Protein sequence comparison based on the wavelet transform approach

A protein's chemical properties, the chain conformation, the function of the protein and its species specificity are determined by the information contained in the amino acid sequence. Proteins of similar functions have at some level sequential identical amino acid sequences. The closer the phylogenetic relationship, the more similar are the sequences. To find the similarities between two or more protein sequences is of great importance for protein sequence analysis. The differences in the amino acid sequences permit the construction of a family tree of evolution. In this work, a comparison method was devised that is capable of analysing a protein sequence 'hierarchically', i.e. it can examine a protein sequence at different spatial resolutions. Based on a wavelet decomposition of protein sequences and a cross-correlation study, a sequence-scale similarity concept is proposed for generating a similarity vector, which renders the comparison of two sequences feasible at different spatial resolutions (scales). This new similarity concept is an expansion of the conventional sequence similarity, which only takes into account the local pairwise amino acid match and ignores the information contained in coarser spatial resolutions.     

Auricular mild errors of morphogenesis: epidemiological study with application of a targeted computer program

Mild errors of morphogenesis (MEMs) are findings of great importance in multiple fields because of their predictive value in identification of major malformations, specific genetic syndromes, mental retardation and childhood malignancy. In order to evaluate the contribution of auricular MEMs, we constructed a computerized program, especially designed for the recording of auricular MEMs in connection with nonauricular MEMs, personal data, family, pregnancy and birth history, presence of major malformation and postural defects. The program is easy to use and delivers, at request, immediate information regarding prevalence of each auricular MEM and percentage associations between all variables. In the period from January 1, 1999 to December 31, 1999, we screened 3107 consecutively born neonates and recorded auricular and nonauricular MEMs, detected by surface examination. The general prevalence of auricular MEMs was 43.2% and the most frequent MEMs and their associates were of postural-type. This computerized program offers many advantages for the study of ear MEMs.     

Dual effect of fluid shear stress on volume-regulated anion current in bovine aortic endothelial cells

The key mechanism responsible formaintaining cell volume homeostasis is activation ofvolume-regulated anion current (VRAC). The role of hemodynamicshear stress in the regulation of VRAC in bovine aortic endothelialcells was investigated. We showed that acute changes in shear stresshave a biphasic effect on the development of VRAC. A shear stress stepfrom a background flow (0.1 dyn/cm²) to 1 dyn/cm² enhanced VRAC activation induced by an osmoticchallenge. Flow alone, in the absence of osmotic stress, did not induceVRAC activation. Increasing the shear stress to 3 dyn/cm²,however, resulted in only a transient increase of VRAC activity followed by an inhibitory phase during which VRAC was gradually suppressed. When shear stress was increased further (5-10dyn/cm²), the current was immediately strongly suppressed.Suppression of VRAC was observed both in cells challenged osmoticallyand in cells that developed spontaneous VRAC under isotonic conditions. Our findings suggest that shear stress is an important factor inregulating the ability of vascular endothelial cells to maintain volume homeostasis.

     

Salt induction of fatty acid elongase and membrane lipid modifications in the extreme halotolerant alga Dunaliella salina

In studies of the outstanding salt tolerance of the unicellular green alga Dunaliella salina, we isolated a cDNA for a salt-inducible mRNA encoding a protein homologous to plant beta-ketoacyl-coenzyme A (CoA) synthases (Kcs). These microsomal enzymes catalyze the condensation of malonyl-CoA with acyl-CoA, the first and rate-limiting step in fatty acid elongation. Kcs activity, localized to a D. salina microsomal fraction, increased in cells transferred from 0.5 to 3.5 M NaCl, as did the level of the kcs mRNA. The function of the kcs gene product was directly demonstrated by the condensing activity exhibited by Escherichia coli cells expressing the kcs cDNA. The effect of salinity on kcs expression in D. salina suggested the possibility that salt adaptation entailed modifications in the fatty acid composition of algal membranes. Lipid analyses indicated that microsomes, but not plasma membranes or thylakoids, from cells grown in 3.5 M NaCl contained a considerably higher ratio of C18 (mostly unsaturated) to C16 (mostly saturated) fatty acids compared with cells grown in 0.5 M salt. Thus, the salt-inducible Kcs, jointly with fatty acid desaturases, may play a role in adapting intracellular membrane compartments to function in the high internal glycerol concentrations balancing the external osmotic pressure.     

Egg yolk platelet proteins from Xenopus laevis are amyloidogenic

The association of amphibian (Xenopus laevis) egg yolk platelet proteins, represented predominantly by lipovitellin, was studied as a model of the formation of amyloid deposits. Two kinds of molecular organization formed by this protein material - native and heat-denatured - were found to exhibit amyloid properties although they differ significantly in structural organization. The first consisted in protein molecules arranged in the natural, physiological, net-like platelet organization, with a tendency to orient uni-directionally. The second was obtained by the gradual removal of Congo red from lipovitellin denatured by heating in an excess of dye. This procedure produced the twisted fibrillar organization of molecules typical for amyloids, represented predominantly by end-to-end associated major polypeptide chains of lipovitellin. Both native and denatured structural forms bind Congo red and produce a green birefringence effect, confirming the near parallel alignment of the complexed Congo red molecules. However, a dye(1,4-bis(1-amino-4-sulfonaphtyl-2-azo)phenylene) closely related to Congo red but with a very weak self-assembling tendency appeared inactive when the spectral shift was studied in a cross-polarization system, indicating in this way that dye supramolecularity is an extra factor which may determine binding to amyloid proteins and specific spectral effects.     

Renal injuries induced by chronic intoxication with microcystins

The microcystins (MCs) LR and YR are hepatotoxins produced by some species of freshwater cyanobacteria. Only a few reports on the acute nephrotoxicity of MCs have been published to date. Here we investigate the effects on rat kidneys of chronic administration of relatively low doses of MC LR and MC YR. Male Wistar rats were injected every second day for 8 months with MC LR (10 microg/kg i.p., n=5) and MC YR (10 microg/kg i.p., n=5). The control group was treated with vehicle, a mixture containing 0.8 % ethanol and 0.2 % methanol in 0.9 % saline (3.7 ml/kg i.p., n=5). We found that MCs could induce damage to the kidney cortex and medulla. The lesions mainly consisted of damaged and dilated tubuli filled with homogenous eosinophil material. We conclude that long-term exposure to relatively low doses of MCs poses a considerable risk for kidney injury.     

Testing cms-P-linked AFLPs for selection of rye hybrid components

Application of AFLPs linked to pollen fertility restoration and non-performing genes evaluated in the C394-F2 hybrid was studied using a set of male sterile lines in the sterilising Pampa cytoplasm, several restorers and maintainer lines and, finally, two inbred lines backcrossed into cms-P, cms-R, cms-S and cms-C cytoplasms each. The set of male sterile lines based on the Pampa cytoplasm exhibited gradual variation in their ability to restore pollen fertility (starting from low and closing with high) in crosses with three unrelated restorers. Variations in the AFLPs between the analysed materials were observed, however, no clustering of the lines according to their sterile and fertile phenotypes was observed. The same markers, when applied to the population restorer (cv. Walet) that formed the C394-F2 cross permitted identification of plants with genotypes that could be recognized as restorers.     

Enhancing effect of melatonin on chemiluminescence accompanying decomposition of hydrogen peroxide in the presence of copper

The oxidation of melatonin (MEL) using the Cu(II) + H₂O₂ + HO⁻ (the Fenton-like reaction) system was investigated by chemiluminescence (CL), fluorescence, spectrophotometric, and EPR spin trapping techniques. The reaction exhibits CL in the 400–730 nm region. The light emission from the Fenton-like reaction was greatly enhanced in the presence of MEL and was strongly dependent on its concentration. The spectrum measured with cut-off filters revealed maxima at around 460, 500, 580–590, 640–650, and 690–700 nm. The band at 460 nm may be due to the excited cleavage product, N¹-acetyl-N²-formyl-5-methoxykynuramine, whereas the bands at 500, 580–590, 640–650, and 700 nm were similar to those observed for singlet molecular oxygen (¹O₂). The effect of reactive oxygen species (ROS) scavengers on the light emission was studied. The CL was strongly inhibited by the ¹O₂ scavengers in a dose-dependent manner; at concentration 1 mM the potency of ¹O₂ scavenging was 5,5-dimethylcyclohexandione-1,3 > methionine > histidine > hydroquinone. The potency of HO^• scavenging by thiourea, tryptophan, cysteine at concentration 5 mM was 79–94%, by 1 mM glutathione and trolox 75 and 94%, respectively, and by 10 mM cimetidine 18%. Specific acceptors of O₂^•− such as p-nitroblue tetrazolium chloride and 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron) at concentration 5 mM decreased the CL by 51 and 95%, respectively, whereas superoxide dismutase (SOD) does not reduce the emission at concentration 2.8 U/ml. At higher concentration SOD substantially enhanced the light emission. Addition of 1360 U/ml catalase and 100 μM desferrioxamine strongly inhibited CL (96 and 90%, respectively). The increased generation of ¹O₂ from the Cu/H₂O₂ system in the presence of MEL was confirmed using the spectrophotometric method based on the bleaching of p-nitrosodimethylaniline and by trapping experiments with 2,2,6,6-tetramethylpiperidine (TEMP) and subsequent electron paramagnetic (EPR) spectroscopy. These findings suggest the increased production of reactive oxygen species (O₂^•−, HO^•, ¹O₂) from the Fenton-like reaction in the presence of MEL. This means that the hormone is not able to act as classical chain-breaking antioxidant even at low concentration, and may show clear prooxidant activity at higher concentrations. In addition, long-lived carbonyl product of the MEL transformation in the triplet state can also be toxic by transferring its energy to organelles and causing a photochemical process.     

Paraoxonase activity and concentration of indicators of lipids status in the serum of cardiac patients

In this research, we measured the activity of paraoxonase (basal and activated) enzyme, and components of lipid status components (total cholesterol, LDL cholesterol, HDL cholesterol and Apo A I) in the serum of patients, undergoing bypass surgery. We also tested how the applied EKC affected changes of defined indicators. Measuring of all the given parameters was conducted prior to the operation, 90 minutes, 1.5 hour, 6 hours, 24 hours and 72 hours, on 29 patients (11 of them did undergo myocardium revascularization with the application of EKC, while the rest of them did not). Activity of paraoxonase (both basal and activated) changes significantly during the postoperative period, in relation to pre-operative values, p < 0.05. Total cholesterol concentration is reduced in both examined groups, regardless of the application of EKC. This trend is also accompanied by LDL cholesterol concentration. On the other hand, HDL cholesterol concentration during post-operative period does not indicate any significant statistical change in relation to pre-operative values, while we noticed difference with regard to EKC application, 90 minutes after surgery. This change of lipid status indicator is partly due to heparin, a stimulator of lipoprotein lipase that was applied during the surgery. Our conclusion is that lipid profile changes significantly after the bypass surgery, mostly regardless of the application of EKC.     

Shear stress increases expression of a KATP channel in rat and bovine pulmonary vascular endothelial cells

We have shown previously that acute ischemia leads to depolarization of pulmonary microvascular endothelial cells that is prevented with cromakalim, suggesting the presence of ATP-sensitive K⁺ (K_ATP) channels in these cells. Thus K_ATP channel expression and activity were evaluated in rat pulmonary microvascular endothelial cells (RPMVEC) by whole cell current measurements, dot blot (mRNA), and immunoblot (protein) for the inwardly rectifying K⁺ channel (K_IR) 6.2 subunit and fluorescent ligand binding for the sulfonylurea receptor (SUR). Low-level expression of a K_ATP channel was detected in endothelial cells in routine (static) culture and led us to examine whether its expression is inducible when endothelial cells are adapted to flow. Channel expression (mRNA and both K_IR6.2 and SUR proteins) and inwardly rectified membrane current by patch clamp increased significantly when RPMVEC were adapted to flow at 10 dyn/cm² for 24 h in either a parallel plate flow chamber or an artificial capillary system. Induction of the K_ATP channel with flow adaptation was also observed in bovine pulmonary artery endothelial cells. Flow-adapted but not static RPMVEC showed cellular plasma membrane depolarization upon stop of flow that was inhibited by a K_ATP channel opener and prevented by addition of cycloheximide to the medium during the flow adaptation period. These studies indicate the induction of K_ATP channels by flow adaptation in pulmonary endothelium and that the expression and activity of this channel are essential for the endothelial cell membrane depolarization response with acute decrease in shear stress. flow adaptation; K_IR 6.2; sulfonylurea receptor; fluorescent glyburide; pulmonary microvascular endothelial cells