Characterization of two new multiforms of Trametes pubescens laccase

Electrochemical properties of two multiforms of laccase from Trametes pubescens basidiomycete (LAC1 and LAC2) have been studied. The standard redox potentials of the T1 sites of the enzymes were found to be 746 and 738 mV vs. NHE for LAC1 and LAC2, respectively. Bioelectroreduction of oxygen based on direct electron transfer between each of the two forms of Trametes pubescens laccase and spectrographic graphite electrodes has been demonstrated and studied. It is concluded that the T1 site of laccase is the first electron acceptor, both in solution (homogeneous case) and when the enzymes are adsorbed on the surface of the graphite electrode (heterogeneous case). Thus, the previously proposed mechanism of oxygen bioelectroreduction by adsorbed fungal laccase was additionally confirmed using two forms of the enzyme. Moreover, the assumed need for extracellular laccase to communicate directly and electronically with a solid matrix (lignin) in the course of lignin degradation is discussed. In summary, the possible roles of multiforms of the enzyme based on their electrochemical, biochemical, spectral, and kinetic properties have been suggested to consist in broadening of the substrate specificity of the enzyme, in turn yielding the possibility to dynamically regulate the process of lignin degradation according to the real-time survival needs of the organism.     

Ca2+ channel currents and contraction in CaVbeta3-deficient ileum smooth muscle from mouse

Voltage activated L-type Ca(2+) channels are the principal Ca(2+) channels in intestinal smooth muscle cells. They comprise the ion conducting Ca(V)1 pore and the ancillary subunits alpha(2)delta and beta. Of the four Ca(V)beta subunits Ca(V)beta(3) is assumed to be the relevant Ca(V)beta protein in smooth muscle. In protein lysates isolated from mouse ileum longitudinal smooth muscle we could identify the Ca(V)1.2, Ca(V)alpha(2), Ca(V)beta(2) and Ca(V)beta(3) proteins, but not the Ca(V)beta(1) and Ca(V)beta(4) proteins. Protein levels of Ca(V)1.2, Ca(V)alpha(2) and Ca(V)beta(2) are not altered in ileum smooth muscle obtained from Ca(V)beta(3)-deficient mice indicating that there is no compensatory increase of the expression of these channel proteins. Neither the Ca(V)beta(2) nor the other Ca(V)beta proteins appear to substitute for the lacking Ca(V)beta(3). L-type Ca(2+) channel properties including current density, inactivation kinetics as well as Cd(2+)- and dihydropyridine sensitivity were identical in cells of both genotypes suggesting that they do not require the presence of a Ca(V)beta(3) protein. However, a key hallmark of the Ca(V)beta modulation of Ca(2+) current, the hyperpolarisation of channel activation is slightly but significantly reduced by 4 mV. In addition to L-type Ca(2+) currents T-type Ca(2+) currents could be recorded in the murine ileum smooth muscle cells, but T-type currents were not affected by the lack of Ca(V)beta(3). Both proteins, Ca(V)beta(2) and Ca(V)beta(3) are localized near the plasma membrane and the localization of Ca(V)beta(2) is not altered in Ca(V)beta(3) deficient cells. Spontaneous contractions and potassium and carbachol induced contractions are not significantly different between ileum longitudinal smooth muscle strips from mice of both genotypes. In summary the data show that in ileum smooth muscle cells, Ca(V)beta(3) has only subtle effects on L-type Ca(2+) currents, appears not to be required for spontaneous and potassium induced contraction but might have a function beyond being a Ca(2+) channel subunit.     

Biocatalytic ketone reduction—a powerful tool for the production of chiral alcohols—part I: processes with isolated enzymes

Traditional cultivation-based methods to quantify microbial abundance are not suitable for analyses of microbial communities in environmental or medical samples, which consist mainly of uncultured microorganisms. Recently, different cultivation-independent quantification approaches have been developed to overcome this problem. Some of these techniques use specific fluorescence markers, for example ribosomal ribonucleic acid targeted oligonucleotide probes, to label the respective target organisms. Subsequently, the detected cells are visualized by fluorescence microscopy and are quantified by direct visual cell counting or by digital image analysis. This article provides an overview of these methods and some of their applications with emphasis on (semi-)automated image analysis solutions.     

The bovine seminal plasma protein PDC-109 extracts phosphorylcholine-containing lipids from the outer membrane leaflet

The bovine seminal plasma protein PDC-109 modulates the maturation of bull sperm cells by removing lipids, mainly phosphatidylcholine and cholesterol, from their cellular membrane. Here, we have characterized the process of extraction of endogenous phospholipids and of their respective analogues. By measuring the PDC-109-mediated release of fluorescent phospholipid analogues from lipid vesicles and from biological membranes (human erythrocytes, bovine epididymal sperm cells), we showed that PDC-109 extracts phospholipids with a phosphorylcholine headgroup mainly from the outer leaflet of these membranes. The ability of PDC-109 to extract endogenous phospholipids from epididymal sperm cells was followed by mass spectrometry, which allowed us to characterize the fatty acid pattern of the released lipids. From these cells, PDC-109 extracted phosphatidylcholine and sphingomyelin that contained an enrichment of mono- and di-unsaturated fatty acids as well as short-chain and lyso-phosphatidylcholine species. Based on the results, a model explaining the phospholipid specificity of PDC-109-mediated lipid release is presented. Astrid Tannert and Anke Kurz have contributed equally to this work. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Citric acid production from sucrose using a recombinant strain of the yeast Yarrowia lipolytica

The yeast Yarrowia lipolytica is able to secrete high amounts of several organic acids under conditions of growth limitation and carbon source excess. Here we report the production of citric acid (CA) in a fed-batch cultivation process on sucrose using the recombinant Y. lipolytica strain H222-S4(p67ICL1) T5, harbouring the invertase encoding ScSUC2 gene of Saccharomyces cerevisiae under the inducible XPR2 promoter control and multiple ICL1 copies (10–15). The pH-dependent expression of invertase was low at pH 5.0 and was identified as limiting factor of the CA-production bioprocess. The invertase expression was sufficiently enhanced at pH 6.0–6.8 and resulted in production of 127–140 g l⁻¹ CA with a yield Y _CA of 0.75–0.82 g g⁻¹, whereas at pH 5.0, 87 g l ⁻¹ with a yield Y _CA of 0.51 gg⁻¹ were produced. The CA-productivity Q _CA increased from 0.40 g l ⁻¹ h⁻¹ at pH 5.0 up to 0.73 g l ⁻¹ h⁻¹ at pH 6.8. Accumulation of glucose and fructose at high invertase expression level at pH 6.8 indicated a limitation of CA production by sugar uptake. The strain H222-S4(p67ICL1) T5 also exhibited a gene–dose-dependent high isocitrate lyase expression resulting in strong reduction (<5%) of isocitric acid, a by-product during CA production.     

A Thr94Ala mutation in human liver fatty acid-binding protein contributes to reduced hepatic glycogenolysis and blunted elevation of plasma glucose levels in lipid-exposed subjects

Liver fatty acid-binding protein (L-FABP) is a highly conserved key factor in lipid metabolism. Amino acid replacements in L-FABP might alter its function and thereby affect glucose metabolism in lipid-exposed subjects, as indicated by studies in L-FABP knockout mice. Amino acid replacements in L-FABP were investigated in a cohort of 1,453 Caucasian subjects. Endogenous glucose production (EGP), gluconeogenesis, and glycogenolysis were measured in healthy carriers of the only common Thr(94)-to-Ala amino acid replacement (Ala/Ala(94)) vs. age-, sex-, and BMI-matched wild-type (Thr/Thr(94)) controls at baseline and after 320-min lipid/heparin-somatostatin-insulin-glucagon clamps (n = 18). Whole body glucose disposal was further investigated (subset; n = 13) using euglycemic-hyperinsulinemic clamps without and with lipid/heparin infusion. In the entire cohort, the only common Ala/Ala(94) mutation was significantly associated with reduced body weight, which is in agreement with a previous report. In lipid-exposed, individually matched subjects there was a genotype vs. lipid-treatment interaction for EGP (P = 0.009) driven mainly by reduced glycogenolysis in Ala/Ala(94) carriers (0.46 +/- 0.05 vs. 0.59 +/- 0.05 mgxkg(-1)xmin(-1), P = 0.013). The lipid-induced elevation of plasma glucose levels was smaller in Ala/Ala(94) carriers compared with wild types (P < 0.0001). Whole body glucose disposal was not different between lipid-exposed L-FABP genotypes. In summary, the Ala/Ala(94)-mutation contributed significantly to reduced glycogenolysis and less severe hyperglycemia in lipid-exposed humans and was further associated with reduced body weight in a large cohort. Data clearly show that investigation of L-FABP phenotypes in the basal overnight-fasted state yielded incomplete information, and a challenge test was essential to detect phenotypical differences in glucose metabolism between L-FABP genotypes.     

Luminal antigens access late endosomes of intestinal epithelial cells enriched in MHC I and MHC II molecules: in vivo study in Crohn's ileitis

In contrast to healthy conditions, intestinal epithelial cells (IECs) stimulate proinflammatory CD4+ and CD8+ T cells during Crohn's disease (CD). The underlying regulatory mechanisms remain unknown. Here we investigated the epithelial expression of major histocompatibility complex (MHC) I and MHC II and its interference with endocytic pathways, in vivo. During ileoscopy, ovalbumin (OVA) was sprayed onto ileal mucosa of CD patients (ileitis and remission) and controls. The epithelial traffic of OVA and MHC I/II pathways were studied in biopsies using fluorescence and electron microscopy. We found MHC I and MHC II to accumulate within multivesicular late endosomes (MVLE) of IECs. Faint labeling for these molecules was seen in early endosomes and lysosomes. MVLE were entered by OVA 10 min after exposure. Exosomes carrying MHC I, MHC II, and OVA were detected in intercellular spaces of the epithelium. OVA trafficking and labeling patterns for MHC I and MHC II in IECs showed no differences between CD patients and controls. Independent of inflammatory stimuli, MHC I and MHC II pathways intersect MVLE in IECs, which were efficiently targeted by luminal antigens. Similar to MHC II-enriched compartments in professional antigen presenting cells, these MVLE might be critically involved in MHC I- and MHC II-related antigen processing in IECs and the source of epithelial-released exosomes. The access of luminal antigens to MHC I in MVLE might indicate that the presentation of exogenous antigens by IECs must not be restricted to MHC II but might also occur as "cross-presentation" via MHC I to CD8+ T cells.     

Fasting induces basolateral uptake transporters of the SLC family in the liver via HNF4alpha and PGC1alpha

Fasting induces numerous adaptive changes in metabolism by several central signaling pathways, the most important represented by the HNF4alpha/PGC-1alpha-pathway. Because HNF4alpha has been identified as central regulator of basolateral bile acid transporters and a previous study reports increased basolateral bile acid uptake into the liver during fasting, we hypothesized that HNF4alpha is involved in fasting-induced bile acid uptake via upregulation of basolateral bile acid transporters. In rats, mRNA of Ntcp, Oatp1, and Oatp2 were significantly increased after 48 h of fasting. Protein expression as determined by Western blot showed significant increases for all three transporters 72 h after the onset of fasting. Whereas binding activity of HNF1alpha in electrophoretic mobility shift assays remained unchanged, HNF4alpha binding activity to the Ntcp promoter was increased significantly. In line with this result, we found significantly increased mRNA expression of HNF4alpha and PGC-1alpha. Functional studies in HepG2 cells revealed an increased endogenous NTCP mRNA expression upon cotransfection with either HNF4alpha, PGC-1alpha, or a combination of both. We conclude that upregulation of the basolateral bile acid transporters Ntcp, Oatp1, and Oatp2 in fasted rats is mediated via the HNF4alpha/PGC-1alpha pathway.     

Cross-reactivity and 1.4-A crystal structure of Malassezia sympodialis thioredoxin (Mala s 13), a member of a new pan-allergen family

We have identified thioredoxins (Trx) of Malassezia sympodialis, a yeast involved in the pathogenesis of atopic eczema, and of Aspergillus fumigatus, a fungus involved in pulmonary complications, as novel IgE-binding proteins. We show that these Trx, including the human enzyme, represent cross-reactive structures recognized by serum IgE from individuals sensitized to M. sympodialis Trx. Moreover, all three proteins were able to elicit immediate-type allergic skin reactions in sensitized individuals, indicating a humoral immune response based on molecular mimicry. To analyze structural elements involved in these reactions, the three-dimensional structure of M. sympodialis Trx (Mala s 13) has been determined at 1.4-A resolution by x-ray diffraction analysis. The structure was solved by molecular replacement and refined to a crystallographic R factor of 14.0% and a free R factor of 16.8% and shows the typical Trx fold. Mala s 13 shares 45% sequence identity with human Trx and superposition of the solved Mala s 13 structure with those of human Trx reveals a high similarity with a root mean square deviation of 1.11 A for all Calpha atoms. In a detailed analysis of the molecular surface in combination with sequence alignment, we identified conserved solvent-exposed amino acids scattered over the surface in both structures which cluster to patches, thus forming putative conformational B cell epitopes potentially involved in IgE-mediated cross- and autoreactivity.