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In vitro it was studied the isoform spectra of the intracellular and secreted alpha-L-fucosidase from skin fibroblasts of patients with Fabry disease (glycolipidosis), Hurler and Sanfilippo D diseases (mucopolysaccharodosis, types I and III) and in the normal state was studied. It was shown that the multiple form profile of secreted alpha-L-fucosidase in patients fibroblasts was changed as compared to that in control: the pathological cells were characterized by expression of more basic isoforms of alpha-L-fucosidase. The changes were similar to those in sucrose-loaded normal cells, modelling storage disease. The data obtained allow the suggestion that the intracellular accumulation of compounds whose hydrolysis was disturbed on a hereditary deficiency of enumerated glycosidases can influence the posttranslational processing of alpha-L-fucosidase, the enzyme which is not primary affected in these disorders. These data allow the conclusion that the high phenotypic heterogenity of lysosomic storage diseases is possibly due to the influence of so-called epigenetic factors involving the changes in properties of such glycosidases as are not associated with a primary hereditary defect.

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Activity and multiple forms of intracellular and secreted alpha-L-fucosidases from human skin fibroblasts were studied in normal and sucrose-loaded cells--modelling intralysosomal nonhydrolyzable products' accumulation in vitro. Certain differences in secretion levels were found after sucrose load for alpha-L-fucosidases from embryonal and postnatal cell lines. Different effects of sucrose load on multiple forms' spectra of the intracellular and secreted alpha-L-fucosidases from embryonal and postnatal fibroblasts were revealed.

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The conditions for assay of beta-mannosidase activity in human chorionic villi (CV) were studied using the fluorogenic substrate 4-methylumbelliferyl-beta-D-mannopyranoside. A comparison of the biochemical properties of the CV beta-mannosidase with those of the enzyme from human cultured fibroblasts showed their similarity. Like the enzyme from skin fibroblasts, the CV beta-mannosidase had rather high activity. Both enzymes had virtually the same pH optimum (4.2-4.7) and Km value. The data presented suggest that chorion biopsy specimens can be used for prenatal determination of beta-mannosidase activity at an early stage of development.

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Conditions for assay of beta-mannosidase activity in human chorionic villi were studied using the fluorogenic substrate 4-methylumbelliferyl-beta-D-mannopyranoside. Comparison of the biochemical properties of the chorionic villi beta-mannosidase with those of the enzyme from human cultured fibroblasts showed their similarity. Like the enzyme from skin fibroblasts, the chorionic villi beta-mannosidase had rather high activity. Both enzymes had virtually the same pH optimum (4.2-4.7) and Km value. The data presented suggest that chorion biopsy specimens can be used for prenatal determination of beta-mannosidase activity at the early stage of development.

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The organization of the system of the vimentin intermediate filaments (IFs) in human fibroblasts in lysosomal storage diseases (Fabry's disease, mannosidosis) and their modelling has been studied in vitro. It was shown that during accumulation of nonhydrolyzable compounds, hypertrophy of the lysosomal compartment is accompanied by formation of ring-shaped bundles IFs, surrounding apparently these increased organelles. The changed organization of IFs is characteristic of polarised pathological cells in monolayer, and after repassage it is retained only at the spreading state; on transition from the discoid to extended cellular form there occurred the centrifugal shift of ring-shaped structures of IFs to active cell border and gradual restoration of radial fibrillar state of IFs. It is suggested that on intralysosomal storage of unsplit compounds reorganization of the vimentin-type IFs in ring-shaped structure is necessary for optimal distribution and stabilization into the cytoplasm of large amounts of increased lysosomes with exo- and endogenous contents. In condition of free spreading (i. e. with diminished cell density) the restoration of normal fibrillar IF organization may be due to the loss of considerable number of hypertrophied lysosomes; the involvement of lysosomal membrane in formation of active cellular border is not to be ruled out.

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Presented are the results of measurements of pH in cytoplasm and lysosomes of skin fibroblasts of healthy donors and patients with lysosomal storage diseases, mannosidosis, Fabry, Krabbe disease. The pH value was estimated in the stationary phase of growth using neutral red (lysosomes) and fluorescein diacetate (cytoplasm). It was shown that the cytoplasmic pH value in pathological cells didn't virtually differ from the control values. The intralysosomal pH value in fibroblasts of patients with mannosidosis and Fabry disease was essentially increased, which correlated with the size increase of these organelles upon the accumulation of unsplit compounds. This led to the decrease in pH gradient between the cytoplasm and lysosomes in the pathological cells, an increase in intralysosomal pH along with hereditary deficiency of enzymes could bring about the retardation of catabolic processes in lysosomes.

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Measurements were made of the pH of cytoplasm and lysosomes of cultured skin fibroblasts from healthy donors and from patients with lysosomal storage diseases (mannosidosis, Fabry's disease, and Krabbe's disease), and the effects of sucrose loading on normal fibroblasts were studied. The cytoplasmic pH of the pathological cells did not differ from control values, but the intralysosomal pH was significantly higher in sucrose-loaded normal fibroblasts and in cells from a patient with mannosidosis and from another with Fabry's disease. The change in pH observed accorded with an increase in size of the organelles, owing to accumulation of nonhydrolyzable compounds. In fibroblasts from a patient with Krabbe's disease, which do not store nonhydrolyzable compounds, there was no increase either in intralysosomal pH or in lysosome size. It is suggested that the decrease in the pH difference between the cytoplasm and lysosomes in the pathological cells leads to inhibition of catabolic processes in lysosomes and to some changes in the intracellular transport of these vesicles.

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The changes in intralysosomal pH were measured in the stationary phase of normal human embryonic fibroblast growth under sucrose loading over a period of 6 to 120 hours and in cells with a typical lysosomal storage pathology, Fabry's disease, using a vital indicator dye, neutral red. It was shown that long-term hypertrophy of the lysosomal compartment during intracellular accumulation of non-hydrolysable compounds is concomitant with a pH increase, on the average, by 0.4 units. The highest values of pH (7.0-7.2) were seen in large-sized heterogeneic lysosomes of pathological cells. It is suggested that an increase in intralysosomal pH during accumulation of non-hydrolysable compounds leads to deterioration of conditions that are favourable for the acidic hydrolase function.

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Intracellular activation of lysosomal glycosidases from human skin fibroblasts (alpha-L-fucosidase, beta-D-hexosaminidase, beta-D-galactosidase and beta-D-glucuronidase) was shown to occur on the 3rd-6th days of cultivation in media containing 0.04 M sucrose. The increase in the enzyme activity ranged from 40 to 300% depending on cell strain, nature of enzyme and cultivation time. Among pre- and postnatal fibroblast strains, those with a high and low response to sucrose load were identified. The maximal intracellular activation was observed in beta-D-galactosidase, the minimal one--in beta-D-glucuronidase. In pathological cells (Krabbe's disease) the highest activation by sucrose load was observed, as in normal cells, with beta-D-galactosidase, whereas the lowest one--with beta-D-glucuronidase. Secretion of lysosomal glycosidase is selective and noncoordinated. The maximal secretion of alpha-L-fucosidase and beta-D-hexosaminidase was observed within the first 24 hours (intensive sucrose endocytosis), but was considerably decreased at later times, i. e., by the 3rd and 6th days. The enzymes secreted during the 1st and 3rd days differed significantly in stability (37 degrees C, pH 7.0).

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The amount of alpha-L-fucosidase secreted by normal human fibroblasts was higher in the medium containing 10% bovine serum than in the medium containing 0.1% bovine serum. Glycosidase secretion was twice higher at the advanced than at the initial stage of subcultivation. Extracellular activity of alpha-L-fucosidase from 3 different fibroblast strains differed insignificantly in the medium containing 0.1% bovine serum, while intracellular activity of the enzyme in these strains was altogether different. The results suggest that the lysosomal glycosidase secretion is determined by the level of cellular endocytosis.

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The intracellular activities of four lysosomal glycosidases (alpha-L-fucosidase, beta-D-hexosaminidase, beta-D-galactosidase and beta-D-glucuronidase) in human skin fibroblasts cultured in a medium with 0.1% serum increased in a greater degree than that in a medium with 10% serum. Only two glycosidases (alpha-L-fucosidase and beta-D-hexosaminidase) were secreted by fibroblasts in the culture medium. The extracellular activity of alpha-L-fucosidase and beta-D-hexosaminidase was equivalent to 80 and 25% of their intracellular activity in serum-sufficient fibroblasts and 40 and 15%--in serum-restricted fibroblasts. These results suggest that the observer phenomena are controlled by the levels of autophagy, endocytosis and membrane recycling.

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A study was made of the activity of 3 lysosomal glycosidases -beta-D-galactosidase (K. P. 3.2.1.23), alpha-L-fucosidase (K. P. 3.2.1.51), N-acetyl-beta-D-hexosoaminidase (K. P. 3.2.1.52) depending on the time after subcultivation and duration of the passage of human skin embryonal and postembryonal fibroblasts. It was established that changes in the specific activity of the enzymes should be calculated with reference to the cell rather than to protein whose amount might vary considerably. It was also found that for measuring the specific activity of enzymes, of great importance are the procedures of cell removal from the base layer (by mechanical scraping off or by trypsin solution) and the regimen of the homogenization of cell preparations.

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The number of cells with fragmented nuclei (FN) (mainly, multilobate) increased with aging of ascites hepatoma 22A (AH22A) as follows: 15 +/- 9.3% in the 6-day AH22A, 196 +/- 53% in the 14-day tumor and 453 +/- 51% in the delayed (18-day) AH22A. The basic way of FN formation was amitotic. About 150 and 170% of cells with FN in the 14- and 18-day AH22A were at the reversible resting R1 stage (or at the very long G1 period, more than 4 days). The rest of the cells, 50 and 230%, respectively, quit the mitotic cycle irreversibly and apparently undergo the involution that is faster during passage-stimulated division.

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At early stages after partial hepatectomy (17 hours after the operation) binuclear cells become involved in proliferation in much lesser numbers, and 37 and 53 hours after the operation--in much greater numbers relative to their part in the population. New formation of binuclear cells (the presence of labeled binuclear cells 20 hours after the thymidine H3 administration) was the most intensive at the early regeneration stages (16--36 hours after the operation), when about 20% of mitoses are acytokinetic and lead to the formation of binuclear cells. At later periods only 8% of mitoses result in formation of binuclear cells.

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The authors studied growth peculiarities of the ascitic hepatoma 22A at the terminal stage of development. As shown by the autoradiography method, some of the cells could be at the G1-period or at the R1-period for 2 or even 4 days; the average duration of the G2-period constituted 16 hours. 55--60% of the cells were at the much prolonged G1- and R1-period, 7%--at the S-period, and 9%--at the G2-period. The rest 25--30% of cells apparently leave the mitotic cycle irreversibly since they take no part in proliferation after the division stimulation.

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Repeated stimulation of division soon after the primary one (2--3 days later) induces a shorteining of the prereplicative phase of the mitotic cylce of hepatocytes to 9--10 hours in the regenerating rat liver. The cells dividing repeatedly after one stimulation were found to pass through the G1-phase of the second mitotic cycle during the same period. It may be suggested that the cells with the minimal duration of the prereplicative phase failed to pass through the transformation period. With increase of the time interval between the successive stimulation of divisions (to 4--5 days) increases the resting time state for most hepatocytes, and they lose the capacity to pass through the reduced prereplicative phases.

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A study was made of the progress rate of cells of the ascitic hepatoma 22A of different age during the iirst mitotic cycle after the stimulation of division. The "ageing" (11-day), terminal (14-day), and "delayed" (4 days older than the terminal stage) ascitic fluids were used. The maximal values of the labeled nuclei index was found to be reached by 9--12 hours (it was mainly due to the transtion of the quiescent to the S-period) and the maximal mitotic index--by 18--21 hours after the inoculation, independently of the tumour age. These results suggest that the duration of both the prereplicative (G1) period and of the whole first mitotic cycle after the stimulation were independent of the time during which the cells of the ascitic hepatoma 22A were at the resting stage or at the very prolonged G1-period.

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A study was made of the recurrent growth of ascite hepatoma 22A, occurring at transplantation of 11-12-day old tumours to new hosts. The mitotic activity of the hepatoma was found to increase by 3 to 4 times (12-15 hours after inoculation). This increased cell proliferation is due mainly to a sharp shortening of all the periods of mitotic cycle. During the recurrent growth, the resting R1 cells resume their mitotic cycle. No resumption of the mitotic cycle by the resting R2 cells was observed

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