Effect of Helminthosporic Root Rot on the Lipid Content in Wheat Seedlings

The concentration of dry matter and the content of esterified fatty acids in total lipids of roots and etiolated shoots of 3- to 10-day-old seedlings of wheat (Triticum aestivum L.) infected with Bipolaris sorokiniana (Sacc.) Schoemaker, the agent of helminthosporic root rot, were determined in the course of germination. At the onset of germination, fungal infection caused a considerable increase in the dry matter concentration in both roots and shoots due to the enhanced mobilization of seed reserves. However, after the 7th day of germination, dry matter concentration fell below the level of noninfected control seedlings as a result of infection. The content of total lipids rose immediately after infection and always exceeded the control index up to the end of germination, in spite of a continuous decrease in this index in both control and infected seedling. It is concluded that an increase in the content of cellular lipids is a characteristic response of both shoots and roots to the root rot infection of wheat seedlings.     

Structure of the Cytosolic Portion of the Motor Protein Prestin and Functional Role of the STAS Domain in SLC26/SulP Anion Transporters

Prestin is the motor protein responsible for the somatic electromotility of cochlear outer hair cells and is essential for normal hearing sensitivity and frequency selectivity of mammals. Prestin is a member of mammalian solute-linked carrier 26 (SLC26) anion exchangers, a family of membrane proteins capable of transporting a wide variety of monovalent and divalent anions. SLC26 transporters play important roles in normal human physiology in different tissues, and many of them are involved in genetic diseases. SLC26 and related SulP transporters carry a hydrophobic membrane core and a C-terminal cytosolic portion that is essential in plasma membrane targeting and protein function. This C-terminal portion is mainly composed of a STAS (sulfate transporters and anti-sigma factor antagonist) domain, whose name is due to a remote but significant sequence similarity with bacterial ASA (anti-sigma factor antagonist) proteins. Here we present the crystal structure at 1.57 Å resolution of the cytosolic portion of prestin, the first structure of a SulP transporter STAS domain, and its characterization in solution by heteronuclear multidimensional NMR spectroscopy. Prestin STAS significantly deviates from the related bacterial ASA proteins, especially in the N-terminal region, which—although previously considered merely as a generic linker between the domain and the last transmembrane helix—is indeed fully part of the domain. Hence, unexpectedly, our data reveal that the STAS domain starts immediately after the last transmembrane segment and lies beneath the lipid bilayer. A structure-function analysis suggests that this model can be a general template for most SLC26 and SulP anion transporters and supports the notion that STAS domains are involved in functionally important intramolecular and intermolecular interactions. Mapping of disease-associated or functionally harmful mutations on STAS structure indicates that they can be divided into two categories: those causing significant misfolding of the domain and those altering its interaction properties.     

Cardioprotective activity of a novel and potent competitive inhibitor of lactate dehydrogenase

Alkaline incubation of NADH results in the formation of a very potent inhibitor of lactate dehydrogenase. High resolution mass spectroscopy along with NMR characterization clearly showed that the inhibitor is derived from attachment of a glycolic acid moiety to the 4-position of the dihydronicotinamide ring of NADH. The very potent inhibitor is competitive with respect to NADH. The inhibitor added in submicromolar concentrations to cardiomyocytes protects them from damage caused by hypoxia/reoxygenation stress. In isolated mouse hearts, addition of the inhibitor results in a substantial reduction of myocardial infarct size caused by global ischemia/reperfusion injury.     

Sequence Insertions and ITS Data Provide Congruent Information on Roccella canariensis and R. tuberculata (Arthoniales, Euascomycetes) Phylogeny

Four Roccella species, R. canariensis, R. fimbriata, R. montagnei, and R. tuberculata, were found to possess sequence insertions in up to four locations in the first half of the SSU rDNA. Insertions from one of these positions have been classified as group I introns, while the others may represent degenerative forms of group I introns or messenger RNA introns. Two of the insertion-containing taxa, R. canariensis and R. tuberculata, differ only in their dispersal strategy: R. canariensis is sexual, producing only fruiting bodies and R. tuberculata is sterile, producing only vegetative propagules, i.e., soredia. Because insertions occurred in specimens of both taxa, they were used to examine the phylogenetic relationships between and within the two species. The sequence insertions from each of the four positions were aligned and cladistically analyzed separately. Internal transcribed spacers (ITS) were additionally sequenced to study the phylogeny of all R. canariensis and R. tuberculata specimens. Three other Roccella species (R. babingtonii, R. fimbriata, and R. montagnei) and Dirina catalinariae were used as outgroups in this parsimony analysis. Sequence insertions were found to be potentially useful in phylogenetic studies, although due to the sequence dissimilarity, homology relations were difficult to establish above the species level and in some cases even within the species. The phylogenies obtained from the insertion matrices were totally consistent with the ITS data and the insertions were concluded to have been inherited. When the insertion and ITS data were combined for total evidence, R. canariensis and R. tuberculata did not form distinct lineages in the phylogenetic tree, but appeared mixed in well-supported groups containing both sorediate and fertile specimens.     

Effects of vitamin B6 supplementation in rats during lactation on vitamin B6 concentration and transaminase activities in the offspring

The aim of the present investigation was to study the effect of a varying maternal vitamin B₆ supplementation during lactation period on vitamin B₆ levels in blood, liver and total body, and on the activity of two transaminase enzymes in the offspring. Therefore, eighty female Sprague‐Dawley rats were fed a semi‐synthetic diet (0.2 mg vitamin B₆ per kg) which was supplemented during gravidity with 5 mg vitamin B₆ per kg diet. During the following lactation period the rats were assigned to one of 10 vitamin B₆ treatment groups (supplementation of 0, 3, 6, 9, 12, 15, 18, 36, 360, 3600 mg vitamin B₆ per kg diet). At day 14 of lactation the pubs of all dams were decapitated and blood, liver, and carcass were used for analysis of vitamin B₆ concentration, activities of two transaminases, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in plasma, erythrocytes, and liver, and of haematological parameters.

While the liver and total body wet weights as well as the haematological parameters (red blood cells, haemoglobin concentration, hematocrit, middle corpuscular cell volume, middle corpuscular haemoglobin, middle corpuscular haemoglobin concentration) did not differ within the experimental groups, the present data clearly show that in blood, liver and total body of the offspring exists a slight dose‐response relationship between the maternal dietary vitamin B₆ supplementation and the vitamin B₆ concentration. Concerning the activities of the transaminases a dietary supplementation above 3mg vitamin B₆ per kg diet had no influence on the AST and ALT activities in offspring plasma. In the erythrocytes no statistical significant influence of the vitamin B₆ supplementation during lactation on the activities of AST and ALT was found. The activities of ALT and AST in liver were not consistently altered by the vitamin B₆ supplementation of the dams during lactation. In conclusion these results indicate that a minimal maternal dietary vitamin B₆ supply of 3.1 mg per kg diet is necessary with regard to health and development of their offspring. But not all of the analysed parameters as the liver and total body weights, the activities of AST and ALT in the erythrocytes, and the haematological parameters were influenced by a deficient maternal dietary vitamin B₆ supply.     

The activities of MMP‐9 and total gelatinase respond differently to substrate coating and cyclic mechanical stretching in fibroblasts and myoblasts

The current study was designed to investigate whether the activities of TGC (total gelatinase and collagenase) as well as MMP‐9 (matrix metalloproteinase‐9, gelatinase B) secreted by the cultured fibroblasts and myoblasts were influenced by the specific extracellular substrates and by cyclic mechanical strain. Fibroblasts (Rat 2) and myoblasts (C2C12) were cultured with either fibronectin, laminin or collagen type I for 24 h and applied with or without a biaxial deformation at 1 Hz using the Flexcell FX‐4000 system. MMP‐9 activity was increased in fibroblasts when the cells were in contact with fibronectin and laminin, while in myoblasts, enhanced activity of the secreted enzyme was only observed when collagen was present. TGC activity expressed from myoblasts was increased in cells growing on all three types of extracellular proteins in response to the mechanical stimulation, but in fibroblasts, such an increase was only observed in cells grown on the laminin coating. In summary, our data demonstrate that the activities of MMP‐9 synthesized by fibroblasts tend to be regulated by the specific extracellular protein the cells are in contact with, whereas the gelatinolytic actions of proteases produced by myoblasts are more responsive to the mechanical deformation.     

Glutathione cycle in stable chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation and oxidant/antioxidant imbalance. Glutathione is the most abundant cellular low‐molecular weight thiol and the glutathione redox cycle is the fundamental component of the cellular antioxidant defence system. Concentration of total glutathione and catalytic activities of glutathione peroxidase and glutathione reductase were determined in peripheral blood of patients (n = 109) and healthy subjects (n = 51). Concentration of total glutathione in patients was not changed in comparison to healthy controls. However, we found statistically significant difference between patients with moderate and severe disease stages. Glutathione reductase activity was increased, while glutathione proxidase activity was decreased in the patients with COPD, when compared to healthy controls. We found no significant difference in glutathione peroxidase and glutathione reductase activities between stages. Patients who smoked had lower concentration of total glutathione compared with former smokers and never‐smoking patients. Lung function parameters were inversely associated with glutathione level. Evidence is presented for differential modulation of glutathione peroxidase and glutathione reductase activities in peripheral blood of patients with stable COPD. We suppose that in addition to glutathione biosynthesis, glutathione reductase‐dependent regulation of the glutathione redox state is vital for protection against oxidative stress. Copyright © 2010 John Wiley & Sons, Ltd.     

Significance of magnetic resonance image and blood manganese measurement for the assessment of brain manganese during total parenteral nutrition in rats

In this study, we report on the influence of trace elements (TE) on signal intensities of nuclear magnetic resonance images (MRI), both in vivo and in vitro. Optimal parameters for the assessment of Mn concentration in the brain of rats on total parenteral nutrition were established. For the in vitro study, Mn and trace element solutions, one containing Zn, Cu, Fe, and I (TE-4) and another containing the above elements plus Mn (TE-5), were diluted with physiological saline or with rat brain homogenate and used to measure signal intensities in MRI. Concentration-dependent signal hyperintensity was observed in both cases in the Mn and the TE-5 solutions, but no effect was observed with the TE-4 solution. The signal increase was greater for brain tissue homogenates. In the in vivo study, the experimental animals were maintained under total parenteral nutrition (TPN) with a standard clinical dose of TE-5 and/or with 10-fold the clinical dose of TE-4 and TE-5 for 1 wk. Only rats that were receiving the increased TE-5 dose showed signal hyperintensity on MRI. Positive correlations were observed among the signal hyperintensity, the blood Mn concentrations, and that of the rat brain. Our results suggest that Mn in TE preparations may be the cause of signal hyperintensity on MRI in a concentration-dependent fashion, and that MRI and measurement of blood Mn may be used to estimate Mn accumulation in brain tissue.     

How to Arm a Supervillin: Designing F-Actin Binding Activity into Supervillin Headpiece

Villin-type headpiece domains are compact motifs that have been used extensively as model systems for protein folding. Although the majority of headpiece domains bind actin, there are some that lack this activity. Here, we present the first NMR solution structure and ¹⁵N-relaxation analysis of a villin-type headpiece domain natively devoid of F-actin binding activity, that of supervillin headpiece (SVHP). The structure was found to be similar to that of other headpiece domains that bind F-actin. Our NMR analysis demonstrates that SVHP lacks a conformationally flexible region (V-loop) present in all other villin-type headpiece domains and which is essential to the phosphoryl regulation of dematin headpiece. In comparing the electrostatic surface potential map of SVHP to that of other villin-type headpiece domains with significant affinity for F-actin, we identified a positive surface potential conserved among headpiece domains that bind F-actin but absent from SVHP. A single point mutation (L38K) in SVHP, which creates a similar positive surface potential, endowed SVHP with specific affinity for F-actin that is within an order of magnitude of the tightest binding headpiece domains. We propose that this effect is likely conferred by a specific buried salt bridge between headpiece and actin. As no high-resolution structural information exists for the villin-type headpiece F-actin complex, our results demonstrate that through positive mutagenesis, it is possible to design binding activity into homologous proteins without structural information of the counterpart's binding surface.