Generation of lipase-containing static emulsions in silicone spheres for synthesis in organic media

Because of the broad versatility of lipases as biocatalysts, interest has for some years been focused on the improvement of the economy of processes using these enzymes, especially by appropriate immobilisation. In this study, a method was developed to emulsify aqueous solutions of lipase A of Candida antarctica (CALA) and lipase of Thermomyces lanuginosa (TLL) in silicone elastomers yielding elastic beads. The persistent water-organic interface created by this static emulsion enabled an improved performance of the immobilised lipases due to the well known fact that from a kinetic point of view these enzymes show a higher efficiency in biphasic than in monophasic systems. The entrapped lipases catalysed the esterification of octanol and caprylic acid in hexane with an activity that, related to the free enzyme, was enhanced about 31-fold for CALA and 250-fold for TLL. Comparison to the activity of the same enzymes in sol–gels revealed that for CALA immobilisation in static emulsion was the only method yielding active biocatalysts, whereas activation of TLL was in the same range in static emulsion and sol–gels. However, apparent activity of TLL in static emulsion was considerably higher than in sol–gels due to the feasible high enzyme loading. The results indicate that immobilising lipases as static emulsion is a technique suitable for biotechnological application. Moreover, a transfer to enzymes of other classes seems possible.     

Psychological Stress-Induced,IDO1-Dependent Tryptophan Catabolism: Implications on Immunosuppression in Mice and Humans

It is increasingly recognized that psychological stress influences inflammatory responses and mood. Here, we investigated whether psychological stress (combined acoustic and restraint stress) activates the tryptophan (Trp) catabolizing enzyme indoleamine 2,3-dioxygenase 1(IDO1) and thereby alters the immune homeostasis and behavior in mice. We measured IDO1 mRNA expression and plasma levels of Trp catabolites after a single 2-h stress session and in repeatedly stressed (4.5-days stress, 2-h twice a day) naïve BALB/c mice. A role of cytokines in acute stress-induced IDO1 activation was studied after IFNγ and TNFα blockade and in IDO1^−/− mice. RU486 and 1-Methyl-L-tryptophan (1-MT) were used to study role of glucocorticoids and IDO1 on Trp depletion in altering the immune and behavioral response in repeatedly stressed animals. Clinical relevance was addressed by analyzing IDO1 activity in patients expecting abdominal surgery. Acute stress increased the IDO1 mRNA expression in brain, lung, spleen and Peyer''s patches (max. 14.1±4.9-fold in brain 6-h after stress) and resulted in a transient depletion of Trp (−25.2±6.6%) and serotonin (−27.3±4.6%) from the plasma measured 6-h after stress while kynurenine levels increased 6-h later (11.2±9.3%). IDO1 mRNA up-regulation was blocked by anti-TNFα and anti-IFNγ treatment. Continuous IDO1 blockade by 1-MT but not RU486 treatment normalized the anti-bacterial defense and attenuated increased IL-10 inducibility in splenocytes after repeated stress as it reduced the loss of body weight and behavioral alterations. Moreover, kynurenic acid which remained increased in 1-MT treated repeatedly stressed mice was identified to reduce the TNFα inducibility of splenocytes in vitro and in vivo. Thus, psychological stress stimulates cytokine-driven IDO1 activation and Trp depletion which seems to have a central role for developing stress-induced immunosuppression and behavioral alteration. Since patients showed Trp catabolism already prior to surgery, IDO is also a possible target enzyme for humans modulating immune homeostasis and mood.     

Efficacy of different types of aerobic exercise in fibromyalgia syndrome: a systematic review and meta-analysis of randomised controlled trials

Introduction  

The efficacy and the optimal type and volume of aerobic exercise (AE) in fibromyalgia syndrome (FMS) are not established. We therefore assessed the efficacy of different types and volumes of AE in FMS.     

Peroxidase cytochemistry and ultrastructure of resident macrophages in fetal rat liver: A developmental study

The peroxidase cytochemistry and the ultrastructural characteristics of resident macrophages in fetal rat liver have been investigated. Livers of 10-, 11-, 14-, 17-, and 20-day-old fetuses were fixed by immersion or perfusion, incubated for peroxidase, and processed for transmission electron microscopy. Some 17- and 20-day-old fetuses were injected prior to sacrifice with carbon or 0.8-μm latex particles through the umbilical vein. Some livers were additionally processed for scanning electron microscopy (SEM). The endogenous peroxidase was present in the nuclear envelope (NE) and endoplasmic reticulum (ER) of fetal macrophages with a negative reaction in the Golgi apparatus, a distribution pattern identical to that in Kupffer cells of adult rat liver. Such peroxidase-positive cells avidly took up the injected latex and carbon particles and were the only cell type in fetal liver involved in erythrophagocytosis. Furthermore, they were associated with erythropoietic elements, forming close contacts with such cells, especially normoblasts. The peroxidase pattern in leukopoietic cells differed at all stages of maturation from that in macrophages. By SEM the macrophages exhibited ruffles and lamellopodia on their surfaces and protruded often into the lumen of fetal sinusoids. Macrophages in fetal liver underwent mitotic divisions. The macrophages were first seen on gestation day 11, whereas the first mature monocytes were found on gestation day 17. These observations suggest that resident macrophages in fetal rat liver form a self-replicating cell line independent of the monocytopoietic series, although they may both arise from a common precursor cell.     

Distribution, morphology and projections of nitrergic and non-nitrergic submucosal neurons in the pig small intestine

 Sequential nitric oxide synthase immunohistochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry in pig small intestinal wholemounts revealed a complete colocalisation of the two nitrergic markers in submucous neurons. The external submucous plexus (ESP) contained nitrergic neurons throughout. In the internal submucous plexus (ISP) we found a moderate number of nitrergic neurons in the duodenum, while they were rare in the jejunum and nearly absent in the ileum. Combined NADPHd histochemistry and silver impregnation showed morphological ESP type III and VI neurons to be NADPHd positive whereas ESP type II, IV and V neurons were NADPHd negative. Axons of ESP type III, IV and VI neurons were often observed to enter interconnecting strands directed abluminally. ESP type II neurons projected mainly to the ISP. In special silver-impregnated wholemounts containing both external muscle layers and the abluminal part of the submucous layer, i.e. the myenteric plexus and the ESP, the great majority of impregnated axons within the interconnecting strands were observed to run between both plexuses and did not enter the circular muscle layer. We conclude that ESP type III and VI neurons are nitrergic while ESP type II, IV and V neurons are non-nitrergic. Furthermore, we assume that ESP type III, IV and VI neurons may represent a submucosal input to the myenteric plexus. Accepted: 26 August 1997     

IL-15 augments antitumoral activity of an ErbB2/HER2 cancer vaccine targeted to professional antigen-presenting cells

Targeted delivery of tumor-associated antigens to professional antigen-presenting cells (APC) is being explored as a strategy to enhance the antitumoral activity of cancer vaccines. Here, we generated a cell-based system for continuous in vivo production of a CTLA-4-ErbB2 fusion protein as a therapeutic vaccine. The chimeric CTLA-4-ErbB2 molecule contains the extracellular domain of CTLA-4 for specific targeting to costimulatory B7 molecules on the surface of APC, genetically fused to residues 1-222 of human ErbB2 (HER2) as an antigenic determinant. In wild-type BALB/c mice, inoculation of syngeneic epithelial cells continuously secreting the CTLA-4-ErbB2 fusion vaccine in the vicinity of subcutaneously growing ErbB2-expressing renal cell carcinomas resulted in the rejection of established tumors, accompanied by the induction of ErbB2-specific antibodies and cytotoxic T cells. In contrast, treatment with CTLA-4-ErbB2 vaccine-secreting producer cells alone was insufficient to induce tumor rejection in ErbB2-transgenic WAP-Her-2 F1 mice, which are characterized by pronounced immunological tolerance to the human self-antigen. When CTLA-4-ErbB2 producer cells were modified to additionally secrete interleukin (IL)-15, antigen-specific antitumoral activity of the vaccine in WAP-Her-2 F1 mice was restored, documented by an increase in survival, and marked inhibition of the growth of established ErbB2-expressing, but not antigen-negative tumors. Our results demonstrate that continuous in vivo expression of an APC-targeted ErbB2 fusion protein results in antigen-specific immune responses and antitumoral activity in tumor-bearing hosts, which is augmented by the pleiotropic cytokine IL-15. This provides a rationale for further development of this approach for specific cancer immunotherapy.     

Structural basis for budding by the ESCRT-III factor CHMP3

The vacuolar protein sorting machinery regulates multivesicular body biogenesis and is selectively recruited by enveloped viruses to support budding. Here we report the crystal structure of the human ESCRT-III protein CHMP3 at 2.8 A resolution. The core structure of CHMP3 folds into a flat helical arrangement that assembles into a lattice, mainly via two different dimerization modes, and unilaterally exposes a highly basic surface. The C terminus, the target for Vps4-induced ESCRT disassembly, extends from the opposite side of the membrane targeting region. Mutations within the basic and dimerization regions hinder bilayer interaction in vivo and reverse the dominant-negative effect of a truncated CHMP3 fusion protein on HIV-1 budding. Thus, the final steps in the budding process may include CHMP protein polymerization and lattice formation on membranes by employing different bilayer-recognizing surfaces, a function shared by all CHMP family members.     

Structure of the Ni(II) complex of Escherichia coli peptide deformylase and suggestions on deformylase activities depending on different metal(II) centres

Crystal structures of polypeptide deformylase (PDF) of Escherichia coli with nickel(II) replacing the native iron(II) have been solved with chloride and formate as metal ligands. The chloro complex is a model for the correct protonation state of the hydrolytic hydroxo ligand and the protonated status of the Glu133 side chain as part of the hydrolytic mechanism. The ambiguity that recently some PDFs have been identified with Zn²⁺ ion as the active-site centre whereas others are only active with Fe²⁺ (or Co²⁺, Ni²⁺) is discussed with respect to Lewis acid criteria of the metal ion and substrate activation by the CD loop.