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Biomedical subjects

R S Decker

Publications and source records attributed to R S Decker.

At least 55 records · Page 3Linked to original sources
PubMed1985

Cell proliferation-associated expression of a recently evolved isozyme of triosephosphate isomerase.

An electrophoretically unique, thermolabile isozyme of triosephosphate isomerase (TPI; EC 5.3.1.1) accounts for 10-30% of the enzymatic activity in a range of mitotically active human cells and tissues. This type 2 form (subunit) of human TPI appears in two isozymes, an anodally migrating, relative to the constitutive TPI-1/1 homodimer, TPI-2/2 homodimer and the TPI-1/2 heterodimer with an intermediate mobility. Human cell types expressing the induced isozyme, which is the product of the same structural locus as the constitutive isozyme, include mitogen-stimulated lymphocytes, virally transformed B-lymphoblastoid cells, leukemia-derived T-lymphoblastoid cells, HeLa cells, both normal and transformed fibroblasts, and placental tissue. Extracts of nondividing or terminally differentiated human cells/tissues, such as erythrocytes, striated muscle, peripheral lymphocytes, and platelets, contain high levels of the constitutive TPI-1/1 isozyme but little or undetectable levels of the TPI-1/2 or TPI-2/2 isozyme. The cell division-associated TPI-1/2 and -2/2 isozymes are distinct in electrophoretic mobility from the deamidated forms of the constitutive isozyme. Extracts of dividing gorilla fibroblasts display an isozyme pattern identical to that of proliferating human cells, but various proliferating cells derived from the African green monkey, rabbit, and chicken express only the constitutive isozyme. Thus, expression of the cell division-associated isozyme of TPI is restricted to the hominoids, suggesting a recently evolved modification mechanism which is specifically activated in proliferating cells.

Animals↗
PubMed1985

Responses of cultured cardiac myocytes to lysosomotropic compounds and methylated amino acids.

Cardiac myocytes whose lysosomes had been pre-labelled with acridine orange were exposed to either L-amino acid methyl esters (L-leucine or methionine methyl ester) or to 'lysosomotropic' weak bases (chloroquine, methylamine, and NH4Cl) for 1 h. Both types of interventions dilated lysosomes equally and inhibited proteolysis to varying degrees. The weak bases produced no apparent alterations in the acridine orange staining, whereas the methylated amino acids induced a marked redistribution of the fluorescent dye from lysosomes into the myoplasm, suggesting that they may have provoked a change in lysosomal membrane permeability. A brief exposure to weak bases failed to enhance acid proteinase secretion into the culture medium but apparently inactivated cellular cathepsin B activity. In contrast, methylated amino acids induced no alterations in acid proteinase activity or the cellular distribution of the two proteolytic enzymes. Lastly, weak bases markedly elevated intralysosomal pH as measured with fluorescein dextran, while only modest rises were observed after amino acid methyl ester treatment. The present observations imply that amino acid methyl esters represent a new class of reagents with actions distinctly different from those of chloroquine and NH4Cl, and they may provide a unique and valuable means of studying secondary lysosomal function in cell culture.

Ammonium Chloride↗
PubMed1984

Methylated amino acids and lysosomal function in cultured heart cells.

Methylated amino acids inhibit lysosomal function in cultured rat heart myocytes more effectively than the classically employed lysosomotropic weak bases. Moreover, L-leucine methyl ester (L-Leu-OMe) or L-methionine methyl ester (L-Meth-OMe) do not alter lysosomal pH or inactivate lysosomal cysteine proteinases, but do inhibit protein degradation more efficiently than either chloroquine or NH4Cl. These observations suggest that amino acid methyl esters are more effective probes to investigate lysosomal function in cultured myocytes than chloroquine or NH4Cl.

Ammonium Chloride↗
PubMed1983

Dinitrophenyl-pepstatins as active-site-directed localization reagents for cathepsin D.

1. N-Pepstatinyl-N'-dinitrophenyl-1,6-diaminohexane, a potential active-site-directed localization reagent for cathepsin D, was found to bind non-specifically to immuno-precipitates containing cathepsin D. 2. Three new water-soluble localization reagents were synthesized, by using NN'-bis-(3-aminopropyl)piperazine, 3-oxa-1,5-diamino-pentane or 3,6-dioxa-1,8-diamino-octane, as spacer arms between the pepstatin and dinitrophenyl moieties. 3. The hydrophilic dinitrophenyl-pepstatins were all tight-binding inhibitors of cathepsin D at pH 3.5, but showed little or no binding to immuno-precipitates containing the inactive enzyme at pH 7.4. 4. Gel-chromatographic experiments showed that, at pH 5.0, all the dinitrophenyl-pepstatins were bifunctional reagents able to bind cathepsin D and anti-dinitrophenyl antibody at the same time. Enzyme-inhibitor-antibody complexes were not formed at pH 7.4, thus confirming that the reagents were active-site-directed. 5. Cultured human synovial cells were fixed and incubated with the dinitrophenyl-pepstatins at pH 5.0 or pH 7.4. After washing briefly, the cells were incubated at the appropriate pH value with anti-dinitrophenyl antibody labelled with fluorescein. When examined by fluorescence microscopy the cells stained at pH 5.0 showed fluorescent perinuclear granules, which were not seen in the cells treated at pH 7.4. The distribution of cathepsin D, determined by indirect immuno-fluorescence at pH 7.4, closely resembled that revealed by the dinitrophenyl-pepstatins at pH 5.0. 7. NN'-(3-Pepstatinylaminopropyl-3'-dinitrophenylaminopropyl)piperazine gave the most intense lysosomal staining and showed no non-specific binding. We conclude that this reagent is suitable for the subcellular localization of the active conformation of cathepsin D.

Binding Sites↗
PubMed1983

Influence of chlorpromazine on lysosomal alterations during myocardial ischaemia.

Ligation of the circumflex artery of anaesthetised, open-chest rabbits caused a progressive increase in nonsedimentable cathepsin D activity in severely ischaemic myocardium and an anatomical redistribution of the enzyme from lysosomes into the cytosol, along with progressive ultrastructural signs of cellular damage and necrosis. Chlorpromazine pretreatment (15 mg X kg-1 intravenously) reduced the increase in nonsedimentable cathepsin D activity slightly, but no appreciable protective effect on the anatomical redistribution of the enzyme or the development of ultrastructural signs of necrosis could be detected. It is concluded that in this experimental model of myocardial infarction, high concentrations of chlorpromazine have a mild stabilising action on lysosomes, but the drug has minimal if any effect in protecting the heart from ischaemic damage.

Animals↗
PubMed1982

Cardiac catabolic factors: the degradation of heart valve intercellular matrix.

Cultures of porcine heart valves and aorta secrete a factor that stimulates the degradation of cartilage matrix in a fashion similar to that displayed by synovial catabolin. The heart valve factor also induces the release of chondroitin sulfate and hydroxyproline from isolated heart valve cultures. The present observations support the hypothesis that tissues producing catabolic factors (catabolins) may well be responsive to them and that these messengers may play a role in the cellular regulation of the degradation of intercellular macromolecules.

Animals↗
PubMed1982

Identification of several electrophoretic variants of human ceruloplasmin including CpMichigan, a new polymorphism.

Two new electrophoretic variants of human ceruloplasmin (Cp) were detected in studies of a population from Ann Arbor, Mich., USA. The Cp Michigan variant occurs as a polymorphism in both Caucasians and Negroes while the Cp C-Ann Arbor-1 variant was identified in a single family. During reexamination of Cp variants previously detected among Amerindian tribes, a new variant Cp A-Wap-1 was identified in the Macushi and Wapishana tribes as being different from the variant observed in the Xavante and Cayapo tribes.

Adult↗
PubMed1981

Bimane-labelled pepstatin, a fluorescent probe for the subcellular location of cathepsin D.

1. Pepstatinyl-cystamine was synthesized. The disulphide bond was cleaved and the pepstatin-bound thiol was made to react with monobromobimane. The fluorescent N-pepstatinyl-S-bimanyl-2-aminoethanethiol was purified. 2. Human cathepsin D showed tight binding of the bimane-labelled pepstatin at pH 3.5. The titration curves were used to determine the apparent dissociation constant, KD; values of approx. 1 x 10(-10) M were obtained. 3. Gel-chromatographic experiments showed that, like that of pepstatin, the binding of N-pepstatinyl-S-bimanyl-1-aminoethanethiol to cathepsin D was strongly pH-dependent. Binding was seen at pH 5.0, but could not be demonstrated at pH 7.4. 4. Cultured human synovial cells were fixed and incubated with the fluorescent inhibitor at pH 5.0 or pH 7.4. When examined by fluorescence microscopy the cells stained at pH 5.0 showed a punctate perinuclear distribution of bimane fluorescence. By contrast, the cells stained at pH 7.4 showed no fluorescence. 5. The distribution of cathepsin D, determined by indirect immunofluorescence at pH 7.4, closely resembled that of the fluorescent inhibitor seen at pH 5.0. 6. We conclude that N-pepstatinyl-S-bimanyl-2-aminoethanethiol is a fluorescent probe selective for the active conformation of cathepsin D.

Cathepsin D↗
PubMed1981

Intracellular disruption of rat heart lysosomes by leucine methyl ester: effects on protein degradation.

Perfusion of rat hearts with Krebs--Henseleit medium containing 10 mM L-leucine methyl ester leads to swelling of lysosomes and loss of lysosomal integrity within 30-60 min. No morphological changes can be detected in the nuclei, mitochondria, sarcoplasmic reticulum, or Golgi complex as a result of the treatment with leucine methyl ester, and the hearts continue to beat normally during the treatment period. Homogenates of rat hearts perfused with the methyl ester exhibit a decrease in the sedimentability of cathepsin D activity compared to controls, thus providing additional evidence for a loss of lysosomal integrity. Swelling and disruption of the lysosomes presumably occurs because of the extensive accumulation of leucine within the organelles resulting from the intralysosomal hydrolysis of the freely permeating methyl ester. The lysosomal dysfunction that occurs with exposure to leucine methyl ester produces a 30% decrease in cardiac protein degradation. These results provide an estimate of the contribution of lysosomes to total protein degradation in the rat heart, and they also suggest that the enzymes released as a result of lysosomal disruption are relatively inactive in hydrolyzing cellular constituents under the perfusion conditions used here. The use of amino acid methyl esters to produce rapid, specific loss of lysosomal integrity in situ provides an approach to the study of lysosomal function in intact cells.

Animals↗
PubMed1981

Immunofluorescent localization of cathepsins B and D in human fibroblasts.

The proteinase cathepsin B has been localized in human fibroblasts using a monospecific antiserum that reacts in a precipitating manner with the denatured enzyme only. The distribution of staining as revealed by both direct and indirect immunofluorescence indicates a typical lysosomal localization similar to that observed for cathepsin D. Using dual localization methods employing fluorescein- and tetramethylrhodamine-labeled affinity purified antibodies both proteinases were usually observed in the same lysosome, indicating a combined role in intracellular digestion.

Cathepsin B↗
PubMed1981

Lysosomal alterations in heart, skeletal muscle, and liver of hyperthyroid rabbits.

Daily administration of L- or D-thyroxine for 1 week produced hypertrophy of the heart and atrophy of skeletal muscle and liver. The myocardial hypertrophy was accompanied by a rise in the activity of cathepsin D but not of cathepsin B; this was correlated with an increase in cathepsin-D-rich interstitial cells while the number of cathepsin-D-positive lysosomes in myocytes was decreased, as assessed from immunohistochemistry. In atrophying skeletal muscle (soleus and tibialis anterialis), large increases in the activities of cathepsins B and D were present. Immunohistochemical localization of cathepsin D revealed that in thyrotoxic striated muscle cells this acid proteinase had become localized diffusely in the paranuclear myoplasm. The atrophying liver of thyrotoxic rabbits also developed large increases in cathepsin D activity, but in this organ the increase was correlated with an increased number of cathepsin-D-positive secondary lysosomes without diffuse extralysosomal deposits. These observations indicate that changes in lysosomes and lysosomal enzyme activities elicited by thyrotoxicosis are tissue-specific. In some organs, the changes may be associated with net changes in protein balance or with tissue injury, but the exact functional significance of the lysosomal alterations remains uncertain.

Animals↗
PubMed1981

Origin of the triosephosphate isomerase isozymes in humans: genetic evidence for the expression of a single structural locus.

A genetic approach is used to ascertain that a single structural locus for triosephosphate isomerase (TPI) (E.C.5.3.1.1.) is expressed in rapidly dividing human lymphoblasts. This approach is made possible through the identification of a rare electrophoretic variant of human TPI. The variant phenotype is expressed by the TPI-B isozyme in both erythrocytes and peripheral lymphocytes. The variant phenotype is also expressed in the thermostability and electrophoretic pattern of the TPI-A isozyme in mitogen-stimulated lymphoblasts, indicating that TPI-A and TPI-B are products of the same structural locus. These findings are in contrast to the recent conclusions of Yuan et al. based upon structural analysis, suggesting that the TPI-A and TPI-B isozymes are products of distinct structural loci.

Carbohydrate Epimerases↗