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Biomedical subjects

E Knecht

Publications and source records attributed to E Knecht.

At least 55 records · Page 3Linked to original sources
PubMed1986

Electron microscopic localization of glutamate dehydrogenase in rat liver mitochondria by an immunogold procedure and monoclonal and polyclonal antibodies.

Glutamate dehydrogenase (GDH) was localized in rat liver by indirect electron microscopic immunogold, using different sizes of gold particles and monoclonal and polyclonal antibodies. Using the protein A-gold technique in double immunocytochemical experiments, both antibodies, at their optimal dilutions, gave similar results. A novel assessment of the distribution of GDH was made by measurements of the number of gold particles per square micrometer of cross-sectional images of individual mitochondria. The data indicate intracellular homogeneity among mitochondria in individual parenchymal cells. The enzyme is almost absent in non-parenchymal cells. Finally, GDH was found mainly in association with the mitochondrial inner membrane.

Animals↗
PubMed1986

Regulatory mechanisms of intracellular proteolysis in mammalian cells.

Low molecular weight phosphoryl compounds, such as carbamoyl phosphate, 2,3-diphosphoglycerate and phytic acid protect, to different extents, mitochondrial and cytosolic proteins such as ornithine transcarbamoylase (OTC), carbamoyl phosphate synthetase (CPS), glutamate dehydrogenase (GDH) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), from proteolytic inactivation (rat liver lysosomal extracts, pronase, elastase). Given the wide variety and common occurrence of low molecular weight reagents such as typified here, it seems that this kind of inhibition may be important in the regulation of protein turnover. Regulation of intracellular proteolysis can also occur via the proteolytic systems. Immunocytochemical procedures for mitochondrial enzymes (CPS, GDH, OTC), show intracellular homogeneity, but intercellular heterogeneity in rat liver, compatible with a role of the autophagic-lysosomal system in degrading these proteins. However, degradation of short-lived proteins occurs by other mechanisms. Using centrifugation of cultured cells, we find that the Golgi apparatus takes part in the degradation of these proteins, probably by controlling the traffic of proteins or proteases to the degradation site.

Animals↗
PubMed1984

Homogeneity among mitochondria revealed by a constant proportion of their enzymes.

The homogeneity or heterogeneity at the enzyme level of mitochondria has not been directly demonstrated and is important for many studies. To clarify this point, carbamoyl phosphate synthase (ammonia), glutamate dehydrogenase and mitochondrial adenosine triphosphatase (F1) were located in rat liver by immunolabeling using protein A-gold. Measurements of the number of gold particles per square micron of cross sectional images of mitochondria permit to assess the relative molecular concentration of the three enzymes and, most interestingly, it presents the first evidence that different mitochondria in rat liver cells have the same relative proportion of the three enzymes. Since they have vastly different half-lives, bulk or unregulated autophagy as the main mechanism regulating the turnover of these enzymes seems unlikely.

Animals↗
PubMed1984

Regulation of lysosomal autophagy in transformed and non-transformed mouse fibroblasts under several growth conditions.

The role of the lysosomal system in accelerated protein degradation was investigated in 3T3 mouse fibroblasts and in the SV40 virus-transformed derivative, SV3T3. Rates of protein degradation and quantitative electron microscopic alterations in the lysosomal system were compared under four different growing conditions: exponential growth, confluent phase, serum deprivation, and confluent phase together with serum deprivation. We found a significant correlation between increases in rates of proteolysis of long-lived proteins and fractional volume of lysosomes, suggesting a causal relationship between the two, as well as a morphological explanation for the differences in rates of protein degradation in transformed and non-transformed cultured cells. The increase in lysosomal fractional volume resulted from an increase in dense bodies only (in serum-deprived exponential or confluent cultures) or from an increase in autophagic vacuoles and dense bodies (in serum-supplemented confluent cultures).

Animals↗
PubMed1983

The precursor of rat liver mitochondrial glutamate dehydrogenase has enzymatic activity.

The cytosolic precursor for the mitochondrial glutamate dehydrogenase of rat liver was synthesized in a cell-free reticulocyte lysate using messenger RNA from rat liver. To check whether this precursor had enzymatic activity, a highly sensitive fluorimetric method, which can measure picogram quantities of enzyme, was used together with competitive dissociation of the precursor from an immunoprecipitate with inactive glutamate dehydrogenase. Glutamate dehydrogenase activity, corresponding to that estimated from incorporation of [35S]-methionine, was detected in the precursor. The significance of this finding is discussed.

Animals↗
PubMed1983

Exit of proteins and fragments thereof from mitochondria is accelerated by the import of cytosolic synthesized proteins.

Most mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria. Incubation of 35S-methionine labeled mitochondria from rat hepatocytes with proteins synthesized in a cell-free system, using messenger RNA from rat liver, dramatically increased the release of mitochondrial proteins and fragments thereof into the medium. Since the synthesized proteins include cytosolic precursors of mitochondrial proteins, our results strongly suggest that import of proteins from the cytosol into mitochondria influences the half-life of proteins in these organelles. The use of this simple approach--i.e. combining the study of protein import and exit with mitochondria--to further clarify intracellular protein turnover and its regulation is suggested.

Animals↗
PubMed1982

Degradation of short-lived proteins is decreased by centrifugation.

We have examined the effects of enucleation and of inhibitors of mRNA synthesis (actinomycin D and cordycepin) on protein turnover of HeLa cells. Enucleation markedly inhibited the rate of protein degradation for short-lived proteins. However, cells centrifuged in the absence of cytochalasin B at the speed required to obtain cytoplasts showed protein degradation rates identical to those of cytoplasts, while inhibitors of mRNA synthesis did not affect the process. Although enucleation may affect degradation of specific proteins, these results suggest that centrifugation is largely responsible for the inhibition of protein degradation in cytoplasts.

Dactinomycin↗
PubMed1982

A comparative study of complex mitochondrial DNA in human lymphocytes transformed by Epstein-Barr virus and PHA.

Mitochondrial DNA (mt DNA) molecules, isolated from normal human lymphocytes, lymphocytes stimulated with phytohemagglutinin (72 h) and a cultured Burkitt's lymphoma cell line, were examined by electron microscopy. Only monomeric forms of mt DNA were found in normal human lymphocytes, whereas in the other cell types, catenated forms were also observed. Unicircular complex forms were apparently absent in all the cell types studied, suggesting that these forms are not a common malignant transformation in human cells. No clear correlation between mitochondrial modifications--as detected by stereological analysis--and the presence of catenated forms could be established. It is suggested that other mechanisms rather than replication of mt DNA would appear to be the mechanism responsible for the formation of these forms.

Cell Line↗
PubMed1982

Degradation of proteins synthesized in the presence of cycloheximide in normal and transformed fibroblasts.

Monolayer cultures of 3T3 and WI-38 fibroblasts were pulse labeled with radioactive leucine in the presence of cycloheximide. The rate of protein degradation was measured and compared with that of SV-40 virus transformed cells. The results clearly show that normal and transformed cells have essentially identical rates of degradation of proteins synthesized in the presence of cycloheximide. These findings indicate that the lower rates of protein degradation observed in some transformed cells is not a general rule.

Animals↗
PubMed1981

Effects of different fixative solutions on labeling of concanavalin-A receptor sites in human T-lymphocytes.

We have studied the effect of several fixative solutions on the number of Concanavalin-A (Con-A) receptor sites of human peripheral blood T-lymphocytes. Cells treated with different fixative solutions (glutaraldehyde (G); formaldehyde (F) G+F; osmium tetroxide (Os); Os+G; Os+F; and Os+G+F) were labeled with a Con-A gold labeled horseradish peroxidase (HRP) complex and the number of gold particles on the lymphocytic surface was evaluated. Comparison of cells treated with the different fixatives used showed significant differences in the density of labeling. After G fixation the number of gold particles was lower than after fixation with Os or F. Moreover, G used in combination with F or Os reduced the labeling obtained when the two latter fixatives were used alone.

Fixatives↗
PubMed1980

Transmitter-related retrograde labeling in the pigeon optic lobe: a high resolution autoradiographic study.

High resolution light microscopic and electron microscopic autoradiography in a restricted area of the pigeon subtectal nucleus isthmi, pars parvocellularis (Ipc) 30 min after onset of a tectal [3H]glycine injection shows labeling mainly of the following elements: neuronal perikarya, initial axon segments and dendrites. Rapid labeling of such intrinsic Ipc elements and not of synaptic terminals possibly of tectal origin strongly suggests a fast retrograde migration of radioactivity within Ipc-tectal neurons. Fixation experiments indicate a soluble nature of this radioactive material. This type of retrograde labeling in the glycinergic Ipc-tectal pathway seems to be related to the transmitter specificity of the system.

Animals↗
PubMed1980

Autophagy of ferritin incorporated into the cytosol of Hela cells by liposomes.

Ferritin was trapped in negatively charged liposomes consisting of phosphatidyl choline and phosphatidyl serine in a molar ratio of 9:1. Unilamellar vesicles were prepared by ultrasonic treatment. HeLa cells were incubated with these liposomes for 2h and chased for 0, 2, 14 and 20 h. The migration pathway of ferritin in the cells was followed by quantitative electron microscopy. Ferritin was detected exclusively in cytosol and lysosomes. Evidence is presented that free ferritin in cytosol enters lysosomes.

Autophagy↗
PubMed1979

Transmitter-specific retrograde labeling in the striato-nigral and raphe-nigral pathways.

Injecting radioactive transmitters into the rat substantia nigra led to retrograde neuronal labeling either in the dorsal raphe nucleus, after 3H-labeled serotonin injection, or in the caudoputamen, after 3H-labeled gamma-aminobutyric acid injection. This differential labeling in projections whose transmitter has been established provides the basis for a histochemical tracing method indicating both connectivity and transmitter specificity of neural pathways.

Animals↗