Showing papers on "Gel electrophoresis published in 1999"

Correlation between Protein and mRNA Abundance in Yeast

Abstract: The description of the state of a biological system by the quantitative measurement of the system constituents is an essential but largely unexplored area of biology. With recent technical advances including the development of differential display-PCR (21), of cDNA microarray and DNA chip technology (20, 27), and of serial analysis of gene expression (SAGE) (34, 35), it is now feasible to establish global and quantitative mRNA expression profiles of cells and tissues in species for which the sequence of all the genes is known. However, there is emerging evidence which suggests that mRNA expression patterns are necessary but are by themselves insufficient for the quantitative description of biological systems. This evidence includes discoveries of posttranscriptional mechanisms controlling the protein translation rate (15), the half-lives of specific proteins or mRNAs (33), and the intracellular location and molecular association of the protein products of expressed genes (32). Proteome analysis, defined as the analysis of the protein complement expressed by a genome (26), has been suggested as an approach to the quantitative description of the state of a biological system by the quantitative analysis of protein expression profiles (36). Proteome analysis is conceptually attractive because of its potential to determine properties of biological systems that are not apparent by DNA or mRNA sequence analysis alone. Such properties include the quantity of protein expression, the subcellular location, the state of modification, and the association with ligands, as well as the rate of change with time of such properties. In contrast to the genomes of a number of microorganisms (for a review, see reference 11) and the transcriptome of Saccharomyces cerevisiae (35), which have been entirely determined, no proteome map has been completed to date. The most common implementation of proteome analysis is the combination of two-dimensional gel electrophoresis (2DE) (isoelectric focusing-sodium dodecyl sulfate [SDS]-polyacrylamide gel electrophoresis) for the separation and quantitation of proteins with analytical methods for their identification. 2DE permits the separation, visualization, and quantitation of thousands of proteins reproducibly on a single gel (18, 24). By itself, 2DE is strictly a descriptive technique. The combination of 2DE with protein analytical techniques has added the possibility of establishing the identities of separated proteins (1, 2) and thus, in combination with quantitative mRNA analysis, of correlating quantitative protein and mRNA expression measurements of selected genes. The recent introduction of mass spectrometric protein analysis techniques has dramatically enhanced the throughput and sensitivity of protein identification to a level which now permits the large-scale analysis of proteins separated by 2DE. The techniques have reached a level of sensitivity that permits the identification of essentially any protein that is detectable in the gels by conventional protein staining (9, 29). Current protein analytical technology is based on the mass spectrometric generation of peptide fragment patterns that are idiotypic for the sequence of a protein. Protein identity is established by correlating such fragment patterns with sequence databases (10, 22, 37). Sophisticated computer software (8) has automated the entire process such that proteins are routinely identified with no human interpretation of peptide fragment patterns. In this study, we have analyzed the mRNA and protein levels of a group of genes expressed in exponentially growing cells of the yeast S. cerevisiae. Protein expression levels were quantified by metabolic labeling of the yeast proteins to a steady state, followed by 2DE and liquid scintillation counting of the selected, separated protein species. Separated proteins were identified by in-gel tryptic digestion of spots with subsequent analysis by microspray liquid chromatography-tandem mass spectrometry (LC-MS/MS) and sequence database searching. The corresponding mRNA transcript levels were calculated from SAGE frequency tables (35). This study, for the first time, explores a quantitative comparison of mRNA transcript and protein expression levels for a relatively large number of genes expressed in the same metabolic state. The resultant correlation is insufficient for prediction of protein levels from mRNA transcript levels. We have also compared the relative amounts of protein and mRNA with the respective codon bias values for the corresponding genes. This comparison indicates that codon bias by itself is insufficient to accurately predict either the mRNA or the protein expression levels of a gene. In addition, the results demonstrate that only highly expressed proteins are detectable by 2DE separation of total cell lysates and that therefore the construction of complete proteome maps with current technology will be very challenging, irrespective of the type of organism.

A sampling of the yeast proteome

Abstract: In this study, we examined yeast proteins by two-dimensional (2D) gel electrophoresis and gathered quantitative information from about 1,400 spots. We found that there is an enormous range of protein abundance and, for identified spots, a good correlation between protein abundance, mRNA abundance, and codon bias. For each molecule of well-translated mRNA, there were about 4,000 molecules of protein. The relative abundance of proteins was measured in glucose and ethanol media. Protein turnover was examined and found to be insignificant for abundant proteins. Some phosphoproteins were identified. The behavior of proteins in differential centrifugation experiments was examined. Such experiments with 2D gels can give a global view of the yeast proteome.

Quantification of bias related to the extraction of DNA directly from soils.

Abstract: In recent years, several protocols based on the extraction of nucleic acids directly from the soil matrix after lysis treatment have been developed for the detection of microorganisms in soil. Extraction efficiency has often been evaluated based on the recovery of a specific gene sequence from an organism inoculated into the soil. The aim of the present investigation was to improve the extraction, purification, and quantification of DNA derived from as large a portion of the soil microbial community as possible, with special emphasis placed on obtaining DNA from gram-positive bacteria, which form structures that are difficult to disrupt. Furthermore, we wanted to identify and minimize the biases related to each step in the procedure. Six soils, covering a range of pHs, clay contents, and organic matter contents, were studied. Lysis was carried out by soil grinding, sonication, thermal shocks, and chemical treatments. DNA was extracted from the indigenous microflora as well as from inoculated bacterial cells, spores, and hyphae, and the quality and quantity of the DNA were determined by gel electrophoresis and dot blot hybridization. Lysis efficiency was also estimated by microscopy and viable cell counts. Grinding increased the extracellular DNA yield compared with the yield obtained without any lysis treatment, but none of the subsequent treatments clearly increased the DNA yield. Phage λ DNA was inoculated into the soils to mimic the fate of extracellular DNA. No more than 6% of this DNA could be recovered from the different soils. The clay content strongly influenced the recovery of DNA. The adsorption of DNA to clay particles decreased when the soil was pretreated with RNA in order to saturate the adsorption sites. We also investigated different purification techniques and optimized the PCR methods in order to develop a protocol based on hybridization of the PCR products and quantification by phosphorimaging.

A Microfabricated Device for Sizing and Sorting DNA Molecules

Abstract: We have demonstrated a microfabricated single-molecule DNA sizing device This device does not depend on mobility to measure molecule size, is 100 times faster than pulsed-field gel electrophoresis, and has a resolution that improves with increasing DNA length It also requires a million times less sample than pulsed-field gel electrophoresis and has comparable resolution for large molecules Here we describe the fabrication and use of the single-molecule DNA sizing device for sizing and sorting DNA restriction digests and ladders spanning 2-200 kbp

Identification and Characterization of Major Lipid Particle Proteins of the Yeast Saccharomyces cerevisiae

Abstract: Lipid particles of the yeast Saccharomyces cerevisiae were isolated at high purity, and their proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major lipid particle proteins were identified by mass spectrometric analysis, and the corresponding open reading frames (ORFs) were deduced. In silicio analysis revealed that all lipid particle proteins contain several hydrophobic domains but none or only few (hypothetical) transmembrane spanning regions. All lipid particle proteins identified by function so far, such as Erg1p, Erg6p, and Erg7p (ergosterol biosynthesis) and Faa1p, Faa4p, and Fat1p (fatty acid metabolism), are involved in lipid metabolism. Based on sequence homology, another group of three lipid particle proteins may be involved in lipid degradation. To examine whether lipid particle proteins of unknown function are also involved in lipid synthesis, mutants with deletions of the respective ORFs were constructed and subjected to systematic lipid analysis. Deletion of YDL193w resulted in a lethal phenotype which could not be suppressed by supplementation with ergosterol or fatty acids. Other deletion mutants were viable under standard conditions. Strains with YBR177c, YMR313c, and YKL140w deleted exhibited phospholipid and/or neutral lipid patterns that were different from the wild-type strain and thus may be further candidate ORFs involved in yeast lipid metabolism.

Export of Virulence Genes and Shiga Toxin by Membrane Vesicles of Escherichia coli O157:H7

Abstract: Membrane vesicles released by Escherichia coli O157:H7 into culture medium were purified and analyzed for protein and DNA content. Electron micrographs revealed vesicles that are spherical, range in size from 20 to 100 nm, and have a complete bilayer. Analysis of vesicle protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates vesicles that contain many proteins with molecular sizes similar to outer membrane proteins and a number of cellular proteins. Immunoblot (Western) analysis of vesicles suggests the presence of cell antigens. Treatment of vesicles with exogenous DNase hydrolyzed surface-associated DNA; PCR demonstrated that vesicles contain DNA encoding the virulence genes eae, stx1 and stx2, and uidA, which encodes for β-galactosidase. Immunoblot analysis of intact and lysed, proteinase K-treated vesicles demonstrate that Shiga toxins 1 and 2 are contained within vesicles. These results suggest that vesicles contain toxic material and transfer experiments demonstrate that vesicles can deliver genetic material to other gram-negative organisms.

Advances in protein solubilisation for two‐dimensional electrophoresis

Abstract: Two-dimensional (2-D) electrophoresis remains the highest resolution technique for protein separation and is the method of choice when complex samples need to be arrayed for characterisation, as in proteomics. However, in current proteome projects the total number of proteins identified from 2-D gels is often only a small percentage of the predicted proteome. In addition, there is an almost complete lack of hydrophobic proteins on 2-D gels, especially those using immobilised pH gradients. Recently there have been a number of publications reporting reagents which improve protein solubilisation prior to isoelectric focusing. The improved solubilization possible with these reagents has increased the total number of proteins able to be visualised on 2-D gels and also allowed the separation of hydrophobic proteins, such as integral membrane proteins.

Molecular Typing of S-alleles through Identification, Characterization and cDNA Cloning for S-RNases in Sweet Cherry

Abstract: This report identifies S-RNases of sweet cherry (Prunus avium L.) and presents information about cDNA sequences encoding the S-RNases, which leads to the development of a molecular typing system for S-alleles in this fruit tree species. Stylar proteins of sweet cherry were surveyed by two dimensional polyaclylamide gel electrophoresis (2D- PAGE) to identify S-proteins associated with gametophytic self-incompatibility. Glycoprotein spots linked to S-alleles were found in a group of proteins which had Mr and pI similar to those of other rosaceous S-RNases. These glycoproteins were present at highest concentration in the upper segment of the mature style and shared immunological characteristics and N-terminal sequences with those of S-RNases of other plant species. cDNAs encoding these glycoproteins were cloned based on the N-terminal sequences. Genomic DNA and RNA blot analyses and deduced amino acid sequences indicated that the cDNAs encode S-RNases; thus the S-proteins identified by 2D-PAGE are S-RNases. Although S 1 to S 6 -alleles of sweet cherry cultivars could be distinguished from each other with the genomic DNA blot analysis, a much simpler method of PCR-based typing system was developed for the six S-alleles based on the DNA sequence data obtained from the cDNAs encoding S-RNases.

Recent developments in two-dimensional gel electrophoresis with immobilized pH gradients: wide pH gradients up to pH 12, longer separation distances and simplified procedures.

Abstract: Wide-range immobilized pH 3—12 and 6—12 gradients were generated. Depending on the extraction method of sample preparation, proteins with pIs up to pH 11.7 were resolved. Highly reproducible protein patterns, focused to the steady-state with round-shaped spots up to the basic end were obtained. Moreover, because a strong water transport from cathode to anode (reverse electroendosmotic flow) inherent to narrow immobilized pH gradients (IPGs) exceeding pH 11, such as IPG 10—12, was negligible, the wide-range IPGs 3—12 and 6—12 could be run under standard conditions as originally described by Gorg et al. (Electrophoresis 1988, 9, 531—546). The wide-range immobilized pH gradient 3—12 proved to be perfectly suited for an overview separation of total cell extracts. Resolution could be increased by extending the separation distance from 18 to 24 cm. Furthermore, two-dimensional gel electrophoresis with IPGs (IPG-Dalt) was simplified by the use of an integrated system (IPGphor) where sample application by in-gel rehydration and isoelectric focusing (IEF) are performed automatically in a one-step procedure, overnight, without human assistance.

Interactions of grape seed tannins with salivary proteins.

Abstract: To evaluate the amount and type of condensed tannins binding salivary proteins, which are supposed to be involved in astringent sensation, model systems allowing further analyses of proteins and condensed tannins were developed. The precipitates formed after addition of grape seed tannins to salivary proteins indicate that a binding interaction occurs. Dissociation of insoluble complexes was achieved by sodium dodecyl sulfate treatment. Thiolysis reaction allowed the quantification and characterization of proanthocyanidins on both the resulting pellet and the supernatant. Binding proteins were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The higher polymerized tannins predominantly precipitated together with the salivary proteins. The condensed tannins remaining in solution were low molecular weight polymers.

Self-assembly of DNA-streptavidin nanostructures and their use as reagents in immuno-PCR

Abstract: The self-assembly of bis-biotinylated double-stranded DNA and the tetravalent biotin-binding protein streptavidin (STV) have been studied by non-denaturing gel electrophoresis and atomic force microscopy (AFM). The rapid self-assembly reproducibly generated populations of individual oligomeric complexes. Most strikingly, the oligomers predominantly contained bivalent STV molecules bridging two adjacent DNA fragments to form linear nanostructures. Trivalent STV branch points occurred with a lower frequency and the presence of tetravalent STV was scarce. However, valency distribution, size and the exchange dynamics of the supramolecular aggregates were highly sensitive to stoichiometric variations in the relative molar coupling ratio of bis-biotinylated DNA and STV. The largest aggregates were obtained from equimolar amounts while excess STV led to the formation of smaller oligomers appearing as fingerprint-like band patterns in electrophoresis. Excess DNA, however, induces a complete breakdown of the oligomers, likely a consequence of the instability of STV conjugates containing more than two biotinylated DNA fragments. It was demonstrated that the oligomers can further be functionalized, for instance by the coupling of biotinylated immunoglobulins. Both pure and also antibody-modified DNA-STV oligomers were used as reagents in immuno-PCR (IPCR), a highly sensitive detection method for proteins and other antigens. Employment of the supramolecular reagents led to an approximately 100-fold enhanced sensitivity compared to the conventional IPCR procedure.

Identification of a Novel Group of Bacteria in Sludge from a Deteriorated Biological Phosphorus Removal Reactor

Abstract: The microbial diversity of a deteriorated biological phosphorus removal reactor was investigated by methods not requiring direct cultivation. The reactor was fed with media containing acetate and high levels of phosphate (P/C weight ratio, 8:100) but failed to completely remove phosphate in the effluent and showed very limited biological phosphorus removal activity. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA was used to investigate the bacterial diversity. Up to 11 DGGE bands representing at least 11 different sequence types were observed; DNA from the 6 most dominant of these bands was further isolated and sequenced. Comparative phylogenetic analysis of the partial 16S rRNA sequences suggested that one sequence type was affiliated with the alpha subclass of the Proteobacteria, one was associated with the Legionella group of the gamma subclass of the Proteobacteria, and the remaining four formed a novel group of the gamma subclass of the Proteobacteria with no close relationship to any previously described species. The novel group represented approximately 75% of the PCR-amplified DNA, based on the DGGE band intensities. Two oligonucleotide rRNA probes for this novel group were designed and used in a whole-cell hybridization analysis to investigate the abundance of this novel group in situ. The bacteria were coccoid and 3 to 4 microm in diameter and represented approximately 35% of the total population, suggesting a relatively close agreement with the results obtained by the PCR-based DGGE method. Further, based on electron microscopy and standard staining microscopic analysis, this novel group was able to accumulate granule inclusions, possibly consisting of polyhydroxyalkanoate, inside the cells.

Acetone preservation: a practical technique for molecular analysis

Abstract: In attempts to establish a convenient and reliable method for field collection and archival preservation of insects and their endosymbiotic microorganisms for molecular analysis, acetone, ethanol, and other organic solvents were tested for DNA preservability of the pea aphid Acyrthosiphon pisum and its intracellular symbiotic bacterium Buchnera sp. After 6 months' storage, not only the band of high-molecular-size DNA but also the bands of rRNA were well preserved in acetone, ethanol, 2-propanol, diethyl ether and ethyl acetate. Polymerase chain reaction (PCR) assays confirmed that the DNA of both the insects and their symbionts was well preserved in these solvents. In contrast, methanol and chloroform showed poor DNA preservability. When water-containing series of acetone and ethanol were examined for DNA preservability, acetone was apparently more robust against water contamination than ethanol. Considering that most biological materials contain high amounts of water, acetone may be a more recommendable preservative for DNA analysis than ethanol which has been widely used for this purpose. The DNA of various insects could be preserved in acetone at room temperature in good condition for several years. In addition to the DNA of the host insects, the DNA of their endosymbionts, including Buchnera and other mycetocyte symbionts, Wolbachia, and gut bacteria, was amplified by PCR after several years of acetone storage. The RNA and protein of the pea aphid and its endosymbiont were also preserved for several years in acetone. After 2 years' storage in acetone, proteins of A. pisum could be analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, and the endosymbiotic bacteria were successfully detected by immunohistochemistry and in situ hybridization on the tissue sections.

Proteome Research: Two-Dimensional Gel Electrophoresis and Identification Methods

Abstract: 1 Introduction: The Virtue of Proteomics.- 2 Solubilization of Proteins in 2D Electrophoresis.- 1 Introduction.- 2 Rationale of Solubilization-Breaking Molecular Interactions.- 3 Initial Solubilization.- 4 Solubility During IEF.- 5 Conclusions: Current Limits and How to Push Them.- References.- 3 Two-Dimensional Electrophoresis with Carrier Ampholytes.- 1 Introduction.- 2 Sample Preparation.- 3 First Dimension. Standard Isoelectric Focusing.- 4 Second Dimension. Standard Slab Gel Electrophoresis.- 5 Modification of the Standard Two-Dimensional Gel Method.- 6 Visualization of Separated Proteins: Silver Staining.- References.- Appendix: Problems and Troubleshooting.- 4 Two-Dimensional Electrophoresis with Immobilized pH Gradients.- 1 Introduction.- 2 Sample Preparation.- 3 First Dimension: IEF with IPGs.- 4 Equilibration of IPG Strips.- 5 Second Dimension: SDS-PAGE.- References.- Appendix A: General Troubleshooting.- Appendix B: Troubleshooting for IPG and Horizontal PAGE.- 5 Detection of Proteins on Two-Dimensional Electrophoresis Gels.- 1 Introduction.- 2 Detection by Organic Dyes.- 3 Detection by Differential Precipitation of Salts.- 4 Detection by Metal Ion Reduction (Silver Staining).- 5 Detection by Fluorescence.- 6 Detection of Radioactive Isotopes.- 7 Conclusions and Future Prospects.- References.- 6 Blotting and Immunoaffinity Identification of Two-Dimensional Electrophoresis-Separated Proteins.- 1 Introduction.- 2 Protein Transfer onto Membranes.- 3 Membrane Staining.- 4 Immunodetection.- 5 Assignment of Two-Dimensional Immunoreactive Spots by Matching.- References.- 7 Identification of Proteins by Amino Acid Composition After Acid Hydrolysis.- 1 Introduction.- 2 Blotting of Proteins from Gels to PVDF Membranes.- 3 Hydrolysis of PVDF-bound Proteins.- 4 Extraction of Amino Acids from PVDF Membranes.- 5 Derivatization and Chromatography.- 6 Amino Acid Analysis Troubleshooting Guide.- 7 Protein Identification by Database Matching.- 8 Identification by N-Terminal Sequence Tags and Amino Acid Composition.- 9 Conclusions.- References.- 8 Identification by Amino Acid Composition Obtained from Labeling.- 1 Introduction.- 2 Choice and Labeling Amino Acids.- 3 Single Labeling Method.- 4 Double Labeling Method Based on 35S Decay.- 5 Double Labeling Method using Scintillation Counting.- 6 Determination of pI and Mr.- 7 Construction of the Database.- 8 Search in a Protein Database.- 9 Result Analysis.- 10 Conclusion.- References.- 9 Identification of Proteins by Amino Acid Sequencing.- 1 Purpose of Protein Sequencing.- 2 N-Terminal Sequence Analysis by Edman Chemistry.- 3 Other Possibilities for Generation of Amino Acid Sequences.- 4 The Interface from Two-Dimensional Gel.- 5 Conclusion.- 6 Perspectives.- References.- 10 Identification of Proteins by Mass Spectrometry.- 1 Introduction.- 2 Protein Preparation Methods Compatible with MS.- 3 Enzymatic Digestion of Proteins.- 4 Sample Clean-up for ESI-MS and On-line Liquid Chromatography.- 5 Sample Clean-up for MALDI-MS.- 6 Mass Spectrometers.- 7 Protein Identification by Correlating MS DATA with Sequence Databases.- References.- 11 Mass Spectrometry of Intact Proteins from Two-Dimensional PAGE.- 1 Introduction.- 2 Elution of Proteins from Gels for Mass Spectrometry.- 3 Mass Spectrometry of Proteins Electroblotted onto Polymer Membranes.- 4 UV-MALDI Mass Spectrometry of Proteins Directly from Polyacrylamide Gels.- 5 Conclusion.- References.

The staphylococcal transferrin-binding protein is a cell wall glyceraldehyde-3-phosphate dehydrogenase.

Abstract: Staphylococcus aureus and Staphylococcus epidermidis possess a 42-kDa cell wall transferrin-binding protein (Tpn) which is involved in the acquisition of transferrin-bound iron. To characterize this protein further, cell wall fractions were subjected to two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis blotted, and the N-terminus of Tpn was sequenced. Comparison of the first 20 amino acid residues of Tpn with the protein databases revealed a high degree of homology to the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Analysis of staphylococcal cell wall fractions for GAPDH activity confirmed the presence of a functional enzyme which, like Tpn, is regulated by the availability of iron in the growth medium. To determine whether Tpn is responsible for this GAPDH activity, it was affinity purified with NAD+ agarose. Both S. epidermidis and S. aureus Tpn catalyzed the conversion of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate. In contrast, Staphylococcus saprophyticus, which lacks a Tpn, has no cell wall-associated GAPDH activity. Native polyacrylamide gel electrophoresis of the affinity-purified Tpn revealed that it was present in the cell wall as a tetramer, consistent with the structures of all known cytoplasmic GAPDHs. Furthermore, the affinity-purified Tpn retained its ability to bind human transferrin both in its native tetrameric and SDS-denatured monomeric forms. Apart from interacting with human transferrin, Tpn, in common with the group A streptococcal cell wall GAPDH, binds human plasmin. Tpn-bound plasmin is enzymatically active and therefore may contribute to the ability of staphylococci to penetrate tissues during infections. These studies demonstrate that the staphylococcal transferrin receptor protein, Tpn, is a multifunctional cell wall GAPDH.

A Novel Lipolytic Enzyme Located in the Outer Membrane of Pseudomonas aeruginosa

Abstract: A lipase-negative deletion mutant of Pseudomonas aeruginosa PAO1 still showed extracellular lipolytic activity toward short-chain p-nitrophenylesters. By screening a genomic DNA library of P. aeruginosa PAO1, an esterase gene, estA, was identified, cloned, and sequenced, revealing an open reading frame of 1,941 bp. The product of estA is a 69.5-kDa protein, which is probably processed by removal of an N-terminal signal peptide to yield a 67-kDa mature protein. A molecular mass of 66 kDa was determined for 35S-labeled EstA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The amino acid sequence of EstA indicated that the esterase is a member of a novel GDSL family of lipolytic enzymes. The estA gene showed high similarity to an open reading frame of unknown function located in the trpE-trpG region of P. putida and to a gene encoding an outer membrane esterase of Salmonella typhimurium. Amino acid sequence alignments led us to predict that this esterase is an autotransporter protein which possesses a carboxy-terminal β-barrel domain, allowing the secretion of the amino-terminal passenger domain harboring the catalytic activity. Expression of estA in P. aeruginosa and Escherichia coli and subsequent cell fractionation revealed that the enzyme was associated with the cellular membranes. Trypsin treatment of whole cells released a significant amount of esterase, indicating that the enzyme was located in the outer membrane with the catalytic domain exposed to the surface. To our knowledge, this esterase is unique in that it exemplifies in P. aeruginosa (i) the first enzyme identified in the outer membrane and (ii) the first example of a type IV secretion mechanism.

Purification and characterization of an extracellular protease from the fish pathogen Yersinia ruckeri and effect of culture conditions on production.

Abstract: A novel protease, hydrolyzing azocasein, was identified, purified, and characterized from the culture supernatant of the fish pathogen Yersinia ruckeri. Exoprotease production was detected at the end of the exponential growth phase and was temperature dependent. Activity was detected in peptone but not in Casamino Acid medium. Its synthesis appeared to be under catabolite repression and ammonium control. The protease was purified in a simple two-step procedure involving ammonium sulfate precipitation and ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified protein indicated an estimated molecular mass of 47 kDa. The protease had characteristics of a cold-adapted protein, i.e., it was more active in the range of 25 to 42°C and had an optimum activity at 37°C. The activation energy for the hydrolysis of azocasein was determined to be 15.53 kcal/mol, and the enzyme showed a rapid decrease in activity at 42°C. The enzyme had an optimum pH of around 8. Characterization of the protease showed that it required certain cations such as Mg2+ or Ca2+ for maximal activity and was inhibited by EDTA, 1,10-phenanthroline, and EGTA but not by phenylmethylsulfonyl fluoride. Two N-methyl-N-nitro-N-nitrosoguanidine mutants were isolated and analyzed; one did not show caseinolytic activity and lacked the 47-kDa protein, while the other was hyperproteolytic and produced increased amounts of the 47-kDa protein. Azocasein activity, SDS-PAGE, immunoblotting by using polyclonal anti-47-kDa-protease serum, and zymogram analyses showed that protease activity was present in 8 of 14 strains tested and that two Y. ruckeri groups could be established based on the presence or absence of the 47-kDa protease.

CspI, the ninth member of the CspA family of Escherichia coli, is induced upon cold shock.

Abstract: Escherichia coli contains the CspA family, consisting of nine proteins (CspA to CspI), in which CspA, CspB, and CspG have been shown to be cold shock inducible and CspD has been shown to be stationary-phase inducible. The cspI gene is located at 35.2 min on the E. coli chromosome map, and CspI shows 70, 70, and 79% identity to CspA, CspB, and CspG, respectively. Analyses of cspI-lacZ fusion constructs and the cspI mRNA revealed that cspI is cold shock inducible. The 5′-untranslated region of the cspI mRNA consists of 145 bases and causes a negative effect on cspI expression at 37°C. The cspI mRNA was very unstable at 37°C but was stabilized upon cold shock. Analyses of the CspI protein on two-dimensional gel electrophoresis revealed that CspI production is maximal at or below 15°C. Taking these results together, E. coli possesses a total of four cold shock-inducible proteins in the CspA family. Interestingly, the optimal temperature ranges for their induction are different: CspA induction occurs over the broadest temperature range (30 to 10°C), CspI induction occurs over the narrowest and lowest temperature range (15 to 10°C), and CspB and CspG occurs at temperatures between the above extremes (20 to 10°C).

Heterologous Expression of a Plant Small Heat-Shock Protein Enhances Escherichia coli Viability under Heat and Cold Stress

Abstract: A small heat-shock protein (sHSP) that shows molecular chaperone activity in vitro was recently purified from mature chestnut (Castanea sativa) cotyledons. This protein, renamed here as CsHSP17.5, belongs to cytosolic class I, as revealed by cDNA sequencing and immunoelectron microscopy. Recombinant CsHSP17.5 was overexpressed in Escherichia coli to study its possible function under stress conditions. Upon transfer from 37°C to 50°C, a temperature known to cause cell autolysis, those cells that accumulated CsHSP17.5 showed improved viability compared with control cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cell lysates suggested that such a protective effect in vivo is due to the ability of recombinant sHSP to maintain soluble cytosolic proteins in their native conformation, with little substrate specificity. To test the recent hypothesis that sHSPs may be involved in protection against cold stress, we also studied the viability of recombinant cells at 4°C. Unlike the major heat-induced chaperone, GroEL/ES, the chestnut sHSP significantly enhanced cell survivability at this temperature. CsHSP17.5 thus represents an example of a HSP capable of protecting cells against both thermal extremes. Consistent with these findings, high-level induction of homologous transcripts was observed in vegetative tissues of chestnut plantlets exposed to either type of thermal stress but not salt stress.

Analysis of membrane proteins by two-dimensional electrophoresis: comparison of the proteins extracted from normal or Plasmodium falciparum-infected erythrocyte ghosts.

Abstract: Parasite-encoded membrane proteins translocated to the surface of infected erythrocytes or in specialized vesicles underneath (Maurer's clefts) play a key role in the asexual life cycle of Plasmodium falciparum (a malaria-causing protozoan), by mediating key steps such as red blood cell invasion, sequestration of infected cells in microcapillaries, and red blood cell rupture. A large-scale analysis of these membrane proteins would therefore be of great help to gain knowledge of the different stages of the Plasmodium falciparum life cycle. In order to be able to detect and identify parasite-encoded proteins directed to the red blood cell membrane, we first defined the conditions required for optimal extraction and separation of normal red blood cell ghost proteins by two-dimensional gel electrophoresis. These conditions included the use of urea, thiourea and new zwitterionic detergents in the extraction and isoelectric focusing media. The optimized conditions were then applied to analyze normal and P. falciparum-infected red blood cell ghosts. Several protein spots were found only in infected ghosts and are expected to represent parasite-encoded proteins. These proteins are currently under investigation.

Two‐dimensional map of human brain proteins

Abstract: Samples of human brain from the parietal cortex lobe were analyzed by two-dimensional gel electrophoresis, using immobilized pH gradient strips covering the various pH regions. The protein spots were visualized with colloidal Coomassie blue stain and identified by matrix-assisted laser desorption/ionization mass spectrometry. Approximately 400 spots were identified, corresponding to 180 different brain proteins. The list of identified proteins includes a large number of structural proteins and of enzymes or enzyme subunits with various catalytic activities. The majority of proteins are localized in the cytoplasma and in mitochondria. The two-dimensional map may be useful as a reference database to study changes in the protein level caused by various disorders, such as Alzheimer's disease, major depression and schizophrenia.

Single cell gel electrophoresis: detection of DNA damage at different levels of sensitivity.

Abstract: Single cell gel electrophoresis, also known as the comet assay, is widely used for the detection and measurement of DNA strand breaks With the addition of a step in which DNA is incubated with specific endonucleases recognising damaged bases, these lesions can be measured, too In the standard protocol, electrophoresis is carried out at high pH If, instead, electrophoresis is in neutral buffer, the effect of DNA damage seems to be much reduced--either because alkaline conditions are needed to reveal certain lesions, or because the effect of the same number of breaks on DNA migration is greater at high pH A lower sensitivity can be useful in some circumstances, as it extends the range of DNA damage levels over which the assay can be used Here we compare the performance of standard and modified techniques with a variety of DNA-damaging agents and offer possible explanations for the differences in behaviour of DNA under alternative electrophoretic conditions

Hydrolysis of Pork Muscle Sarcoplasmic Proteins by Lactobacillus curvatus and Lactobacillus sake

Abstract: Lactobacillus curvatus CECT 904 and Lactobacillus sake CECT 4808 were selected on the basis of their proteolytic activities against synthetic substrates. Further, the effects of whole cells, cell extracts, and a combination of both enzymatic sources on muscle sarcoplasmic proteins were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase high-performance liquid chromatography analyses. Strains of both species displayed proteinase activities on five sarcoplasmic proteins. The inoculation of whole cells caused a degradation of peptides, whereas the addition of cell extracts resulted in the generation of both hydrophilic and hydrophobic peptides. This phenomenon was remarkably more pronounced when L. curvatus was involved. Whole cells also consumed a great amount of free amino acids, while the addition of intracellular enzymes contributed to their generation. L. sake accounted for a greater release of free amino acids. In general, cell viability and also proteolytic events were promoted when cell suspensions were provided with cell extracts as an extra source of enzymes.

Glycosylphosphatidylinositol‐anchored cell‐surface proteins from Arabidopsis

Abstract: Remodeling of the plant cell surface occurs during the establishment of cell polarity, cellular differentiation, and organ development. This report demonstrates the existence of multiple glycosylphosphatidylinositol (GPI)-anchored proteins in the model plant Arabidopsis. Using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), we also show that GPI-anchored proteins are a relatively abundant class of protein and that they are present at the plant plasma membrane. Furthermore, some of these proteins are released into the extracellular matrix. At least one of these is an arabinogalactan protein (AGP), a class of proteins known to be associated with cellular differentiation. Analysis of the amino acid sequences of two novel AGP-like proteins from Arabidopsis predicts that these proteins contain consensus signals for GPI-anchor addition. These findings support a model where GPI-anchored proteins are involved in the generation of specialized cell surfaces and extracellular signaling molecules.

Association and dissociation characteristics of polymer/DNA complexes used for gene delivery.

Abstract: Purpose. The DNA association/dissociation properties of water-soluble cationic methacrylate polymers with closely related structures (poly(2-dimethylamino)ethyl methacrylate) [p(DMAEMA)], poly(2-(trimethylamino)ethyl methacrylate chloride) [p(TMAEMA)]) and the frequently used transfectant poly(L-lysine) were studied to gain a better insight into their transfection characteristics. Methods. Association of DNA with different polymers and dissociation of the complexes, achieved by adding an excess of anionic polymers or salt, were studied by using spectroscopic techniques (fluorescence, circular dichroism (CD)), agarose gel electrophoresis and an enzymatic assay (DNase I treatment). The transfection efficiency of the polyplexes was evaluated in tissue culture with OVCAR-3 cells. Results. Plasmid DNA complexed with either poly(L-lysine) or p(DMAEMA) was protected against digestion by DNase I. Fluorescence and CD spectroscopy as well as gel electrophoresis revealed that p(DMAEMA) with a relatively high molecular weight and poly(L-lysine) have similar DNA association/dissociation characteristics. Therefore, differences in transfection potential of the polyplexes cannot be ascribed to differences in binding characteristics, but are probably caused by other factors. As compared with the other polymers, p(TMAEMA) has a high affinity for DNA as was concluded from the observation that poly(aspartic acid) was unable to fully dissociate complexes containing this polymer. This fact might very well explain the low transfection efficiency of these polyplexes. p(DMAEM A) with a relatively low molecular weight probably has a low affinity for DNA, which might explain both the formation of DNA aggregates Ψ-DNA) and the low transfection potential obtained when using this polymer. Conclusions. DNA association/dissociation studies shed light on the preferred characteristics of polymeric transfectants.