Autophagy in hepatocytes and erythropoietic cells isolated from the twenty-one day old rat embryo.

Liver cells of the twenty-one day old rat embryo are isolated by a modified method and autophagy is studied in them by electron microscopic morphology and morphometry. Immediately after isolation or 2.5 h incubation in nutrient-free medium, embryonic hepatocytes contain high amount of glycogen and only very few autophagic vacuoles. In contrast, all glycogen is lost and 15% of the cytoplasmic volume is occupied by late autophagic vacuoles in hepatocytes after 18 h in the same medium. Presence of 3-methyladenine in the latter case inhibits both the loss of glycogen and the appearance of autophagic vacuoles while enlarging the multivesicular body compartment. Our findings reveal major differences between isolated embryonic and adult hepatocytes concerning autophagy. Several types of autophagic vacuoles are described in the cell types of the erythropoietic cell lineage. This means that autophagy is an integral part of erythropoiesis not only in bone marrow, but also in embryonic liver that is investigated here for the first time from this point of view. The presence of unclosed isolation membranes and the predominance of early autophagic vacuoles in reticulocytes indicates that the molecular machinery of segregation is still active in this functionally and structurally highly reduced cell type.

Schineria larvae gen. nov., sp. nov., isolated from the 1st and 2nd larval stages of Wohlfahrtia magnifica (Diptera: Sarcophagidae).

Four bacterial strains were isolated from the fly larvae of an obligate parasitic fly, Wohlfahrtia magnifica (Diptera: Sarcophagidae). These isolates were characterized by a polyphasic approach and represent a new lineage of gamma-Proteobacteria as their closest relative is Xylella fastidiosa (87.1% 16S rDNA similarity). The four strains are identical at the 16S rDNA level, the level of similarity between them, based on DNA-DNA hybridization, is high (97.8-102.5%) and they are similar in their physiological and biochemical characteristics, although they differ in their utilization of different sole carbon sources. All produce chitinase. They are obligately aerobic: no growth is detected under anaerobic conditions, even in the presence of NO3- as terminal electron acceptor. Their predominant respiratory quinone is Q-8. The G+C content of their DNA is 42 mol%. Their cell membrane contains phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine and two unknown polar lipids. Their main fatty acids are C18:1, C16:0 and C14:0. To accommodate these bacteria, a new genus, Schineria gen. nov., with the type species Schineria larvae sp. nov., is proposed.

Autophagy in the epithelial cells of murine seminal vesicle in vitro. Formation of large sheets of nascent isolation membranes, sequestration of the nucleus and inhibition by wortmannin and 3-ethyladenine.

The spontaneous autophagic activity in epithelial cells of isolated tissue slices of murine seminal vesicle is strongly enhanced by short (5 min) pretreatment in a medium containing 0.03% Triton X-100. In addition to the significant increase in the cytoplasmic volume fraction and the mean size of autophagic vacuoles, the appearance of shorter or longer smooth membrane pairs located between cisterns of rough endoplasmic reticulum (RER) and in the vicinity of nucleus is also greatly stimulated. Their morphological features observed after application of various fixation methods, freeze-substitution and freeze-fracture techniques show that they are unclosed nascent isolation membranes, representing a unique class of intracellular membranes. They may grow around the nucleus, leading to its complete autophagic sequestration and degradation, which is observed here for the first time. Treatment with 3-methyladenine or wortmannin inhibits the formation of autophagosomes, leading to their regression with a halving time of 7 min. In contrast, these inhibitors cause extremely fast shrinking of nascent isolating membranes, leading to their complete disappearance within 7 min. We propose that the early events of autophagy involve three main steps: initiation, growth and closure, and suggest that the growth of nascent isolation membranes is reversible i.e. the membranes may be subject to disassembly before their closure is completed. Bending and closure of the isolation membrane and the stability of neighbouring cellular structures appear as important determinants of size regulation. These early steps of autophagy are good candidates for very fast accommodation to changing conditions and subtle regulation by phosphoinositide kinases as indicated by wortmannin sensitivity.

Intracellular protein degradation and autophagy in isolated pancreatic acini of the rat.

Simultaneous investigation of protein degradation and autophagy of isolated exocrine pancreatic cells is carried out here for the first time in a systematic way by a complex biochemical, morphological and morphometrical approach. Protein degradation proceeds with a decreasing rate of 4-1.5 per cent per h over a 4-h period indicating a comparatively low degradation capacity. Cells in freshly isolated acini do not contain autophagic vacuoles but the latter appear within an hour in vitro and their quantity remains close to a steady state during the subsequent 3 h. Both traditional inhibitors of the autophagic-lysosomal pathway, e.g. vinblastine, leupeptin, and lysosomotropic amines together with the recently introduced 3-methyladenine, inhibit degradation to a similar maximal extent, offering the possibility of the estimation of the ratio of lysosomal/non-lysosomal degradation. In pancreatic acinar cells autophagic sequestration is unaffected and protein degradation is inhibited inside secondary lysosomes by leupeptin and lysosomotropic amines, while 3-methyladenine prevents the formation of autophagosomes. Vinblastine seems to act by inhibiting the fusion of autophagosomes with lysosomes and there is no evidence for the stimulation of autophagic sequestration by vinblastine in the present system. The effect of inhibitors of protein breakdown on protein synthesis is variable and does not correlate with their influence on degradation. Amino acids strongly stimulate protein synthesis, but in contrast to what is found in liver cells, they do not seem to affect protein degradation or autophagy significantly, thus indicating major regulatory differences of these processes between pancreatic acinar cells and hepatocytes.

Carrier design: new generation of polycationic branched polypeptides containing OH groups with prolonged blood survival and diminished in vitro cytotoxicity.

For the construction of macromolecule-drug conjugates, it is important to provide rational basis to the selection of proper carrier. With respect to the importance of the side-chain structure and charge of the branched polypeptides in biological properties, we have prepared a new class of branched polypeptides with single or multiple hydroxyl groups and studied their solution conformation, in vitro cytotoxicity, biodistribution, and immunoreactivity. For comparative studies, polypeptides were designed to contain serine at various positions of the side chains, varying also the number. Ser was attached to the end of oligo(DL-Ala) side chains grafted to polylysine resulting polypeptides with the general formula poly[Lys(Ser(i)-DL-Ala(m))], (SAK). Ser was also coupled directly to the polylysine backbone poly[Lys(Ser(i))] (S(i)K) and then elongated by polymerization of N-carboxy-DL-Ala anhydride resulting poly[Lys(DL-Ala(m)-Ser(i))] (ASK). An additional polymer was also prepared, but instead of the oligo(DL-Ala) branches, oligo(DL-Ser) side chains were introduced (poly[Lys(DL-Ser(m))], SK). The presence of hydroxyl groups resulted in compounds with improved of water solubility. CD spectra of polypeptides showed significant differences correlating with the position and numbers of Ser residues in the side chains. Under physiological conditions, polycationic polypeptides assumed ordered secondary structure (S(i)K and LSK) or partially unordered conformation (SK, SAK, and ASK). Data of selected polymers demonstrate that these polycationic compounds are essentially nontoxic in vitro on normal rat liver or mouse spleen cells and have no cytostatic effect on mouse colorectal carcinoma C26 cells. The blood clearance and biodistribution of these derivatives were greatly dependent on the position and number of Ser residues in the branches and possess a rather extended blood survival in mice. Polypeptides were taken up predominantly by the liver and kidney (S(i)K, LSK, and ASK) or kidney and lung (SK and SAK). The best survival in the blood was found with SAK, representing the first polycationic branched polypeptide, which show extended blood clearance. The relative position of Ser residue had also a marked influence on the immunogenicity of polypeptides. The characteristics of the antibody response to polypeptide containing Ser at the end of the branches (SAK) or adjacent to the polylysine backbone (ASK) was also dependent on the genetic background of the mouse strains. We also found that these compounds have no effect on to the SRBC-specific humoral immune response, indicating the lack of nonspecific immunostimulatory potential. In conclusion, these studies suggest that synthetic branched polypeptides with Ser can be considered as candidates for constructing suitable conjugates for drug/epitope delivery. It is not only due to the presence of hydroxyl group to be used for oxime chemistry but also to their beneficial biological features.

Complete dissociation of rat pancreas into acini by a perfusion-incubation method: measurement of protein synthesis and the degradation of endogenous proteins.

A new method has been developed for the dissociation of rat pancreas into acinar suspension by introducing an in situ flow-through perfusion with a Ca(2+)-chelating buffer before in vitro enzymatic digestion with collagenase. As a result of the perfusion step and other minor modifications, a high-quality and uniform suspension of acini and small acinar complexes is obtained that offers the possibility to work with 50 parallel samples from a single rat for protein synthesis and degradation measurements. Initial cellular viability is 95-99% and remains > 85% even after 6 h in vitro. Electron-microscopic observations show that the cells in isolated acini retain their polarity, tight junctions remain intact, and desmosomes and basement membranes disappear. Protein synthesis shows strong dependence on the extracellular supply of amino acids and 30-50% stimulation by insulin. With the help of the new system of isolated acini, data on the degradation of intracellular proteins of the exocrine pancreatic tissue is presented for the first time in the literature. After a 2-h labeling period with [14C]valine, 2-4% of the radioactive protein is degraded per hour. Of this protein breakdown, 30-40% appears to take place in lysosomes, as measured in the presence of leupeptin, an inhibitor of lysosomal degradation.

Inhibition of hepatocytic autophagy by adenosine, adenosine analogs and AMP.

Autophagy, measured in isolated rat hepatocytes as the sequestration of electroinjected [3H]raffinose, was moderately (17%) inhibited by adenosine (0.4 mM) alone, but more strongly (85%) in the presence of the adenosine deaminase inhibitor, 2'-deoxycoformycin (50 microM), suggesting that metabolic deamination of adenosine limited its inhibitory effectiveness. The adenosine analogs, 6-methylmercaptopurine riboside and N6,N6-dimethyladenosine, inhibited autophagy by 89% and 99%, respectively, at 0.5 mM, probably reflecting the adenosine deaminase-resistance of their 6-substitutions. 5-Iodotubercidin (10 microM), an adenosine kinase inhibitor, blocked the conversion of adenosine to AMP and largely abolished the inhibitory effects of both adenosine and its analogs, indicating that AMP/nucleotide formation was required for inhibition of autophagy. Inhibition by adenosine of autophagic protein degradation, measured as the release of [14C]valine from prelabelled protein, was similarly potentiated by deoxycoformycin and prevented by iodotubercidin. Inhibition of autophagy by added AMP, ADP or ATP (0.3-1 mM) was, likewise, potentiated by deoxycoformycin and prevented by iodotubercidin, suggesting dephosphorylation to adenosine and intracellular re-phosphorylation to AMP. Suppression of autophagy by AMP may be regarded as a feedback inhibition of autophagic RNA degradation, or as an aspect of the general down-regulation of energy-requiring processes that occurs under conditions of ATP depletion, when AMP levels are high.

Time course of vinblastine-induced autophagocytosis and changes in the endoplasmic reticulum in murine pancreatic acinar cells: a morphometric and biochemical study.

The time course of the vinblastine(-sulfate; 10 mg/kg body weight, single injection)-induced enlargement and subsequent regression of the autolysosomal compartment was studied by electron microscopic morphometrical and cell biochemical methods in order to gain information concerning some key problems of this major route of intralysosomal degradation of the cell's endogenous macromolecules and structures. Detailed analysis of the dynamics of the total autophagic vacuole (AV) compartment and its different subcompartments (early, advanced, late, and fused AVs), as well as of changes of rough-surfaced endoplasmic reticulum (RER) showed: 1. Pancreatic acinar cells react to vinblastine biphasically, i.e. two expansion phases of the AV compartment, the first in the 0 to 90 min and the second in the 2 to 8 h post-injectional periods, were detected. 2. Fusions of AVs are not inhibited by vinblastine, at least during the second expansion phase when cytoplasmic volume fraction (CVF) of fused AVs steadily increased until the 12th h. Fusion of early, advanced and late AVs or composition of fused complex vacuoles (AVc) are somehow regulated, as the proportion of the three AV stages from the CVF of AVc, was maintained constant throughout the second expansion phase. 3. Stimulation of autophagosome formation and resulting substrate overload seems to be the primary mode of action by which vinblastine causes the enormous expansion of the autolysosomal compartment. 4. Degranulation of the rough-surfaced endoplasmic reticulum (RER) membranes occurs in a biphasic fashion, similarly to the volume and surface changes of the AV compartment, thus supporting our previous hypothesis, that labilization or change of RER may have a role in the formation of autophagosomes. 5. Vinblastine-induced autophagocytosis is a selective process, as mitochondria, Golgi elements and zymogen granules are very much underrepresented, whereas RER is more than twice overrepresented in the volume of early AVs, when compared to their volume fraction in the whole cytoplasm. 6. Immunogold electron microscopy revealed the presence of ubiquitinylated proteins in advanced and late, but not in early AVs.

X-irradiation-induced changes of the prelysosomal and lysosomal compartments and proteolysis in HT-29 cells.

As a consequence of external and internal ionizing radiation, lysosome-like bodies have been observed to increase both in size and number in some cell types. We investigated this process by morphological methods (electron microscopy, cationized ferritin uptake, acid phosphatase histochemistry, morphometry) in cultured HT-29 cells. In parallel with these studies, we measured the rate of protein degradation on the basis of 14C-valine release from prelabeled cellular proteins. We found that at 2 and 4 Gy doses of X-irradiation the volume of the vacuolar (probably lysosomal) compartment increased without detectable changes of acid phosphatase activity. A 2 Gy irradiation dose did not change protein degradation rate. However, 4 Gy caused a significant inhibition of 14C-valine release from prelabeled proteins. Our results indicate, that the radiation induced expansion of the lysosomal compartment is not necessarily accompanied by increased lytic activity of HT-29 cells.

Mechanism and dynamics of macroautophagy in murine exocrine pancreatic cells. A review of vinblastine-induced changes.

Autophagy is a major pathway of lysosomal degradation of cellular macromolecules. The paper summarizes the results obtained in the studies on macroautophagy using the exocrine pancreatic acinar cell as model system and vinblastine as inducer. Current knowledge about the origin and properties of the limiting membranes of autophagic vacuoles, and the results of quantitative morphological studies into the dynamics and kinetics of vinblastine-induced autophagocytosis, as well as recent achievements in isolation and characterization of subclasses of autophagic vacuoles (autophagosomes and autolysosomes) are reviewed.

Time course of quantitative morphological changes of the autophagic-lysosomal compartment of murine seminal vesicle epithelial cells under the influence of vinblastine.

Changes of the autophagic-lysosomal compartment (ALC) of the murine seminal vesicle epithelial cells were monitored by electron microscopic morphometry during 36 h following a single 10 mg/kg bw dose of vinblastine sulfate (VBL), a widely used tool to cause an accumulation of the autophagic vacuoles (AVs). Three morphologically distinct subcompartments of the ALC, i.e. early autophagic vacuoles (AV1) being presumably prelysosomal autophagosomes, advanced AVs (AV2) containing material under degradation and dense bodies (DB) were defined. The ALC and its subcompartments expanded after VBL in a two-phase reaction. The first subcompartments to react significantly were AV1 and AV2 (at 30 min) followed by DBs with a 30 min delay. The ALC then ceased to grow until the 90th min when a second expansion phase started peaking around 8 h with a cytoplasmic volume fraction 15 times larger than in the untreated control. This second growth was entirely brought about by the expansion of the two AV subcompartments. After 8 h the volume fraction of both AV1 and AV2 decreased to cause a gradual regression of the ALC. AV1, however, already ceased to expand as early as after 6 h, i.e. during the last 2 h of the expansion phase of the ALC. Comparison of this time curve with the one we previously measured in mouse liver shows considerable differences between the two cell types. The growth curves of the AV subcompartments in our experiment along with others' kinetic data obtained in steady state cells not treated with VBL show that segregation (= formation of AV1) is possibly stimulated by VBL in our system.

Time course of the quantitative changes in the autophagic-lysosomal and secretory granule compartments of murine liver cells under the influence of vinblastine.

The dynamics of the transient expansion of the autophagic-lysosomal (ALC) and secretory granule (SGC) compartments in mouse liver cells were monitored by electron microscopic morphometry after a single injection of 10 mg/kg b.w. vinblastine sulfate (VBL). Initially (first phase) the cytoplasmic volume fractions of the total ALC and its subcompartments, as well as of the SGC increased by an order of magnitude and peaked at the second h. In the second phase, all the aforementioned compartments regressed gradually, approaching their normal size between 12 and 36 h after VBL injection. Analysis of the dynamic changes in fractional volumes of subcompartments of the ALC showed that early autophagic vacuoles (AV1) were the first to enlarge. Advanced AVs (AV2) reacted 30 min later and a further 30 min time lag was required before late autolysosomes, appearing as dense bodies (DB), started to expand. We regard these data as kinetic proof that the bodies of the later reacting subcompartments developed from the earlier reacting ones. The time lag between the expansion of AV1 and AV2 subcompartments may be explained by a period of retardation of conversion of nascent autophagosomes (AV1) to autolysosomes (AV2) which is known to occur normally by fusion of AV1 with enzyme-carrying lysosomes. However, transformation of AV1 to AV2 and later to DB resumed after the respective time lags. Moreover, our quantitative data lend support to the view that segregation of cytoplasmic portions into newly-formed autophagosomes was stimulated by VBL, at least in the first 2 h of treatment. The expansion of ALC accelerated during this period and led to an obvious overload of the lysosomal apparatus.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental characterization of the autophagic-lysosomal pathway in isolated rat hepatocytes.

When isolated rat hepatocytes are incubated under amino-acid-free conditions autophagy is maximally activated and overall lysosomal proteolysis is greatly enhanced. To facilitate this, cytoplasmic material is sequestered by autophagic membranes, and the resulting autophagosomes enter the lysosomes by a fusion process to have their contents degraded. The various steps in the autophagic-lysosomal pathway have been investigated by utilizing reversible electropermeabilization to introduce radioactive sugar probes into the hepatocyte cytosol. The inert disaccharide sucrose and the inert trisaccharide raffinose have been used as sequestration probes to study the sequestrational step of autophagy, while the hydrolysable disaccharide lactose has been used to study the fusion and intralysosomal hydrolysis steps. Sucrose and lactose have been used to map the extent of convergence between the autophagic and endocytic (heterophagic) pathways. Sequestration of native cytosolic enzymes has been measured to investigate the question of selectivity in the autophagic process. Finally, the effect of insulin on the sequestrational step has been evaluated.

Combined effects of fasting and vinblastine treatment on serum insulin level, the size of autophagic-lysosomal compartment, protein content and lysosomal enzyme activities of liver and exocrine pancreatic cells of the mouse.

1. The volume fraction of autophagic vacuoles in liver parenchymal and exocrine pancreatic cells was smallest and the serum insulin level highest in the 24 hr prestarved mouse immediately after 3 hr feeding period. 2. The size of the autophagic vacuole and lysosome (dense body) compartments increased in both types of cells during 2-72 hr fasting parallel with decreasing serum insulin levels. 3. The protein content of the cells decreased and the DNA-based activity of acid phosphatase showed little change throughout fasting. The activity of cathepsin D increased during days 2 and 3 of food deprivation. 4. Vinblastine (50 mg/kg body wt) applied for the last 2 hr of different periods (2, 12, 24, 48 and 72 hr) of fasting decreased serum insulin level and increased the fractional cytoplasmic volume of autophagic vacuoles and dense bodies. This increase was smaller when the drug was applied shortly after feeding and much larger after prolonged fasting. The increase was more pronounced in the pancreatic than in the liver cells. 5. Our data show that the effect of vinblastine on the size of the autophagic-lysosomal compartment depends on the feeding status of the animals.

Regression of autophagic vacuoles in pancreatic acinar, seminal vesicle epithelial, and liver parenchymal cells: a comparative morphometric study of the effect of vinblastine and leupeptin followed by cycloheximide treatment.

Treatment of mice with both leupeptin (0.06 mg/g body wt) and vinblastine (0.05 mg/g body wt) for 2 h caused a many-fold enlargement of the autophagic-lysosomal compartment of pancreatic acinar, seminal vesicle epithelial, and liver parenchymal cells. In all three types of cells a predominance of large, dense bodies was seen after leupeptin treatment and that of typical autophagic vacuoles were seen after vinblastine treatment. An exponential decrease of the volume fraction of autophagic vacuoles was observed in leupeptin-treated cells after the administration of cycloheximide (0.2 mg/g body wt). The half-life of autophagic vacuoles estimated from the decay curve was 5.3, 5.7, and 6.6 min for pancreatic, seminal vesicle, and liver cells, respectively. Our data suggest that sequestered cytoplasmic material rapidly enters the lysosomes in leupeptin-treated cells and accumulates in this compartment. In contrast, no regression of the autophagic vacuole compartment of pancreatic and seminal vesicle cells was observed after the administration of cycloheximide to animals pretreated with vinblastine, and only a slight decrease was seen in liver cells. These observations show that the lifetime of autophagic vacuoles is prolonged by vinblastine resulting in their accumulation in the cells. However, our measurements also lend support to the view that in addition to the accumulatory effect on undegraded cytoplasmic material, stimulation of sequestration may play a role in the enlargement of the autophagic lysosomal compartment after treatment with leupeptin as well as with vinblastine in all three types of cells investigated.

Temperature dependence of protein degradation, autophagic sequestration and mitochondrial sugar uptake in rat hepatocytes.

Lysosomal (propylamine-sensitive) protein degradation as well as the energy-dependent (chymostatin-sensitive) part of the non-lysosomal protein degradation was found to be strongly affected by temperature in isolated rat hepatocytes, the activation energy (Ea) being about 25 kcal/mol for both processes. In contrast, the energy-independent (chymostatin-resistant) part of the non-lysosomal degradation had an Ea of approx. 10 kcal/mol only. Sequestration of electroinjected [14C]sucrose into sedimentable organelles showed a pronounced temperature dependence. By means of digitonin extraction it was possible to distinguish between a moderately temperature-sensitive mitochondrial sugar uptake (Ea approx. 12 kcal/mol) and a strongly temperature-dependent autophagic sequestration (Ea approx. 22 kcal/mol). There was no significant autophagic sequestration below 20 degrees C. The sequestration process is more temperature-sensitive than, for example, the early steps of endocytosis, and is likely to represent the major controlling step in the overall autophagic-lysosomal pathway.

Morphometric evaluation of the turnover of autophagic vacuoles after treatment with Triton X-100 and vinblastine in murine pancreatic acinar and seminal vesicle epithelial cells.

Large numbers of autophagic vacuoles were found in murine pancreatic acinar and seminal vesicle epithelial cells following the administration of Triton X-100 or vinblastine for 4 h. The autophagic vacuoles disappeared rapidly from the cells after the administration of cycloheximide to animals pretreated with Triton X-100. The decay in seminal vesicle cells appeared to follow first-order kinetics with an estimated t1/2 of 8.7 min. The regression in pancreatic cells was equally rapid and less than half the initial volume of autophagic vacuoles was found at the 12th min after cycloheximide injection. This time, the decay curve appeared to be linear rather than exponential. Our data, together with the work of others, support the view that the average half-life of autophagic vacuoles is a fairly constant parameter kept within the range of 6-9 min in various types of mouse and rat cell when the late steps of autophagocytosis (i.e. the fusion of autophagosomes and lysosomes and the degradation within lysosomes) are not affected. The regression of autophagic vacuoles was slow in mice pretreated with vinblastine (t1/2 of about 27-30 min) suggesting that this drug slows down the turnover of autophagic vacuoles. Morphometric evaluation of the regression of the autophagic vacuole compartment after cycloheximide treatment can be used as a tool to distinguish between treatments which elevate the amount of autophagic vacuoles within the cells by increasing the rate of sequestration from those which expand the autophagic vacuole compartment by causing accumulation of autophagic vacuoles as a result of blockade of the late steps of the autophagic process.

Immunomodulatory effect of synthetic branched polypeptides. II.

A comparative investigation of various polypeptides was carried out in order to elucidate structure-activity correlations. The immunoadjuvant properties of the chemically well-characterized branched polypeptides, poly[Lys-(DL-Alam)] (Lys:Ala = 1:2.95) (AK), poly[Lys-(D-Leui-DL-Alam)] (Lys:Ala:Leu = 1:3.0:0.95) (D-LAK), and poly[Lys-Hisi-DL-Alam)] (Lys:Ala:His = 1:2.95:0.85) (HAK), were investigated. D-LAK and HAK were able to augment the antibody response of BDF inbred mice to immunization with sheep red blood cells (SRBC), as assessed by the hemolytic plaque-forming cell assay, whereas AK had no similar effect. The stimulating effect of D-LAK and HAK was dependent on dose and timing of treatment relative to SRBC immunization. However, the optimal dose levels were lower and the effective dose interval more restricted as compared to the previously described poly[Lys-(Leui-DL-Alam)] (LAK). Like LAK, both HAK and D-LAK were able to compensate for the immunosuppressive effect of the cytotoxic drugs dianhydrogalactitol, vincristine, and 5-fluorouracil, which all have different mechanisms of action, provided that combined treatment by polypeptide and drug was applied repeatedly before the SRBC immunization.

The turnover of autophagic vacuoles: evaluation by quantitative electron microscopy.

Accumulation of autophagic vacuoles was induced in mouse liver, exocrine pancreas and seminal vesicle cells by intraperitoneal injection of vinblastine, leupeptin, Triton X100 or estron acetate and the turnover of the autophagic vacuole content was estimated by morphometry from the time course of regression of autophagic vacuoles following the administration of cycloheximide to the animals. The volume fractions of autophagic vacuoles increased manifold following the administration of any of the drugs in each cell type investigated. The autophagic vacuoles regressed rapidly after administration of cycloheximide to the animals pretreated with Triton X100, estron acetate and leupeptin and the decay appeared to follow first-order kinetics; the estimated half life of autophagic vacuoles was about 6-9 min. Regression of autophagic vacuoles was very slow (apparent half life 30 min or longer) in mice, pretreated with vinblastine, indicating the inhibitory action of this drug on the turnover of autophagic vacuoles in vivo.

Effect of amino acids and cycloheximide on changes caused by vinblastine, leupeptin and methylamine in the autophagic/lysosomal system of mouse hepatocytes in vivo.

The number of autophagic vacuoles in hepatocytes of 24 h fasted mice in vivo increased manyfold following the administration of vinblastine, leupeptin and methylamine. The effect of each chemical is characterized by the predominance of a certain kind of vacuole. Vinblastine treatment is accompanied by a large proportion of vacuoles containing morphologically unaltered organelles, leupeptin causes preferential accumulation of dense and complex vacuoles, methylamine administration produces mostly large, electron-lucent, swollen vacuoles. The amounts of segregated and accumulated cytoplasmic material, expressed as percentage cytoplasm per hour, were 0.84%, 2.08% and 0.74% following vinblastine, leupeptin and methylamine treatment respectively. The actual rate of segregation was probably higher than this. Inhibition of degradation of the sequestered cytoplasmic material is proposed to be a main factor in the increase in the size of the autophagic/lysosomal compartment. Treatment with cycloheximide or exogenously added mixture of amino acids cut down the size of the autophagic/lysosomal system in control cells and strongly inhibited the accumulation caused by vinblastine, leupeptin and methylamine.