Expression of α1-adrenergic receptor subtypes in heart cell culture

Three ₁AR subtypes have been cloned so far and are designated as _1a, _1b, and _1d. Organspecific distribution pattern and subtype-specific effects are known but not fully understood. To address a cell-type specific expression pattern in the heart we investigated expression pattern of ₁AR subtypes on RNA and proteinlevel in heart tissue, cultured cardiomyocytes and nonmyocytes of the rat. Each ₁ARsubtype mRNA was present in neonatal and adult rat heart culture but the relative distribution pattern was significantly different. While the _1aAR subtype is preferentially expressed in adult cardiomyocytes, the _1bAR subtype was preferentially expressed in the nonmyocyte cell fraction. The RTPCR results were confirmed by Westernblotting (_1b) and immunocytochemical studies. Incubation with an ₁agonist (phenylephrine) for 72 h led to a significant reduction of the _1bAR in neonatal heart cell culture on both mRNA and protein level. In contrast, incubation with an ₁antagonist (prazosin) induced a 1.6 fold upregulation of the _1aAR mRNA without significant effects on radioligand binding and functional assay. The results indicate a distribution pattern of the ₁AR subtype which is specific for cell type and ontogeny of the rat heart and may be regulated by adrenergic agents.     

Molecular and functional characterisation of mild MCAD deficiency

We report a novel mild variant of medium-chain acyl-CoA dehydrogenase deficiency (MCADD) diagnosed in four infants who, in neonatal screening, showed abnormal acylcarnitine profiles indicative of MCADD. Three patients showed completely normal urinary organic acids and phenylpropionic acid loading tests were normal in all four patients. Enzyme studies showed residual MCAD activities between "classical" MCADD and heterozygotes. ACADM gene analysis revealed compound heterozygosity for the common mutation K329E and a novel mutation, Y67H, in two cases, and homozygosity for mutation G267R and the novel mutation S245L, respectively, in two children of consanguineous parents. As in other metabolic disorders, the distinction between "normal" and "disease" in MCAD deficiency is blurring into a spectrum of enzyme deficiency states caused by different mutations in the ACADM gene potentially influenced by factors affecting intracellular protein processing.     

Classification accuracy in multiple color fluorescence imaging microscopy

BACKGROUND: The discriminatory power and imaging efficiency of different multicolor FISH (M-FISH) analysis systems are key factors in obtaining accurate and reproducible classification results. In a recent paper, Garini et al. put forth an analytical technique to quantify the discriminatory power ("S/N ratio") and imaging efficiency ('excitation efficiency') of multicolor fluorescent karyotyping systems. METHODS: A parametric model of multicolor fluorescence microscopy, based on the Beer-Lambert law, is analyzed and reduced to a simple expression for S/N ratio. Parameters for individual system configurations are then plugged into the model for comparison purposes. RESULTS: We found that several invalid assumptions, which are used to reduce the complex mathematics of the Beer-Lambert law to a simple S/N ratio, result in some completely misleading conclusions about classification accuracy. The authors omit the most significant noise source, and consider only one highly abstract and unrepresentative situation. Unwisely chosen parameters used in the examples lead to predictions that are not consistent with actual results. CONCLUSIONS: The earlier paper presents an inaccurate view of the M-FISH situation. In this short communication, we point out several inaccurate assumptions in the mathematical development of Garini et al. and the poor choices of parameters in their examples. We show results obtained with different imaging systems that indicate that reliable and comparable results are obtained if the metaphase samples are well-hybridized. We also conclude that so-called biochemical noise, not photon noise, is the primary factor that limits pixel classification accuracy, given reasonable exposure times. Copyright Wiley-Liss, Inc.     

Extrusion of wheat or sorghum and/or addition of exogenous enzymes to pig diets influences the large intestinal microbiota but does not prevent development of swine dysentery following experimental challenge

A study was made of dietary influences on the large intestinal microbiota of pigs and on the incidence of swine dysentery (SD) after experimental infection with Brachyspira hyodysenteriae, the aetiological agent of SD. Animals were fed diets based either on wheat (expts 1 and 2) or sorghum (expt 2). Grains were ground and fed either raw or after high temperature and pressure extrusion and/or after addition of exogenous enzymes to the whole diet to reduce the starch and soluble non-starch polysaccharides available for fermentation in the large intestine. Limiting fermentation creates conditions that apparently reduce the incidence of SD after infection with B. hyodysenteriae. The diets were fed to weaned pigs for 4-6 weeks, then half the animals on each diet were killed and gut samples collected for microbiology. The treatments had little effect on bacterial numbers. In expt 1, dietary extrusion of wheat reduced lactobacilli in the large intestine. Addition of enzymes to extruded wheat-based diets in expt 2 reduced facultative anaerobes and increased non-sporing anaerobes. Addition of enzymes to a raw sorghum diet in expt 3 decreased numbers of facultative anaerobes, while extrusion of sorghum increased total anaerobes. Bacteroides spp. and Fusobacterium spp., which act in synergy with B. hyodysenteriae in SD, were isolated at a higher percentage in pigs fed the untreated wheat diet than in pigs fed the treated wheat diets. Following experimental infection the incidence of SD amongst pigs fed treated wheat diets was slightly lower than those fed the untreated diet, but with sorghum-based diets the opposite was found. Overall, the different dietary treatments used did not significantly reduce SD.     

SUT2, a putative sucrose sensor in sieve elements

In leaves, sucrose uptake kinetics involve high- and low-affinity components. A family of low- and high-affinity sucrose transporters (SUT) was identified. SUT1 serves as a high-affinity transporter essential for phloem loading and long-distance transport in solanaceous species. SUT4 is a low-affinity transporter with an expression pattern overlapping that of SUT1. Both SUT1 and SUT4 localize to enucleate sieve elements of tomato. New sucrose transporter-like proteins, named SUT2, from tomato and Arabidopsis contain extended cytoplasmic domains, thus structurally resembling the yeast sugar sensors SNF3 and RGT2. Features common to these sensors are low codon bias, environment of the start codon, low expression, and lack of detectable transport activity. In contrast to LeSUT1, which is induced during the sink-to-source transition of leaves, SUT2 is more highly expressed in sink than in source leaves and is inducible by sucrose. LeSUT2 protein colocalizes with the low- and high-affinity sucrose transporters in sieve elements of tomato petioles, indicating that multiple SUT mRNAs or proteins travel from companion cells to enucleate sieve elements. The SUT2 gene maps on chromosome V of potato and is linked to a major quantitative trait locus for tuber starch content and yield. Thus, the putative sugar sensor identified colocalizes with two other sucrose transporters, differs from them in kinetic properties, and potentially regulates the relative activity of low- and high-affinity sucrose transport into sieve elements.     

Preparation of selenoanhydro- and telluroanhydroglycofuranosides and some corresponding nucleosides

Methyl 2,3-anhydro-alpha-D-ribofuranoside (3a) was transformed into methyl 2-seleno-2,5-anhydro-alpha-D-arabinofuranoside (5a) and methyl 3-seleno-3,5-anhydro-alpha-D-xylofuranoside (6a) in two steps via the reaction of the C-5 mesylate of 3a, methyl 2,3-anhydro-5-O-mesyl-alpha-D-ribofuranoside (4a), with sodium hydrogen selenide. The corresponding beta anomer of 3a yielded methyl 3-seleno-3,5-anhydro-beta-D-xylofuranoside as the main product and only traces of methyl 2-seleno-2,5-anhydro-beta-D-arabinofuranoside. Sodium hydrogen telluride transformed 4a into methyl 2-telluro-2,5-anhydro-alpha-D-arabinofuranoside. Starting from 5a we prepared 1-(2-seleno-2,5-anhydro-alpha-D-arabinofuranosyl)uracil and the analogous thymidine nucleoside. Compound 6a could not be transformed into nucleosides.     

Identification and characterization of ecologically significant prokaryotes in the sediment of freshwater lakes: molecular and cultivation studies

The aim of this review is to interpret recent studies in which molecular methods were used to identify and characterize prokaryotes in lake sediments and related habitats. In the first part studies based on the phylogenetic diversity of prokaryotes found in lacustrine habitats are summarized. The application of various cultivation-independent methods for the characterization of distinct groups of sediment bacteria is exemplified with morphologically conspicuous, colorless sulfur bacteria in the second part of this review. Finally, traditional and recently developed methods are described which could be used for linking the function of microbial populations with their identification. The potential of these approaches for the study of lake sediments is discussed in order to give a perspective for future studies in this habitat.     

Meningeal cells stimulate and direct the migration of cerebellar external granule cells in vitro

The external granular layer is a secondary proliferative zone that arises from the caudolateral margin of the cerebellar ventricular zone and then spreads beneath the pial surface, eventually covering the entire cerebellar anlage. Here, both a part of the Bergmann glia and granule cells are generated. Selective destruction of the leptomeningeal cell layer during development in vivo disrupts the subpial extension of the external granular layer and the laminar deposition of its descendant cells. The mechanisms by which meningeal fibroblasts exert their controlling influence on cortical development have remained unclear but could involve diffusible factors and/or interactions mediated by direct cellular contacts. In order to test these assumptions, we have co-cultivated cerebellar slice explants with meningeal cells with and without interposition of a microfilter barrier. In this setup, meningeal cells by a diffusible factor stimulated the emigration of immature neurons exclusively from the external granular layer. This effect could also be elicited by fibroblasts from other tissues but not by nonfibroblastic cells such as, e.g., astroglia. In the Boyden chamber assay, the migration of undifferentiated neurons isolated from the external granular layer was chemotactically oriented towards the source of meningeal cell-conditioned media. In comparison, neurons from the internal granular layer did not respond to this stimulus. The attraction of immature neurons towards the pial surface could (1) represent a mechanism for the establishment of (subpial) secondary proliferative zones and (2) hypothetically also play a role in the outward-directed migration of postmitotic cells, e.g., in the isocortical anlage.     

Strategies to Rescue the Consequences of Inducible Arginase-1 Deficiency in Mice

Arginase-1 catalyzes the conversion of arginine to ornithine and urea, which is the final step of the urea cycle used to remove excess ammonia from the body. Arginase-1 deficiency leads to hyperargininemia in mice and man with severe lethal consequences in the former and progressive neurological impairment to varying degrees in the latter. In a tamoxifen-induced arginase-1 deficient mouse model, mice succumb to the enzyme deficiency within 2 weeks after inducing the knockout and retain <2 % enzyme in the liver. Standard clinical care regimens for arginase-1 deficiency (low-protein diet, the nitrogen-scavenging drug sodium phenylbutyrate, ornithine supplementation) either failed to extend lifespan (ornithine) or only minimally prolonged lifespan (maximum 8 days with low-protein diet and drug). A conditional, tamoxifen-inducible arginase-1 transgenic mouse strain expressing the enzyme from the Rosa26 locus modestly extended lifespan of neonatal mice, but not that of 4-week old mice, when crossed to the inducible arginase-1 knockout mouse strain. Delivery of an arginase-1/enhanced green fluorescent fusion construct by adeno-associated viral delivery (rh10 serotype with a strong cytomegalovirus-chicken β-actin hybrid promoter) rescued about 30% of male mice with lifespan prolongation to at least 6 months, extensive hepatic expression and restoration of significant enzyme activity in liver. In contrast, a vector of the AAV8 serotype driven by the thyroxine-binding globulin promoter led to weaker liver expression and did not rescue arginase-1 deficient mice to any great extent. Since the induced arginase-1 deficient mouse model displays a much more severe phenotype when compared to human arginase-1 deficiency, these studies reveal that it may be feasible with gene therapy strategies to correct the various manifestations of the disorder and they provide optimism for future clinical studies.