A Bethlem myopathy Gly to Glu mutation in the von Willebrand factor A domain N2 of the collagen alpha3(VI) chain interferes with protein folding.

A single G1679E mutation in the amino-terminal globular domain N2 of the alpha3 chain of type VI collagen was found in a large family affected with Bethlem myopathy. Recombinant production of N2 ( approximately 200 residues) in transfected mammalian cells has now been used to examine the possibility that the mutation interfered with protein folding. The wild-type form and a G1679A mutant were produced at high levels and shown to fold into a stable globular structure. Only a small amount of secretion was observed for mutants G1679E and G1679Q, which apparently were efficiently degraded within the cells. Homology modeling onto the related von Willebrand factor A1 structure indicated that substitution of G1679 by the bulky E or Q cannot be accommodated without considerable changes in the folding pattern. This suggests protein misfolding as a molecular basis for this particular mutation in Bethlem myopathy, in agreement with radioimmunoassay data showing reduced levels of domain N2 in cultured fibroblasts from two patients.     

Anwendung der automatisierten Festphasenextraktion bei der Bestimmung von Ochratoxin A

For some foodstuffs, determination of the mycotoxin ochratoxin A (OTA) requires time consuming clean up by means of solid phase extraction (SPE). Therefore a system for automated SPE was tested for cleaning up roasted coffee as a possible way of shortening preparation time. Validation of the method in accordance to the so called “Concept '98” led to a LOD of 0.2 μg/kg and a recovery rate of 92%. By using the described procedure with samples of roasted coffee the OTA contents varied between the LOD and 3.4 μg/kg. This method was also used to determine ochratoxin A in liquorice roots, ginger and valerian.

Presented at the 26^th Mykotoxin Workshop in Herrsching, Germany, May 17–19, 2004     

Use of cyanuric chloride-activated paper for detection of subpicogram quantities of specific DNA sequences and its application to linked restriction fragment length polymorphism analysis in a Duchenne muscular dystrophy affected family

Conditions for the optimal use of cyanuric chloride-activated (CCA) paper in Southern transfer hybridization experiments of genomic DNA were investigated. They depend critically on pH and ionic strength during transfer and on the composition of the hybridization solution. Simplified hybridization conditions using a SSC/dextran sulfate system at 65 degrees C without sodium dodecyl sulfate and the complex Denhardt's solution are applied. CCA paper allows repeated use in hybridization experiments. Under optimized conditions CCA paper allows a more sensitive detection of single-copy gene sequences in the subpicogram range than do nylon membranes. Application of these transfer and hybridization conditions with our newly developed CCA paper to carrier determination and prediction of the healthy male haplotype demonstrates its usefulness for prenatal counseling of a Duchenne muscular dystrophy family.     

Clustering of halophytic species from Cyprus based on ionic contents

This paper presents the work conducted on the chemical constituents of some common and widely distributed halophyte taxa from Cyprus with the aim that these studies will help in the evaluation of halophytes for different economical purposes. The plant species of Crithmum maritimum L., Limbarda crithmoides (L.) Dumort, Atriplex portulacoides L., Salsola kali L., Atriplex halimus L., Limonium oleifolium Mill., L. meyeri (Boiss.) Kuntze; and Tetraena alba (L.f.) Beier & Thulin were collected in the middle of July. The shoot tissue and leaf samples were collected from the natural habitats and left for drying under air circulation followed by placing them in oven at 60 °C for 96 hours. The material was crushed using mortar and pestle and subjected to an analysis of macro- and micro-nutrients and biochemical compounds. K⁺/Na⁺ in the leaf tissues of the dicot species showed relatively high values depicting their behavior as Na+ includes but very low Cl- levels were recorded. Out of the species investigated here in 4 TFAA content was rather high. Values ranging from 0.5% to 1% dry weight were exhibited in one species. However, only 3 species showed very low TFAA values. Later may be due to low nitrogen availability in their environment. The phenetic analyses of eight halophyte species performed on the data matrix using Ntsys-pc program version 2.1 revealed that, cluster analysis of the overall results obtained here leads to 2 clusters. This discrimination appears to be as a result of their different abilities to accumulate either proline or glycine betaine.     

Surface hydrolysis of sphingomyelin by the outer membrane protein Rv0888 supports replication of Mycobacterium tuberculosis in macrophages

Sphingomyelinases secreted by pathogenic bacteria play important roles in host–pathogen interactions ranging from interfering with phagocytosis and oxidative burst to iron acquisition. This study shows that the Mtb protein Rv0888 possesses potent sphingomyelinase activity cleaving sphingomyelin, a major lipid in eukaryotic cells, into ceramide and phosphocholine, which are then utilized by Mtb as carbon, nitrogen and phosphorus sources, respectively. An Mtb rv0888 deletion mutant did not grow on sphingomyelin as a sole carbon source anymore and replicated poorly in macrophages indicating that Mtb utilizes sphingomyelin during infection. Rv0888 is an unusual membrane protein with a surface‐exposed C‐terminal sphingomyelinase domain and a putative N‐terminal channel domain that mediated glucose and phosphocholine uptake across the outer membrane in an M. smegmatis porin mutant. Hence, we propose to name Rv0888 as SpmT (sp hingomyelinase of M ycobacterium t uberculosis). Erythrocyte membranes contain up to 27% sphingomyelin. The finding that Rv0888 accounts for half of Mtb's hemolytic activity is consistent with its sphingomyelinase activity and the observation that Rv0888 levels are increased in the presence of erythrocytes and sphingomyelin by 5‐ and 100‐fold, respectively. Thus, Rv0888 is a novel outer membrane protein that enables Mtb to utilize sphingomyelin as a source of several essential nutrients during intracellular growth.     

Predicting the response of the deep-ocean microbiome to geochemical perturbations by hydrothermal vents

Submarine hydrothermal vents perturb the deep-ocean microbiome by injecting reduced chemical species into the water column that act as an energy source for chemosynthetic organisms. These systems thus provide excellent natural laboratories for studying the response of microbial communities to shifts in marine geochemistry. The present study explores the processes that regulate coupled microbial-geochemical dynamics in hydrothermal plumes by means of a novel mathematical model, which combines thermodynamics, growth and reaction kinetics, and transport processes derived from a fluid dynamics model. Simulations of a plume located in the ABE vent field of the Lau basin were able to reproduce metagenomic observations well and demonstrated that the magnitude of primary production and rate of autotrophic growth are largely regulated by the energetics of metabolisms and the availability of electron donors, as opposed to kinetic parameters. Ambient seawater was the dominant source of microbes to the plume and sulphur oxidisers constituted almost 90% of the modelled community in the neutrally-buoyant plume. Data from drifters deployed in the region allowed the different time scales of metabolisms to be cast in a spatial context, which demonstrated spatial succession in the microbial community. While growth was shown to occur over distances of tens of kilometers, microbes persisted over hundreds of kilometers. Given that high-temperature hydrothermal systems are found less than 100 km apart on average, plumes may act as important vectors between different vent fields and other environments that are hospitable to similar organisms, such as oil spills and oxygen minimum zones.