestinal response, we analyzed gene expression changes in the rat jejunum during Trichinella infection with a whole-genome microarray. The rats were euthanized on day five of infection, and their jejunal mucosa was sampled for microarray analysis. In addition, intestinal histology and hematology were examined. Against our expectations, the gene expression changes were similar in both T.nativa- and T. spiralis-infected groups. The two groups were hence pooled, and in the combined Trichinella-infected group, 551 genes were overexpressed and 427 underexpressed when compared to controls (false discovery rate ?0.001 and fold change at least 2 in either direction). Pathway analysis identified seven pathways significantly associated with Trichinella infection (p < 0.05). The microarray data suggested nonspecific damage and an inflammatory response in the jejunal mucosa. Histological findings, including hyperemia, hemorrhage and a marked infiltration of inflammatory cells, supported the microarray data. Trichinella infection caused complex gene expression changes that indicate a host response to tissue damage in the mucosa of the jejunum, but the changes were not notably dependent on the studied species of Trichinella.     

Synthesis of functionalized amino acid derivatives as new pharmacophores for designing anticancer agents

A new series of functionalized amino acid derivatives N-substituted 1-N-(tert-butoxycarbonyl)-2,2-dimethyl-4-phenyl-5-oxazolidine carboxamide (1-17) and 1-N-substituted-3-amino-2-hydroxy-3-phenylpropane-1-carboxamide (18-34) were synthesized and evaluated for their in vitro cytotoxicity against human cancer cell lines. Compound 6 has shown interesting cytotoxicity (IC₅₀ = 5.67 μm) in ovarian cancer, while compound 10 exhibited promising cytotoxicity in ovarian (IC₅₀ = 6.1 μm) and oral (IC₅₀ = 4.17 μm) cancers. These compounds could be of use in designing new anti-cancer agents.     

Effects of long-term open-air exposure to fluoride,nitrogen compounds and SO2 on visible symptoms,pollutant accumulation and ultrastructure of Scots pine and Norway spruce seedlings

 Effects of SO₂, aqueous fluoride (NaF) and a solution of nitrogen compounds (NH₄NO₃) on the visible symptoms, pollutant accumulation and ultrastructure of Scots pine (Pinus sylvestris L.) and Norway spruce [Picea abies (L.) Karst.] seedlings were studied in an open-air experiment lasting for 3 consecutive years. Visible injury symptoms were most pronounced in combination exposures and whenever F was applied. Visible symptoms correlated well with needle pollutant concentrations. Exposure to NaF increased needle F contents particularly when F was applied with SO₂ or NH₄NO₃. This suggests that a reduction in N or SO₂ emissions, in F polluted areas, could improve the condition of conifers via decreased accumulation of phytotoxic F in the needles. Norway spruce needles accumulated 2 – 10 times as much S and F as those of Scots pine. Microscopic observations showed various changes in the needle mesophyll cell ultrastructure. In both species, exposure to SO₂ increased significantly the amount of cytoplasmic vacuoles, suggesting detoxification of excess sulphate or low pH. F treatments resulted in a significant enlargement of plastoglobuli in Scots pine and a darkening of plastoglobuli in Norway spruce. All exposures enhanced the accumulation of lipid bodies. An increased portion of translucent plastoglobuli was most pronounced in N treatments. Many of the ultrastructural changes and visible symptoms appeared only as number of years exposed increased, indicating that long-term experiments are needed. Both visible symptoms and ultrastructural changes pointed to the more pronounced sensitivity of Norway spruce compared to Scots pine. Ultrastructural results mostly supported earlier qualitative observations of F, N and SO₂ effects on needle mesophyll cell ultrastructure. However, no reduction of thylakoids in SO₂ containing exposure or curling of thylakoids in F exposure could be detected in the present study. Received: 5 December 1994 / Accepted: 28 April 1995     

Amber Mutants of Bacteriophages T3 and T7 Defective in Phage-directed Deoxyribonucleic Acid Synthesis

Amber mutants of the related phages T3 and T7 were isolated and tested for their ability to restore-as the wild type does-thymidine incorporation in ultraviolet (UV)-irradiated, UV-sensitive, nonpermissive host bacteria (Escherichia coli B(s-1)). Most amber mutants had this ability. However, in both T3 and T7, mutants unable to promote thymidine incorporation under these conditions were found and classified into two well-defined complementation groups: T3DO-A and T3DO-B, T7DO-A and T7DO-B. Infection of B(s-1) cells with representatives of groups DO-A had the following characteristics: (i) phage-directed uridine uptake in UV-irradiated cells was reduced to less than 20% of normal; (ii) breakdown of host deoxyribonucleic acid (DNA) was delayed and incomplete; (iii) no serum-blocking antigens appeared; (iv) no cell lysis occurred; (v) the ability to exclude the heterologous wild type was impaired. Amber mutants of the DO-B groups, infecting B(s-1), were able to: (i) promote an efficient phage-directed uridine uptake in UV-irradiated cells; (ii) bring about rapid breakdown of host DNA; (iii) synthesize serum-blocking antigens; (iv) lyse the host cells, generally after the normal latent period; (v) exclude efficiently the heterologous wild type. Although physiological similarities between the respective DO-A mutants or DO-B mutants of T3 and T7 were evident, no physiological cross-complementation occurred, and genetic crosses gave no evidence of genetic homologies between groups of T3 and T7.