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Biomedical subjects

R J Klebe

Publications and source records attributed to R J Klebe.

At least 73 records · Page 4Linked to original sources
PubMed1980

Somatic cell genetic studies of the cystic fibrosis mucociliary inhibitor.

A series of cystic fibrosis homozygous fibroblast X mouse cell hybrids were constructed in order to determine the chromosome which carries the gene controlling the cystic fibrosis mucociliary inhibitor (CFMI) phenotype. Several hybrids and their subclones retained the CFMI phenotype for long periods in cell culture. Correlation of the CFMI phenotype with the presence of specific human chromosomes indicated that possible linkage existed between CFMI and human chromosomes 2, 4, 6, 10, and 18. The strongest chance of linkage existed for chromosome 4.

Animals↗
PubMed1979

Bioautographic visualization of aminoacylase-1: assignment of the structural gene ACY-1 to chromosome 3 in man.

A bioautographic assay was developed for the visualization of aminoacylase-1 (N-acylamino acid aminohydrolase, ACY-1; EC 3.5.1.14) after zone electrophoresis. Bioautography and species differences in electrophoretic mobility of ACY-1 made it possible to investigate the chromosome assignment of the gene for human ACY-1 using human--mouse somatic cell hybrids. Human ACY-1 segregated concordantly with beta-galactosidase-A (beta GALA; EC 3.2.1.23) but showed discordant segregation with 32 other markers representing 23 linkage groups. The beta GALA gene has been previously assigned to chromosome 3. From this evidence and confirming chromosome analyses, ACY-1 has been assigned to chromosome 3. A genetic polymorphism in the electrophoretic mobility of ACY was observed in mouse strains, demonstrating that this enzyme can be mapped in genetic crosses of Mus musculus.

Amidohydrolases↗
PubMed1979

Characterization of naturally occurring auxotrophic mammalian cells.

In a previous study, several cultured cell lines were detected which are naturally occurring auxotrophs. In this investigation, the enzyme deficienceis involved are described. It is demonstrated that the Chinese hamster cell lines CHO(K1), YH21, RJK-36, and CHW-1102 are deficient in cystathionase and argininosuccinate synthetase. In addition, CHO (K1) and CHW-1102 were found to lack argininosuccinate lyase. CHW-1102 cells were also found to be unable to proliferate in medium containing branched-chain alpha-keto acids in place of the corresponding L-amino acids since CHW-1102 cells lack branched-chain aminotransferase.

Amino Acid Isomerases↗
PubMed1978

Localization of the binding site for cell attachment in the alpha1(I) chain of collagen.

Certain cells such as CHO (Chinese hamster ovary) and fibroblasts attach to a substrate of type I collagen via proteins that link the cell surface to collagen. Serum contains a glycoprotein, c-CAP (collagen cell attachment protein), that can mediate this adhesion. Previously, we established that alpha1(I)-CB7 (cyanogen bromide peptide 7), which lies within residues 552 to 822 of collagen, contains a major binding site for c-CAP(alpha1(I)-CB7 has been renumbered to account for an additional triplet recently identified at residue 613 (P. P. Fietzek and R. W. Glanville, manuscript in preparation). Now we have examined the ability of various peptides derived by proteolytic digestion of alpha1(I)-CB7 to bind to c-CAP based upon their ability to inhibit cell attachment to collagen. The binding site lies within residues 757 to 791. It is likely that this is the sole binding site in the alpha1(I) chain since cleavage of the bond between residues 775 and 776 in the alpha1(I) chain destroys cell attachment activity.

Amino Acid Sequence↗
PubMed1978

Argininosuccinic aciduria: assignment of the argininosuccinate lyase gene to the pter to q22 region of human chromosome 7 by bioautography.

Argininosuccinic aciduria, an autosomal recessive disorder of the urea cycle in humans, is associated with a deficiency of argininosuccinate lyase (ASL; L-argininosuccinate arginine-lyase, EC 4.3.2.1). ASL activity was visualized on gels after electrophoresis by a new method, termed bioautography. Bioautography involves the use of mutant bacteria to visualize the location of mammalian enzymes after zone electrophoresis. By this technique, human ASL migrated to a position different from mouse ASL, while a survey of mouse strains, tissues, and tissue culture cell extracts demonstrated the same electrophoretic form and no genetic variants of mouse ASL. Identifying human ASL, by bioautography in human-mouse somatic cell hybrids has made it possible to regionally locate the ASL gene on human chromosome 7. The human ASL phenotype segregated concordantly with the human enzyme beta-glucoronidase (GUS; beta-D-glucoronide glucuronosohydrolase, EC 3.2.1.31) in cell hybrids, but showed discordant segregation with 32 other enzyme markers representing 23 linkage groups. The gene for GUS has been assigned to chromosome 7 in humans, and cosegregation (synteny) of ASL and GUS demonstrates the assignment of ASL to chromosome 7. Regional location of ASL and GUS to the pter to q22 region of chromosome 7 was achieved in hybrids segregating a 7/9 translocation.

Amino Acid Metabolism, Inborn Errors↗
PubMed1977

Bioautography: a general method for the visualization of isozymes.

A new method has been developed for visualization of isozymes which are difficult or impossible to detect with standard histochemical or autoradiographic methods. The principle of this method, bioautography, is the use of a microbial reagent to locate an enzyme after gel electrophoresis. When bioautography was compared to other staining procedures, the bioautographic method yielded identical results to those observed by the histochemical method for lactate dehydrogenase (LDH) or by the autoradiographic method for the adenine phosphoribosyltransferase (APRT). Using the bioautographic method, stains for enzymes which could not be visualized by any other procedure have been developed: argininosuccinate lyase and branched-chain aminotransferase. By employing appropriately genetically marked bacterial strains, it should be possible to develop new isozyme stains for a large number of unstudied isozymes.

Adenine Phosphoribosyltransferase↗
PubMed1976

Carbohydrate preferences of mammalian cells.

Carbohydrate preferences of mammalian cells can be utilized to biochemically distinguish between different cell lines. Ninety-three carbohydrates were examined of which (a) 15 supported cell proliferation and (b) 42 were toxic or growth inhibitory. The present investigation has employed an enzymatic system to eliminate trace glucose levels from reagents and glucose generated by serum enzymes.

Alcohols↗
PubMed1976

Biochemical selection systems for mammalian cells: the essential amino acids.

The essential amino acid requirement of cultured mammalian cells can be satisfied by 19 amino acid derivatives. This finding (a) confirms the results of animal nutritional studies and (b) identifies 19 essential amino acid derivatives and should permit the isolation of a new class of auxotrophic mutants. Five naturally occurring auxotrophic markers have been detected in this survey; namely, inability to utilize cystathionine and citrulline in Chinese hamster ovary (CHO) cells, inability to metabolize citrulline by HTC+hepatoma cells, and confirmation of Eagle's observation that KB cells can utilize homocystine in place of methionine or cystine and D-cystine in place of L-cystine.

Amino Acids↗
PubMed1976

Somatic cell genetic studies of cystic fibrosis.

The expression of the gene coding for the cystic fibrosis ciliary inhibitor has been examined in somatic cell hybrid clonal lines. Hybrids were produced by the fusion of cultured skin fibroblasts from cystic fibrosis patients with mouse RAG and LM (TK-) cell lines. The ciliary inhibitor was detected by the oyster ciliary assay in several primary hybrid clones but not detected in others, indicating the segregation of an associated nuclear gene.

Animals↗
PubMed1975

Cell attachment to collagen: the requirement for energy.

It has previously been demonstrated that cell attachment to collagen depends on the presence of a serum-derived cell attachment factor. Two steps in the cell attachment process have been defined. First, the cell attachment factor can bind to collagen in the absence of divalent cations. Secondly, either Ca++ or Mg++ are required for cells to attach to the collagen-cell attachment factor complex. A third requirement for cell attachment to collagen is demonstrated here; namely, cellular metabolic energy. A simple method for the assay of ATP in tissue culture cells is presented.

Adenosine Triphosphate↗
PubMed1975

A simple method for the quantitation of isozyme patterns.

Isozyme patterns may be analyzed quantitatively, without the use of a densitometer, by performing serial twofold dilutions of a sample to a visual end point. The specific activity (S) of a given dehydrogenase isozyme can be assessed in the presence of other isozymes catalyzing the same reaction, by (1) determining the isozyme titer (T) (defined as mg protein/ml in the last visible band) and (2) applying the formula S = K/T, where K is 1.6 X 10(-3) units/ml in the last visible band. The units/ml (U) in the starting material can be calculated from the equation U = K (2)n-1, where n is the number of the slot producing the last visible band.

Animals↗