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L Marzella

Publications and source records attributed to L Marzella.

53 records · Page 3Linked to original sources
PubMed1982

Isolation of autophagic vacuoles from rat liver: morphological and biochemical characterization.

The induction of autophagy caused by vinblastine (VBL) has been found to be concomitant with a stimulation of proteolysis in a mitochondrial-lysosomal (ML) fraction from the rat liver (Marzella and Glaumann, 1980, Lab. Invest., 42: 8-17. Marzella and Glaumann, 1980, Lab. Invest., 42:18-27). In this fraction the enhanced proteolysis is associated with a threefold increase in the relative fractional volume of autophagic vacuoles (AVs). In an attempt to isolate the AVs, we subfractionated the ML suspension at different intervals after the induction of autophagy by VBL by centrifugation on a discontinuous Metrizamide gradient ranging from 50% to 15%. The material banding at the 24 to 20% and the 20 to 15% interphases was collected. Morphological analysis reveals that 3 h after induction of autophagy these fractions consist predominantly (approximately 90%) of intact autophagic vacuoles. These autophagic vacuoles contain cytosol, mitochondria, portions of endoplasmic reticulum, and occasional very low density lipoprotein, particles either free or in Golgi apparatus derivatives, in particular secretory granules. The sequestered materials show ultrastructural signs of ongoing degradation. In addition to containing typical autophagic vacuoles, the isolated fractions consist of lysosomes lacking morphologically recognizable cellular components. Contamination from nonlysosomal material is only a few percent as judged from morphometric analysis. Typical lysosomal "marker" enzymes are enriched 15-fold, whereas the proteolytic activity is enriched 10- to 20-fold in the isolated AV fraction as compared to the homogenate. Initially, the yield of nonlysosomal mitochondrial and microsomal enzyme activities increases in parallel with the induction of autophagy but, later on, decreases with advanced degradation of the sequestered cell organelles. Therefore, in the case of AVs the presence of nonlysosomal marker enzymes cannot be used for calculation of fraction purity, since newly sequestered organelles are enzymatically active. Isolated autophagic vacuoles show proteolytic activity when incubated in vitro. The comparatively high phospholipid/protein ratio (0.5) of the AV fraction suggests that phospholipids are degraded more slow than proteins. Is it concluded that AVs can be isolated into a pure fraction and are the subcellular site of enhanced protein degradation in the rat liver after induction of autophagy.

Animals↗
PubMed1982

Uptake and degradation of proteins by isolated rat liver lysosomes. Suggestion of a microautophagic pathway of proteolysis.

The aim of this work was to study possible uptake of electron-dense marker particles and uptake and degradation of proteins in vitro by two types of lysosomes, namely, secondary lysosomes and iron-laden "residual" bodies. The sedimentability and latency of lysosomal enzymes were measured to evaluate whether or not the different lysosomes were suitable for in vitro incubation experiments. Residual bodies displayed leakage and enzyme inactivation with increasing incubation times, whereas secondary lysosomes were stable. The two types of lysosomes were incubated in vitro with isotopically labeled methemoglobin or insulin. Secondary lysosomes, but not iron-laden residual bodies, were active in the degradation of the exogenously added proteins. Control experiments were performed to evaluate possible extra-lysosomal proteolysis of the substrates due to leakage of lysosomal enzymes during incubation. The enzymes released during incubation could only account for a minor portion of the degradation of the added labeled proteins. Secondary lysosomes were preincubated with radiolabeled protein, followed by washing and reincubation. The radioactivity in the lysosomal pellet decreased during reincubation concomitant with an increase in trichloroacetic acid-soluble label. Chloroquine decreased the release of degradation products from the lysosomes. Further support for the interpretation that lysosomes take up proteins was gained from ultrastructural observations. Secondary lysosomes were incubated with electron-dense Percoll particles in vitro. Morphologic analysis revealed Percoll particles inside intralysosomal vesicles which appeared to be formed during incubation. The results indicate that lysosomes are able to internalize their membranes during in vitro incubation followed by degradation of the micropinocytosed proteins.

Acid Phosphatase↗
PubMed1981

Autophagy, heterophagy, microautophagy and crinophagy as the means for intracellular degradation.

It is generally accepted that the lysosomal compartment plays an important role in the degradation of cellular components. In this communication we discuss various experimental models which have been used to study mechanisms of intralysosomal degradation and also discuss the evidence obtained in support of the following proposals: 1. The autophagosomes can be isolated into high purity and are the subcellular locus of induced protein degradation. 2. Different membrane components such as proteins and lipids are degraded at different rates inside the lysosomes. Intralysosomal hydrolysis is not the rate limiting step in degradation. 3. Lysosomes take up soluble material in vitro by invagination and pinching off of their membranes (microautophagy). 4. Secretory vesicles can degrade their secretory contents by fusing with the lysosomes.

Animals↗
PubMed1980

Increased degradation in rat liver induced by vinblastine. I. Biochemical characterization.

The administration of vinblastine sulfate to rats causes an increase in degradation of liver proteins and lipids as measured in vitro by the release of trichloroacetic acid-soluble products. In mitochondrial-lysosomal fractions both lipolysis and proteolysis increase. Postmicrosomal supernatants (cytosol) also show an increase in lipolysis. Little or no enhancement of degradation occurs in the microsomal fraction. The stimulation of degradation by vinblastine is dose-related and is associated with increased lysosomal fragility. Degradation in vitro in the vinblastine model persists longer than in the control. No alterations either in the recovery of cell fractions or in the distribution of lysosomal enzymes are seen after vinblastine treatment. The increase in proteolysis induced by vinblastine can be completely prevented by pretreating the rats with either low or high doses of cycloheximide. Biochemical characterization by use of an inhibitor (iodoacetamide) of cathepsins and by use of ammonium chloride suggests that the increased proteolysis seen after vinblastine is related to increased sequestration of substrate into the lysosomal pool. Our studies do not support the notion that in the liver vinblastine impairs fusion between autophagosomes and preexisting primary or secondary lysosomes during the time points studied. The stimulation of both proteolysis and lipolysis in the liver lysosomes of rats treated with vinblastine provides an experimental model for the studies on mechanisms of intralysosomal degradation of cell constituents.

Animals↗
PubMed1977

Studies on the pathogenesis of ischemic cell injury. VII. Proton gradient and respiration of renal tissue cubes, renal mitochondrial and submitochondrial particles following ischemic cell injury.

Electron transport in tissue cubes, isolated mitochondria and submitochondria particles were examined as a function of ischemic time. It was found that electron transport remains active in all systems beyond the 2 hour ischemic time interval. The NADH stimulated respiration, however, declined after 2 hours of ischemia in ASU (Ammonia-Sephadex-Urea) particles followed by respiration with matrix-located dehydrogenases tested by substrates such as glutamate, alpha-ketoglutarate and pyruvate plus malate. Succinate dependent respiration remains active at control levels. In contrast proton gradient reveals changes in two phases: Phase A is characterized by gradually increasing gradient without valinomycin and by a rapidly declining gradient with valinomycin in the medium. Phase B is characterized by a declining proton gradient with or without valinomycin. It is suggested that the alteration of the proton gradient between 1 and 2 hours ischemia is an important factor contributing to irreversible cell injury.

Animals↗
PubMed1976

Effect of storage and in vitro ischemia on the ultrasture of microsomal membranes and on microsomal enzymes.

Pieces of liver (in vitro ischemia) and isolated microsomes were subjected to incubation at 4 degrees C and 37 degrees C for various time intervals. The effects on microsomal protein, phospholipids, and cholesterol and on microsomal phosphatases and electron transport enzymes were followed as a functional of time and temperature. NADH-cytochrome c reductase was very labile and was completely inactivated by 1 h, whereas G6Pase lost 50% of its activity after 2 h at 37 degrees C. IDPase and NADPH-cyt. c red. were of intermediate susceptibility whereas cytochromes b5 and P-450 were the most stable enzymes assayed. After 24 h of incubation of isolated microsomes at 37 degrees C there was no significant detachment of membrane components (protein, PLP or cholesterol), indicating that the inactivation of the enzymes was not primarily attributable to their solubilization. Instead, experiments with 14C-leucine and 14C-glycerol prelabeled microsomes demonstrated that the proteins detached from microsomes during incubation originated mainly from the intravesicular space due to repture of the microsomal membranes. The addition of a lysosomal extract during incubation did not alter either the rate of inactivation of the enzymes or the proportion of solubilized membrane components indicating that attack from the outside by proteolytic enzymes is not the mechanism for enzyme inactivation. There was no apparent correlation between the rates of inactivation of enzymes in vitro and their calculated half-lives in vivo or their postulated intramembranous localization. Ultrastructurally, enzyme inactivation was initially associated with alterations of the microsomal membranes, such as vesicle aggregation, membrane rupture, loss of unit membrane structure, and subsequently, thickening of membranes and transformation of the microsomes into nonrecognizable amorphous material.

Animals↗
PubMedDate not supplied

Follicular atresia as an apoptotic process: atresia-associated increase in the ovarian expression of the putative apoptotic marker sulfated glycoprotein-2.

OBJECTIVE: To evaluate the possibility that morphologically confirmed/hypophysectomy-induced ovarian follicular atresia, a putative apoptotic process, is coupled to alterations in the steady-state levels of ovarian sulfated glycoprotein-2 (SGP-2) transcripts. METHODS: Hypophysectomy-induced follicular atresia in immature rats, morphologically confirmed at the light and electron microscopic levels, was correlated with alterations in the steady-state levels of ovarian SGP-2 transcripts as assessed by a solution hybridization/RNase protection assay. Cellular localization was accomplished by in situ hybridization technology. RESULTS: Hypophysectomy of the 24-day-old immature rat, an established precipitant of follicular atresia, led (3 days later) to a significant (P < .05) increase (up to 3.3-fold) in the relative abundance of densitometrically quantified ovarian SGP-2 transcripts compared with age-matched intact controls. Detailed time-course analysis after hypophysectomy revealed significantly (P < .05) increased ovarian SGP-2 mRNA expression as early as 2 days after hypophysectomy; no further increments were noted on days 4 or 8. Light microscopic analysis of comparable ovarian material 4 days after hypophysectomy revealed increased numbers of atretic follicles displaying large numbers of degenerating granulosa cells. Electron microscopic analysis of the degenerating cells of atretic follicles (from hypophysectomized rats) disclosed nuclear condensation and cytoplasmic shrinkage as well as apoptotic bodies at all levels of the granulosa cell layer. In situ hybridization established the granulosa cell of the intact untreated rat as the somatic cell concerned with SGP-2 gene expression. In turn, hypophysectomy led to an increase in SGP-2 expression at the level of the theca-interstitial cell, an effect prevented by the concurrent provision of pregnant mare serum gonadotropin (PMSG). The hypophysectomy-induced increase in ovarian SGP-2 transcripts was similarly reversed (54% inhibition by day 27) by the concomitant provision of FSH, an established antiatretic principle. The delayed administration (day 26) of a single dose of PMSG to rats hypophysectomized on day 24 eliminated the hypophysectomy-induced increase in ovarian SGP-2 transcripts as assessed on day 28. Qualitatively similar but quantitatively more pronounced increments in ovarian SGP-2 gene expression were obtained when atresia was induced by hypophysectomy of PMSG-primed immature rats. CONCLUSIONS: These observations establish the immature rat ovary as a site of SGP-2 gene expression and reveal hypophysectomy-induced follicular atresia to result in the up-regulation of ovarian (specifically, theca-interstitial) SGP-2 gene expression, an effect prevented by the concurrent provision of FSH or PMSG. To the extent that SGP-2 is an acceptable apoptotic marker, the present findings support the hypothesis that ovarian follicular atresia may be an apoptotic process.

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