Modulation of the voltage-dependent anion-selective channel by cytoplasmic proteins from wild type and the channel depleted cells of Saccharomyces cerevisiae

It is well known that effective exchange of metabolites between mitochondria and the cytoplasm is essential for cell physiology. The key step of the exchange is transport across the mitochondrial outer membrane, which is supported by the voltage-dependent anion-selective channel (VDAC). Therefore, it is clear that the permeability of VDAC must be regulated to adjust its activity to the actual cell needs. VDAC-modulating activities, often referred to as the VDAC modulator, were identified in the intermembrane space of different organism mitochondria but the responsible protein(s) has not been identified as yet. Because the VDAC modulator was reported to act on VDAC of intact mitochondria when added to the cytoplasmic side it has been speculated that a similar modulating activity might be present in the cytoplasm. To check the speculation we used mitochondria of the yeast Saccharomyces cerevisiae as they constitute a perfect model to study VDAC modulation. The mitochondria contain only a single isoform of VDAC and it is possible to obtain viable mutants devoid of the channel (Deltapor1). Moreover, we have recently characterised a VDAC-modulating activity located in the intermembrane space of wild type and Deltapor1 S. cerevisiae mitochondria. Here, we report that the cytoplasm of wild type and Deltapor1 cells of S. cerevisiae contains a VDAC-modulating activity as measured in a reconstituted system and with intact mitochondria. Since quantitative differences were observed between the modulating fractions isolated from wild type and Deltapor1 cells when they were studied with intact wild type mitochondria as well as by protein electrophoresis it might be concluded that VDAC may influence the properties of the involved cytoplasmic proteins. Moreover, the VDAC-modulating activity in the cytoplasm differs distinctly from that reported for the mitochondrial intermembrane space. Nevertheless, both these activities may contribute efficiently to VDAC regulation. Thus, the identification of the proteins is very important.     

Preserved Tissue Allografts in Reconstructive Surgery

Results of treatment with three various kinds of allografts: lyophilized bone, deep frozen bone and cartilage preserved in physiological solution, all of them radiation-sterilized are presented. We believe that this presentation may be helpful in estimating the tissue bank's allografts and in establishing indications and contraindications in the application of allografts in surgery. The ‘indices of coincidence’ were compared in a group of 1014 patients after bone (lyophilized and radiation-sterilized) transplantation. It seems that such a variable as ‘rebuilding of graft’ may be of prognostic value in analysing the results of treatment in this group. The application of frozen and radiation-sterilized allogenic bone grafts for reconstructions is also described. An analysis of the results of treatment in 1125 patients reveals that the use of preserved bone reduces the extent and duration of surgery. Almost total substitution of grafts may be seen in 3–8 months after surgery. Allogenic, preserved cartilage is often used in facial reconstructions of face. Human costal cartilage, preserved in 0.9% NaCl and radiation-sterilized, was used for reconstruction. The patients were examined 24–190 months after surgery (in several clinical units) and results were collected in a special questionnaire by the team that performed surgery. In an analysed group of 437 patients after cartilage transplantation, 42.2% were operated because of posttraumatical changes, 29.0% because of congenital malformations and in 16.7% non-specific inflammations were the cause of reconstructive operations. Malformations were located mainly in the nose (59%), the ear concha (16.5%) and 10.9% were mandible. The results of treatment were compared with ages of patients, diagnosis and the locations of the changes. Very good results were achieved in 33.5% of the patients, and satisfactory in 41.8% of the patients. However, in 19.9% of the patients the result of treatment was unsatisfactory. Correlation between some clinical and biological characteristics and the result of treatment is under discussion. This revised version was published online in July 2006 with corrections to the Cover Date.     

Changes in antioxidant status of heart muscle tissue in experimental diabetes in rabbits

The present study was designed to evaluate the oxidative stress-related parameters in alloxan-induced diabetes in rabbits. After 3, 6, 12 and 24 weeks of hyperglycaemia the enzymatic and non-enzymatic factors were measured in heart tissue of diabetic and control groups. Superoxide dismutase and glutathione peroxidase activities and the contents of total sulfhydryl compounds significantly increased at all time intervals. Catalase activity increased initially (after 3 and 6 weeks), decreased after 12 weeks and increased again at the 24th week of the experiment. Glutathione reductase activity increased initially (at 3rd week), decreased below control level after 6 and 12 weeks, then increased again. Ascorbic acid concentration decreased after 3 and 6 weeks, and increased at the 12th and 24th weeks. The level of lipid peroxidation products was reduced after 3, 6 and 12 weeks of the experiment. After 24 weeks it was significantly elevated. These data suggest that hyperglycaemia induces oxidative stress in the heart but the defense mechanisms in the heart tissue are fairly efficacious against oxidative injury.     

New liquid chromatographic assay with electrochemical detection for the measurement of amifostine and WR1065

A high-performance liquid chromatographic method (HPLC) was developed for the analysis of the radio- and chemo-protectant, amifostine and its active metabolite-WR1065 in deproteinized human whole blood and plasma. The two compounds were quantified by measuring WR1065 after two different sample pretreatment procedures. During these procedures, amifostine was quantitatively converted into WR1065, by incubating the sample at 37 degrees C for 4 h at pH<1.0. The resulting amounts of WR1065 were determined by HPLC with coulometric detection (analytical cell: E(1)=200 mV and E(2)=600 mV; guard cell: E(G)=650 mV). The WR1065 standard curve ranged from 0.37 to 50.37 microM. The lower limit of quantitation of WR1065 was 0.25 microM. The within- and between-day precisions were < or = 4.3% and < or = 6.0% for amifostine, < or = 4.4% and < or = 3.8% for WR1065, respectively. The within- and between-day accuracy ranged from 95.4 to 97.7% and 95.4 to 97.8% for amifostine, and from 97.1 to 101.7% and 97.2 to 99.7% for WR1065, respectively. This method minimizes WR1065 loss during sample preparation, and allows for rapid analysis of both compounds on one system. Furthermore, the application of a coulometric electrode is more efficient and requires less maintenance than previously published methods for the two compounds.     

Influence of anther pretreatment on the efficiency of androgenesis in barley

The change in the developmental pathway of microspores from gametophytic to sporophytic is induced by stress during pretreatment of spikes and anthers. In our experiments, anther culture of three barley cultivars was tested with regard to the effect of chilling at 4 degrees C for 28 days, starvation in 0.3 M mannitol solution for 4 days, and a combination of both methods. Chilling was shown to increase embryo/callus formation, while mannitol treatment favoured plant development, including development of green plants; simultaneous application of the two stress factors for 4 days proved to be ineffective. The tested cultivars exhibited a similar ability (calculated per 100 transferred embryos/calli) to develop plants without pretreatment; however, their responses to stress varied greatly. The collected data indicate that mannitol pretreatment, as compared to chilling, is more efficient in responsive cultivars.     

Deciphering endocytosis in Caenorhabditis elegans

We discuss in this review recent studies using the worm Caenorhabditis elegans to decipher endocytic trafficking in a multicellular organism. Recent advances, including in vivo assay systems, new genetic screens, comparative functional analysis of conserved proteins, and RNA-mediated interference (RNAi) in C. elegans, are being used to study the functions of known membrane trafficking factors and to identify new ones. The ability to monitor endocytosis in vivo in worms allows one to test current endocytosis models and to demonstrate the physiological significance of factors identified by genetic and biochemical methods. The available human genome sequence facilitates comparative studies where human homologs of new factors identified in C. elegans can be quickly assayed for similar function using traditional cell biological methods in mammalian cell systems. New studies in C. elegans have used a combination of these techniques to reveal novel metazoan-specific trafficking factors required for endocytosis. Many more metazoan-specific trafficking factors and insights into the mechanisms of endocytosis are likely to be uncovered by analysis in C. elegans .     

Combined expression of S-VSPalpha in two different organelles enhances its accumulation and total lysine production in leaves of transgenic tobacco plants

Soybean vegetative storage proteins (S-VSPs) accumulate to high levels in vacuoles of both wild types and heterologous plants. Here it is shown that directing S-VSPalpha to two different organelles-chloroplasts and vacuoles-in a single transgenic plant significantly increased its accumulation. Accumulation of S-VSPalpha in heterologous plants correlated with total soluble lysine. Using this approach with essential amino-acid-rich transgene proteins may lead to a breakthrough in improving plant nutritional quality.     

Modeling of the phase equilibria of polystyrene in methylcyclohexane with semi-empirical quantum mechanical methods I

A method for calculating interaction parameters traditionally used in phase-equilibrium computations in low-molecular systems has been extended for the prediction of solvent activities of aromatic polymer solutions (polystyrene+methylcyclohexane). Using ethylbenzene as a model compound for the repeating unit of the polymer, the intermolecular interaction energies between the solvent molecule and the polymer were simulated. The semiempirical quantum chemical method AM1, and a method for sampling relevant internal orientations for a pair of molecules developed previously were used. Interaction energies are determined for three molecular pairs, the solvent and the model molecule, two solvent molecules and two model molecules, and used to calculated UNIQUAC interaction parameters, a _ij and a _ji . Using these parameters, the solvent activities of the polystyrene 90,000 amu+methylcyclohexane system, and the total vapor pressures of the methylcyclohexane+ethylbenzene system were calculated. The latter system was compared to experimental data, giving qualitative agreement. Figure Solvent activities for the methylcylcohexane(1)+polystyrene(2) system at 316 K. Parameters a _ij (blue line) obtained with the AM1 method; parameters a _ij (pink line) from VLE data for the ethylbenzene+methylcyclohexane system. The abscissa is the polymer weight fraction defined as ₂(x ₁)=(1–x ₁)M ₂/[x ₁ M ₁+(1–x ₁)M ₂], where x ₁ is the solvent mole fraction and M _i are the molecular weights of the components.An erratum to this article can be found at     

Elimination of Fc receptor-dependent effector functions of a modified IgG4 monoclonal antibody to human CD4

Several CD4 mAbs have entered the clinic for the treatment of autoimmune diseases or transplant rejection. Most of these mAbs caused CD4 cell depletion, and some were murine mAbs which were further hampered by human anti-mouse Ab responses. To obviate these concerns, a primatized CD4 mAb, clenoliximab, was generated by fusing the V domains of a cynomolgus macaque mAb to human constant regions. The heavy chain constant region is a modified IgG4 containing two single residue substitutions designed to ablate residual Fc receptor binding activity and to stabilize heavy chain dimer formation. This study compares and contrasts the in vitro properties of clenoliximab with its matched IgG1 derivative, keliximab, which shares the same variable regions. Both mAbs show potent inhibition of in vitro T cell responses, lack of binding to complement component C1q, and inability to mediate complement-dependent cytotoxicity. However, clenoliximab shows markedly reduced binding to Fc receptors and therefore does not mediate Ab-dependent cell-mediated cytotoxicity or modulation/loss of CD4 from the surface of T cells, except in the presence of rheumatoid factor or activated monocytes. Thus, clenoliximab retains the key immunomodulatory attributes of keliximab without the liability of strong Fcgamma receptor binding. In initial clinical trials, these properties have translated to a reduced incidence of CD4+ T cell depletion.