Structural interpretation of DNA-protein hydroxyl-radical footprinting experiments with high resolution using HYDROID.

Hydroxyl-radical footprinting (HRF) is a powerful method for probing structures of nucleic acid-protein complexes with single-nucleotide resolution in solution. To tap the full quantitative potential of HRF, we describe a protocol, hydroxyl-radical footprinting interpretation for DNA (HYDROID), to quantify HRF data and integrate them with atomistic structural models. The stages of the HYDROID protocol are extraction of the lane profiles from gel images, quantification of the DNA cleavage frequency at each nucleotide and theoretical estimation of the DNA cleavage frequency from atomistic structural models, followed by comparison of experimental and theoretical results. Example scripts for each step of HRF data analysis and interpretation are provided for several nucleosome systems; they can be easily adapted to analyze user data. As input, HYDROID requires polyacrylamide gel electrophoresis (PAGE) images of HRF products and optionally can use a molecular model of the DNA-protein complex. The HYDROID protocol can be used to quantify HRF over DNA regions of up to 100 nucleotides per gel image. In addition, it can be applied to the analysis of RNA-protein complexes and free RNA or DNA molecules in solution. Compared with other methods reported to date, HYDROID is unique in its ability to simultaneously integrate HRF data with the analysis of atomistic structural models. HYDROID is freely available. The complete protocol takes ~3 h. Users should be familiar with the command-line interface, the Python scripting language and Protein Data Bank (PDB) file formats. A graphical user interface (GUI) with basic functionality (HYDROID_GUI) is also available.

Acute phase proteins in serum and cerebrospinal fluid in healthy cattle: possible use for assessment of neurological diseases / Proteínas de fase aguda no soro e no líquido cefalorraquidiano de bovinos hígidos: possíveis usos na avaliação de doenças neurológicas

Use of acute-phase proteins (APPs) for assessment of health and disease in animals has increased greatly within the last decade. The objective was to determine the normal concentration of APPs in the serum and cerebrospinal fluid (CSF) of healthy cattle by polyacrylamide gel electrophoresis. Fifty crossbred animals (350±70kg of BW and 18±1.2 months of age), 25 heifers and 25 steers were used. CSF samples were collected from atlanto-occipital (AO) site and blood samples were obtained from the jugular vein. CSF and serum protein electrophoresis were performed by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Thirty-seven proteins with molecular weights ranging from 7 and 37kDa were identified in CSF of all animals. These eight were nominally identified with immunoglobulin A and G, celuloplasmin, transferrin, albumin, α1-antitripsin, acidic glycoprotein, and haptoglobin. All protein fractions in CSF did not differ between heifers and steers. In sera, 34 proteins with molecular weights between 7 and 244kDa were identified in heifers and steers. Similar proteins were nominally identified in the sera, but only the CSF presented α1-antitripsin. The serum values of acidic glycoprotein and immunoglobulin G were significantly higher in steers compared with heifers. In conclusion, measurement of CSF acute phase protein concentrations can be useful in diagnosing and monitoring the progression of bovine neurological diseases, perhaps even to guide therapeutic procedures. The CSF electrophoretic profile of healthy cattle does not change depending on gender.(AU)

O uso de proteínas de fase aguda (PFAs) para a avaliação da saúde e da doença em animais de produção tem aumentado consideravelmente na última década. O objetivo deste estudo foi determinar a concentração normal de PAFs no soro e no líquido cefalorraquidiano (LCR) de bovinos sadios por meio da eletroforese em gel de poliacrilamida. Foram avaliados cinquenta animais mestiços (350±70kg de PV e 18±1,2 meses de idade), 25 novilhas e 25 novilhos. As amostras de LCR foram colhidas no espaço atlanto-occipital (AO) e as amostras de sangue obtidas da veia jugular. As PAFs do soro e do LCR foram determinadas através da eletroforese em gel poliacrilamida. Trinta e sete proteínas com pesos moleculares que variaram entre 7 e 37kDa foram identificadas no LCR de todos os animais, independente do sexo. Estas oito proteínas foram nominalmente identificadas como imunoglobulina A e G, ceruloplasmina, transferrina, albumina, α1-antitripsina, glicoproteína ácida, e haptoglobina. As frações de proteínas presentes no LCR não diferiram entre novilhas e novilhos. No soro de machos e fêmeas, 34 proteínas com pesos moleculares entre 7 e 244 kDa foram identificadas. As proteínas do soro foram similarmente identificadas, entretanto a α1-antitripsina foi identificada somente no LCR. Os valores séricos de glicoproteína ácida e imunoglobulina G foram significativamente mais elevados nas novilhas em comparação aos novilhos. Em conclusão, a determinação das concentrações de proteínas de fase aguda presentes do LCR pode ser útil no diagnóstico e monitoramento da progressão de doenças neurológicas bovinas, talvez possa ainda direcionar procedimentos terapêuticos. O perfil eletroforético do LCR de bovinos hígidos não se altera em função do sexo.(AU)

[Correction of the electrophoretic shift in virtual 2D SDS-PAGE electrophoresis]. / Korrektsiia élektroforeticheskogo sdviga v virtual'nom 2D SDS-PAGE élektroforeze.

Virtual electrophoresis in proteomics can be used to search localization of proteins and their proteoforms (especially those existing in low concentrations), to identify proteoforms found in experiments etc. Although the problem of predicting the isoelectric point is well studied, the need of electrophoretic shift correction is usually ignored. Researchers simply use the brutto molecular weight of the protein. In this study four data sets taken from the literature sources and the SWISS-2DPAGE database have been used to build correction equations for prediction of the electrophoretic shift (123, 72, 118 and 470 points, respectively). Two groups of models were built. The first model was based on the amino acid composition of proteins, the second one, on analysis of parameters calculated by amino acid sequences (theoretical molecular weight, hydrophobicity, charge distribution, ability to form helix structures). The coefficient of determination ranged from 0.35 to 0.75 in each single set, but cross-prediction between samples did not gave satisfactory results. At the same time, the direction of correction was predicted correctly in 74% of cases. After combining of the samples and dividing pooled data into 2 representative sets, the coefficient of determination during in the process of learning ranged from 0.44 to 0.51, and R2 of predictions were not less than 0.39. The direction of correction was predicted correctly in 80% of cases. This prediction models have been integrated into the program pIPredict v.2, freely available at http://www.ibmc.msk.ru/LPCIT/pIPredict.

SSCP screening of the dihydropyrimidine dehydrogenase gene polymorphisms of the Japanese population using a semi-automated electrophoresis unit.

The single-strand conformation polymorphism (SSCP) procedure has been applied in routine testing for hereditary diseases. Temperature, running buffer, gel composition, and fragment length can influence its sensitivity. Mutation detection in the clinical setting depends on the development of automated technology, especially for large genes, such as the dihydropyrimidine dehydrogenase (DPYD) gene, which codes the initial, rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU). The authors have optimized the condition of SSCP with an automated system (GenePhor system, GE Healthcare UK Ltd.) to screen genetic polymorphisms in the DPYD gene. The efficiency of the method was evaluated using 21 positive controls (DNA samples with polymorphisms in the DPYD gene, previously characterized) and DNA samples from 35 Japanese. Results showed that the use of three different running buffers (pH 7.4, 8.3, and 9.0) in combination with other optimized conditions (10% polyacrylamide gel, 60-90 min at constant 900 V at 5 degrees C) resulted in a high polymorphism detection rate (95.3%), which was considered appropriate for routine screening. Therefore, this strategy could be useful for pharmacogenetic studies on 5FU.

Modeling ssDNA electrophoretic migration with band broadening in an entangled or cross-linked network.

We use a coarse-grained model proposed by Graham and Larson based on the temporary network model by Schieber et al.. [1] to simulate the electrophoretic motion of ssDNA and corresponding band broadening due to dispersion. With dimensionless numbers reflecting the experimental physical properties, we are able to simulate ssDNA behavior under weak to moderate electric field strengths for chains with 8-50 entanglements per chain ( approximately 1000-8500 base pairs), and model smoothly the transition from reptation to oriented reptation. These results are fitted with an interpolation equation, which allows the user to calculate dimensionless mobilities easily from input parameters characterizing the gel matrix, DNA molecules, and field strengths. Dimensionless peak widths are predicted from mobility fluctuations using the central limit theorem and the assumption that the mobility fluctuations are Gaussian. Using results from previous studies of ssDNA physical properties (effective charge xiq and Kuhn step length b(K)) and sieving matrix properties (pore size or tube diameter a), we give scaling factors to convert the dimensionless values to "real" experimental values, including the mobility, migration distance, and time. We find that the interpolation equation fits well the experimental data of ssDNA mobilities and peak widths, supporting the validity of the coarse-grained model. The model does not account for constraint release and hernia formation, and assumes that the sieving network is a homogeneous microstructure with no temperature gradients and no peak width due to injection. These assumptions can be relaxed in future work for more accurate prediction.

Model carbyne knots vs ideal knots.

The structure and stability of model carbyne knots built from 60 to 120 carbon atoms with 0, 3, 4,., 7 crossings have been estimated by semiempirical AM1 calculations. The calculations have shown an increase of the knot-cycle energy difference (deltaE) with an increasing number of knot crossings and a decrease of deltaE with an increasing number of atoms constituting the molecule. The deltaE changes nonlinearly with the characteristics of the corresponding ideal knots such as the average crossing number (ACN) and the length-to-diameter ratio (L/D). The molecular mechanic strain energy of carbyne knots correlates similarly with ACN and L/D of ideal knots. The calculated energy of the model carbyne knots correlates also with the electrophoretic mobility or sedimentation coefficient of DNA knots. Thus, similarly to characteristics of ideal knots, the energy of carbyne knots is a rather easily available parameter which can be used for further correlations with some characteristics of DNA knots.

SDS-PAGE is underutilized as a tool for investigating renal patients.

SDS-PAGE is an excellent single test for investigating proteinuria. It can provide much useful information on the underlying renal problem. Yet the literature hardly report a SDS-PAGE result in the management of renal patients. To examine how closely SDS-PAGE results may reflect biopsy findings, we investigated 11 patients scheduled for renal biopsy. Urine samples were taken at the same time for SDS-PAGE analysis using the PhastSystem (Pharmacia, Sweden). Comparing biopsy findings and SDS-PAGE results, the data show consistency in the revelation of tubular dysfunction and/or glomerular damage in all 11 patients. We concluded that the SDS-PAGE test is underutilized and suggest that its role for the management of renal patients be fully explored particularly in its potential for reducing the need for renal biopsy in certain patient groups.

Increased BAEP latency and reduced brain cytochrome c oxidase activity observed in the Suzuki model of Alzheimer's disease [abstract]

Cytochrome c oxidase (COX), the terminal respiratory enzyme, is reported to be deficient and to exhibit reduced activity in a number of neurodegenerative disorders. The Alzheimer's rabbit model is based on the intracerebral injection of Holt's adjuvant, an aluminum salt solution. This model was used to compare the brain activity and the activity of brain COX in treated and controlled rabbits. Of a total of 26 New Zealand white rabbits, thirteen were injected intracerebrally with Holt's adjuvant solution, five with physiologically saline solution and the remainder were kept as controls. The auditory responses (BAEP) of the rabbits were monitored for 14 days before injections and 14 days thereafter. The animals were then sacrificed and brain mitochondrial extracts were used for SDS-PAGE analysis, difference spectra spectrophotometry and polarographic assays. The results of SDS-PAGE showed that there were no differences in the protein composition of the brain mitochondria of the three groups. Similarly, difference spectra (reduced minus oxidized, 400-630 nm) from both treated groups were identical to that of the control with characteristic maxima centered around 434, 550 and 604 nm. Polarographic assays, however, showed that while the enzyme from both treated groups displayed the characteristic biphasic kinetics, there was reduced activity in the enzymes from the brain of rabbits receiving the adjuvant but not the saline solution. This result is significant in light of the fact that several researchers have reported reduced activity of COX from Alzheimer's disease (AD) patients. Of particular significance, BAEP results reveal an increase in the interpeak latency between peaks III and V of the Holt's injected but not the saline injected or control rabbits. This latter result suggests that monitoring the BAEP might provide a simple non-invasive method for confirming AD. (AU)

Automated detection of differently expressed fragments in mRNA differential display.

We present a novel method for the automated detection of fragments showing dissimilar expression in mRNA differential display. The analysis is based on aligning the numerical electrophoretic lane data in respect of a given distance function defined on a set of fragments, or signal peaks in general. We presume that significant dissimilarities between peaks result in extreme score values computed for aligned peak pairs. Whereas in sequence comparison, an overall sequence similarity score is conventionally used, the current method defines a special dissimilarity score for searching the peak pairs showing the largest relative differences between the lanes. The output of the analysis is a highly reduced list of peak pairs, along with a set of associated features extracted from the lanes. Only the peaks of this list need to be visually confirmed instead of the vast amount of peaks in the original electrophoretic results. The results obtained by the algorithm correlate well with results of visual evaluation of the same electropherograms. The current algorithm may be applied to the study of complex expression patterns in multiple lanes and, in general, to automated recognition of variously defined patterns of quantitative electrophoretic data.

The use of polyacrylamide gel electrophoresis in animal systematics, phylogeny, and ecophysiology.

In the 1980s, the use of polyacrylamide gel electrophoresis (PAGE) was popular. But more recent developments in other electrophoretic techniques have resulted in this method being less widely used. However, it is adequate for the needs of naturalists for issues which do not require high-performance methods, such as in systematics, phylogeny, studies of intraspecific or clinal variability, ecology and ecophysiology. We illustrate the application of PAGE by the results of a research program on marine and terrestrial invertebrates which was conducted from 1979 to 1992, but which is still used to initiate graduate students to the research. Thus, issues faced by the zoologist can be clarified by this method, not yet obsolete and still useful in natural history despite considerable advances in other electrophoretic methods.

Identification of human lung and skin proteins conjugated with hexamethylene diisocyanate in vitro and in vivo.

Diisocyanates are asthma-causing chemicals used in the commercial production of polyurethane. We have previously shown that human lung epithelial cell proteins can become conjugated with hexamethylene diisocyanate (HDI) and may be biologically important in diisocyanate-induced asthma. The objective of this study was to identify specific human lung and skin proteins that become conjugated with diisocyanate after in vitro and in vivo exposure. Following in vitro exposure of human airway epithelial cells (A549), keratin 18, the 78-kD glucose-regulated protein, trans-1, 2-dihyrobenzene-1,2-diol dehydrogenase, and actin were identified as prominent diisocyanate-conjugated proteins through use of a combination of immunocytochemical and mass spectrometric techniques. Following in vivo inhalation of an HDI aerosol, keratin 18 was also identified as the predominant diisocyanate-conjugated protein in human endobronchial biopsy samples, whereas albumin was the predominant diisocyanate-conjugated protein in bronchoalveolar lavage fluid. Keratin was also identified as a predominant diisocyanate-conjugated protein in human skin biopsy samples after epicutaneous exposure to liquid-phase HDI, although the major skin diisocyanate-conjugated protein (56-kD) differed from the predominant lung diisocyanate-conjugated keratin (47-kD). The data from this study identify keratin and other proteins as potential "carriers" for diisocyanates in vivo, and suggest that HDI conjugation of these proteins may play a role in the pathogenesis of diisocyanate-induced asthma.

[An assessment of the degree of chromophore photo damage to the heme and globin components in UV-irradiated molecules and electrophoretic fractions of human carboxyhemoglobin]. / Otsenka stepeni fotopovrezhdeniia khromoforov gemovogo i globinovogo komponentov UF-obluchennykh molekul i élektroforeticheskikh fraktsii karboksigemoglobina cheloveka.

The contribution of hem and globin components of electrophoretic fractions of UV-irradiated human carboxyhemoglobin to photodestruction of the protein was studied. The changes observed are the result of summation of some processes unequal in intensity and direction that take place in microheterogeneous media of photomodified protein. Photosensitivity of hemoproteid in electrophoretic fraction depends on apoprotein condition, whereas the hem photoresistance cannot be the evidence of the photostability of the whole molecule.

[The genetic interpretation of the electrophoregrams of the helianthin in F1 sunflower seeds]. / Henetychna interpretatsiia elektroforehram heliantynu nasinnia F1 soniashnyka.

It has been shown, that in most cases the fertile plants of maternal line and F1 plants of respective hybrid are present among sterile plants of sunflower female lines in crossing plots. As a result, 16 various genotypes of seeds are ascertained in crossing plots at monogenic differences in marker trait. Only two classes are F1 seeds. In such cases specific share of 5 genotype groups may be distinguished by phenotypes of heliantin electrophoregrammes. Seeds of biological admixture may be attributed to the 6th distinguishing type.

[A comparison of the yield of Vi and O antigens during the adaptation of Salmonella typhi strains to cultivation under starvation conditions]. / Sravnenie vykhoda Vi- i O-antigenov v protsesse adaptatsii shtammov Salmonella typhi k kul'tivirovaniiu v usloviiakh golodaniia.

S. typhi strains Ty(2)4446 and Vi-1S underwent multiple passages in f synthetic liquid starvation culture medium consisting of water with salts and glucose added. In the process of the adaptation of the cultures to these stress conditions (starvation stress) the increasing yield of biomass from passage to passage was observed. Differences in the accumulation of Vi- and O-antigens were noted in two strains under study. In the cultures of strain Ty(2)4446 an insignificant increase in the antigen content from passage to passage was observed, while in the cultures of strain Vi-1S an increase in the content of Vi- and O-antigens was 4- to 5-fold. With the adaptation of the culture the Vi-antigen to O-antigen ratio changed from 1:57 to 1:20 for strain Ty(2)4446 and from 1:2.7 tp 1:2.2 for strain Vi-1S. Strain Ty(2)4446 had an advantage over strain Vi-1S with respect to the synthesis of Vi-antigen. These data are indicative of the expediency of using not only strain Ty(2)4446, but also strain Vi-1S for the preparation of typhoid vaccine, especially the one based on Vi-antigen.

Precipitation of specific proteins by freeze-thawing of human saliva.

Frozen saliva samples demonstrate a variable amount of precipitate on thawing depending on the type of secretion [submandibular-sublingual (SML) greater than parotid]. This precipitate has been resuspended using EDTA or removed by centrifugation by some workers and others do not mention it. Yet others collect the salivas into EDTA or centrifuge them before freezing. To determine the adsorption of proteins to hydroxyapatite, prior treatment with EDTA would be disadvantageous. The aim here was to determine if the protein pattern in parotid and SML saliva as demonstrated by sodium dodecyl sulphate gel electrophoresis is affected by the formation of precipitates. Portions of parotid and SML saliva were thawed and treated in the following ways: (a) mixed vigorously with a vortex mixer; (b) centrifuged to remove the precipitate; (c) mixed with EDTA (1 and 5 mmol final concentration for parotid and SML samples, respectively) to resuspend the precipitate. The samples were loaded on to gradient (5-20%) SDS gels and, following electrophoresis, the gels were stained with Coomassie brilliant blue R-250. The protein patterns obtained for (a) and (c) were the same. The centrifuged samples demonstrated loss of a specific band of less than 14 kDa, although this was less obvious in the parotid samples. The SML samples also showed a reduction in other lower molecular-weight proteins. This study demonstrates that precipitates in thawed frozen salivas contain specific proteins and that these samples require careful handling to avoid any alteration in the overall protein composition.

Multiple forms of the major phenylalanine specific protease in Treponema denticola.

The 160, 190 and 270 kDa outer sheath proteases of Treponema denticola ATCC 35404 were found to be multiple forms of the major 91 kDa phenylalanine protease (PAP) by immunoblotting using anti-91 kDa specific antibodies. Multiple forms of the phenylalanine protease were also found in 2 other T. denticola strains studied, ATCC 33520 and the clinical isolate GM-1. Protein, proteolytic and Western blot analyses using antibodies against the PAP and the major outer sheath protein (MSP) indicated that the 190 and 270 kDa proteases were protein complexes formed by the MSP and the PAP. These complexes dissociated by storage in 0.3% or higher SDS concentrations. The purified PAP was found to completely degrade keratin, but was unable to degrade native actin either in its monomeric or polymerized form. The association of the MSP adhesin with a protease capable of degrading host native proteins may benefit the obtention of protein-based nutrients necessary to support the growth of these treponemes. These complexes may also play a role in the structural organization of T. denticola outer sheath.

Capsaicin induces cystatin S-like substances in submandibular saliva of the rat.

Irritating dietary substances such as tannin and papain have been reported to alter the morphology of salivary glands and their secretions. Such alterations can be one line of protection from toxic or irritating substances in food. We investigated the effects of dietary capsaicin (a pungent ingredient of hot red pepper) on the rat submandibular gland and its secretions. Several groups of animals were offered either control diets or diets containing capsaicin (from 0.0001 to 0.1%) for seven days. Higher concentrations suppressed food consumption for two days, after which only the highest concentration continued to reduce intake. The relative weight of the salivary glands in capsaicin-diet groups increased in a dose-dependent fashion, and new proteins appeared in the submandibular saliva. Chromatographic and electrophoretic properties of these proteins were identical or similar to those of isoproterenol-induced proteins. After affinity chromatography of the new protein fraction on a Cm-papain Sepharose 4B column, SDS-electrophoresis of the eluate revealed three major bands (15,500, 16,500, and 28,000 kDa). Hydrolysis of N-benzoyl-D,L-arginine-p-nitroanilide by papain (a cysteine protease) decreased in the presence of the new protein fraction, suggesting that these proteins have cystatin-like activity (inhibition of cysteine protease). Denervation of the glossopharyngeal nerve suppressed induction of these proteins. The results suggest that dietary capsaicin induces cystatin S-like substances in submandibular saliva by stimulating the reflex arc involving the glossopharyngeal nerve. These proteins likely facilitate ingestion of diets containing the irritating substance.

Abnormal NF-kappa B activity in T lymphocytes from patients with systemic lupus erythematosus is associated with decreased p65-RelA protein expression.

Numerous cellular and biochemical abnormalities in immune regulation have been described in patients with systemic lupus erythematosus (SLE), including surface Ag receptor-initiated signaling events and lymphokine production. Because NF-kappa B contributes to the transcription of numerous inflammatory genes and has been shown to be a molecular target of antiinflammatory drugs, we sought to characterize the functional role of the NF-kappa B protein complex in lupus T cells. Freshly isolated T cells from lupus patients, rheumatoid arthritis (RA) patients, and normal individuals were activated physiologically via the TCR with anti-CD3 and anti-CD28 Abs to assess proximal membrane signaling, and with PMA and a calcium ionophore (A23187) to bypass membrane-mediated signaling events. We measured the NF-kappa B binding activity in nuclear extracts by gel shift analysis. When compared with normal cells, the activation of NF-kappa B activity in SLE patients was significantly decreased in SLE, but not in RA, patients. NF-kappa B binding activity was absent in several SLE patients who were not receiving any medication, including corticosteroids. Also, NF-kappa B activity remained absent in follow-up studies. In supershift experiments using specific Abs, we showed that, in the group of SLE patients who displayed undetectable NF-kappa B activity, p65 complexes were not formed. Finally, immunoblot analysis of nuclear extracts showed decreased or absent p65 protein levels. As p65 complexes are transcriptionally active in comparison to the p50 homodimer, this novel finding may provide insight on the origin of abnormal cytokine or other gene transcription in SLE patients.

Mobility of acetylated histones in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

We describe an altered mobility for acetylated histone isoforms in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoforms of histones H3 and H4 with a higher acetylation degree have a slightly faster electrophoretic mobility. Since acetylation neutralizes the positive charge of the epsilon-amino group of lysine, without significantly changing the molecular mass of the protein, the acetylation-dependent mobility shift could be explained by the increase of the net negative charge of the SDS-histone complexes. A possible consequence of this differential mobility for the acetylation site determination by protein microsequencing from SDS gels is discussed.