Uptake of epidermal growth factor into a lysosomal enzyme-deficient organelle: correlation with cell's mitogenic response and evidence for ubiquitous existence in fibroblasts

The internalization process following cell surface receptor binding by epidermal growth factor (EGF) was studied. It was found that EGF is taken up into a dense, membranous organelle. This organelle is deficient in lysosomal enzyme activity and is biochemically dissimilar to the major lysosomal fraction. The uptake of EGF by this organelle was demonstrated in three different cell types representing three different species. Each of these cell types is highly responsive to the mitogenic action of EGF. These results indicate that EGF is endocytosed and delivered to a dense, possibly nonlysosomal, organelle which is ubiquitous in fibroblasts. Furthermore, we demonstrate a close, positive correlation between EGF uptake into this fraction and the ability of cells to respond to the mitogen. A negative relationship between uptake into the subcellular fraction containing lysosomal enzymes and EGF-stimulated DNA synthesis was observed. Using numerous incubation conditions no exceptions to the correlation between EFG uptake into the lysosomal enzyme-deficient fraction and EGF-induced DNA synthesis were observed. These results suggest a role for this dense organelle in the production of a mitogenic signal.     

Ultrastructural enzyme-cytochemical study of the intrinsic glandular cells in the corpus cardiacum of Locusta migratoria: relation to the secretory and endocytotic pathways,and to the lysosomal system

Summary Ultrastructural aspects of the secretory and the endocytotic pathways and the lysosomal system of corpus cardiacum glandular cells (CCG cells) of migratory locusts were studied using morphological, marker enzyme, immunocytochemical and tracer techniques. It is concluded that (1) the distribution of marker enzymes of trans Golgi cisternae and trans Golgi network (TGN) in locust CCG cells corresponds to that in most non-stimulated vertebrate secretory cell types; (2) the acid phosphatase-positive TGN in CCG cells is involved in sorting and packaging of secretory material and lysosomal enzymes; (3) these latter substances are produced continuously; (4) at the same time, superfluous secretory granules and other old cell organelles are degraded; (5) the remarkable endocytotic activity in the cell bodies and the minor endocytotic activity in cell processes are coupled mainly to constitutive uptake of nutritional and/or regulatory (macro)molecules, rather than to exocytosis; (6) plasma membrane recycling occurs mainly by direct fusion of tubular endosomal structures with the plasma membrane and little traffic passes the Golgi/TGN; and (7) so-called cytosomes arise mainly from autophagocytotic vacuoles and represent a special kind of complex secondary lysosomes involved in the final degradation of endogenous (cell organelles) and exogenous material.     

Apical and basolateral 4F2hc and the amino acid exchange of L-DOPA in renal LLC-PK1 cells

Summary. The present study aimed to examine the presence and define the role of 4F2hc, a glycoprotein associated with the LAT2 amino acid transporter, in L-DOPA handling by LLC-PK₁ cells. For this purpose we have measured the activity of the apical and basolateral inward and outward transport of [¹⁴C] L-DOPA in cell monolayers and examined the influence of 4F2hc antisense oligonucleotides on [¹⁴C] L-DOPA handling. The basal-to-apical transepithelial flux of [¹⁴C] L-DOPA progressively increased with incubation time and was similar to the apical-to-basal transepithelial flux. The spontaneous and the L-DOPA-stimulated apical fractional outflow of [¹⁴C] L-DOPA were identical to that through the basal cell side. The L-DOPA-induced fractional outflow of [¹⁴C] L-DOPA through the apical or basal cell side was accompanied by marked decreases in intracellular levels of [¹⁴C] L-DOPA. In cells treated with an antisense oligonucleotide complementary to 4F2hc mRNA for 72 h, [¹⁴C] L-DOPA inward transport and 4F2hc expression were markedly reduced. Treatment with the 4F2hc antisense oligonucleotide markedly decreased the spontaneous fractional outflow of [¹⁴C] L-DOPA through the apical or the basal cell side. It is likely that the Na⁺-independent and pH-sensitive uptake of L-DOPA include the hetero amino acid exchanger LAT2/4F2hc, which facilitates the trans-stimulation of L-DOPA and its outward transfer at both the apical and basal cell sides.     

Germination growth response of different plant species to the allelochemical L-3,4-dihydroxyphenylalanine (L-DOPA)

The aim of this study was to investigate the seed germination response of different plant families to L-3,4-dihydroxyphenylalanine (L-DOPA), one of the strongest allelochemicals in nature. Three types of responses in terms of colouration changes on filter paper were obtained; black and gray (Gramineae and Compositae), no change (Leguminosae, Brassicaceae, and Cucurbitaceae) and an obstructed-circle around the seeds with black colouration on the outer side of the circle (Hydrophyllaceae) when L-DOPA solution was applied during seed germination. Radicle growth in the Gramineae and Leguminosae families was inhibited less by a single treatment of L-DOPA solution (250 g/ml) than in the other families. However, continuous treatment with L-DOPA demonstrated that the Gramineae family was less affected in terms of the inhibition of radicle growth than the Leguminosae family. When more seeds were added to the L-DOPA solution less inhibition of radicle growth was observed in all plants tested. The EC₅₀ of L-DOPA for bluebell (Hydrophyllaceae), white clover (Leguminosae), and lettuce (Compositae) was approximately 200, 100, and 50 g/ml, respectively. However, in perennial ryegrass (Gramineae) no EC₅₀ was observed even at 250 g/ml L-DOPA. In the Gramineae family, addition of more seeds into the L-DOPA solution increased the colouration on the filter paper. These results demonstrated that each seed functions to oxidize or dissolve L-DOPA. In the Gramineae, Leguminosae, Compositae, and Hydrophyllaceae, increasing the number of seeds imbibed in the L-DOPA solution increased the rate of L-DOPA disappearance from the petri-dish. Of the Grammaceous plants tested, only perennial ryegrass, which showed fairly weak allelopathic activity, metabolised L-DOPA to dopamine. Although the relationships between the changes in colouration of the filter paper and the inhibition of radicle growth in these experiments are still unknown, there appears to be a strong response in each species to protect the cell from L-DOPA damage.     

Adeno-associated virus-mediated gene transfer of human aromatic L-amino acid decarboxylase protects mixed striatal primary cultures from L-DOPA toxicity

Although L-DOPA is the drug of choice for Parkinson's disease, prolonged L-DOPA therapy results in decreased drug effectiveness and the appearance of motor complications. This may be due in part to the progressive loss of the enzyme, aromatic L-amino acid decarboxylase (AADC). We have developed an adeno-associated virus vector (AAV-hAADC) that contains human AADC cDNA under the control of the cytomegalovirus promoter. Infusion of this vector into the striatum of parkinsonian rats and monkeys improves L-DOPA responsiveness by improving AADC-mediated conversion of L-DOPA to dopamine. This is now the basis of a proposed therapy for advanced Parkinson's disease. A key concern has been that over-production of dopamine in striatal neurons could cause dopamine toxicity. To investigate this possibility in a controlled system, mixed striatal primary rat neuronal cultures were prepared. Exposure of cultures to high concentrations of L-DOPA induced the following changes: cell death in nigral and striatal neurons, aggregation of neurofilaments and focal axonal swellings, abnormal expression of DARPP-32, and activation of astroglia and microglial cells. Transduction of cultures with AAV-hAADC resulted in efficient and sustained neuronal expression of the AADC protein and prevented all the L-DOPA-induced toxicities. The protective effects were due primarily to AADC-dependent conversion of L-DOPA to dopamine and an increase in induction of vesicular monoamine transporter resulting in dopamine storage in cultured cells. These results suggest a neuroprotective role for AADC gene transfer against L-DOPA toxicity.     

The locus coeruleus is directly implicated in L-DOPA-induced dyskinesia in parkinsonian rats: an electrophysiological and behavioural study

Despite being the most effective treatment for Parkinson's disease, L-DOPA causes a development of dyskinetic movements in the majority of treated patients. L-DOPA-induced dyskinesia is attributed to a dysregulated dopamine transmission within the basal ganglia, but serotonergic and noradrenergic systems are believed to play an important modulatory role. In this study, we have addressed the role of the locus coeruleus nucleus (LC) in a rat model of L-DOPA-induced dyskinesia. Single-unit extracellular recordings in vivo and behavioural and immunohistochemical approaches were applied in rats rendered dyskinetic by the destruction of the nigrostriatal dopamine neurons followed by chronic treatment with L-DOPA. The results showed that L-DOPA treatment reversed the change induced by 6-hydroxydopamine lesions on LC neuronal activity. The severity of the abnormal involuntary movements induced by L-DOPA correlated with the basal firing parameters of LC neuronal activity. Systemic administration of the LC-selective noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine did not modify axial, limb, and orolingual dyskinesia, whereas chemical destruction of the LC with ibotenic acid significantly increased the abnormal involuntary movement scores. These results are the first to demonstrate altered LC neuronal activity in 6-OHDA lesioned rats treated with L-DOPA, and indicate that an intact noradrenergic system may limit the severity of this movement disorder.     

Effects of insulin on cardiac lysosomes and protein degradation

Hearts perfused in the absence of added insulin had 1) accelerated rates of protein degradation, as assessed by release of phenylalanine and tyrosine; 2) increased rates of release of seven other amino acids; 3) decreased lysosomal latency and sedimentable lysosomal enzyme activity; 4) increased numbers of autophagic vacuoles in cardiac muscle cells; and 5) decreased activity of beta-N-acetylglucosaminidase in dense lysosomes (1.06-1.09 g/ml), as compared to hearts perfused in the presence of the hormone. After 3 h of perfusion in the absence of insulin, the changes that developed in protein degradation, lysosomal latency, and sedimentability, and in enzyme activity in dense lysosomes, were reversed by insulin addition during 90 min of subsequent perfusion. These studies suggest a role for insulin in controlling the activity of the lysosomal system and the involvement of this system in protein degradation, particularly in insulin-deprived tissue.     

Cathepsin E deficiency induces a novel form of lysosomal storage disorder showing the accumulation of lysosomal membrane sialoglycoproteins and the elevation of lysosomal pH in macrophages

Cathepsin E, an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system, has an important role in immune responses. However, little is known about the precise roles of cathepsin E in this system. Here we report that cathepsin E deficiency (CatE(-/-)) leads to a novel form of lysosome storage disorder in macrophages, exhibiting the accumulation of the two major lysosomal membrane sialoglycoproteins LAMP-1 and LAMP-2 and the elevation of lysosomal pH. These striking features were also found in wild-type macrophages treated with pepstatin A and Ascaris inhibitor. Whereas there were no obvious differences in their expression, biosynthesis, and trafficking between wild-type and CatE(-/-) macrophages, the degradation rates of these two membrane proteins were apparently decreased as a result of cathepsin E deficiency. Because there was no difference in the vacuolar-type H(+)-ATPase activity in both cell types, the elevated lysosomal pH in CatE(-/-) macrophages is most likely due to the accumulation of these lysosomal membrane glycoproteins highly modified with acidic monosaccharides, thereby leading to the disruption of non-proton factors controlling lysosomal pH. Furthermore, the selective degradation of LAMP-1 and LAMP-2, as well as LIMP-2, was also observed by treatment of the lysosomal membrane fraction isolated from wild-type macrophages with purified cathepsin E at pH 5. Our results thus suggest that cathepsin E is important for preventing the accumulation of these lysosomal membrane sialoglycoproteins that can induce a new form of lysosomal storage disorder.     

The Role of the Subthalamic Nucleus in L-DOPA Induced Dyskinesia in 6-Hydroxydopamine Lesioned Rats

L-DOPA is the most effective treatment for Parkinson's disease (PD), but prolonged use leads to disabling motor complications including dyskinesia. Strong evidence supports a role of the subthalamic nucleus (STN) in the pathophysiology of PD whereas its role in dyskinesia is a matter of controversy. Here, we investigated the involvement of STN in dyskinesia, using single-unit extracellular recording, behavioural and molecular approaches in hemi-parkinsonian rats rendered dyskinetic by chronic L-DOPA administration. Our results show that chronic L-DOPA treatment does not modify the abnormal STN activity induced by the 6-hydroxydopamine lesion of the nigrostriatal pathway in this model. Likewise, we observed a loss of STN responsiveness to a single L-DOPA dose both in lesioned and sham animals that received daily L-DOPA treatment. We did not find any correlation between the abnormal involuntary movement (AIM) scores and the electrophysiological parameters of STN neurons recorded 24 h or 20-120 min after the last L-DOPA injection, except for the axial subscores. Nonetheless, unilateral chemical ablation of the STN with ibotenic acid resulted in a reduction in global AIM scores and peak-severity of dyskinesia. In addition, STN lesion decreased the anti-dyskinetogenic effect of buspirone in a reciprocal manner. Striatal protein expression was altered in dyskinetic animals with increases in ΔFosB, phosphoDARPP-32, dopamine receptor (DR) D3 and DRD2/DRD1 ratio. The STN lesion attenuated the striatal molecular changes and normalized the DRD2/DRD1 ratio. Taken together, our results show that the STN plays a role, if modest, in the physiopathology of dyskinesias.     

Melanin granules prevent the cytotoxic effects of L-DOPA on retinal pigment epithelial cells in vitro by regulation of NO and superoxide radicals

Inasmuch as the nitrogen cycle elicits the direct reduction of N2 to NH3 through enzymatic reactions, and inasmuch as L-DOPA (L-dihydroxyphentlalamine), a catecholamine, can be a source of nitric oxide (NO), it is possible that melanin granules in the eye affect the generation of NO, which causes damage to the retinal pigment epithelial (RPE) cells during the oxidation of L-DOPA. In order to confirm this possibility, we analyzed the correlations of NO generation, cell growth, and superoxide dismutase (SOD) activities in two types (melanotic and amelanotic) of bovine RPE cells following exposure to L-DOPA. NO generation from L-DOPA was determined using an NO detector that is reliant on redox currents. The concentration of NO was measured in terms of diffusion currents run between a working electrode and a counter electrode, both being set in culture medium placed in a Petri dish. For the assays, L-DOPA was added to the medium at various concentrations (5, 29.9, 79.4, 152.7 or 249 microM), and 6 min after addition, an NO-trapping agent 2,4-carboxyphenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (carboxy-PTIO) was also added. The melanotic and amelanotic types of RPE cells were cultured separately in medium with L-DOPA under an atmosphere containing 20, 10 or 5% oxygen. Cell numbers were counted using a Coulter counter, and SOD activities were determined following incubation for 24, 48 or 72 hr using a modification of the luminol assay. The results obtained indicated that: (a) NO was produced from L-DOPA in a concentration-dependent manner and was trapped quantitatively by carboxy-PTIO; (b) the generation of NO was inhibited more markedly in the melanotic cell line than in the amelanotic one, suggesting an increased tolerance to L-DOPA-derived cytotoxicity in the former; and (c) the SOD activities were more affected by oxygen concentration in the melanotic cells than in the amelanotic ones. From these results, it is concluded that melanin granules in RPE cells have a role in preventing the cytotoxicity derived from L-DOPA and in regulating the generation of NO and superoxide radicals.     

Molecular mechanisms of pathogenesis in a glycosphingolipid and a glycoprotein storage disease

The lysosomal system comprises a specialized network of organelles crucial for the sorting, digestion, recycling and secretion of cellular components. With their content of hydrolytic enzymes, lysosomes regulate the degradation of a multitude of substrates that reach these organelles via the biosynthetic or the endocytic route. Gene defects that affect one or more of these hydrolases lead to LSDs (lysosomal storage diseases). This underscores the apparent lack of redundancy of these enzymes and the importance of the lysosomal system in cell and tissue homoeostasis. Some of the lysosomal enzymes may form multiprotein complexes, which usually work synergistically on substrates and, in this configuration, may respond more efficiently to changes in substrate load and composition. A well-characterized lysosomal multienzyme complex is the one comprising the glycosidases β-gal (β-galactosidase) and NEU1 (neuramidase-1), and of the serine carboxypeptidase PPCA (protective protein/cathepsin A). Three neurodegenerative LSDs are caused by either single or combined deficiency of these lysosomal enzymes. Sialidosis (NEU1 deficiency) and galactosialidosis (combined NEU1 and β-gal deficiency, secondary to a primary defect of PPCA) belong to the glycoprotein storage diseases, whereas GM1-gangliosidosis (β-gal deficiency) is a glycosphingolipid storage disease. Identification of novel molecular pathways that are deregulated because of loss of enzyme activity and/or accumulation of specific metabolites in various cell types has shed light on mechanisms of disease pathogenesis and may pave the way for future development of new therapies for these LSDs.     

Effect of L-DOPA on the Behavioral Activity of Wistar and Spontaneously Hypertensive (SHR) Rats in the Open-Field Test

We studied the effects of i.p. injections of L-DOPA (100 mg/kg) on the behavioral activity of Wistar rats and spontaneously hypertensive rats (SHR) in the open-field test, as well as on the content of catecholamines in the blood plasma of these animals. Prior to the administration of L-DOPA, the total level of locomotor/research activity in SHR was higher, as compared with that in Wistar rats. This was manifested in significantly greater values of the duration of moving of the animals in the center and on the periphery of the field and also in a greater number of rearings on the periphery of this field. At the same time, the episodes of grooming and sitting in Wistar rats were longer. After injections of L-DOPA, interstrain differences increased and became significant for most (9/10) indices of behavioral activity examined in our study. Injections of L-DOPA resulted in significant modifications of the behavioral activity of rats of the above strains, which is evidenced by changes in the number of visits to the peripheral squares of the open field and of rearings in the same field zones. Over repetitive test sessions, interstrain differences between most indices of behavioral activity (except the duration of research activity on the periphery and that of sitting) decreased. Injections of L-DOPA resulted in a significant increase in the content of this agent and dopamine in the blood plasma of rats of both strains; the level of noradrenaline in SHR also increased. The importance of a hereditary factor-determined increase in the activity of catecholaminergic brain systems (first of all, the dopaminergic system) in SHR for the specificity of locomotor behavior of these animals is discussed.     

Lysosomes in anterior pituitary corticotrophs of the rat. A combined immunocytochemical and enzyme cytochemical study

E600 resistant non-specific esterase activity or acid phosphatase activity were localized in corticotrophic cells identified by postembedding immunocytochemistry (PAP of protein A-immunogold techniques). The lysosomal system of this cell type consists of dense bodies, of a population of small lysosomes mostly situated at the cell periphery in the vicinity of secretory granules as well as of tubular structures. These latter were located either in the central part of the cytoplasm and probably belonged to the Golgi apparatus or at the cell periphery, partly in the extensions. Small lysosomes occurred to be in continuity with enzyme-containing tubules. In a few structures lysosomal enzyme activity and ACTH immunoreactivity overlapped. Some autophagic vacuoles seemed to contain secretory granule matrix. It is suggested that the concept of crinophagy can be extended to the corticotrophs, though the lysosomal system may be involved in the specific function of this cell type by other mechanisms as well.     

Fine structure of the mouse anterior pituitary maintained in a short-term incubation system

Summary Anterior pituitaries of mice were incubated for periods up to four hours in Krebs-Ringer bicarbonate glucose gassed with 95% O₂5% CO₂. The incubated explants survived and retained a fine structure that approximated the condition in situ. The few necrotic cells were sharply localized, and were found to be due to initial mechanical damage to the tissue.Some cells of the six granulated types exhibited slight but significant changes attributable to the liberation from the hypothalamic control: in LTH cells there was a release of preexisting granules and a development of cell organelles, whereas in other cell types there was an inhibition of release of granules and an enhanced digestion of the accumulated granules by the lysosomal system.Follicular cells responded uniquely to the changed environment by hypertrophy of the cytoplasm and were found to phagocytize cell debris. A part of non-epithelial elements of the gland showed a tendency to modulate cytologically.The author would like to express his appreciation to Mr. T. Anzai and Mr. S. Terada for their excellent technical assistance.     

Enhancing Effect of Manganese on L-DOPA-Induced Apoptosis in PC12 Cells

L-DOPA and manganese both induce oxidative stress-mediated apoptosis in catecholaminergic PC12 cells. In this study, exposure of PC12 cells to 0.2 mM MnCl2 or 10-20 microM L-DOPA neither affected cell viability, determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, nor induced apoptosis, tested by flow cytometry, fluorescence microscopy, and the TUNEL technique. L-DOPA (50 microM) induced decreases in both cell viability and apoptosis. When 0.2 mM MnCl2 was associated with 10, 20, or 50 microM L-DOPA, a concentration-dependent decrease in cell viability was observed. Apoptotic cell death also occurred. In addition, manganese inhibited L-DOPA effects on dopamine (DA) metabolism (i.e., increases in DA and its acidic metabolite levels in both cell lysate and incubation medium). The antioxidant N-acetyl-L-cysteine significantly inhibited decreases in cell viability, apoptosis, and changes in DA metabolism induced by the manganese association with L-DOPA. An increase in autoxidation of L-DOPA and of newly formed DA is suggested as a mechanism of manganese action. These data show that agents that induce oxidative stress-mediated apoptosis in catecholaminergic cells may act synergistically.     

Autophagy impairment with lysosomal and mitochondrial dysfunction is an important characteristic of oxidative stress-induced senescence

Macroautophagy/autophagy has profound implications for aging. However, the true features of autophagy in the progression of aging remain to be clarified. In the present study, we explored the status of autophagic flux during the development of cell senescence induced by oxidative stress. In this system, although autophagic structures increased, the degradation of SQSTM1/p62 protein, the yellow puncta of mRFP-GFP-LC3 fluorescence and the activity of lysosomal proteolytic enzymes all decreased in senescent cells, indicating impaired autophagic flux with lysosomal dysfunction. The influence of autophagy activity on senescence development was confirmed by both positive and negative autophagy modulators; and MTOR-dependent autophagy activators, rapamycin and PP242, efficiently suppressed cellular senescence through a mechanism relevant to restoring autophagic flux. By time-phased treatment of cells with the antioxidant N-acetylcysteine (NAC), the mitochondria uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and ambroxol, a reagent with the effect of enhancing lysosomal enzyme maturation, we found that mitochondrial dysfunction plays an initiating role, while lysosomal dysfunction is more directly responsible for autophagy impairment and senescence. Interestingly, the effect of rapamycin on autophagy flux is linked to its role in functional revitalization of both mitochondrial and lysosomal functions. Together, this study demonstrates that autophagy impairment is crucial for oxidative stress-induced cell senescence, thus restoring autophagy activity could be a promising way to retard senescence.     

L-DOPA is an endogenous ligand for OA1

Albinism is a genetic defect characterized by a loss of pigmentation. The neurosensory retina, which is not pigmented, exhibits pathologic changes secondary to the loss of pigmentation in the retina pigment epithelium (RPE). How the loss of pigmentation in the RPE causes developmental defects in the adjacent neurosensory retina has not been determined, but offers a unique opportunity to investigate the interactions between these two important tissues. One of the genes that causes albinism encodes for an orphan GPCR (OA1) expressed only in pigmented cells, including the RPE. We investigated the function and signaling of OA1 in RPE and transfected cell lines. Our results indicate that OA1 is a selective L-DOPA receptor, with no measurable second messenger activity from two closely related compounds, tyrosine and dopamine. Radiolabeled ligand binding confirmed that OA1 exhibited a single, saturable binding site for L-DOPA. Dopamine competed with L-DOPA for the single OA1 binding site, suggesting it could function as an OA1 antagonist. OA1 response to L-DOPA was defined by several common measures of G-protein coupled receptor (GPCR) activation, including influx of intracellular calcium and recruitment of beta-arrestin. Further, inhibition of tyrosinase, the enzyme that makes L-DOPA, resulted in decreased PEDF secretion by RPE. Further, stimulation of OA1 in RPE with L-DOPA resulted in increased PEDF secretion. Taken together, our results illustrate an autocrine loop between OA1 and tyrosinase linked through L-DOPA, and this loop includes the secretion of at least one very potent retinal neurotrophic factor. OA1 is a selective L-DOPA receptor whose downstream effects govern spatial patterning of the developing retina. Our results suggest that the retinal consequences of albinism caused by changes in melanin synthetic machinery may be treated by L-DOPA supplementation.     

Lysosomal membrane proteins: life between acid and neutral conditions

Whereas we have a profound understanding about the function and biogenesis of the protein constituents in the lumen of the lysosomal compartment, much less is known about the functions of proteins of the lysosomal membrane. Proteomic analyses of the lysosomal membrane suggest that, apart from the well-known lysosomal membrane proteins, additional and less abundant membrane proteins are present. The identification of disease-causing genes and the in-depth analysis of knockout mice leading to mutated or absent membrane proteins of the lysosomal membrane have demonstrated the essential role of these proteins in lysosomal acidification, transport of metabolites resulting from hydrolytic degradation and interaction and fusion with other cellular membrane systems. In addition, trafficking pathways of lysosomal membrane proteins are closely linked to the biogenesis of this compartment. This is exemplified by the recent finding that LIMP-2 (lysosomal integral membrane protein type-2) is responsible for the mannose 6-phosphate receptor-independent delivery of newly synthesized β-glucocerebrosidase to the lysosome. Similar to LIMP-2, which could also be linked to vesicular transport processes in certain polarized cell types, the major constituents of the lysosomal membrane, the glycoproteins LAMP (lysosome-associated membrane protein)-1 and LAMP-2 are essential for regulation of lysosomal motility and participating in control of membrane fusion events between autophagosomes or phagosomes with late endosomes/lysosomes. Our recent investigations into the role of these proteins have not only increased our understanding of the endolysosomal system, but also supported their major role in cell physiology and the development of different diseases.     

Cytochemical manifestations of short- and long-term activation of the dopaminergic system of the rat brain]

In order to find correlation between changes in morpho-chemical characteristics of neurons of certain brain structures and changes of the functional state of rats with high horizontal motional activity in "open field" under activation of dopaminergic system caused by short-term and long-term L-DOPA injection aminopeptidase activity and protein content were studied. It was shown that in spite of similar effects of short- and long-term injection on the behaviour of animals, the nature of morpho-chemical changes in the brain differs significantly depending on the duration of the drug injection.