Biomedical subjects

J Klose

Publications and source records attributed to J Klose.

At least 37 records · Page 2Linked to original sources

PubMed1993

Adult phenotype in the mouse can be affected by epigenetic events in the early embryo.

Major epigenetic modifications apparently occur during early development in the mouse. The factors that induce such modifications are complex and may involve the various components of a zygote. We have started to explore whether changes in the nucleocytoplasmic composition brought about by micromanipulation can induce phenotypic effects through epigenetic modifications. Nucleocytoplasmic hybrids were therefore prepared by transplanting a female pronucleus into a recipient egg from a different genotype. As a result, the maternal genome was of a different genetic background as compared with the egg cytoplasm. Specifically, experimental zygotes had cytoplasm from the inbred strain C57BL/6, a maternal genome from DBA/2, and a paternal genome from C57BL/6 (termed BDB hybrids). The mirror-image combination, termed DBD, was also made. The reconstituted zygotes were transferred to recipients and allowed to develop to term. Mice born from manipulated zygotes showed transcriptional repression and DNA methylation of major urinary protein genes in their liver, as well as growth deficiency resulting in reduced adult body weight. No altered phenotype was observed in controls in which the maternal pronucleus was simply transplanted back into another zygote of the same genetic background. These results clearly demonstrate phenotypic as well as molecular effects on DNA methylation and expression of at least one gene. Phenotype was therefore no longer predicted by genotype as a result of epigenetic modifications in experimental embryos. What precisely triggers the phenotypic and epigenetic changes is unknown, but presumably, nucleocytoplasmic interactions in hybrid zygotes may be partly responsible.

Animals↗

PubMed1992

Identification of tissue proteins by amino acid analysis after purification by two-dimensional electrophoresis.

Mouse brain proteins were separated by two-dimensional electrophoresis (2-DE). The proteins of a section of the 2-DE pattern were blotted onto hydrophobic membranes and 43 of them were excised and hydrolyzed by liquid-phase hydrolysis. The amino acid composition of these proteins was determined by orthophthaldialdehyde precolumn derivatization and compared with the compositions of known proteins stored in the NBRF sequence database. An identification program named ASA was developed for this purpose. The ASA program includes correction and weighting factors, data reduction by molecular weight windows, and exclusion or inclusion of certain organisms as desired. As a control, eight test proteins and five well-known proteins from mouse brain, all separated by 2-DE, were correctly identified by the program. Out of the 43 brain proteins selected, 19 were identified with high confidence.

Amino Acids↗

PubMed1992

Two-dimensional electrophoretic protein patterns of reciprocal hybrids of the mouse strains DBA and C57BL.

Two-dimensional electrophoretic patterns of cytoplasmic liver proteins of the mouse were investigated in reciprocal hybrids of the two inbred mouse strains DBA and C57BL in order to establish whether strain-specific protein variants reveal a mode of inheritance compatible with the concept of genomic imprinting. Variants of this type were found to account for about 11% of approximately 200 variant protein spots scrutinized. Transmission of the maternal form of a variant protein was more frequent than transmission of the paternal form. Maternal/paternal transmission was observed only for proteins showing strain variations in their amount. The results are discussed in terms of the frequency of imprinted genes.

Animals↗

PubMed1990

Blotting efficiency investigated by using two-dimensional electrophoresis, hydrophobic membranes and proteins from different sources.

Purification and chemical characterization of proteins may be achieved by combining two-dimensional electrophoresis (2-DE) and microsequencing or amino acid analysis. To enable this combination, the protein has to be transferred as completely as possible from the gel into the sequencer. In this study hydrophobic membranes were used as support for the transfer and proteins were transferred from the gels onto the membranes by semidry blotting. Blotting conditions were optimized to obtain high blotting efficiencies for as many proteins of a complex 2-DE pattern as possible. Under optimized conditions, blotting efficiencies between 60% and 100% were obtained for five marker proteins; the mean values from four regions of a 2-DE pattern from 29 unknown proteins of a complex protein mixture from mouse brain were between 60% and 79%. The four commercially available hydrophobic membranes that were compared showed only slight differences in protein amount on the membranes after blotting for whole protein patterns, whereas single proteins occurred with higher amounts on either one or the other membrane. The results of the blotting optimization allowed us to suggest a blotting mechanism with which systematic improvement of the blotting conditions is possible for problematic proteins.

Animals↗

PubMed1989

Dye ligand chromatography and two-dimensional electrophoresis of complex protein extracts from mouse tissue.

A complex protein fraction of mouse brain was subjected to dye ligand chromatography with various dye ligands. The proteins that were bound by the dye-gel matrix and also the non-binding proteins were separated by high-resolution two-dimensional electrophoresis. The protein patterns obtained were compared. The results show that a large number of different protein species bind to dye ligands and do not occur in the eluate. Red A was the most efficient dye in isolating an individual protein class from a complex tissue extract. Moreover, we found that many of the binding proteins did not cross-react among different types of dye ligands. Orange A and Blue B were the most unrelated dyes among those compared. Our investigation shows that dye ligand chromatography can be used as a means (among others employed previously) of fractionating and classifying the enormous number of different protein species in a mammalian tissue when combined with high-resolution two-dimensional electrophoresis.

Animals↗

PubMed1989

Systematic analysis of the total proteins of a mammalian organism: principles, problems and implications for sequencing the human genome.

High-resolution two-dimensional electrophoresis (2-DE) has reached a technological level that allows us to resolve most of the numerous unknown protein species of a mammalian organism if appropriate strategies are used. We will discuss the problems of classification and characterization of proteins and propose a systematic approach to the analysis of the total protein complex. Both a comprehensive as well as a pragmatic approach towards systematic analysis have been considered. A "complex protein database" is suggested and considered with regard to various uses. A systematic analysis of the mouse proteins has been started and some of the preliminary results are summarized here. In particular, genetic properties of the proteins were investigated and are presented in order to demonstrate the significance of a systematic analysis of proteins for research and practical application (e.g. mutagenicity testing). A concept is presented for sequencing the coding DNA of mouse and man, starting with a systematic analysis of mouse proteins and then using two recently developed methods - microsequencing of proteins from spots of 2-DE protein patterns, and utilization of the relatively short N-terminal sequences obtained - to produce the corresponding cDNA's of these proteins.

Animals↗

PubMed1989

Composition and genetic variability of proteins from nuclear fractions of mouse (DBA/2J and C57BL/6J) liver and brain.

Proteins from nuclear plasma of mouse liver and brain and from the nuclear membranes of mouse liver were separated by two-dimensional electrophoresis. For the purpose of comparison, liver cytosol proteins were also investigated. The protein samples were prepared from two inbred strains of the mouse (DBA/2J, C57BL/6J) and their hybrids. The patterns obtained were compared with regard to the composition and genetic variability (qualitative and quantitative variants) of proteins from different nuclear fractions and organs. The percentage (greater than 30%) of spots common to different organs (liver, brain), but from the same nuclear fraction (plasma) was greater than the percentage (less than 20%) of spots common to different cell and nuclear fractions (cytosol, nuclear plasma and nuclear membranes) of the same organ (liver). Quantitative genetic variants occurred much more frequently than qualitative genetic variants (5.1% vs. 0.2%; liver nuclear plasma). The incidence of genetic variants was much higher in liver (5.3%) than in brains (0.0%), and higher in solubilized nuclear proteins (5.3%) than in structure-bound nuclear proteins (2.1%).

Animals↗

PubMed1989

Radioactive labeling of proteins in cultured postimplantation mouse embryos. I. Influence of the embryo preparation method.

Conditions for optimum incorporation of radioactive amino acids into proteins of cultured postimplantation mouse embryos were investigated under the aspect of using these proteins for two-dimensional electrophoretic separations followed by fluorography. The aim was to obtain highly radioactive proteins under conditions as physiological as possible. Embryos at Days 10, 11, and 12 of gestation were prepared in different ways and incubated for 4 h in Tyrode's solution containing [3H]amino acids (mixture) at a concentration of 27 microCi/ml medium. The preparations were: a) yolk sac opened, placenta and blood circulation intact; b) yolk sac and amnion opened, placenta and blood circulation intact (Day 10 embryos only); c) placenta, yolk sac, and amnion removed (embryo "naked"); d) naked embryos cut randomly into pieces (Day 10 embryos only). After incubation whole embryos or certain parts (tail, liver, rest body) were investigated by determining the radioactivity taken up by the protein. The results are given in dpm per mg protein per embryo. Radioactivity of proteins was about 3 times higher in naked embryos than in embryos left in their yolk sacs. This was true for all three stages investigated. However, the degree of radioactivity in the various parts of naked embryos differed by a factor of 15, whereas radioactivity was evenly distributed in embryos incubated in their yolk sacs. Therefore, embryos prepared according to the first method (see above) fulfilled the conditions required at the best.

Amino Acids↗

PubMed1989

Radioactive labeling of proteins in cultured postimplantation mouse embryos. II. Dose and time dependency.

The conditions for optimum incorporation of radioactive amino acids into proteins of cultured postimplantation mouse embryos were investigated under the aspect of using these proteins for two-dimensional electrophoretic separations and fluorography. The aim was to obtain highly radioactively labeled proteins under conditions as physiological as possible. Mouse embryos of Days 8, 10, and 11 of gestation were cultured in Tyrode's solution. Incubation time and concentration of [3H (or 14C)]amino acids in the culture medium were varied over a broad range. Embryos were prepared with placenta and yolk sac or without any embryonic envelopes. After culturing, the physiologic-morphologic state of the embryos was registered on the basis of several criteria. The radioactivity taken up by the total protein of each embryo was determined and calculated in disintegrations per minute per milligram protein per embryo. To approach our aim, embryos of different developmental stages had to be cultured under different conditions. A good compromise for Day-8, Day-10, and Day-11 embryos was: embryos prepared with yolk sac (opened) and placenta, 150 microCi radioactive amino acids added per milliliter medium, incubation for 4 to 5 h. For maximum labeling of proteins it is advisable to culture Day-10 embryos without embryonic envelopes under particular conditions.

Amino Acids↗

PubMed1988

Identification of mouse brain proteins after two-dimensional electrophoresis and electroblotting by microsequence analysis and amino acid composition analysis.

Two-dimensional electrophoretic separation and immobilization of proteins onto inert membranes for subsequent amino acid sequence and amino acid composition analysis is described as a rapid procedure for the identification or characterization of proteins from complex mixtures. This method avoids the drawbacks of classical purification and isolation methods which involve time-consuming operations with low resolution and, often, insufficient yields. Excellent overall yields of minor amounts (in the low microgram range) using this method allow for sequence determination of yet inaccessible proteins. Solubilized cell proteins of mouse brain were separated by high resolution two-dimensional electrophoresis and electroblotted onto a siliconized glass fiber membrane. The immobilized proteins were stained with Coomassie Brilliant Blue R-250, and twelve proteins spots were then submitted to both Edman degradation and amino acid analysis. Proteins were identified by comparison of the experimentally determined amino acid composition with a dataset derived from the Protein Identification Resource (PIR) protein sequence database. Eight out of twelve proteins tested were identified by amino acid analysis and confirmed by N-terminal sequence determination.

Amino Acid Sequence↗

PubMed1988

An NMR blood test for cancer: a critical assessment.

An evaluation of the reproducibility and accuracy of the NMR human blood test for cancer described by Fossel, E. T., Carr, J. M. and McDonagh, J., (New England Journal of Medicine 315, 1369-1376) in 1986 has been conducted jointly at the National Cancer Institute-Frederick Cancer Research Facility, Frederick, MD (NCI-FCRF) and the National Research Council, Ottawa, Canada (NRC). The influences on the test of the following were studied: (a) subject fasting; (b) sample collection, storage and handling; (c) use of plasma or serum; (d) variations of test results from the same individual with time; (e) NMR observation parameters including field strength and temperature; and (f) variations in obtaining the Fossel Index (FI) (a number defined by Fossel and co-workers as the average of the widths at half height of the regions in the NMR spectrum of human plasma at 1.3 and 0.88 ppm) by different people from the same plotted spectrum. This test was found to be reproducible but not accurate for screening a general asymptomatic population. The accuracy is defined in terms of the sensitivity, specificity, and predictive values of the test. The accuracy of the test results from our laboratories is compared with the accuracies from other laboratories including Fossel's. The correlation of the Fossel Index with total triglyceride content in the serum has been confirmed by analysing blood components using the following technologies: KBr density gradient centrifugation, high resolution agarose gel electrophoresis, high performance gel permeation chromatography, and chemical analysis.

Blood Chemical Analysis↗

PubMed1987

Human dirofilariasis with reactive arthritis--case report and review of the literature.

A 43-year-old German man suffered from an insect bite, which was followed by a severe local inflammation and lymphadenitis. A year later, he developed dry cough, chest pain, and an oligoarthritis of the ankle and knee joints. While other causes of the symptoms could be excluded, serologic findings suggested a dirofilarial infection with reactive arthritis due to the parasite. The observation points out, that in cases of unclassifiable mono- or oligoarthritis, reactive arthritis due to parasites should be considered in the spectrum of differential diagnoses. The disease is rare at present, but its frequency might be underestimated because of the diagnostic difficulties. Dirofilarial infection should be borne in mind after a stay abroad. The present case, however, shows, that the disease also occurs in patients who had not travelled to regions of risk in recent years.

Antibodies↗

PubMed1987

Analysis of protein patterns from different organs and cell fractions of trisomy 19 mice.

Proteins were extracted from liver, brain, and skin of 6-day-old mice with trisomy (Ts) 19 and fractionated into solubilized cell proteins and structure-bound cell proteins. The proteins were separated by two-dimensional electrophoresis, and protein patterns were compared in the combinations Ts/normal and normal/normal. Analysis of the protein patterns revealed protein spots (variants) with densities higher (h-type) or lower (l-type) in trisomies than in normal mice. Some of these variants were found in all Ts individuals investigated for a particular protein class. These variants, termed regular Ts-variants, constituted 0.8%-1.6% of the total number of spots. The proteins of the regular Ts variants were in most cases organ-nonspecific. However, in almost all cases a given quantitative variation was expressed in only one of the three organs investigated. To explain our results, we have presented models for the control of protein levels on the basis of gene regulation. New aspects in the conception of studies on trisomies in man could be gained.

Animals↗

PubMed1987

Qualitative and quantitative variability in different classes of proteins: comparison of mouse and rat.

Proteins of membranes and cytosols were extracted from the livers and brains of mice (inbred strain DBA/6J) and rats (inbred strain DA/Han) and separated by two-dimensional electrophoresis (2-DE). The 2-DE patterns were compared with regard to qualitative (spot position) and quantitative (spot intensity) characteristics of the proteins of these two species. The following results were obtained: Brain had more (higher percentage) conservative proteins (proteins found in both mice and rats) than liver; plasma membranes had more conservative proteins than the cytosols; organ-unspecific proteins contained more conservative proteins than relatively organ-specific proteins; the pattern of distribution of genetic variability among different classes of proteins represented by findings 1-3 was the same for the qualitative and quantitative characteristics of the proteins; and some observations indicated that quantitative variability occurred more frequently among proteins than did qualitative variability. Our conclusion is that regulatory sequences in the DNA (regulatory genes) are subjected to functional constraints that differ in strength among different classes of proteins by the same ratios as the constraints acting on the structural genes. The overall effect of the selective pressure is, however, less stringent for regulatory genes than for structural genes. The results obtained here by comparing two different species are very similar to previous results we obtained by studying different subspecies (inbred strains of the mouse). From this finding arises a new concept: the study of molecular evolution on the basis of different classes of proteins. Our results were compared with data from the literature that were obtained in part from studies on cultured cells. The comparison suggested that cultured cells have lost their tissue-specific proteins, and so generate predominantly extremely conservative proteins.

Animals↗

PubMed1986

Composition and genetic variability of heparin-sepharose CL-6B protein fractions obtained from the solubilized proteins of mouse organs.

The solubilized proteins of liver and brain from mice of two inbred strains (C57BL/6J and DBA/2J) and their hybrids were subfractionated by heparin Sepharose (H-S) CL-6B affinity chromatography. The H-S binding and nonbinding proteins were separated by two-dimensional electrophoresis. The protein patterns obtained were analyzed with regard to their protein composition and their genetic variability (qualitative and quantitative variants). Eighty to ninety percent of the H-S binding proteins were unique to this class of proteins. This class was rich in organ-specific proteins. Compared to the nonbinding proteins the portion of basic proteins was only slightly increased, suggesting that most of the H-S binding proteins interact specifically with heparin. The frequency of qualitative protein variants revealed that H-S binding proteins are more conservative than H-S nonbinding proteins. The quantitative genetic variability was higher in liver than in brain. Quantitative protein variants occurred more frequently than qualitative variants.

Animals↗

PubMed1985

Genetic variability of proteins from mitochondria and mitochondrial fractions of mouse organs.

Proteins of whole mitochondria from mouse liver and brain and proteins of liver mitochondrial fractions (plasma and rough membrane fraction) were separated by two-dimensional electrophoresis. Protein patterns of two inbred strains of mouse, C57BL/6J and DBA/2J, and of F1 mice of these two strains were studied. The protein patterns obtained from the different mitochondrial materials were analyzed with regard to their protein composition and the genetic variability of proteins (qualitative and quantitative protein variants). Included in this analysis are data previously obtained from the cytosols and plasma membranes of the same organs and mouse strains. The results showed the following. Mitochondria and organelle-free cell components (cytosol and plasma membranes) have only a few percent of their proteins in common, while two organs, liver and brain, reveal up to approximately 50% organ-nonspecific proteins. The frequency of proteins common to solubilized and structure-bound proteins ranges below 20%. Genetic variability in protein amount occurs much more frequently than genetic variability in protein structure. Liver proteins reveal more genetic variants than brain proteins. Proteins solubilized in the cell show more genetic variation than structure-bound proteins. Furthermore, the results show that with regard to the composition and the genetic variability of proteins, liver and brain differ more in their mitochondria than in their cytosol and plasma membranes.

Animals↗

PubMed1984

Separation of polychlorinated biphenyls (Aroclor 1254) by high-performance liquid chromatography.

The separation of Aroclor 1254 on various reversed-phase columns was investigated. The results show that the cyano- and phenyl-bonded columns performed poorly, but C18 bonded columns gave better results. It was also found that the absorption wavelength at which the effluent is monitored has great effect on the detection of these isomers. The optimum conditions for the separation and detection are: C18, 5 micron; reversed-phase column, 20 cm X 4.6 mm I.D.; acetonitrile-water (60-40); flow-rate 1.5 ml/min; detection at 210 nm or 205 nm.

Aroclors↗

PubMed1984

An attempt to resolve all the various proteins in a single human cell type by two-dimensional electrophoresis: I. Extraction of all cell proteins.

A concept is presented for estimation of the total number of different proteins in a single human cell type (exemplified here by Hep cells) by use of two-dimensional electrophoresis (2DE). This concept includes three problems, the first, investigated in this study, being the transfer of all protein species of the cells into a sample useful for separation by 2DE. Five different extraction media containing--in various combinations--urea, Nonidet P-40, Zwittergent, mercaptoethanol, dithiothreitol, and sodium dodecyl sulfate were used step by step in three different extraction procedures to extract the cell proteins. The amount of radiolabeled proteins in each extract was measured. Each extract was subjected to 2DE. From the total mass of cell proteins, 99.99% could be extracted in two steps: 96% were extracted with urea/beta-mercaptoethanol solution, the remaining 4% with sodium dodecyl sulfate/urea/beta-mercaptoethanol solution. A special class of proteins assumed to be present in the latter fraction was not detected. Thus this fraction can be omitted from the further analysis of all cell proteins by 2DE. Protein classes that possibly remain undetected by the described extraction procedures are mentioned.

Cell Line↗