EST analysis in barley defines a unigene set comprising 4,000 genes.

We report the generation of 13,109 EST (Expressed Sequence Tag) sequences from barley as a first step towards the generation of a unigene set for this organism. Sequences were generated from three libraries encompassing 7,568 cDNA clones. Comparisons to nucleic acid and protein sequence databases enabled the assignment of putative functions to the mRNAs. The results of the searches against protein databases were parsed and built into a regularly updated database, available over the World Wide Web. The Stack_Pack clustering system has been applied to survey the level of redundancy, which was calculated to amount to 69%, thus we identified 4,000 different barley genes. To prove the usability of the results of the clustering process for further experiments, we subjected alignments with sequences similar to elongation factor 1 alpha to additional analysis. These sequences represented the largest group with identical putative functions (228 members) and clustering based on the analysis of 3; sequences subdivided the group into five different assemblies. Alignments of the consensus sequences facilitated the development of PCR assays suitable for genetic mapping of four of the different gene-family members, which reside on chromosomes 2H, 4H and 5H, thus demonstrating the suitability of the cluster-results as a basis for in-depth analyses of barley gene families.

Effects of selenium deficiency on the rat myocardial protein pattern-- investigation by two-dimensional gel electrophoresis.

Dietary selenium deficiency represents an etiological factor in "Keshan disease", a distinct form of an endemic cardiomyopathy. The biochemical effects of selenium depletion in the myocardium are, however, not yet known. Therefore, we investigated the changes in the myocardial protein pattern in rats after long-term selenium deficiency. The myocardial proteins were analyzed in samples from five selenium-depleted rats (Se-deficient group) and five rats supplied with adequate amounts of the element (Se-adequate group). Isoelectric focusing (IEF) with carrier ampholytes on large 2-DE gels was used for the separation of proteins in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the second dimension. The protein patterns were evaluated by means of a computer-assisted gel analysis system. The biochemical identification of the proteins of interest was achieved by matrix-assisted laser desorption/ionization mass spectrometry (MALDI) or immunoblotting. On average, 588 +/- 68 protein spots were found on the gels. No significant difference in spot numbers existed between the groups. A pattern of 270 spots with identical positions was found on every gel; 247 of these spots were not saturated and used for quantitative comparison. Thirty-five, i.e., 14 %, differed significantly in their relative intensity in the two groups. Twenty-eight protein spots were decreased in the Se-deficient group and seven were increased. Sarcomeric creatine kinase M chain, alpha-myosin heavy chain (alpha-MHC) and myosin light chain 1 and 2 (MLC 1 and 2) were largely decreased in Se-deficiency. Three protein spots were increased by more than twofold or appeared only in the Se-deficient group. A mitochondrial creatine kinase was identified in this group. The results suggest that selenium deficiency affects myocardial energy metabolism and contractile proteins. These changes probably reflect non-specific alterations in heart failure.

Proteomics in human disease: cancer, heart and infectious diseases.

In recent years, genomics has increased the understanding of many diseases. Proteomics is a rapidly growing research area that encompasses both genetic and environmental factors. The protein composition represents the functional status of a biological compartment. The five approaches presented here resulted in the detection of disease-associated proteins. Calgranulin B was upregulated in colorectal cancer, and hepatoma-derived aldose reductase-like protein was reexpressed in a rat model during hepatocarcinogenesis. In these two investigations, attention was focused on one protein, obviously differing in amount, directly after two-dimensional electrophoresis (2-DE). Additional methods, such as enzyme activity measurements and immunohistochemistry, confirmed the disease association of the two candidates resulting from 2-DE subtractive analysis. The following three investigations take advantage of the holistic potential of the 2-DE approach. The comparison of 2-DE patterns from dilated cardiomyopathy patients with those of controls revealed 25 statistically significant intensity differences, from which 12 were identified by amino acid analysis, Edman degradation or matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). A human myocardial 2-DE database was constructed, containing 3300 protein spots and 150 identified protein species. The number of identified proteins was limited by the capacity of our group, rather than by the principle of feasibility. Another field where proteomics proves to be a valuable tool in identifying proteins of importance for diagnosis is proteome analysis of pathogenic microorganisms such as Borrelia burgdorferi (Lyme disease) and Toxoplasma gondii (toxoplasmosis). Sera from patients with early or late symptoms of Lyme borreliosis contained antibodies of various classes against about 80 antigens each, containing the already described antigens OspA, B and C, flagellin, p83/100, and p39. Similarly, antibody reactivity to seven different marker antigens of T. gondii allowed differentiation between acute and latent toxoplasmosis, an important diagnostic tool in both pregnancy and immunosuppressed patients.

New algorithmic approaches to protein spot detection and pattern matching in two-dimensional electrophoresis gel databases.

Protein spot identification in two-dimensional electrophoresis gels can be supported by the comparison of gel images accessible in different World Wide Web two-dimensional electrophoresis (2-DE) gel protein databases. The comparison may be performed either by visual cross-matching between gel images or by automatic recognition of similar protein spot patterns. A prerequisite for the automatic point pattern matching approach is the detection of protein spots yielding the x(s),y(s) coordinates and integrated spot intensities i(s). For this purpose an algorithm is developed based on a combination of hierarchical watershed transformation and feature extraction methods. This approach reduces the strong over-segmentation of spot regions normally produced by watershed transformation. Measures for the ellipticity and curvature are determined as features of spot regions. The resulting spot lists containing x(s),y(s),i(s)-triplets are calculated for a source as well as for a target gel image accessible in 2-DE gel protein databases. After spot detection a matching procedure is applied. Both the matching of a local pattern vs. a full 2-DE gel image and the global matching between full images are discussed. Preset slope and length tolerances of pattern edges serve as matching criteria. The local matching algorithm relies on a data structure derived from the incremental Delaunay triangulation of a point set and a two-step hashing technique. For the incremental construction of triangles the spot intensities are considered in decreasing order. The algorithm needs neither landmarks nor an a priori image alignment. A graphical user interface for spot detection and gel matching is written in the Java programming language for the Internet. The software package called CAROL (http://gelmatching.inf.fu-berlin.de) is realized in a client-server architecture.

A two-dimensional electrophoresis database of rat heart proteins.

More than 3000 myocardial protein species of Wistar Kyoto rat, an important animal model, were separated by high-resolution two-dimensional gel electrophoresis (2-DE) and characterized in terms of isoelectric point (pI) and molecular mass (Mr). Currently, the 2-DE database contains 64 identified proteins; forty-three were identified by peptide mass fingerprinting (PMF) using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), nine by exclusive comparison with other 2-DE heart protein databases, and in only 12 cases of 60 attempts N-terminal sequencing was successful. We used the Make2ddb software package downloaded from the ExPASy server for the construction of a rat myocardial 2-DE database. The Make2ddb package simplifies the creation of a new 2-DE database if the Melanie II software and a Sun workstation under Solaris are available. Our 2-DE database of rat heart proteins can be accessed at URL http://gelmatching.inf.fu-berlin.de/pleiss/2d.

Effects of renovascular hypertension on myocardial protein patterns: analysis by computer-assisted two-dimensional gel electrophoresis.

Hypertensive heart disease caused by renovascular hypertension reflects the response of the heart to an increased afterload and neurohormonal stimulation. We hypothesized that in this condition the composition of the myocardial proteins of rats was altered. To identify yet unknown quantitative and qualitative differences in myocardial proteins in renovascular hypertensive heart disease, we analyzed protein patterns by computer-assisted two-dimensional polyacrylamide large gel electrophoresis. Renovascular hypertension was induced by placing a silver clip on the left renal artery in 9-week-old rat siblings. Sham-operated animals served as controls. Systolic blood pressure (197 +/- 19 mm Hg) and heart/body weight ratios (0.36 +/- 0.04%) were significantly increased in the hypertensive animals. Twenty protein patterns from the left ventricle of five hypertensive and five control rats were compared. The molecular mass and isoelectric point (pI) of proteins spots ranging from 13 to 100 kDa and from 4.5 to 8.5, respectively, were determined using marker proteins. In total, 761 +/- 88 protein spots were resolved in all twenty gels. For the quantitative data analysis a univariate (Mann-Whitney test) as well as a multivariate statistical approach (correspondence analysis) were applied. Only one myocardial protein spot (molecular mass = 41.3 kDa; pI = 6.3) was decreased by more than twofold (p < 0.05) in renovascular hypertension. The vast majority of spots did not indicate a significant alteration of intensity. Left ventricular hypertrophy in early renovascular hypertension induces a form of myocardial hypertrophy that conserves the naturally occurring protein expression pattern.

Dilated cardiomyopathy-associated proteins and their presentation in a WWW-accessible two-dimensional gel protein database.

High resolution two-dimensional electrophoresis (2-DE) and computer-assisted image analysis were used to screen 13 patients suffering from dilated cardiomyopathy (DCM) versus 15 control patients for quantitative and qualitative differences in their myocardial protein expression. Right atrial tissue samples were obtained from end-stage failing explanted hearts and control hearts. Fifty-two spots differed significantly in average intensity between the DCM and the control groups. Myosin light chain 2, ventricular (MLC2) and heat shock protein HSP 27 were identified by protein microsequencing and gel map comparison with other databases. These proteins were found to be characteristic protein markers for DCM in the right atrium. In DCM patients, the spot intensity (protein abundance) of MLC2 is increased to 336% and HSP 27 is decreased to 59%, compared to the control group. The HEART-2DPAGE, a World Wide Web-accessible 2-DE database, was used and extended for the presentation of these disease-associated proteins. Retrievable via Internet we present a list of disease-associated proteins, their altered level of expression in DCM, and their position on a right atrial protein pattern. The accession number to protein sequence databases confers a connection to databases like SWISS-PROT to obtain a detailed functional and structural description of disease-associated proteins. New DCM-associated proteins are detected and their presentation in a 2-DE gel protein database is described.

Towards design and comparison of World Wide Web-accessible myocardial two-dimensional gel electrophoresis protein databases.

In addition to the recently published HEART-2DPAGE--a myocardial World Wide Web-accessible 2-DE gel protein database--the usage and installation of software tools are described with regard to the hard- and software environments. Further, access to the HEART-2DPAGE from other two-dimensional electrophoresis (2-DE) databases using name or accession code of a protein is now available. Moreover, database images, published in the myocardial HSC-2DPAGE and HEART-2DPAGE databases are compared. Using the warping tool of the common image processing system Khoros the database images are matched and added in order to visualize the effects of warping. The application of such image processing tools is aimed at improving the comparability of protein spot patterns of different gel images available through the net.

Representation of amino acid sequences as two-dimensional point patterns.

An algorithm for the representation of amino acid sequences as two-dimensional point patterns (2-D plot) is described. The algorithm is based on chaos game representation (CGR) for DNA sequences and was extended for amino acid sequences. The 2-D plot depicts the sequentiality of amino acids and the amino acid composition of a protein. Changes in a protein sequence as insertion, deletion and repeats of amino acids are characterized by specific geometrical properties and changes in the 2-D plots. The 2-D plot may be considered as a two-dimensional "fingerprint" of a protein. The properties of the algorithm are explained by user-defined amino acid sequences. As an example the 2-D plots of two selected heart proteins are generated. The sequences of these proteins are obtained from the protein sequence database SWISS-PROT.

The construction of the World Wide Web-accessible myocardial two-dimensional gel electrophoresis protein database "HEART-2DPAGE": a practical approach.

The construction of the two-dimensional electrophoresis (2-DE) gel protein database "HEART-2DPAGE" for human heart proteins, accessible via the World Wide Web (WWW), is described. This database provides the opportunity to retrieve descriptive information interactively by mouse clicking on a protein spot within a 2-DE gel image or to retrieve the position of a protein if its protein name or a search expression is given. The realization of searching for proteins, the creation of clickable spots, marked by crosses or textual information, and interactive viewing opportunities are discussed from a software-oriented point of view. The database takes into account that some human heart proteins are chamber specific. Several Common Gateway Interface (CGI) scripts are described and some practical experience is discussed.

Chamber-specific expression of human myocardial proteins detected by two-dimensional gel electrophoresis.

High resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), followed by computer-assisted image analysis (PDQUEST) was used to screen atrial and ventricular protein patterns for quantitative and qualitative differences in protein expression. Myocardial proteins from left ventricular (LV) and right atrial (RA) samples from end-stage, failing explanted hearts and from a healthy donor heart (control) were separated by 2-D large gel electrophoresis. Ten RA versus ten LV gels from explanted dilated cardiomyopathic (DCM) hearts were analyzed for quantitative differences in their spot patterns. Of the 197 spots matched to every gel, 40 spots differed significantly in intensity between RA and LV for DCM patients. A larger number of atrial and ventricular gels (20 RA, 20 LV) from DCM patients and from a healthy donor heart (4 RA, 4 LV gels) were analyzed for qualitative differences in protein expression. Three protein spots (SSP 1120: M(r)/pI:20.5 kDa/4.6; SSP 1119: M(r)/pI:20.6 kDa/4.5; SSP 0117:M(r)/pI:20.7/ < 4.5) that are present in all RA gels for DCM patients are absent in all LV gels. Two protein spots (SSP 0112: M(r)/pI:17.2 kDa,/ < 4.4; SSP 0114:M(r)/pI:17.6 kDa/ < 4.4) occur only in all LV gels but not in the RA gels. These five qualitatively differing spots are identical in DCM patients and in the healthy donor heart. Some of the differing spots were internally sequenced and identified as myosin light chain isoforms (myosin light chain 2, atrial; myosin light chain 2, ventricular; myosin light chain 1, atrial) with the Protein Identification Resource (PIR) accession numbers A44451, S03708, A30881, respectively. Additionally, phosphoglycerate mutase (PIR: JQ0750) and ATP synthase alpha chain (PIR: S17193) were identified. Thus, quantitative and qualitative differences between atrial and ventricular protein patterns were identified by 2-D PAGE. A characteristic distribution of myosin light chains between atrial and ventricular human myocardium was found using our approach.

Characterization of myocardial protein composition in dilated cardiomyopathy by two-dimensional gel electrophoresis.

In order to identify alterations in the myocardial protein pattern that characterize dilated cardiomyopathy (DCM), we compared, by two-dimensional gel electrophoresis, right atrial protein patterns from five patients with DCM and four with normal left ventricular function (two gels per patient). Using computer assisted analysis (PDQUEST, 4.1) we found reproducible protein patterns in the 18 gels (23 x 30 cm, pH 4-9, molecular weight 10-150 kDa). In the two gels from the same patient, 91% of proteins were identical in their position in the pattern and the relative intensities of these protein species correlated with r = 0.85. Three hundred and two +/- 50 protein species were found in several gels, 186 in all 18 gels. Seven proteins in the DCM group were decreased in their relative intensity by > 100%, six were increased by > 100%. Significant quantitative differences between DCM and control patients were found for 25 protein species. Based on seven external marker proteins, a pH and molecular weight value could be calculated for each protein. So far, 30 protein species have been identified by antibodies, amino acid analysis or sequencing procedures. From the 25 proteins that are significantly different between DCM and controls, three have been identified. Expression of the mitochondrial creatine kinase and alpha cristallin B chain was significantly increased in DCM; the malate dehydrogenase family was also significantly decreased in DCM. Two-dimensional electrophoresis appears to be a powerful method for the detection of disease-associated alterations in the myocardial protein pattern.

Dilated cardiomyopathy: computer-assisted analysis of endomyocardial biopsy protein patterns by two-dimensional gel electrophoresis.

In order to identify disease-associated alterations in the myocardial protein patterns in dilated cardiomyopathy, we used 2-dimensional gel electrophoresis to analyse the proteins of endomyocardial biopsies from patients and controls. Proteins (150 micrograms) from biopsies (1-3 mg wet weight) were first separated by isoelectric focusing, then applied to large 2-dimensional gels. A computer-assisted system (PDQUEST) was used for spot detection, quantification and comparison of 2-dimensional protein patterns. From a single endomyocardial biopsy about 1000 different protein species were resolved. The spot pattern was influenced by the concentration of protein during sample preparation, by the amount of protein loaded onto the gels and by the development time of silver staining. Variances of spot position in the first and second dimension and in the long diagonals were less than 5%. Coefficients of variance for the spot quantities in 8 gels were 16 +/- 8%. Contaminating blood proteins could be identified in the biopsy patterns. Computer-assisted comparison between cardiomyopathy (n = 5) and controls (n = 5) over the whole gel revealed that 55 protein spots were increased 100%, 27 protein spots decreased 100%. Four proteins showed significant quantitative differences between the cardiomyopathic hearts and controls. Fourteen proteins were identified by amino acid analysis or microsequencing. An isoelectric point and molecular mass grid was laid over the whole gel based on these identified protein species, resulting in approximate isoelectric point values and molecular masses for all other protein species. Thus, myocardial 2-dimensional protein patterns obtained from endomyocardial biopsies can be used for the characterization of cardiac diseases.

[Fine structure studies and identification of lectin receptors on the viral envelope of two HIV-isolates]. / Feinstrukturuntersuchungen und Identifizierung von Lektinrezeptoren auf der Virushülle zweier HIV-Isolate.

The electron microscopic particle findings were compared with the levels of revertase in corresponding samples over a longer period of time, and a good correlation was found. Comparative investigations of the fine-structure of two HIV isolates did not reveal any morphological differences. It can be assumed, on the basis of the comparative studies on lectin receptors using Helix pomatia lectin, that the viral envelopes of the two isolates are equipped similarly with N-acetyl-d-galactosamine. The differences are not significant with mature particles.

Application of computer-assisted image analysis for studying viral structures.

The application of CAIE has been shown to be useful for analyzing the structures of RNA viruses. Critical assessment of this method is essential for the selection of the micrographs of viruses. In our experience this procedure was helpful for resolving some questions concerning virus morphology.

Studies on the structure of animal ribosomes. VIII. Application of a digital image processing method to the enhancement of electron micrographs of small ribosomal subunits.

Electron micrographs of negatively stained small subunits of rat liver ribosomes have been processed by computer-assisted accumulation. The resulting micrographs are characterized by a significant noise suppression and enhancement of image details.