Two-dimensional gel electrophoresis of Escherichia coli homogenates: the Escherichia coli SWISS-2DPAGE database

We report a patient, MT, who presented a specific, though not isolated, deficit in written calculation. Despite a preserved knowledge of simple arithmetic - single-digit addition and subtraction - he failed systematically in multi-digit subtraction. The nature of errors was consistent across problems and reflected the application of a disturbed underlying algorithm. Moreover, the pattern of error observed mimies a very common finding in developmental studies on arithmetical procedure acquisition (Fuson, 1990, 1992, Young and O'Shea, 1981; VanLehn, 1986, 1990). The data suggest that, within calculation skills, syntax may exist as a system of stable, but inappropriate, rules which are independent of any underlying conceptual knowledge.     

Cholangiocyte-specific rat liver proteins identified by establishment of a two-dimensional gel protein database

The liver is composed of a variety of cells that form a functional unit involved in uptake, synthesis, metabolism, and secretion. Until recently, most studies examining liver function did not analyze the specific proteins expressed or functions performed by the multiple individual cell types that constitute the hepatic mass. In the last decade, novel isolation methods have been developed that allow the purification of liver cell populations highly enriched in one type of liver cell. Here, we present a detailed two-dimensional (2-D) protein map of rat bile duct epithelial cells (i.e., cholangiocytes) using a recently developed isolation procedure. In addition, we identify 27 major cholangiocyte proteins either by comparison to maps of known rat liver proteins (based on pI and Mr) or by tryptic digestion and microsequencing. Finally, we compare the relative abundance of individual proteins present in cholangiocytes to whole liver as well as hepatocyte-specific proteins. Our results show that cholangiocytes express a unique array of individual proteins. The cholangiocyte 2-D protein pattern is markedly different from that of isolated rat hepatocytes or whole rat liver, with high levels of proteins previously known to be expressed by cholangiocytes (e.g., cytokeratins, actins) as well as protein not previously demonstrated to be expressed at high levels (e.g., annexin V, selenium binding protein). We conclude that this cholangiocyte-derived, 2-D protein map will be a crucial resource for studies directed at our understanding of cholangiocyte physiology and pathobiology.     

A comparative two-dimensional gel protein database of the intact and regenerating newt limbs

In this paper we describe a two-dimensional gel database of the regenerating newt limb. Protein synthesis was compared in the intact limb, in the 1-week regenerating limb, representing the dedifferentiation stage, and in the 2-week regenerating limb, representing the formation of the blastema. This comparative database provided data on differential expression of about 800 proteins during the process of limb regeneration. In addition, a map has been generated for these proteins for future guidance in characterizing further new, unknown proteins. The overall expression patterns of the proteins indicated that the dedifferentiation stage was marked by down-regulation of most proteins, while the blastema formation was marked by the appearance of many new proteins. The potential use of such a database in isolating factors involved during limb regeneration is discussed.     

The SWISS-2DPAGE database: what has changed during the last year

SWISS-2DPAGE (http://www.expasy.ch/ch2d/) is an annotated two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) database established in 1993. The current release contains 21 reference maps from human and mouse biological samples, as well as from Saccharomyces cerevisiae, Escherichia coli and Dictyostelium discoideum origin. These reference maps now have 2480 identified spots, corresponding to 528 separate protein entries in the database, in addition to virtual entries for each SWISS-PROT sequence. During the last year, the SWISS-2DPAGE has undergone major changes. Six new maps have been added, and new functions to access the data have been provided through the ExPASy server. Finally, an important change concerns the database funding source.     

Development of a two-dimensional gel electrophoresis database of human lysosomal proteins

Two-dimensional gel electrophoresis databases have been generated for a range of tissue cell and fluid types, from a number of species. A major difficulty in the development of such databases is the large number of proteins present in even a single cell type (> 10,000) and the low levels of many of these proteins. One approach to overcome these difficulties is to fractionate the cell into its organelles and generate individual databases for each subcellular component. This has the added advantage of assigning a cellular location to each protein identified. Here we report the development of a two-dimensional gel electrophoresis database of lysosomal proteins.     

Two-dimensional gel protein database of Saccharomyces cerevisiae

With the systematic sequencing of the yeast genome, yeast biology has entered a new era where novel challenges have to be faced. One challenge is the identification of the function of the several hundred novel genes discovered by genome sequencing. Another is to understand how all yeast genes act in concert to ensure and maintain cell organization. Two-dimensional (2-D) gel electrophoresis is the technique of choice to take up these challenges because it provides the opportunity of obtaining an overall view of genome expression. In prospect of these studies we have undertaken the construction of a yeast 2-D gel protein database that contains information on polypeptides of the yeast protein map. In this paper we report the information presently contained in this database. The reported information includes the identification of 250 protein spots and the characterization of polypeptides corresponding to N-terminal acetylated proteins, mitochondrial proteins, glucose-repressed proteins, heat shock induced proteins and proteins encoded by intron-containing genes. In all, 600 spots are annotated. These data can be accessed on the Yeast Protein Map server through the World Wide Web network.     

The Dictyostelium discoideum proteome--the SWISS-2DPAGE database of the multicellular aggregate (slug)

The cellular slime mold Dictyostelium discoideum is a eukaryotic microorganism which has developmental life stages attractive to the cell and molecular biologist. By displaying the two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) protein map of different developmental stages, the key molecules can be identified and characterised, allowing a detailed understanding of the D. discoideum proteome. Here we describe the preparation of reference gel of the D. discoideum multicellular aggregate, the slug. Proteins were separated by 2-D PAGE with immobilised pH gradients (pH 3.5-10) in the first dimension and sodium dodecyl sulfate (SDS)-PAGE in the second dimension. Micropreparative gels were electroblotted onto polyvinylidene difluoride (PVDF) membranes and 150 spots were visualised by amido black staining. Protein spots were excised and 31 were putatively identified by matching their amino acid composition, estimated isoelectric point (pI) and molecular weight (M(r)) against the SWISS-PROT database with the ExPASy AAcompID tool (http:// expasy.hcuge.ch/ch2d/aacompi.html). A total of 25 proteins were identified by matching against database entries for D. discoideum, and another six by cross-species matching against database entries for Saccharomyces cerevisiae proteins. This map will be available in the SWISS-2DPAGE database.     

Creating a population-based linked health database: a new resource for health services research

Cytologically, the distinction between bronchioalveolar carcinoma and reactive/reparative processes of respiratory epithelium can be difficult. Retrospectively, we have identified 11 consecutive cases of bronchioalveolar carcinoma from the cytology files of University Missouri-Kansas City/Truman Medical Center. On average, a combined 5.71 cytologic/histologic procedures were performed before reaching a definitive diagnosis for this group. An additional seven random cases of reactive/reparative respiratory cases of adult respiratory distress syndrome patients were used as a control. Cytomorphometric analysis was performed. The mean average nuclear diameter for the carcinoma group was 13.76 microns and for the reactive/reparative group was 13.29 microns. There was no statistical difference between the two groups (paired student t test, P > .05). It appears from our data that mean nuclear diameter is not a discriminator for the cytologic distinction between bronchioalveolar carcinoma and reactive/reparative respiratory epithelium and that the accepted cytologic parameters of for bronchioalveolar carcinoma are more valid.     

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.     

Make2ddb: a simple package to set up a two-dimensional electrophoresis database for the World Wide Web

Two-dimensional electrophoresis (2-DE) has become a highly reproducible protein separation technique that currently serves as the main basis for proteome research and in particular for protein identification. Also, the Internet provides large utilities for exchanging data, and we can observe increased interest among scientists to build remote 2-DE databases, since many members of the concerned community are now able to access the data. By preparing the data and programs that are required to create a federated 2-DE database, the Make2ddb package, described here, helps to build such a database on the user own World Wide Web site.     

HOMSTRAD: a database of protein structure alignments for homologous families

We describe a database of protein structure alignments for homologous families. The database HOMSTRAD presently contains 130 protein families and 590 aligned structures, which have been selected on the basis of quality of the X-ray analysis and accuracy of the structure. For each family, the database provides a structure-based alignment derived using COMPARER and annotated with JOY in a special format that represents the local structural environment of each amino acid residue. HOMSTRAD also provides a set of superposed atomic coordinates obtained using MNYFIT, which can be viewed with a graphical user interface or used for comparative modeling studies. The database is freely available on the World Wide Web at: http://www-cryst.bioc.cam. ac.uk/-homstrad/, with search facilities and links to other databases.     

Fluorescence cross-correlation: a new concept for polymerase chain reaction

Infusion of sodium selenite to the occipital cortex of the rat was used for the specific tracing of zinc-rich pathways. Large numbers of labeled somata were found ipsilaterally in the visual, orbital and frontal cortices, and contralaterally in homotopic and heterotopic visual areas. Labeled neurons were also found ipsilaterally in the retrosplenial, parietal, sensory-motor, temporal and perirhinal cortex. In contrast to the cortico-cortical connections, ascending afferents to the visual cortex were not zinc-rich except for a few labeled neurons in the claustrum. Additional injections showed reciprocal zinc-rich connections between the visual cortex and the orbital and frontal cortices. The latter cortices also received ascending zinc-rich afferents from the claustrum. Selenite injections revealed the layered distribution and the morphology of these labeled neurons in the neocortex. Zinc-rich neurons were found in layers II-III, V and VI. However, none was found in layer IV. Zinc-rich somata appeared as pyramidal and inverted neurons. The contrasting chemical properties of cortical and subcortical visual afferents may account for the functional differences between these systems.     

Colloidal carriers for intravenous drug targeting: plasma protein adsorption patterns on surface-modified latex particles evaluated by two-dimensional polyacrylamide gel electrophoresis

Targeting to specific sites of the body via colloidal carriers is sought in order to reduce drug side effects. The adsorption of plasma proteins on intravenously injected particles is regarded as the key factor in explaining their organ distribution: total bound protein, or, more likely, the presence of specific proteins and their conformation, are expected to influence macrophage uptake. Polystyrene beads, 60 nm in diameter, were used as model carriers; their surface was differentially modified by adsorption of increasingly hydrophilic block copolymers, poloxamers 184, 188 and 407. After incubation in plasma, the patterns of protein adsorption onto coated beads were analyzed by high-resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The behavior of some representative proteins was monitored, including albumin, fibrinogen, IgG, factor B and the apolipoproteins, A-I, A-IV, C-III, E and J. The more hydrophobic the particles, the larger the total amount of bound protein. However, this correlation was not valid for all of the analyzed protein species, which proves that it is insufficient to look only at physicochemical data to predict organ distribution. On the contrary, it is essential to use 2-D PAGE to establish the correlation between adsorbed proteins and carrier behavior in vivo.     

Effect of protein application mode and acrylamide concentration on the resolution of protein spots separated by two-dimensional gel electrophoresis

Two-dimensional gel electrophoresis separates large numbers of proteins in two steps on the basis of differences in their pIs and molecular masses. The separation is usually performed on immobilized pH gradient strips, followed by gradient polyacrylamide gels separating proteins with molecular masses between 5-200 kDa. For the first-dimensional separation the protein samples are usually applied near one end of the strip. Using total soluble protein extracts of the bacterium Haemophilus influenzae, we found that simultaneous sample application at both the basic and the acidic ends of the strip resulted in detection of more and stronger protein spots in comparison with sample application at one end only. Because many proteins of an organism have similar pI and Mr values, an overlapping of protein spots is frequently observed in the second-dimensional separation. The soluble protein fraction of H. influenzae was further separated on gels of constant acrylamide concentration between 7.5% and 15.0%. We found that for proteins of molecular mass within certain ranges, the gels of homogeneous acrylamide concentration provided more efficient spot separation than the gradient gels. The observed improvements in spot resolution may be useful in the characterization of proteins from other organisms or cell lines.     

Validity: a concept at the heart of research

This article considers the nature of validity and further explores the relationship between validity and reliability. The different forms of validity are discussed and the importance of developing more valid measures of nursing activity is highlighted.     

Calcifying tendinitis: a new concept of its pathogenesis

To elucidate the pathogenesis of calcifying tendinitis, clinical and morphological investigations were done on 46 surgically treated cases. Contrary to the prevalent concept of degeneration preceding dystrophic calcification, we found no evidence for an active or a healed degenerative process. The affected tendon was transformed into fibrocartilage with a predilection for calcification. The formative phase of calcification was followed in course of time by a resorptive phase during which the deposits were surrounded by phagocytosing cells. There was a concomitant proliferation of vascular channels. We found a significant correlation between severe pain and histological signs of resorption. The pathogenetic mechanism of calcifying tendinitis should be reassessed as a unique disorder of the musculotendinous cuff.     

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.