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Biomedical subjects

P F Lemkin

Publications and source records attributed to P F Lemkin.

16 recordsLinked to original sources
PubMed1995

Two-dimensional protein electrophoresis and multiple hypothesis testing to detect potential serum protein biomarkers in children with fetal alcohol syndrome.

Fetal alcohol syndrome (FAS) surveillance and intervention efforts are hampered by the lack of a specific biochemical test for diagnosis of the syndrome. Based on the hypothesis that abnormalities in growth and development (key features of FAS) involve altered protein metabolism, we analyzed serum proteins by two-dimensional gel electrophoresis and image analysis to search for potential protein biomarkers of FAS. Serum samples from 12 participants in whom FAS had been diagnosed and 8 sex- and age-matched participants whose mothers did not consume alcohol were analyzed in duplicate to determine whether the integrated intensities of matched proteins are significantly altered in children with FAS. Multiple hypothesis testing on 34 of the gels consisting of more than 1700 spots per gel revealed 21 proteins that we classified as potential protein biomarkers of FAS on the basis of significant t-test differences at p < 0.02. We classified 8 of the proteins as candidate biomarkers on the basis of significant concentration differences between case and control subjects at p < 0.01. One of the proteins is clearly an isoform of retinol binding protein; two appear in the area of the gel where alcohol dehydrogenase is expected to appear; one appears to be an isoform of alpha-1-antitrypsin; three appear to be isoforms of the beta-chain of haptoglobin; three may be forms of immunoglobulin light chains; and several others have not been associated with known proteins. No single protein differentiated all case subjects from control subjects, but stepwise canonical discriminant analyses revealed four groups of spots that distinguished between FAS case and control subjects with no misclassifications.(ABSTRACT TRUNCATED AT 250 WORDS)

Biomarkers↗
PubMed1991

Automatic detection of noisy spots in two-dimensional Southern blots. International Electrophoresis Society Meeting, Washington DC, March 16-19, 1991.

One of the problems of automatically quantitating 2D DNA gels, is clearly detecting spots visualized by Southern hybridization blots using DNA probes (Rogan et al. in this conference). Spots appear noisy due to multiple transfer steps. If one applies standard 2D PAGE protein gel spot segmentation methods, spots fragment due to highly textured image noise and a weak radioautograph signal and thus are poorly detected. We have observed that these spots are all of a minimum size. Therefore an image processing filter which both takes minimum spot size into account and has immunity to image texture-type noise should be able to reliably detect this class of spots. The 'Busse' Laplacian filter used in the GELLAB-II system, is a modification of the standard (1-21) digital approximation of the Laplacian. In the Busse Laplacian, the sampling interval is n pixels (n greater than 1) instead of 1. In addition, 3 x 3 averaged 'super pixel' values are used instead of single pixels for each element of the Laplacian convolution. This gives the needed noise immunity by filtering out the high spatial frequency image noise while preserving the low spatial frequency character of the spots. We have used this filter successfully on 2D DNA Southern blot image data.

Blotting, Southern↗
PubMed1989

Identification of proteins secreted from axons of embryonic dorsal-root-ganglia neurons.

Secretion of proteins from the growth cone has been implicated in axon growth and synapse formation and might be involved in the transmission of a variety of axon-derived regulatory signals during neurogenesis. In order to identify axonally secreted proteins, dorsal-root-ganglia neurons from chicken embryos were cultured in a compartmentalized cell culture system that allows separate access to neuronal cell somas and axons. The proteins synthesized by the neurons were metabolically labeled by addition of [35S]methionine to the compartment containing the cell somas; the proteins released from the axons were harvested from the culture medium of the axonal compartment. Two-dimensional gel electrophoresis revealed two axonally secreted proteins with apparent molecular mass of 132-140 kDa and 54-60 kDa; they were termed axonin-1 and axonin-2, respectively. Both axonins were found to be secreted from a variety of neuronal cell cultures, but not from any of the nonneuronal cultures investigated, and hence might be neuron-specific. Virtual absence of these proteins from the axonal protein pattern suggests constitutive secretion. The information acquired on coordinates and spot morphology of these proteins in two-dimensional gel electrophoresis provides a useful assay for their purification.

Animals↗
PubMed1989

Database and search techniques for two-dimensional gel protein data: a comparison of paradigms for exploratory data analysis and prospects for biological modeling.

Two-dimensional (2-D) polyacrylamide gel electrophoresis can detect thousands of polypeptides, separating them by apparent molecular weight (Mr) and isoelectric point (pI). Thus it provides a more realistic and global view of cellular genetic expression than any other technique. This technique has been useful for finding sets of key proteins of biological significance. However, a typical experiment with more than a few gels often results in an unwiedly data management problem. In this paper, the GELLAB-II system is discussed with respect to how data reduction and exploratory data analysis can be aided by computer data management and statistical search techniques. By encoding the gel patterns in a "three-dimensional" (3-D) database, an exploratory data analysis can be carried out in an environment that might be called a "spread sheet for 2-D gel protein data". From such databases, complex parametric network models of protein expression during events such as differentiation might be constructed. For this, 2-D gel databases must be able to include data from other domains external to the gel itself. Because of the increasing complexity of such databases, new tools are required to help manage this complexity. Two such tools, object-oriented databases and expert-system rule-based analysis, are discussed in this context. Comparisons are made between GELLAB and other 2-D gel database analysis systems to illustrate some of the analysis paradigms common to these systems and where this technology may be heading.

Algorithms↗
PubMed1986

Coordinate regulation of the expression of axonal proteins by the axonal microenvironment.

The axonal functions that act in the formation of the neuronal network have been shown to occur in close interdependence with the tissue that surrounds the growing axons. However, little is known about the molecular building blocks underlying axonal functions, although more than 400 axonal proteins have been identified. In view of the existence of such a large number of axonal proteins, we have initiated a project to determine the molecules involved in the implementation of particular axonal functions by a selective approach. On the assumption that plasticity in the expression of axonal functions in response to specific features of the local axonal environment may be based on changes in the expression of particular axonal proteins, the axonal proteins of dorsal root ganglion (DRG) neurons were screened for those whose expression responds to environmental influences. DRG neurons were grown in a compartmental cell system that offers separate access to neuronal somas and to their axons and the axons were locally exposed to different populations of cells from the peripheral or central nervous system. The axonal proteins were metabolically labeled and subjected to two-dimensional gel electrophoresis. Computerized quantitation of the individual axonal proteins revealed that the cocultured cells modulate the synthesis of a few axonal proteins of DRG neurons differentially. The data on the abundance of the newly expressed proteins under varying local environmental conditions were condensed as expression profiles. Comparison of expression profiles and cluster analysis of quantitative gel analysis data revealed that the environmentally modulated proteins subdivide into clusters with common distinct expression profiles under the influence of nonneuronal cells from the peripheral nervous system, nonneuronal cells of the central nervous system, and spinal cord cells, which are composed of neurons and nonneuronal cells. By means of this new, characteristic attribute assigned to environmentally modulated axonal proteins, working hypotheses were made as to their functional role.

Animals↗
PubMed1985

Differential modulation of the expression of axonal proteins by non-neuronal cells of the peripheral and central nervous system.

Axonal behavior during the formation of the neuronal network of the nervous system has been shown to be under environmental control. Hence, as a first step in a project aiming to elucidate the molecular basis of axonal functions, we have identified axonal proteins whose synthesis is subject to environmentally induced changes. Neurons from chicken embryonic dorsal root ganglia (DRG) were grown in a compartmental cell culture system that allows selective examination of axonal proteins. Non-neuronal cells of the peripheral or central nervous system were co-cultured with the DRG axons. The axonal proteins expressed under these different environmental conditions were examined by metabolic labeling and two-dimensional SDS-polyacrylamide gel electrophoresis. Computerized quantification revealed that 12 out of 400 axonal proteins responded to changes in the local axonal environment by a change in their relative abundance. Some proteins changed in response to both types of co-cultures whereas some changed specifically under the influence of either peripheral or central non-neuronal cells.

Animals↗
PubMed1984

Identification of coordinate pairs of polypeptides: a technique for screening of putative precursor product pairs in 2D gels.

Two-dimensional electrophoresis, combined with computerized quantification of individual proteins, provides a sensitive and accurate approach to detection of small differences in the protein pattern. As an extension of "GELLAB," a data base system for the analysis of two-dimensional electrophoretic gels, we report here on a screening algorithm for detection of certain post-translational modification events. It is based on the assumption that, if there is a post-translational modification of a protein in the transition between two functional states of a biological system, in many cases the sum of the protein concentration of a precursor-product pair in one is equivalent to that in the other. In addition to the identification of candidate pairs with a precursor-product relationship, cues to the nature of the post-translational modification that might relate them might also be derived from comparisons of the isoelectric point and apparent molecular mass. This subset of the GELLAB system may be thought of as a screening tool in the search for and investigation of post-translational protein-processing events so that the more time-consuming structural investigations to follow can be performed on a smaller set of proteins.

Animals↗
PubMed1982

Some extensions to the GELLAB two-dimensional electrophoretic gel analysis system.

Several important extensions to the GELLAB 2D electrophoresis gel analysis system are discussed, including concepts that lead to the generation of consistent gel maps for a particular cell line, as illustrated by some recent results for the P388D1 cell system, GELLAB is an interactive graphics computer system that facilitates (a) image accessioning, (b) spot-data extraction from the image with position and density quantitation, (c) pairing of corresponding spots between gels, and (d) spot analysis for a large number of corresponding spots (over 3000 per gel) for up to 128 gels. Multiple gels are accessioned into the system used to create a composite gel data base. These then may be partitioned into multiple classes of gels and searched for statistically significant interclass spot differences, which can be visualized in reference map images of selected spot sets as well as mosaic images of particular spots in all gels. In addition to the visual feedback that is available during the gel analysis, various statistical, numerical, and other displays may be generated in the CGELP interactive program of the GELLAB system.

Animals↗
PubMed1980

Data-base techniques for multiple two-dimensional polyacrylamide gel electrophoresis analyses.

Two-dimensional protein electrophoresis can benefit from a powerful set of computer-supported image processing and data structure/management procedures. Detection of quantitative differences is complicated by local inhomogeneities in the polyacrylamide base; biochemical changes and variations in temperature and preparative technique also make the between-gel density and x-y coordinate correspondences quite imprecise. The program presented here provides local alignment and computer-controlled variable "flicker" rates for multiple gels, with use of an interactive display system. Manual spot densitometry, referred to a National Bureau of Standards density wedge, can be complemented by a set of automatic densitometry routines for previously established lists of spots. The ability to establish a set of local landmarks, either by included standards or user identification, provides a basis for automatic n-way gel comparison for subsets or for the entire set of spots. Automatic segmentatin algorithms allow isolatin of spots and separation of touching and partially overlapping regions. Various analytical and statistical facilities are part of the user's access to the interactively developed data base. The data-structure and image-manipulation techniques developed here allow for user-directed and heuristic comparisons with online presentation of intermediate and final results.

Asbestosis↗