Fungal virulence studies come of age

Sophisticated molecular biological research has revealed many virulence attributes in at least four pathogenic fungi, but the future study of fungal virulence requires investigators to distinguish between molecules that directly interact with the host, molecules that regulate these, and molecules that are always required for fungal growth and survival, independent of the host.     

The redox state of the ascorbate-dehydroascorbate pair as a specific sensor of cell division in tobacco BY-2 cells

The effects of ascorbate (ASC) and dehydroascorbate (DHA) on cell proliferation were examined in the tobacco Bright Yellow 2 (TBY-2) cell line to test the hypothesis that the ASC-DHA pair is a specific regulator of cell division. The hypothesis was tested by measuring the levels of ASC and DHA or another general redox pair, glutathione (GSH) and glutathione disulfide (GSSG), during the exponential-growth phase of TBY-2 cells. A peak in ASC, but not GSH, levels coincided with a peak in the mitotic index. Moreover, when the cells were enriched with ascorbate, a stimulation of cell division occurred whereas, when the cells were enriched with DHA, the mitotic index was reduced. In contrast, glutathione did not affect the mitotic-index peak during this exponential-growth phase. The data are consistent in showing that the ASC-DHA pair acts as a specific redox sensor which is part of the mechanism that regulates cell cycle progression in this cell line.     

Can Zipf's law be adapted to normalize microarrays?

Background  

Normalization is the process of removing non-biological sources of variation between array experiments. Recent investigations of data in gene expression databases for varying organisms and tissues have shown that the majority of expressed genes exhibit a power-law distribution with an exponent close to -1 (i.e. obey Zipf's law). Based on the observation that our single channel and two channel microarray data sets also followed a power-law distribution, we were motivated to develop a normalization method based on this law, and examine how it compares with existing published techniques. A computationally simple and intuitively appealing technique based on this observation is presented.     

Expression of collagen VI α5 and α6 chains in human muscle and in Duchenne muscular dystrophy-related muscle fibrosis

Collagen VI is a major extracellular matrix (ECM) protein with a critical role in maintaining skeletal muscle functional integrity. Mutations in COL6A1, COL6A2 and COL6A3 genes cause Ullrich Congenital Muscular Dystrophy (UCMD), Bethlem Myopathy, and Myosclerosis. Moreover, Col6a1(-/-) mice and collagen VI deficient zebrafish display a myopathic phenotype. Recently, two additional collagen VI chains were identified in humans, the α5 and α6 chains, however their distribution patterns and functions in human skeletal muscle have not been thoroughly investigated yet. By means of immunofluorescence analysis, the α6 chain was detected in the endomysium and perimysium, while the α5 chain labeling was restricted to the myotendinous junctions. In normal muscle cultures, the α6 chain was present in traces in the ECM, while the α5 chain was not detected. In the absence of ascorbic acid, the α6 chain was mainly accumulated into the cytoplasm of a sub-set of desmin negative cells, likely of interstitial origin, which can be considered myofibroblasts as they expressed α-smooth muscle actin. TGF-β1 treatment, a pro-fibrotic factor which induces trans-differentiation of fibroblasts into myofibroblasts, increased the α6 chain deposition in the extracellular matrix after addition of ascorbic acid. In order to define the involvement of the α6 chain in muscle fibrosis we studied biopsies of patients affected by Duchenne Muscular Dystrophy (DMD). We found that the α6 chain was dramatically up-regulated in fibrotic areas where, in contrast, the α5 chain was undetectable. Our results show a restricted and differential distribution of the novel α6 and α5 chains in skeletal muscle when compared to the widely distributed, homologous α3 chain, suggesting that these new chains may play specific roles in specialized ECM structures. While the α5 chain may have a specialized function in tissue areas subjected to tensile stress, the α6 chain appears implicated in ECM remodeling during muscle fibrosis.     

Analysis of GTPases carrying hydrophobic amino acid substitutions in lieu of the catalytic glutamine: implications for GTP hydrolysis

Ras superfamily GTP-binding proteins regulate important signaling events in the cell. Ras, which often serves as a prototype, efficiently hydrolyzes GTP in conjunction with its regulator GAP. A conserved glutamine plays a vital role in GTP hydrolysis in most GTP-binding proteins. Mutating this glutamine in Ras has oncogenic effects, since it disrupts GTP hydrolysis. The analysis presented here is of GTP-binding proteins that are a paradox to oncogenic Ras, since they have the catalytic glutamine (Glncat) substituted by a hydrophobic amino acid, yet can hydrolyze GTP efficiently. We term these proteins HAS-GTPases. Analysis of the amino acid sequences of HAS-GTPases reveals prominent presence of insertions around the GTP-binding pocket. Homology modeling studies suggest an interesting means to achieve catalysis despite the drastic hydrophobic substitution replacing the key Glncat of Ras-like GTPases. The substituted hydrophobic residue adopts a "retracted conformation," where it is positioned away from the GTP, as its role in catalysis would be unproductive. This conformation is further stabilized by interactions with hydrophobic residues in its vicinity. These interacting residues are strongly conserved and hydrophobic in all HAS-GTPases, and correspond to residues Asp92 and Tyr96 of Ras. An experimental support for the "retracted conformation" of Switch II arises from the crystal structures of Ylqf and hGBP1. This conformation allows us to hypothesize that, unlike in classical GTPases, catalytic residues could be supplied by regions other than the Switch II (i.e., either the insertions or a neighboring domain).