A method to identify and characterize Z-DNA binding proteins using a linear oligodeoxynucleotide.

An oligodeoxynucleotide that readily flips to the Z-DNA conformation in 10mM MgCl2 was produced by using Klenow enzyme to incorporate 5-bromodeoxycytosine and deoxyguanosine into a (dC-dG)22 template. During synthesis the oligomer can be labeled with 32P to high specific activity. The labeled oligodeoxynucleotide can be used in bandshift experiment to detect proteins that bind Z-DNA. This allows the binding specificity of such proteins to be determined with high reliability using unlabeled linear and supercoiled DNA competitors. In addition, because the radioactive oligodeoxynucleotide contains bromine atoms, DNA-protein complexes can be readily crosslinked using UV light. This allows an estimate to be made of the molecular weight of the proteins that bind to the radioactive probe. Both techniques are demonstrated using a goat polyclonal anti-Z-DNA antiserum.     

Sequence landmark patterns identify and characterize protein families

The three-dimensional structures of homologous proteins are usually conserved during evolution, as are critical residues in a few short sequence motifs that often constitute the active site in enzymes. The precise spatial organization of such sites depends on the lengths and positions of the secondary structural elements connecting the motifs. We show how members of protein superfamilies, such as kinesins, myosins, and G(alpha) subunits of trimeric G proteins, are identified and classed by simply counting the number of amino acid residues between important sequence motifs in their nucleotide triphosphate-hydrolyzing domains. Subfamily-specific landmark patterns (motif to motif scores) are principally due to inserts and gaps in surface loops. Unusual protein sequences and possible sequence prediction errors are detected.     

Use of inhibitors to identify essential cysteine proteinases of Trichomonas vaginalis

Designing cysteine proteinase inhibitors as antitrichomonal drugs requires knowledge of which cysteine proteinases are essential to the parasite. In an attempt to obtain such information, the effects of a number of cysteine proteinase inhibitors on trichomonad growth in vitro and proteinase activity were investigated. The broad specificity inhibitor trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane (known as E-64) had little effect on growth of Trichomonas vaginalis (27% inhibition at 280 μM, none at 28 μM) even though the addition of 2.8 μM E-64 to growth medium resulted in inhibition of all but two (apparent molecular masses: 35 k and 49 k) of the parasite's proteinases detected by gelatin SDS-PAGE. This shows that many of the parasite's cysteine proteinases are not essential for growth in axenic culture. In contrast, a peptidyl acyloxymethyl ketone, N-benzoyloxycarbonyl-Phe-Ala-CH₂OCO-(2,6,-(CF₃)₂)Ph, at 16 μM killed T. vaginalis and severely inhibited growth of Tritrichomonas foetus. Exposure of Trichomonas vaginalis to 16 μM of this compound for 1 h resulted in both the 35 kDa and 49 kDa proteinases being inhibited, whereas some other proteinases were unaffected. Similar distinctions between the inhibitor sensitivity of the parasite's cysteine proteinases were apparent when a biotinylated peptidyl diazomethyl ketone was used to detect active proteinases. These data suggest that the growth inhibitory effects of the peptidyl acyloxymethyl ketone are through inhibition of cysteine proteinases that are not affected when the parasites are grown in the presence of E-64. At least one of these enzymes, which include the 35 kDa and 49 kDa cysteine proteinases, must be essential and so a suitable target for chemotherapeutic attack.     

Peptide mapping and microsequencing of proteins separated by SDS-PAGE after limited in situ acid hydrolysis.

A method is described for the isolation of peptide fragments from proteins separated by polyacrylamide gel electrophoresis. After completion of the electrophoresis step, gels are stained with Ponceau S or Coomassie Blue. Gel portions containing protein stained with Ponceau S are excised and transferred to borosilicate glass digestion tubes containing 0.9 ml of 1 mM NaOH or 5 mM Na2HPO4. After complete dissociation of the dye from the protein, 0.1 ml of 20% formic acid is added and the protein is hydrolyzed in situ at 112 degrees C for four hours. Subsequently the acid solution is made 10% in acetonitrile and chromatographed as such on a C18 (C4) reversed-phase column using an appropriate large-volume sample loading syringe and injection loop. Proteins stained with Coomassie Blue can be hydrolyzed in situ after complete removal of the dye with an aqueous solution containing 40% acetone, 10% triethylamine and 5% acetic acid. The gel slices are next washed with HPLC-grade water and protein is hydrolyzed in 2% formic acid under standard conditions. Gel-related contaminants do not interfere with the peptide separation under the proper conditions of HPLC analysis.     

Application of microarrays to identify and characterize genes involved in attachment dependence in HeLa cells

The ability to modify cellular properties such as adhesion is of interest in the design and performance of biotechnology-related processes. The current study was undertaken in order to evaluate the effectiveness of modulating cellular adhesion in HeLa cells from a genomics perspective. Using DNA microarrays, differences in gene expression between two phenotypically distinct, anchorage-dependent and anchorage-independent, HeLa cell lines were identified. With the aid of several statistical methods and an extensive literature search, two genes were selected as potential targets for further study: siat7e and lama4. Subsequently, experiments were carried out to investigate the effects of siat7e and lama4 separately, on adhesion in HeLa cells by altering their expression in vivo. Decreasing the expression of siat7e, a type II membrane glycosylating sialyltransferase, in anchorage-independent HeLa cells using short interfering RNA (siRNA) resulted in greater aggregation (i.e. clumping) and morphological changes as compared to untreated anchorage-independent HeLa cells. Similar effects were seen in anchorage-independent HeLa cells when the expression of lama4 which encodes laminin alpha4, a member of the laminin family of glycoproteins, was enhanced as compared to untreated anchorage-independent HeLa cells. Using a shear flow chamber, an attachment assay was developed; illustrating either increased expression of siat7e or decreased expression of lama4 in anchorage-dependent HeLa cells reduced cellular adhesion. Collectively, the results of this study are consistent with the roles siat7e and lama4 play in adhesion processes in vivo and indicate modifying the expression of either gene can influence adhesion in HeLa cells. The strategy of applying bioinformatics techniques to characterize and manipulate phenotypic behaviors is a powerful tool for altering the properties of various cell lines for desired biotechnology objectives.     

Simultaneous in situ hybridization and TUNEL to identify cells undergoing apoptosis

Apoptotic cells in tissue sections can be localized by in situ labelling of partly degraded DNA. In a heterogeneous population of cells, however, the specific identity of cell types undergoing apoptosis often cannot be reliably achieved at the light microscope level because of the marked alterations in cellular morphology that characterize apoptosis. In order to clearly specify cell types undergoing apoptosis, in situ end labelling has been coupled to immunohistochemistry. This method is limited by the availability of antibodies that bind to cell-specific protein markers in tissue sections. In contrast, we describe a method that combines in situ end labelling with in situ hybridization, a technique that specifies cell types based on mRNA expression. Taking advantage of the specific expression of surfactant protein C mRNA in type II alveolar epithelial cells, we demonstrate that this technique has the ability to localize alveolar type II cells undergoing apoptosis in vivo after the intratracheal instillation of an antibody that activates the cell surface Fas protein. The wide availability of cell-specific gene markers suggests that this method can be adapted to define cell types that undergo apoptosis during various physiological and pathological states in vivo. This revised version was published online in November 2006 with corrections to the Cover Date.     

A microquantitative method to characterize lectin-induced cytoagglutination

A microquantitative method to characterize lectin-induced cytoagglutination reactions is described. The assay, which utilizes an electronic particle counter, measures the disappearance of single cells from cell suspensions containing various concentrations of lectin. The method is sensitive, requiring only μg quantities of lectin, reproducible, and amenable to thorough statistical analysis. It was also utilized to quantitate the inhibition of lectin-induced cytoagglutination by various saccharides. The method was employed to characterize ConA-induced agglutination of guinea pig erythrocytes and Novikoff ascites hepatoma cells, and to investigate several parameters which influence lectin-induced cytoagglutination reactions.     

Genomic in situ hybridization to identify alien chromosomes and chromosome segments in wheat

Summary Genomic in situ hybridization was used to identify alien chromatin in chromosome spreads of wheat, Triticum aestivum L., lines incorporating chromosomes from Leymus multicaulis (Kar. and Kir.) Tzvelev and Thinopyrum bessarabicum (Savul. and Rayss) Löve, and chromosome arms from Hordeum chilense Roem. and Schult, H. vulgare L. and Secale cereale L. Total genomic DNA from the introgressed alien species was used as a probe, together with excess amounts of unlabelled blocking DNA from wheat, for DNA:DNA in-situ hybridization. The method labelled the alien chromatin yellow-green, while the wheat chromosomes showed only the orange-red fluorescence of the DNA counterstain. Nuclei were screened from seedling root-tips (including those from half-grains) and anther wall tissue. The genomic probing method identified alien chromosomes and chromosome arms and allowed counting in nuclei at all stages of the cell cycle, so complete metaphases were not needed. At prophase or interphase, two labelled domains were visible in most nuclei from disomic lines, while only one labelled domain was visible in monosomic lines. At metaphase, direct visualization of the morphology of the alien chromosome or chromosome segment was possible and allowed identification of the relationship of the alien chromatin to the wheat chromosomes. The genomic in-situ hybridization method is fast, sensitive, accurate and informative. Hence it is likely to be of great value for both cytogenetic analysis and in plant breeding programmes.     

Strategies to evade false positives in the in situ analysis of peptide antibiotics in SDS-PAGE gels

In situ activity assay is one of the promising techniques for the characterization of peptide antibiotics. This assay was carried out for the peptide purified from a new bacterial isolate Paenibacillus alvei and commercial peptide antibiotic polymixin E. Towards this, the routine and new protocols were tried. Interestingly, the unexpected result of these experiments - the "switch over activity" has led us to have further investigations. Here, we have addressed the potential problem in the methodology of in situ assay and demonstrated a fool proof protocol to evade the false positive results.     

A new method to characterize chemically and topographically nanopatterned surfaces

Surface chemistry of topographically patterned grooved samples with ridges of 150 nm width, adsorbed with self-assembled monolayers (SAMs) of alkanethiols on gold, have been characterized by near edge X-ray absorption fine structure (NEXAFS) spectroscopy. Analysis reveals that NEXAFS may discriminate between different chemistries adsorbed to the tops, sidewalls and grooves of the patterns.     

A controlled silver impregnation method to characterize cultured cardiomyocytes

A morphological characterization of cultured cardiomyocytes was attempted using a modification of a silver impregnation technique originally described for connective tissue. Cardiac cells, obtained from newborn rats and grown as dissociated cultures on plastic surfaces, were fixed in methanol plus 5% glacial acetic acid, treated with potassium permanganate, decolorized in oxalic acid, sensitized with potassium bichromate, impregnated with a silver-ammonium complex, reduced in gelatin-formalin preparation, toned with gold chloride and fixed in sodium thiosulfate. The cultured cardiac cells tended to form a monolayer, although many myocytes remained isolated. Spherical nuclei, sharply stained with silver, were centrally located and surrounded by relatively plentiful cytoplasm packed with well delineated myofibrils. Contaminating fibroblasts were readily distinguished by their spindle-shaped nuclei and the presence of overstained collagen fibers, as well as the absence of myofibrils. In the absence of specific antibody for immunocytochemical identification of cardiomyocytes, morphological characterization of cell type and degree of differentiation by the controlled silver impregnation procedure described here provides a viable alternative, both in short- and long-term studies.     

A flash photolysis method to characterize hexacoordinate hemoglobin kinetics

A flash photolysis method is described for analyzing ligand binding to the new and growing group of hemoglobins which are hexacoordinate in the unligated, ferrous state. Simple analysis of a two exponential fit to time courses for CO rebinding at varying CO concentrations yields rate constants for formation and dissociation of the hexacoordinate complex, and the bimolecular rate constant for CO binding. This method was tested with a nonsymbiotic plant hemoglobin from rice for which these values had not previously been determined. For this protein, dissociation and rebinding of the hexacoordinating amino acid side chain, His(73), is rapid and similar to the rate of CO binding at high CO concentrations. These results indicate that hexacoordination must be taken into account when evaluating the affinity of hexacoordinate hemoglobins for ligands.     

New method to characterize microbial diversity using flow cytometry

The majority of microorganisms have yet to be cultivated and represent a vast uncharacterized and untapped resource. Here, we report the utilization of a combination of flow cytometry, cultivation, and molecular genetics to develop new methodologies to access and characterize biodiversity in microbial samples. We demonstrate that fluorescent dyes and combinations of dyes can selectively stain portions of bacterial populations that can be isolated as sub-populations using fluorescence-activated cell sorting (FACS). Microbial sub-populations obtained by FACS differ substantially from the original microbial population, as demonstrated by denaturing gradient gel electrophoresis and determination of 16S rRNA gene sequences. These sub-populations can subsequently be used to inoculate microbial growth media, allowing the isolation of different microbial species from those that can be readily cultivated from the original sample using the same microbial growth media. When this technique was applied to the analysis of activated-sludge and Yellowstone Lake hydrothermal vent samples, comparative analysis of 16S rDNA sequences revealed that FACS allowed the detection of numerous bacterial species, including previously unknown species, not readily detectable in the original sample due to low relative abundance. This approach may result in a convenient methodology to more thoroughly characterize microbial biodiversity.     

A method to characterize the passive elasticity in contracting muscle bundles

There is concern among researchers whether the passive muscle properties, characterized by purely passive material testing procedures, are an appropriate representation of the actual passive component of the muscle. This aspect is of particular importance in the biomechanical analysis of heart muscle response where it is generally agreed that the so-called parallel elasticity cannot be ignored as is done justifiably in the analysis of skeletal muscle response. In the present article, a method of quantifying the passive elasticity in contracting muscle bundles is presented. The method consists of imposing isometric transients (such as the quick-stretch or quick release) on a muscle bundle during the contraction phase and observing the differences in decayed force levels between a normal twitch and that of a perturbed twitch. The proposed method provides a means of obtaining useful passive properties from contracting muscle bundles and circumvents the difficulty of having to characterize muscle properties from separate experiments on quiescent muscle bundles.     

Genomic subtraction to identify and characterize sequences of Shiga toxin-producing Escherichia coli O91:H21

To identify Shiga toxin-producing Escherichia coli genes associated with severe human disease, a genomic subtraction technique was used with hemolytic-uremic syndrome-associated O91:H21 strain CH014 and O6:H10 bovine strains. The method was adapted to the Shiga toxin-producing E. coli genome: three rounds of subtraction were used to isolate DNA fragments specific to strain CH014. The fragments were characterized by genetic support analysis, sequencing, and hybridization to the genome of a collection of Shiga toxin-producing E. coli strains. A total of 42 fragments were found, 19 of which correspond to previously identified unique DNA sequences in the enterohemorrhagic E. coli EDL933 reference strain, including 7 fragments corresponding to prophage sequences and others encoding candidate virulence factors, such a SepA homolog protein and a fimbrial usher protein. In addition, the subtraction procedure yielded plasmid-related sequences from Shigella flexneri and enteropathogenic and Shiga toxin-producing E. coli virulence plasmids. We found that lateral gene transfer is extensive in strain CH014, and we discuss the role of genomic mobile elements, especially bacteriophages, in the evolution and possible transfer of virulence determinants.     

A modified method to directly detect in SDS-PAGE the bacteriocin of Pediococcus acidilactici

A modified direct detection system was developed to identify the antimicrobial peptide, pediocin AcH in SDS-PAGE. The gel containing pediocin AcH was stained for 30 min, destained for 1.5 h, and then overlaid with indicator bacteria. The pediocin AcH band was identified by a zone of inhibition of bacterial growth surrounding the stained protein band.