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Biomedical subjects

E Olsen

Publications and source records attributed to E Olsen.

At least 55 records · Page 3Linked to original sources
PubMed1994

Use of ferrous sulphate and immunomagnetic separation to recover Salmonella enteritidis from raw eggs.

Contaminated eggs or foods containing eggs have been a source of food borne salmonellosis, with a significant proportion of these outbreaks being attributed to Salmonella enteritidis. Since the level of contamination in individual eggs or a pool of such eggs may be low, enrichment to increase cell numbers can take several days. Pre-enrichment of raw blended eggs which have been supplemented with ferrous sulphate at a concentration of 35 mg/l, for 6 h at 37 degrees C, significantly enhanced the growth of Salmonella. Using Dynabeads Anti-Salmonella (Dynal, Oslo), a new commercial product for selective enrichment of Salmonella from food samples, we have defined a protocol based on immunomagnetic separation, specially for raw eggs. The application of this protocol enables definitive detection of Salmonella enteritidis from eggs within 30 h.

Animals↗
PubMed1994

IMS: a new selective enrichment technique for detection of Salmonella in foods.

In a study designed to evaluate the performance of Dynabeads Anti-Salmonella, immunomagnetic separation (IMS) followed by plating (IMS-Plating) proved far superior to the conventional ISO Salmonella methodology and the Modified Semi-solid Rappaport-Vassiliadis (MSRV) method. Salmonella species were isolated and detected from 135 out of the 180 diverse poultry samples by IMS analysis as against 98 by the conventional method and 33 by the MSRV technique. All results were confirmed biochemically and serologically. It appeared from the data generated that some of the salmonellae isolated using IMS were greatly inhibited in Rappaport-Vassiliadis enrichment broth at 42 degrees C and to a lesser extent in selenite cystine broth at 37 degrees C. A moderately selective plating medium like XLD proved to be better in isolating these possible sensitive wild-type strains of salmonellae than the more selective BGA.

Animals↗
PubMed1994

Psoriasin binds calcium and is upregulated by calcium to levels that resemble those observed in normal skin.

Recently, we described a small molecular weight protein termed psoriasin that showed sequence similarity with the S100 calcium-binding proteins and that is highly upregulated in psoriatic epidermis as well as in primary human keratinocytes undergoing abnormal differentiation. Here we present evidence showing that natural and recombinant psoriasin binds calcium, as judged by the calcium overlay assay, and that it contains all the sequence features characteristic of the S100 family. Furthermore, [35S]-methionine labeling experiments showed that psoriasin synthesis is upregulated by 2 mM Ca++ (ratio Ca++/control at 88 h = 2.56) to levels that resemble those observed in unfractionated keratinocyte populations obtained from normal skin.

Amino Acid Sequence↗
PubMed1994

Recombinant allergens and diagnosis of grass pollen allergy.

To evaluate the use of recombinant allergens for the diagnosis of grass pollen allergies, we examined the levels of IgE antibodies specific for grass pollen as by immunoassays. SDS-PAGE analysis of two batches of Kentucky bluegrass pollen extracts demonstrated that there was considerable variability in allergen content of extracts, which in turn affected quantitation of specific IgE antibodies by different immunoassay procedures. Furthermore, the levels of IgE antibodies in human sera specific for a recombinant grass pollen allergen, rKBG8.3, were examined by enzyme immunoassay. The results demonstrated that quantitation of IgE antibodies specific for even one single allergen may be used to discriminate sera of allergic individuals with respect to IgE specific for grass pollen in general. A positive correlation, r = .82, was found for IgE binding of the recombinant allergen and the crude extracts of grass pollens. It is concluded from these results that a single recombinant allergen or a combination of a few major recombinant allergens can substitute the crude extract for in vitro as well as in vivo diagnostic purposes.

Allergens↗
PubMed1993

The human keratinocyte two-dimensional gel protein database: update 1993.

The master two-dimensional gel database of human keratinocytes currently lists 3038 cellular proteins (2127 isoelectric focusing, IEF; and 911 nonequilibrium pH gradient electrophoresis, NEPHGE) many of which correspond to post-translational modifications. 763 proteins have been identified (protein name, organelle components, etc.) and they are listed both in alphabetical order and with increasing SSP number, together with their M(r), pI, cellular localization and credit to the investigator(s) that aided in the identification. Furthermore we have listed 176 proteins that have been microsequenced so far and that are recorded in this database. We also include synthetic images depicting some interesting sets of proteins identified so far; these include components of hnRNP's, proteasomes or prosomes, ribosomes, as well as assorted organelle markers, GTP-binding proteins, calcium binding proteins, stress proteins, autoantigens, differentiation markers and psoriasis upregulated proteins. The aim of the comprehensive database is to gather, through a systematic study of keratinocytes, qualitative and quantitative information on proteins and their genes that may allow us to identify abnormal patterns of gene expression and ultimately to pinpoint signaling pathways and components affected in various skin diseases, cancer included.

Databases, Factual↗
PubMed1993

Human cellular protein patterns and their link to genome DNA mapping and sequencing data: towards an integrated approach to the study of gene expression.

Analysis of cellular protein patterns by computer-aided two-dimensional gel electrophoresis together with recent advances in protein sequence analysis and expression systems have made possible the establishment of comprehensive two-dimensional gel protein databases that may link protein and DNA mapping and sequence information and that offer an integrated approach to the study of gene expression. With the integrated approach offered by two-dimensional gel protein databases it is now possible to reveal phenotype-specific protein(s), to microsequence them, to search for homology with previous identified proteins, to clone the cDNAs, to assign partial protein sequences to genes for which the full DNA sequence and the chromosome location are known, and to study the regulatory properties and function of groups of proteins that are coordinately expressed in a given biological process. Comprehensive two-dimensional gel protein databases will provide an integrated picture of the expression levels and properties of the thousands of protein components of organelles, pathways, and cytoskeletal systems, both under physiological and abnormal conditions, and are expected to lead to the identification of new regulatory networks. So far, about 20% (600 out of 2,980) of the total number of proteins recorded in the human keratinocyte protein database have been identified and we are actively gathering qualitative and quantitative biological data on all resolved proteins. Given the current improvements on microsequencing as well as the availability of specific antibodies, it seems feasible to expect that most known keratinocyte proteins will be identified in the very near future. This feast will reveal a wealth of new proteins that will become amenable to experimentation both at the biochemical and molecular biology level.

Amino Acid Sequence↗
PubMed1993

On the substitution for carcinogens in the working environment.

The ultimate solution in prevention of occupational cancer risks would be to remove carcinogens from the working environment. Elimination of carcinogenic substances can be achieved by replacement of the carcinogen with non-carcinogen(s) having the same functionality, or by using a different process that produces adequate results without the use of carcinogenic agents. This paper presents selected methods and tools, which have been used for substitution purposes in practice. These are: 1. a strategy for substitution, 2. a tool for creating ideas on how to substitute, 3. a software package, SUBTEC, for solving some technical and industrial hygiene problems, and 4. a proposal for a strategy to deal with organizational and psychological barriers encountered when changes are to be introduced.

Carcinogens, Environmental↗
PubMed1992

The human keratinocyte two-dimensional gel protein database (update 1992): towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.

The master two-dimensional gel database of human keratinocytes currently lists 2980 cellular proteins (2098 isoelectric focusing, IEF; and 882 nonequilibrium pH gradient electrophoresis, NEPHGE) many of which correspond to posttranslational modifications. About 20% of all recorded proteins have been identified (protein name, organelle components, etc.) and they are listed in alphabetical order together with their M(r), pI, cellular localization and credit to the investigator(s) that aided in the identification. Also, we have listed 145 microsequenced proteins that are recorded in this database. As an aid in localizing the polypeptides we have included blow-ups of the master images (IEF, NEPHGE) displaying all the protein numbers. In the long run, the master keratinocyte database is expected to link protein and DNA sequencing and mapping information (Human Genome Program) and to provide an integrated picture of the expression levels and properties of the thousands of proteins that orchestrate various keratinocyte functions both in health and disease.

Cell Differentiation↗
PubMed1992

Mapping of antibody binding epitopes of a recombinant Poa p IX allergen.

Antibody binding epitopes of a recombinant Poa p IX allergen were delineated using recombinant DNA and solid-phase peptide synthesis procedures. The full-length cDNA clone KBG60 and its four overlapping recombinant fragments, KBG60.1, KBG60.2, KBG8.3 and KBG10 which spanned the entire molecule were synthesized in E. coli with aid of the plasmid expression vector, pWR590.1. The antigenic and allergenic sites of these recombinant proteins were analyzed by ELISA using human IgE and murine IgG antibodies. It was thus demonstrated that although the epitopes were found on all the fragments tested, the majority of these were located on a C-terminal fragment, rKBG8.3. Furthermore, synthetic peptides were also employed to identify the epitopes of rKBG60 protein. The use of antisera raised against native KBG pollen extract and the recombinant KBG8.3 protein to scan a total of 56 overlapping deca-penta peptides, covering the entire rKBG60 protein, revealed that 10 positive peptides involved in the antibody-binding site(s). Taken together, the results of these studies indicate that rKBG60 protein possesses at least 10 antibody binding epitopes.

Allergens↗
PubMed1992

Expression and thrombin cleavage of Poa p IX recombinant allergens fused to glutathione S-transferase.

The high-level expression and purification of Poa p IX recombinant grass pollen allergens were examined utilizing a modified pGEX plasmid, designated as pGEX 2T-1. This vector permits frame-1 ligation of lambda gt11 cDNA inserts and cleavage of the recombinant allergenic protein from the fusion partner glutathione S-transferase. The expression of the fusion proteins in water-soluble form varied among the transformants of the same bacterial strain and also between different host strains. Purification of the fusion proteins by affinity chromatography employing glutathione agarose gel revealed that proteases in the bacterial lysate bound to the gel and were co-eluted with the fusion proteins. These proteases, which specifically degraded the recombinant proteins to varying degrees, were inhibited by both of the inhibitors, phenylmethylsulfonyl fluoride and aprotinin. Cleavage by thrombin of the fusion proteins indicated that the structure of the individual protein affected the thrombin accessibility to the cleavage site. Increased concentration of thrombin partly compensated this effect, but resulted in a broader specificity of the enzyme. By contrast, cleavage of the fusion protein when it was still attached to the glutathione gel was convenient and led to purification of the product devoid of proteolytic activity. Since almost all the recombinant allergens have been cloned in lambda gt11 vector, the pGEX 2T-1 vector reported herein will facilitate the synthesis, purification of the corresponding allergenic proteins or their peptides in soluble and biologically active forms.

Allergens↗
PubMed1992

The heart and antiphospholipid antibodies in MRL-lpr/lpr mice.

Our objective in this study was to determine possible associations between antiphospholipid antibodies (aPL) and histologically defined heart valve lesions in the MRL-lpr/lpr mouse, a suitable model for the antiphospholipid syndrome (APS). At monthly intervals, from 2 to 6 months of age, three MRL-lpr/lpr mice (two with anticardiolipin antibodies, one without) and two sex- and age-matched Balb/c mice (controls) were sacrificed for histological studies. Serum binding to phospholipids and DNA was studied at this time. We found thickened heart valves in 68% of MRL-lpr/lpr mice and in 80% of Balb/c mice, and no association with any of the antibodies tested was found. No evidence of coronary vasculitis or thrombi was found in any of the mice studied. Platelet counts in MRL-lpr/lpr mice were significantly lower (640.550 +/- 211.818 x 10(6)/ml) than in Balb/c mice (780.0 + 112.5 x 10(6)/ml) (p < 0.05), and no association was found between platelet counts and aPL. In this model of murine APS, aPL bear no importance in heart valve pathology.

Animals↗
PubMed1991

Identification and characterization of the Poa p IX group of basic allergens of Kentucky bluegrass pollen.

We reported previously the primary structure of three full-length cDNA clones that encode a new group of IgE-binding proteins of Kentucky bluegrass (KBG) pollen, designated as Poa p IX. In the present study we have further characterized the cloned Poa p IX proteins, identified the corresponding proteins in KBG pollen extract, and determined their antigenic relationships with other known grass pollen allergens. A recombinant IgE-binding polypeptide rKBG7.2 that represents the C-terminal fragment, conserved in Poa p IX proteins, appeared to contain epitopes unique to these proteins and served as an immunosorbent for the isolation of the corresponding human IgE antibodies. On two-dimensional PAGE blots these IgE antibodies bound selectively to five distinct KBG pollen proteins with molecular mass 28 to 34 kDa and isoelectric point greater than 9.5. These proteins differ in size and charge from known allergens, but are very similar to those of the recombinant Poa p IX proteins. The rKBG3.1, which represents the N-terminal region of the Poa p IX clone KBG31, as well as the corresponding natural allergens were shown to possess epitopes that crossreact with the acidic group V allergens of Timothy. Comparison of amino acid sequences of recombinant Poa p IX proteins with those of Lol p I isoallergens revealed no significant sequence similarities. In contrast, partial homology was demonstrated between the N-terminal sequences of these proteins and the Phl p V proteins. Our results confirm that the Poa p IX clones represent a distinct and major group of allergens of KBG pollen, and demonstrate structural similarities and antigenic cross-reactivities among different groups of allergenic proteins in grass pollens.

Allergens↗
PubMed1991

Nucleotide sequence analysis of three cDNAs coding for Poa p IX isoallergens of Kentucky bluegrass pollen.

Grass pollen allergens are one of the major causes of type I allergic reactions (allergic rhinoconjunctivitis, allergic bronchial asthma, and hayfever) in temperate climates afflicting 15-20% of a genetically predisposed population. Workers have found considerable physico- and immunochemical heterogeneity within the grass pollen allergens which has made them difficult to purify for both therapeutic uses and further biochemical study. We recently reported the construction of a cDNA library in lambda gt11 using mRNA extracted from dehydrated Kentucky bluegrass (KBG, Poa pratensis). Here, we present the nucleotide and deduced amino acid sequences for three KBG pollen allergen cDNA clones, KBG 41, 60, and 31, which were isolated from the above library using a pool of six sera from grass pollen allergic patients. These clones exhibit an exceptionally high degree of sequence similarity to one another, only minor similarity to other known allergens, and no homologies to other known proteins or genes. The predicted molecular mass for the cloned proteins range from 28.3 to 37.8 kDa with pI values of 9.6-10.2. All three clones appear to possess leader peptides and lack asparagine sequons required for N-glycosylation. Therefore, the molecular mass of the post-translationally modified proteins were calculated to be 28.4-34.9 kDa, which is consistent with the size of the polypeptides revealed in Western blots of pollen proteins using an antiserum to a recombinant peptide encoded by the partial cDNA clone KBG 8.3. Northern blotting analysis indicates that expression of the genes corresponding to these clones is confined to pollen tissue. The results suggest that the clones code for a group of proteins that represent a new and previously uncharacterized group of grass pollen isoallergens, which have been hereby designated as Poa p IX.

Allergens↗
PubMed1991

The master two-dimensional gel database of human AMA cell proteins: towards linking protein and genome sequence and mapping information (update 1991).

The master two-dimensional gel database of human AMA cells currently lists 3801 cellular and secreted proteins, of which 371 cellular polypeptides (306 IEF; 65 NEPHGE) were added to the master images during the last 10 months. These include: (i) very basic and acidic proteins that do not focus under normal running conditions and (ii) low-abundant proteins that can only be detected after prolonged gel exposure. Annotation categories updated in this version include "protein name", "antibody against protein", "cellular localization", and "microsequenced proteins". New entries include "human autoantigens" and "cDNAs". For convenience we have included an alphabetical list of all known proteins recorded in this database. In the long run, the main goal of this database is to link protein and DNA sequencing and mapping information (Human Genome Program) and to provide an integrated picture of the expression levels and properties of the thousands of proteins that orchestrate various cellular functions both under physiological and abnormal conditions.

Cell Line, Transformed↗
PubMed1991

A comprehensive two-dimensional gel protein database of noncultured unfractionated normal human epidermal keratinocytes: towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.

A two-dimensional (2-D) gel database of cellular proteins from noncultured, unfractionated normal human epidermal keratinocytes has been established. A total of 2651 [35S]methionine-labeled cellular proteins (1868 isoelectric focusing, 783 nonequilibrium pH gradient electrophoresis) were resolved and recorded using computer-aided 2-D gel electrophoresis. The protein numbers in this database differ from those reported in an earlier version due to changes in the scanning hardware (Celis et al., Electrophoresis 1990, 11, 242-254). Annotation categories reported include: "protein name" (listing 207 known proteins in alphabetical order), "basal cell markers", "differentiation markers", "proteins highly up-regulated in psoriatic skin", "microsequenced proteins" and "human autoantigens". For reference, we have also included 2-D gel (isoelectric focusing) patterns of cultured normal and psoriatic keratinocytes, melanocytes, fibroblasts, dermal microvascular endothelial cells, peripheral blood mononuclear cells and sweat duct cells. The keratinocyte 2-D gel protein database will be updated yearly in the November issue of Electrophoresis.

Biopsy↗
PubMed1991

Immunologic characterization of a recombinant Kentucky bluegrass (Poa pratensis) allergenic peptide.

A recombinant peptide of Kentucky bluegrass (KBG) pollen was synthesized as a fusion protein (FP) in Escherichia coli by recombinant DNA procedures and was compared with its natural counterparts with respect to its allergenic properties. The FP was demonstrated to bind to the IgE antibodies (Abs) of greater than or equal to 95% of 55 individual sera examined. A positive correlation (r = 0.90) was observed between the levels of IgE Abs corresponding to the FP and the grass-pollen extract(s). With sera of five allergic patients, the IgE binding of three different protein preparations were compared, namely, KBG pollen proteins, 27 to 35 kd gel-purified pollen proteins, and the FP. Results indicated that about 50% of the total IgE binding of KBG pollen proteins was due to the IgE Abs specific to FP. Comparison of the above protein preparations with respect to their abilities to specifically stimulate murine popliteal lymph node cells in vitro indicated that the total pollen proteins stimulated the highest proliferation of lymph node cells. Interestingly, the FP supported higher proliferation of lymph node cells than the gel-purified proteins. Collectively, these results suggest that the recombinant peptide constitutes a major allergenic constituent of grass pollens and may be of diagnostic and therapeutic value.

Allergens↗
PubMed1991

Bias and random errors in historical data of exposure to organic solvents.

The biases and random errors in data from industrial hygiene measurements have been evaluated. Such data are often used for exposure assessment in epidemiologic studies by people who are not professional industrial hygienists. The quality of such data is, therefore, of paramount importance. Data sets from two investigations of the exposure to organic solvents in the same industry have been analyzed. The study shows that the errors and analysis of field samples are larger than errors determined from laboratory data but smaller than errors from other sources. One exception, in which laboratory errors are large, is when data originate from different ways of determining recovery. Very large biases may arise for certain combinations of analytes and extraction liquid when the "equilibrium method" is used. The data sets from the two investigations had a bias, which was as large as a factor of 5 to 10. This bias is believed to be caused by use of two different sampling strategies. In all industrial hygiene data, large random errors are embedded because of day-to-day variations in exposure levels; therefore, the data are mostly used grouped into three classes of low, medium, and high exposure. To do so is meaningful, as a large amount of data will compensate for misclassifications. But a large amount of data cannot compensate for the bias introduced when merging data generated under different sampling strategies. Data describing the same object, but originating from different sampling strategies, should not be pooled without proper tests, which show that data are comparable.(ABSTRACT TRUNCATED AT 250 WORDS)

Bias↗