Functional and morphological evidence for a ventricular conduction system in zebrafish and Xenopus hearts

Zebrafish and Xenopus have become popular model organisms for studying vertebrate development of many organ systems, including the heart. However, it is not clear whether the single ventricular hearts of these species possess any equivalent of the specialized ventricular conduction system found in higher vertebrates. Isolated hearts of adult zebrafish (Danio rerio) and African toads (Xenopus laevis) were stained with voltage-sensitive dye and optically mapped in spontaneous and paced rhythms followed by histological examination focusing on myocardial continuity between the atrium and the ventricle. Spread of the excitation wave through the atria was uniform with average activation times of 20 +/- 2 and 50 +/- 2 ms for zebrafish and Xenopus toads, respectively. After a delay of 47 +/- 8 and 414 +/- 16 ms, the ventricle became activated first in the apical region. Ectopic ventricular activation was propagated significantly more slowly (total ventricular activation times: 24 +/- 3 vs. 14 +/- 2 ms in zebrafish and 74 +/- 14 vs. 35 +/- 9 ms in Xenopus). Although we did not observe any histologically defined tracts of specialized conduction cells within the ventricle, there were trabecular bands with prominent polysialic acid-neural cell adhesion molecule staining forming direct myocardial continuity between the atrioventricular canal and the apex of the ventricle; i.e., the site of the epicardial breakthrough. We thus conclude that these hearts are able to achieve the apex-to-base ventricular activation pattern observed in higher vertebrates in the apparent absence of differentiated conduction fascicles, suggesting that the ventricular trabeculae serve as a functional equivalent of the His-Purkinje system.     

Impairment of TGF-beta signaling in T cells increases susceptibility to experimental autoimmune hepatitis in mice

In autoimmune hepatitis, strong TGF-beta1 expression is found in the inflamed liver. TGF-beta overexpression may be part of a regulatory immune response attempting to suppress autoreactive T cells. To test this hypothesis, we determined whether impairment of TGF-beta signaling in T cells leads to increased susceptibility to experimental autoimmune hepatitis (EAH). Transgenic mice of strain FVB/N were generated expressing a dominant-negative TGF-beta type II receptor in T cells under the control of the human CD2 promoter/locus control region. On induction of EAH, transgenic mice showed markedly increased portal and periportal leukocytic infiltrations with hepatocellular necroses compared with wild-type mice (median histological score = 1.8 +/- 0.26 vs. 0.75 +/- 0.09 in wild-type mice; P < 0.01). Increased IFN-gamma production (118 vs. 45 ng/ml) and less IL-4 production (341 vs. 1,256 pg/ml) by mononuclear cells isolated from transgenic livers was seen. Impairment of TGF-beta signaling in T cells therefore leads to increased susceptibility to EAH in mice. This suggests an important role for TGF-beta in immune homeostasis in the liver and may teleologically explain TGF-beta upregulation in response to T cell-mediated liver injury.     

Mode of action of family 10 and 11 endoxylanases on water-unextractable arabinoxylan

Microbial endo-beta-1,4-xylanases (EXs, EC 3.2.1.8) belonging to glycanase families 10 and 11 differ in their action on water-unextractable arabinoxylan (WU-AX). WU-AX was incubated with different levels of a Thermoascus aurantiacus family 10 and a Sporotrichum thermophile family 11 endoxylanases. At 10 g l(-1) arabinoxylan, enzyme concentrations (KE values) needed to obtain half-maximal hydrolysis rates (V(max) values) were 4.4 nM for the xylanase from T. aurantiacus and 7.1 nM for the xylanase from S. thermophile. Determination of Vmax/KE revealed that the family 10 enzyme hydrolysed two times more efficiently WU-AX than the family 11 enzyme. Molecular weights of the products formed were assessed and separation of feruloyl-oligosaccharides was achieved by anion-exchange and size-exclusion chromatography (SEC). The main difference between the feruloylated products by xylanases of family 10 and 11 concerned the length of the products containing feruloyl-arabinosyl substitution. The xylanase from T. aurantiacus liberated from WU-AX a feruloyl arabinoxylodisaccharide (FAX2) as the shortest feruloylated fragment in contrast with the enzyme from S. thermophile, which liberated a feruloyl arabinoxylotrisaccharide (FAX3). These results indicated that different factors govern WU-AX breakdown by the two endoxylanases.     

Attenuation of blood parameters in smokers and non-smokers after intake of a complex food additive

This report describes an intervention study with healthy volunteers (20 smokers, 28 non-smokers) taking a food additive mainly containing vitamin C (ascorbic acid), vitamin E (alpha-tocopherol), ubiquinone (Q10), vitamin A and zinkoxide for four weeks in a double blind, randomized and placebo controlled manner. Before and after the intervention blood was withdrawn and general blood parameters were analyzed. In addition, lipid soluble antioxidants were analyzed in blood plasma by HPLC and the water soluble antioxidative properties were tested with the enzymic xanthin/xanthinoxidase-reaction. In summary the results show that the smoker-verum group exhibit a significant down regulation of the leukocyte counts. The test for antioxidants show the following significant differences after intervention: Smokers exhibit an increase of both vitamin E and coenzyme Q10 and an attenuation of their (before intervention) clearly increased water soluble-antioxidative potential, non-smokers showed only an increase of vitamin E and trends of an increase of Q10 and water soluble-antioxidative potential. These results may contribute to the discussion of the intrinsic deficiency brought about by smoking and the possible attenuation of part of these deficiency by increasing the intake of certain vitamins or food additives.     

Huntingtin-interacting protein HIP14 is a palmitoyl transferase involved in palmitoylation and trafficking of multiple neuronal proteins

In neurons, posttranslational modification by palmitate regulates the trafficking and function of signaling molecules, neurotransmitter receptors, and associated synaptic scaffolding proteins. However, the enzymatic machinery involved in protein palmitoylation has remained elusive. Here, using biochemical assays, we show that huntingtin (htt) interacting protein, HIP14, is a neuronal palmitoyl transferase (PAT). HIP14 shows remarkable substrate specificity for neuronal proteins, including SNAP-25, PSD-95, GAD65, synaptotagmin I, and htt. Conversely, HIP14 is catalytically invariant toward paralemmin and synaptotagmin VII. Exogenous HIP14 enhances palmitoylation-dependent vesicular trafficking of several acylated proteins in both heterologous cells and neurons. Moreover, interference with endogenous expression of HIP14 reduces clustering of PSD-95 and GAD65 in neurons. These findings define HIP14 as a mammalian palmitoyl transferase involved in the palmitoylation and trafficking of multiple neuronal proteins.     

High phylogenetic diversity of transconjugants carrying plasmid pJP4 in an activated sludge-derived microbial community

Transconjugants of plasmid pJP4, originating from an agar plate mating of a Pseudomonas putida donor with an activated sludge-derived microbial community, were isolated and identified by partial 16S rDNA sequencing. The transconjugant strains belonged to a variety of genera of the alpha-, beta-, gamabeta- and gamma-classes of the Proteobacteria, mostly to the families Rhizobiaceae and Comamonadaceae and the genus Stenotrophomonas. Only P. putida and Delftia spp. strains were able to grow on 2,4-D as the sole carbon source.     

The effects of reduced oxygen tension on swine granulosa cell

Follicular growth is characterized by an augmented vascularization, possibly driven by a fall in the oxygen supply. The present study was undertaken to investigate the effects of hypoxia on swine granulosa cells. At first, we quantified oxygen partial pressure (pO₂) in follicular fluid from different size follicles; the granulosa cells collected from large follicles (>5 mm) were subjected for 18 h to normoxia (19% O₂), partial (5% O₂) or total hypoxia (1% O₂). The effects of these conditions were tested on the main parameters of granulosa cell function, steroidogenesis and cell proliferation, and on vascular endothelial growth factor (VEGF), nitric oxide (NO) and superoxide anion (O₂⁻) production. Oxygen tension in follicular fluid was negatively related to follicular size, pointing out a gradual reduction during follicular growth. Severe hypoxic conditions determined a reduction of both 17β estradiol and progesterone production, while partial hypoxia did not seem to affect them. Hypoxia increased VEGF as well as O₂⁻ production in swine granulosa cells without impairing cell growth; in addition, it decreased NO output.

We may conclude that physiological hypoxia could play a pivotal role in the follicular angiogenic process stimulating VEGF synthesis by granulosa cells. ROS are possibly involved in hypoxic signalling.     

Reconstitution of the type-1 active site of the H145G/A variants of nitrite reductase by ligand insertion

Variants of the copper-containing nitrite reductase (NiR) of Alcaligenes faecalis S6 were constructed by site-directed mutagenesis, by which the C-terminal histidine ligand (His145) of the Cu in the type-1 site was replaced by an alanine or a glycine. The type-1 sites in the NiR variants as isolated, are in the reduced form, but can be oxidized in the presence of external ligands, like (substituted) imidazoles and chloride. The reduction potential of the type-1 site of NiR-H145A reconstituted with imidazole amounts to 505 mV vs NHE (20 degrees C, pH 7, 10 mM imidazole), while for the native type-1 site it amounts to 260 mV. XRD data on crystals of the reduced and oxidized NiR-H145A variant show that in the reduced type-1 site the metal is 3-coordinated, but in the oxidized form takes up a ligand from the solution. With the fourth (exogenous) ligand in place the type-1 site is able to accept electrons at about the same rate as the wt NiR, but it is unable to pass the electron onto the type-2 site, leading to loss of enzymatic activity. It is argued that the uptake of an electron by the mutated type-1 site is accompanied by a loss of the exogenous ligand and a concomitant rise of the redox potential. This rise effectively traps the electron in the type-1 site.