Human mast cells express leptin and leptin receptors

Mast cells are immune cells that produce and secrete a variety of mediators and cytokines that influence various inflammatory and immune processes. Leptin is a cytokine regulating metabolic, endocrine as well as immune functions via the leptin receptor which is expressed by many immune cells. However, there are no data about leptin receptor expression in mast cells. Immunohistochemical and immunofluorescent double stainings showed the expression of leptin and leptin receptors in mast cells in human skin and several parts of the respiratory, gastrointestinal and urogenital tract. Leptin was expressed in mast cells expressing the classification marker chymase, whereas a variable expression was observed in tryptase positive mast cells. For leptin receptors, the expression pattern was tissue dependent and not related to tryptase or chymase expression. Our results demonstrate the expression of leptin and leptin receptors on mast cells, suggesting paracrine and/or autocrine immunomodulatory effects of leptin on mast cells.     

Fusarium graminearum xylanases show different functional stabilities,substrate specificities and inhibition sensitivities

When grown on arabinoxylan as the sole carbon source, the cereal phytopathogen Fusarium graminearum expresses four xylanases. Cloning and heterologous expression of the corresponding xylanase encoding genes and analysis of general biochemical properties, substrate specificities and inhibition sensitivities revealed some marked differences. XylA and XylB are glycoside hydrolase family (GH) 11 xylanases, while XylC and XylD belong to GH10. pH and temperature for optimal activity of the enzymes were between 6.0 and 7.0 and 40 °C, respectively. Interestingly, XylC displayed remarkable pH stability as it retained most of its activity even after pre-incubation at pH 1.0 and 13.0 for 120 min at room temperature. All xylanases hydrolysed xylotetraose, xylopentaose and xylohexaose, but to different extents, while only XylC and XylD hydrolysed xylotriose. The two GH10 xylanases released a higher percentage of smaller products from xylan and xylo-oligosaccharides than did their GH11 counterparts. Analysis of kinetic properties revealed that wheat arabinoxylan is the favoured XylC substrate while XylA and XylB prefer sparsely substituted oat spelt xylan. XylC and XylD were inhibited by xylanase inhibiting protein (XIP), while XylA and XylB were sensitive to Triticum aestivum xylanase inhibitor (TAXI). Because of its pH stability and preference for arabinoxylan, XylC is a valuable candidate for use in biotechnological applications.     

Seasonal influence on heat-resistant proteolytic capacity of Pseudomonas lundensis and Pseudomonas fragi, predominant milk spoilers isolated from Belgian raw milk samples

Psychrotolerant bacteria and their heat-resistant proteases play a major role in the spoilage of UHT-processed dairy products. Summer and winter raw milk samples were screened for the presence of such bacteria. One hundred and three proteolytic psychrotolerant bacteria were isolated, characterized by API tests, rep-PCR fingerprint analysis and evaluated for heat-resistant protease production. Twenty-nine strains (representing 79% of the complete collection) were further identified by 16S rRNA gene sequencing, rpoB gene sequencing and DNA–DNA hybridizations. A seasonal inter- and intra-species influence on milk spoilage capacity (e.g. growth rate and/or protease production) was demonstrated. Moreover, this polyphasic approach led to the identification of Pseudomonas fragi and Pseudomonas lundensis (representing 53% of all isolates) as predominant producers of heat-resistant proteases in raw milk. The role of Pseudomonas fluorescens , historically reported as important milk spoiler, could not unequivocally be established. The use of more reliable identification techniques and further revision of the taxonomy of P. fluorescens will probably result in a different perspective on its role in the milk spoilage issue.     

Seasonal changes in parasite load and a cellular immune response in a colour polymorphic lizard

Permanent colour polymorphisms may be maintained by complex interactions between physiological traits (e.g. immunity) and environmental pressures. In this study we investigate morph specific variation in parasite load and cellular immune response (induced by a Phytohaemagglutinin, PHA injection) in a colour polymorphic population of the Dalmatian wall lizard (Podarcis melisellensis), where adult males have bright white, yellow or orange throats and ventral sides. Orange males have larger heads and can bite harder than the others. To examine seasonal effects, analyses were performed at an early and late stage in the reproductive season (May and September). Infection with mites and ticks did not differ among morphs, but was more severe at the end of the reproductive season. Fewer orange individuals were infected with haemogregarines at the end of the season, but white males were always more infected (higher number of haemogregarines in their blood) than other morphs. White and yellow males showed an increased PHA response towards the end of the season, but PHA response decreased in the orange morph. Finally, across all morphs, a relationship was found between ectoparasite load and PHA response. Our study provides indications of alternative life-history strategies among colour morphs and evidence for an up-regulation of the immune function at the end of the reproductive season.     

The Protein-disulfide Isomerase DsbC Cooperates with SurA and DsbA in the Assembly of the Essential β-Barrel Protein LptD

The assembly of the β-barrel proteins present in the outer membrane (OM) of Gram-negative bacteria is poorly characterized. After translocation across the inner membrane, unfolded β-barrel proteins are escorted across the periplasm by chaperones that reside within this compartment. Two partially redundant chaperones, SurA and Skp, are considered to transport the bulk mass of β-barrel proteins. We found that the periplasmic disulfide isomerase DsbC cooperates with SurA and the thiol oxidase DsbA in the folding of the essential β-barrel protein LptD. LptD inserts lipopolysaccharides in the OM. It is also the only β-barrel protein with more than two cysteine residues. We found that surAdsbC mutants, but not skpdsbC mutants, exhibit a synthetic phenotype. They have a decreased OM integrity, which is due to the lack of the isomerase activity of DsbC. We also isolated DsbC in a mixed disulfide complex with LptD. As such, LptD is identified as the first substrate of DsbC that is localized in the OM. Thus, electrons flowing from the cytoplasmic thioredoxin system maintain the integrity of the OM by assisting the folding of one of the most important β-barrel proteins.     

Pharmacological sensitivity of ATP release triggered by photoliberation of inositol-1,4,5-trisphosphate and zero extracellular calcium in brain endothelial cells

Recently, ATP has gained much interest as an extracellular messenger involved in the communication of calcium signals between cells. The mechanism of ATP release is, however, still a matter of debate. In the present study we investigated the possible contribution of connexin hemichannels or ion channels in the release of ATP in GP8, a rat brain endothelial cell line. Release of ATP was triggered by photoactivation of InsP(3) or by reducing the extracellular calcium concentration. Both trigger protocols induced ATP release significantly above baseline. InsP(3)-triggered ATP release was completely blocked by alpha-glycyrrhetinic acid (alpha-GA), the connexin mimetic peptides gap 26 and 27, and the trivalent ions gadolinium and lanthanum. ATP release triggered by zero calcium was, in addition to these substances, also blocked by flufenamic acid (FFA), niflumic acid, and NPPB. Gap 27 selectively blocked zero calcium-triggered ATP release in connexin-43 transfected HeLa cells, while having no effect in wild-type and connexin-32 transfected cells. Of all the agents used, only alpha-GA, FFA and NPPB significantly reduced gap junctional coupling. In conclusion, InsP(3) and zero calcium-triggered ATP release show major similarities but also some differences in their sensitivity to the agents applied. It is suggested that both stimuli trigger ATP release through the same mechanism, which is connexin-dependent, permeable in both directions, potently blocked by connexin mimetic peptides, and consistent with the opening of connexin hemichannels.     

Protoplast-to-plant regeneration in cotton (Gossypium hirsutum L. cv. Coker 312) using feeder layers

Summary We report the regeneration of protoplasts isolated from two embryogenic cell lines of Gossypium hirsutum L. cv. Coker 312 initiated from hypocotylderived callus. Protoplasts plated on cellulose nitrate filters and placed over feeder layers formed embryogenic callus from which plants were regenerated. Plating efficiency up to 12.8% depended upon the cell line. Addition of phytohormones to the protoplast medium had no stimulating effect on plating efficiency. The influence of feeder cells and conditioned medium on plating efficiency was significantly different for the two cell lines.Abbreviations ACM autoclaved conditioned medium - AFC autoclaved feeder cells - BM basic medium - BM+ basic medium with phytohormones - CM non-autoclaved conditioned medium - FC non-autoclaved feeder cells - FDA fluorescein diacetate - MM maturation medium - NAA 1-naphtaleneacetic acid - PCM protoplast culture medium - PCM+ protoplast culture medium with phytohormones - SC settled cells - 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylamino purine     

Effects of magnesium depletion on myocardial high-energy phosphates and contractility

The effect of prolonged magnesium depletion on contractility, phosphorylating activity, and organic phosphates of spontaneously beating isolated rat atria was studied. Rats were fed a Mg-deficient diet for 8 weeks, during which serum Mg fell from 1.85 +/- 0.02 to 0.52 +/- 0.10 mg/dl. Atrial contractile activity was measured for 1 hr and at the end of this period tissue samples were taken for the determination of the phosphorylated intermediates. Mg depletion was associated with (a) reduced intracellular inorganic phosphorus and adenine nucleotides; (b) elevated creatine phosphate; (c) reduction in contractile force (CF) with no change in atrial beat rate (BR). There were no significant differences in the activities of creatine phosphokinase and adenylate kinase in control and Mg-depleted rat atrial homogenates determined in the presence of 5 mM MgCl2. Addition of various concentrations of MgCl2 to the medium resulted in an immediate reduction in both CF and BR of normal and Mg-depleted rat atria. Intraperitoneal administration of MgCl2 to Mg-depleted rats resulted in complete recovery of CF of isolated atria. This improvement in CF occurred without changes in the levels of inorganic phosphate and adenine nucleotides. The reduced intracellular level of high-energy phosphate or inorganic phosphate cannot therefore be responsible for the impaired contractility seen in Mg-depleted heart muscle. On the other hand, the fact that the creatine phosphate levels were higher in magnesium depletion suggests that myofibrillar utilization of creatine phosphate is more impaired than production, analogous to phenomena seen in postanoxic recovery.