I-cell disease: deficiency of extracellular hydrolase phosphorylation

The content of ³²P-phosphorylated residues of purified extracellular N-acetyl-β-hexosaminidase obtained from the fibroblasts of I-cell disease patients was compared to that of control cells hydrolase. The analyses indicated a 60-fold decrease of the radioactivity per unit enzyme activity in the hydrolase synthesized by the patient's fibroblasts compared to the normal enzyme. Electrofocusing demonstrated again a marked decrease in the ³²P-content of the I-cell hydrolase while the control enzyme showed the presence of radioactivity in both isozymes, namely hexosaminidase A and hexosaminidase B. Most of the radioactivity could be removed from the hydrolase following incubation with alkaline phosphatase, thus indicating its phosphoester linkage.Since phosphorylated sugar residues on lysosomal enzymes function as recognition marker for their transport to the lysosomal compartment and for their specific uptake by fibroblasts, the deficiency of phosphorylated residues on the I-cell hydrolase explains the low intracellular and high extracellular lysosomal enzyme levels observed in this disease.     

Prenatal diagnosis of I-cell disease.

A pregnancy from a family in risk of I-cell disease was monitored. The fetus was diagnosed as having I-cell disease based on the findings that (1) lysosomal enzyme activities except for acid phosphatase and alpha glucosidase were clearly elevated in amniotic fluid and were reduced in cultivated amniotic fluid cells, and (2) cytoplasmic inclusions were seen in cultivated amniotic cells by phase contrast microscopy. The accuracy of prediction was confirmed by cultured skin fibroblast of the aborted fetus.     

Apparently normal extracellular acidic alpha-mannosidase in fibroblast cultures from patients with mannosidosis.

Fibroblasts from patients with mannosidosis, cultured in medium supplemented with fetal calf serum from which acidic alpha-mannosidase (alpha-D-mannoside mannohydrolase, E.C.3.2.1.24) has been removed, secreted a normal amount of apparently unaffected acidic alpha-mannosidase into fetal calf serum-free medium. Both the intracellular and extracellular acidic alpha-mannosidase activities were completely precipitated by antiserum to placenta alpha-mannosidase B. In contrast to the heat-lability of the intracellular acidic alpha-mannosidase and its low affinity for artificial mannoside substrate, the extracellular enzyme exhibited both normal thermostability and normal kinetics. Mixing experiments with the intercellular enzymes suggested that the decreased activity in the patients' fibroblasts is not the effect of an inhibitor or absence of an activator. However, incubation of the mannosidosis extracellular enzyme with either normal or patient cell lysate resulted in a partial loss of activity, whereas an additive value was observed with the normal extracellular enzyme. In contrast to normal culture medium, the medium from mannosidosis cell culture was unable to enhance the rate of reduction of intracellular radioactivity in mucolipidosis type II fibroblasts precultured in the presence of radiolabeled mannose. These findings suggest that the defect in mannosidosis is expressed only after the enzyme has been delivered to lysosomes and presumably undergone some form of processing there.     

Effect of vitamin on 2-deoxy-d-glucose uptake in human fibroblast cultures

Summary 2-Deoxy-d-glucose (2-DOG) uptake was tested in human fibroblast cultures in the presence and absence of vitamin E. Addition of 10 μg/ml vitamin E to the culture medium significantly reduced this uptake for 2-DOG concentrations of 0.005, to 10 mmol/liter (P≤0.01). The decrease of 2-DOG uptake was inversely proportional to the rise in 2-DOG concentration (P≤0.01). The presence of vitamin E reduced by 71% the average cellular level of lipid peroxides (expressed as thiobarbituric acid reactive substances) and caused a small but significant decrease in the cholesterol concentration (P≤0.01). These last results might explain the decrease in 2-DOG uptake observed in the presence of vitamin E.     

Characterization of beta-D-galactosidase isolated from I-cell disease liver.

The residual beta-D-galactosidase activity (10% of normal) present in an autopsy sample of liver derived from an I-cell patient has been characterized. The pH optima for both I-cell and normal acid 4-methylumbelliferyl beta-D-galactoside activities were 4.35. The adsorption and elution profiles of the I-cell enzyme from Con A-Sepharose were similar to those of normal liver beta-D-galactosidase. Although starch gel electrophoresis revealed the presence of beta-D-galactosidase A and B in I-cell disease liver, the A band was more diffuse and migrated less anodally than the A band from normal liver. The electrophoretic mobilities of both I-cell and normal beta-D-galactosidase A appeared to decrease after treatment with neuraminidase. Kinetic studies of the I-cell and normal level beta-D-galactosidase demonstrated similar apparent Km values with respect to the 4-methylumbelliferyl beta-D-galactoside and Gm1 ganglioside, whereas the Vmax values obtained for the I-cell enzyme were 10- to 12-fold lower than those of the normal enzyme for both substrates.     

Tissue origin and extracellular matrix control neutral proteinase activity in human fibroblast three-dimensional cultures

Remodeling of the extracellular matrix by fibroblasts is an important step in the process of wound healing and tissue repair. We compared the behavior of fibroblasts from two different tissues, dermis and gingiva, in three-dimensional lattices made of two different extracellular matrix macromolecules, collagen and fibrin. Cells were grown in monolayer cultures from normal skin or gingiva and seeded in three-dimensional lattices made of either collagen or fibrin. Photonic and scanning electron microscopy did not reveal any morphological differences between the two types of fibroblasts in both sets of lattices. Both types of fibroblasts retracted collagen lattices similarly and caused only a slight degradation of the collagen substratum. By contrast, when seeded in fibrin lattices, gingival fibroblasts completely digested their substratum in less than 8 days, whereas only a slight fibrin degradation was observed with dermal fibroblasts. The ability of gingival but not dermal fibroblasts to express high levels of tissue plasminogen activators (tPA) when cultured in fibrin lattices was assessed on an immunological basis. Also, deprivation of plasminogen-contaminating fibrinogen preparations or use of tPA inhibitors markedly inhibited both fibrinolysis and retraction rates of fibrin lattices by gingival fibroblasts. Casein-zymography confirmed the intense proteolytic activity induced by fibrin in gingival fibroblasts. It was inhibited by aprotinin and phenyl methylsulfonyl fluoride (PMSF), two non-specific inhibitors of serine proteinases, and by η-amino-caproic acid (ηACA), an inhibitor of plasminogen activators. Monolayer cultures exhibited only trace amounts of caseinolytic activity. Our results demonstrate that the expression of proteinases by fibroblasts is dependent not only on their tissue origin but also on the surrounding extracellular matrix. The intense fibrinolytic activity of gingival fibroblasts in fibrin lattices may explain partially the high rate of healing clinically observed in gingiva. © 1996 Wiley-Liss, Inc.     

甘油对粒毛盘菌DP5胞外多酚积累的影响

【目的】探明以甘油为碳源促进粒毛盘菌DP5积累多酚的可能原因。【方法】对碳源种类、甘油浓度、曲酸、抑制剂和前体等对多酚产量和生物量的影响进行分析。【结果】以甘油为碳源,能显著提高粒毛盘菌胞外多酚产量。甘油浓度为20 g/L时,胞外多酚产量最高,达到0.664 g GAE/L,并在发酵液中检测到曲酸,其含量为0.25 g/L。向以蔗糖为碳源的发酵液添加曲酸,胞外多酚含量从0.209 g GAE/L提高至0.376 g GAE/L。以甘油为碳源的发酵液中,酚氧化酶活性较低。粒毛盘菌DP5通过莽草酸途径和聚酮途径合成多酚,甘油有利于莽草酸途径和聚酮途径前体物质的合成。【结论】粒毛盘菌以甘油为碳源合成出曲酸,曲酸抑制多酚向黑色素的转化;甘油促进多酚前体的合成,从而提高了粒毛盘菌胞外多酚的积累量。     

Serum hexosaminidase activity in I-cell disease carriers

Summary Serum heat stable hexosaminidase activities are used to identify 47 I-cell disease heterozygotes in a large kindred. Serum beta-hexosaminidase isozyme patterns in normal individuals, Tay-Sachs disease carriers, I-cell disease carriers and in cord blood samples are compared.     

Influence of cell density on collagen biosynthesis in fibroblast cultures.

Characteristic features of collagen metabolism in human skin fibroblasts were studied in relation to cell density. Measuring peptide-bound hydroxyproline we found that collagen synthesis per cell decreased when cultures approached confluency. On the other hand, the relative rate of collagen synthesis (collagen/total protein) was higher in quiescent than in proliferating cultures. With increasing cell density the proportion of type III collagen in comparison with type I was found to be slightly increased. In addition, in low-density cultures [alpha I(I)]3 collagen trimers were produced in considerable amounts, whereas they were no longer detected in cultures with a high cell density. Although hydroxylation of proline residues was normal in all cell stages, conversion of procollagen into collagen was found to depend strongly on the density at which the cells were investigated. Almost no cleavage of procollagen peptides was observed in rapidly growing cells, whereas highly confluent cell cultures converted most of the newly synthesized procollagen molecules.     

Effect of microtubule-specific drugs upon spatial organization of extracellular matrix in fibroblastic cultures.

The aim of this investigation was to study the effects of microtubule-specific drugs, taxol and colcemid, on the distribution of cell-associated extracellular matrix in dense cultures of fibroblasts. Immunomorphological examination of human seven-day cultures revealed a dense network of fibronectin and tenascin matrix filaments preferentially oriented in parallel with the long axes of cell bodies. Depolymerization of the microtubular system by colcemid and its disorganization by taxol led to rapid and drastic changes in the organization of matrix network: fibronectin and tenascin filaments became disordered and, in particular, lost any orientation. These data show that the microtubular system controls the morphological organization, not only of intracellular cytoskeletal systems, but also of extracellular matrix structures.     

Biochemical heterogeneity in I-cell disease. Sucrose-loading test classifies two distinct subtypes

Since we observed the normalization of intracellular hydrolases in some cell lines of I-cell disease (ICD) by 88 mmol/l sucrose, we have hypothesized that the degree of responses of the hydrolases might be due to biochemical heterogeneity among ICD. In this study the changes of intracellular lysosomal enzymes as well as Golgi enzymes including N-acetylglucosaminyl phosphotransferase (GlcNAcPTase) and extracellular hexosaminidase (HEX) were investigated using normal and ICD fibroblasts. Sucrose loading induced the activities of intracellular HEX and GlcNAcPTase simultaneously only in responding-type ICD cells, and not in nonresponding-type ICD cells, indicating that two biochemical heterogeneous groups exist in ICD.     

Lipids in plant tissue cultures. VI. Effect of temperature on the lipids of Brassica napus and Tropaeolum majus cultures

The lipid contents of callus cultures of rape (Brassica napus) and nasturtium (Tropaeolum majus) increase in response to decreasing the temperature, though to different degree. Irrespective of the incubation temperature the lipids in cultures of both plants contain as predominant classes steryl glycosides, esterified steryl glycosides, sterols, steryl esters and fatty acids and, as minor constituents, various proportions of triacylglycerols, phospholipids and several unidentified fractions.The ratio of phospholipids to triacylglycerols as well as the ratio of diacylglycerophosphorylethanolamines to dicylglycerophosphorylcholines increase with time both in rape cultures incubated at 30°C and in those kept at 5°C.The lipids in rape and nasturtium cultures grown at 30°C contain smaller proportions of polyunsaturated fatty acids than the lipids in cultures incubated at 5°C. Erucic acid, the major constituent fatty acid of the seed lipids, in both plants, occurs only in trace amounts in the lipids of callus cultures. In contrast, linoleic and linolenic acids, which occur only in traces in the seed lipids of nasturtium, are major constituent fatty acids in the lipids of callus cultures derived from seedlings of this plant.The levels of constituent polyunsaturated fatty acids in the diacylglycerophosphorylethanolamines and the diacylglycerophosphorylcholines increase with time whereas in the triacylglycerols only linolenic acid is slightly increased.     

Inhibiting effect of procollagen peptides on collagen biosynthesis in fibroblast cultures.

NH2-terminal extension peptides of type I and type III procollagens were isolated from dermatosparactic and normal fetal calfskin, respectively. Cell culture experiments showed that the globular domains of the tested procollagen peptides were biologically active but that peptides from the helical region of collagen had no effect. The peptides were added to the incubation medium of calf fibroblasts along with radioactive precursor amino acids, and the amount of newly synthesized collagen was determined. The experiments indicated that procollagen peptides exerted a feedback-like inhibitory effect specific for the synthesis of collagen. Neither degradation of collagen, hydroxylation of collagen alpha chains, nor synthesis of noncollagenous proteins were affected. Synthesis of type II collagen by calf chondrocytes was not reduced. In addition, it was shown that procollagen peptides from calf were equally effective when added to human fibroblast cultures, an observation that could be of considerable medical interest.