Novel role of phospholipase C-delta1: regulation of liver mitochondrial Ca2+ uptake

Mitochondrial Ca2+ (mCa2+) handling is an important regulator of liver cell function that controls events ranging from cellular respiration and signal transduction to apoptosis. Cytosolic Ca2+ enters mitochondria through the ruthenium red-sensitive mCa2+ uniporter, but the mechanisms governing uniporter activity are unknown. Activation of many Ca2+ channels in the cell membrane requires PLC. This activation commonly occurs through phosphitidylinositol-4,5-biphosphate (PIP2) hydrolysis and the production of the second messengers inositol 1,4,5-trisphosphate [I(1,4,5)P3] and 1,2-diacylglycerol (DAG). PIP2 was recently identified in mitochondria. We hypothesized that PLC exists in liver mitochondria and regulates mCa2+ uptake through the uniporter. Western blot analysis with anti-PLC antibodies demonstrated the presence of PLC-delta1 in pure preparations of mitochondrial membranes isolated from rat liver. In addition, the selective PLC inhibitor U-73122 dose-dependently blocked mCa2+ uptake when whole mitochondria were incubated at 37 degrees C with 45Ca2+. Increasing extra mCa2+ concentration significantly stimulated mCa2+ uptake, and U-73122 inhibited this effect. Spermine, a uniporter agonist, significantly increased mCa2+ uptake, whereas U-73122 dose-dependently blocked this effect. The inactive analog of U-73122, U-73343, did not affect mCa2+ uptake in any experimental condition. Membrane-permeable I(1,4,5)P3 receptor antagonists 2-aminoethoxydiphenylborate and xestospongin C also inhibited mCa2+ uptake. Although extra mitochondrial I(1,4,5)P3 had no effect on mCa2+ uptake, membrane-permeable DAG analogs 1-oleoyl-2-acetyl-sn-glycerol and DAG-lactone, which inhibit PLC activity, dose-dependently inhibited mCa2+ uptake. These data indicate that PLC-delta1 exists in liver mitochondria and is involved in regulating mCa2+ uptake through the uniporter.     

Standardless screening of chemical warfare agents based on gas chromatographic data

A method for calculation of the retention time of a compound in a temperature-programmed mode of GC analyses is proposed. The method is based on a detailed consideration of thermodynamics of chromatographic process and account of the actual state of a capillary column. The suggested approach permits estimation of probability of a poisonous agent in tests by means of information stored in the database, eliminating a need for reference standard of such an agent.     

Crystal structure of the YffB protein from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> suggests a glutathione-dependent thiol reductase function

Background  

The yffB (PA3664) gene of Pseudomonas aeruginosa encodes an uncharacterized protein of 13 kDa molecular weight with a marginal sequence similarity to arsenate reductase from Escherichia coli. The crystal structure determination of YffB was undertaken as part of a structural genomics effort in order to assist with the functional assignment of the protein.     

Biochemical and heart adaptations to physical training and supplementation with amino acids

This study evaluated the role of amino acids supplementation on the heart's adaptation under extensive training conditions. Sixty active athletes (bicyclists and swimmers) were separated into 2 groups: 30 were given amino acid mixture (1 g per 10 kg of body weight) for a period of 1 month, and the other 30 were given placebo for the same duration (control group). In the same time period, 20 subjects of similar age not engaged in physical training or sports activities were used as the additional control group. Blood concentrations of alanine transaminase (ALT), asparagine transaminase, lactate dehydrogenase (LDH), gamma-glutamil transpeptidase, alkaline phosphatase (ALP), amylase, triglycerides, albumin, interleukin-6 (IL-6), and interleukin-10 (IL-10) were determined for all subjects before and after the intervention period. Concentrations of LDH and ALP were increased, but concentrations of ALT, albumin, and triglycerides were decreased in the blood of trained athletes compared with healthy subjects not engaged in sports activities. In the athletes, some increases in IL-6 levels were noted; however, they were significantly (p < 0.05) lower than in patients with myocardiodystrophy. The values of IL-10 in athletes were higher than concentrations of IL-10 in patients with myocardiodystrophy but still lower than the normal values. The inhibition of IL-10 in blood may play an important role in the induction of apoptosis in cells of the heart muscle. After amino acid supplementation, the athletes' values for albumin, triglycerides, IL-10, LDH, and ALP were significantly increased compared with the post-placebo control groups. Enzyme activities of other enzymes remained unchanged in all groups. Histological data from a secondary study of actual heart tissue showed that the amino acids supplementation may have inhibiting effects on myocardial apoptosis. The criteria of efficiency of the amino acids supplementation were defined by the albumin, IL-6, and IL-10 concentrations.     

Antizyme frameshifting as a functional probe of eukaryotic translational termination

Translation termination in eukaryotes is mediated by the release factors eRF1 and eRF3, but mechanisms of the interplay between these factors are not fully understood, due partly to the difficulty of measuring termination on eukaryotic mRNAs. Here, we describe an in vitro system for the assay of termination using competition with programmed frameshifting at the recoding signal of mammalian antizyme. The efficiency of antizyme frameshifting in rabbit reticulocyte lysates was reduced by addition of recombinant rabbit eRF1 and eRF3 in a synergistic manner. Addition of suppressor tRNA to this assay system revealed competition with a third event, stop codon readthrough. Using these assays, we demonstrated that an eRF3 mutation at the GTPase domain repressed termination in a dominant negative fashion probably by binding to eRF1. The effect of the release factors and the suppressor tRNA showed that the stop codon at the antizyme frameshift site is relatively inefficient compared to either the natural termination signals at the end of protein coding sequences or the readthrough signal from a plant virus. The system affords a convenient assay for release factor activity and has provided some novel views of the mechanism of antizyme frameshifting.     

Structural and functional analysis of the actin binding domain of plectin suggests alternative mechanisms for binding to F-actin and integrin beta4

Plectin is a widely expressed cytoskeletal linker. Here we report the crystal structure of the actin binding domain of plectin and show that this region is sufficient for interaction with F-actin or the cytoplasmic region of integrin alpha6beta4. The structure is formed by two calponin homology domains arranged in a closed conformation. We show that binding to F-actin induces a conformational change in plectin that is inhibited by an engineered interdomain disulfide bridge. A two-step induced fit mechanism involving binding and subsequent domain rearrangement is proposed. In contrast, interaction with integrin alpha6beta4 occurs in a closed conformation. Competitive binding of plectin to F-actin and integrin alpha6beta4 may rely on the observed alternative binding mechanisms and involve both allosteric and steric factors.