Significance of glycosidase patterns in lymphoid cells

Characteristic patterns of cell bound lysosomal glycosidases were found in different lymphoblastoid cell lines derived from Epstein-Barr virus-transformed lymphocytes. The enzyme pattern resembled that found in normal lymphocytes from healthy individuals except for a marked increase in alpha-L-fucosidase. beta-D-Glucuronidase on the contrary markedly decreased in the lymphoblastoid cells. Burkitt's lymphoma cells on the other hand showed glycosidase patterns that were quite distinct from those in lymphoblastoid cells. Each lymphoma cell line showed a characteristic pattern. This is one indication of a heterogeneous origin of these tumors. Glycosidase patterns may be used to roughly distinguish different lymphoid cell lines.     

Verotoxin-resistant cell clones are deficient in the glycolipid globotriosylceramide: differential basis of phenotype

Escherichia coli-derived verotoxin is an extremely toxic protein and is highly selective toward certain primate cells. Two susceptible cell lines are the Daudi cell line (human Burkitt lymphoma) and the Vero cell line (Green African monkey kidney). Both of these cell lines contain significant levels of the verotoxin binding glycolipid globotriosylceramide (Gb3) (1 nmol/10(7) cells and 3 nmol/10(6) cells, respectively). A clone was selected from the Vero cell line for resistance to Verotoxin 2, while a mutant from the Daudi cell line was selected for resistance to Verotoxin 1. Both were found to be deficient in globotriosylceramide with a corresponding increase in the precursor glycolipid lactosylceramide. Cell free assay of alpha-galactosyltransferase activity revealed that the Vero cell clone (VRP) contained significantly reduced enzyme activity, whereas in the case of the Daudi mutant (VT20), no significant decrease in activity was noted in vitro. These observations suggest a complex regulation of Gb3 biosynthesis which is considered in relation to P blood group antigen expression.     

Changes in glycosidase activities during galactoglucomannan oligosaccharide inhibition of auxin induced growth

The inhibition of 2,4-D-induced elongation growth by galactoglucomannan oligosaccharides (GGMOs) in pea stem segments (Pisum sativum L. cv. Tyrkys) after 18 h of incubation results in changes of extracellular, intracellular and cell wall glycosidase activities (beta-D-glucosidase, beta-D-mannosidase, beta-D-galactosidase, beta-D-xylosidase, alpha-D-galactosidase, and alpha-L-arabinosidase). GGMOs lowered the glycosidase activities in the extracellular fraction, while in the cell wall fractions their activities were markedly increased. The intracellular enzyme alpha-d-galactosidase increased while the beta-d-galactosidase decreased in activity in response to the GGMO treatment. Extracellular enzymes showed low values of activities in comparison with intracellular and cell wall glycosidases. It is evident that GGMOs can alter auxin induced elongation and glycosidase activities in different compartments of the cell, however, the mode and site of their action remains unclear.     

Immunoglobulin genes of the kappa light chain type from two human lymphoid cell lines are closely related.

As a first step in our studies of functionally rearranged K genes of man we cloned the germline JK-CK region from placenta DNA employing a mouse JK clone as hybridization probe. Subclones of the human JK-CK region were then used to characterize and clone the rearranged K genes of the lymphoid cell lines Walker and Daudi. The Walker cell line contains one rearranged and one germline K allele (K+,KO; ref. 1). Only one K gene was found in Daudi cells (K+). Restriction mapping and DNA sequencing showed, that the rearranged K genes from both cell lines are closely related. These features make the two cell lines particularly suitable for studies on the chromatin structure of K light chain genes. The 5' flanks of the two genes (388 bp) are identical while there is a 12% divergence between the VK gene segments themselves. This situation may reflect somatic mutation processes and/or gene conversion like events.     

Alkaline phosphatase in hematopoietic tumor cell lines of the mouse: high activity in cells of the B lymphoid lineage

Alkaline phosphatase (EC 3.1.3.1) was assayed in a large number of cultured mouse tumor cell line using p-nitrophenylphosphate as the substrate. Of 19 lines of the B lymphoid lineage, including Abelson pre-B, B lymphoma, and plasma cell tumor lines, all but 1 had substantial activity averaging 407 nmol/min/mg protein (with a range from 5 to 900). Nine T lymphoid and 9 nonlymphoid hematopoietic lines examined had low activity of 0.7 to 4.2 nmol/min/mg protein. The enzyme was markedly enriched in plasma membrane preparations from the B lymphoid cells, but not in those from most T lymphoma cells. The activity of another plasma-membrane-bound enzyme, gamma-glutamyl transferase, did not vary systematically with the type of cell line but was exceptionally high in 1 T lymphoma line. Investigation of pH dependence and susceptibility to inhibition by L-phenylalanine and L-homoarginine indicated similarity of the alkaline phosphatase from B cell lines to the enzyme recoverable from normal mouse kidney, placenta, bone marrow, and lymphoid organs. The enzyme seems to provide a useful marker for tumor lines of the B lymphoid lineage and for their plasma membranes.     

Activity of deoxyribonucleoside-activated nucleotidase in cells from various lymphoid mouse tissues and in concanavalin A-stimulated lymphocytes

The specific activity in cells from lymph nodes, spleen and thymus was 32, 28 and 25 nmol/min per mg of cytosol protein, respectively, whereas that in bone marrow cells was significantly lower (10 units/mg of protein). No difference in specific DAN activity between isolated B- and T-lymphocytes was observed. Two types of lymphoid mouse cell lines (MOPC-31C plasmacytoma cells, S49 Cyc- lymphoma cells) showed specific activities similar to the normal lymphoid cells. In concanavalin A- stimulated spleen lymphocytes in culture there was a rapid increase in DAN activity shortly after maximum DNA synthesis, reaching a plateau 2-3 times the original level. The enzyme (DAN) of mouse tissues possessed the characteristic properties previously detected for the rat enzyme.     

Synergistic antiproliferative effects of glucocorticoids and interferon-alpha on some lymphoid cell lines

The Daudi B lymphoblastoid cell line was previously demonstrated to be highly sensitive to the antiproliferative effect of recombinant interferon-alpha A (rIFN-alpha A). In the present study, glucocorticoid hormones were shown to act synergistically with rIFN-alpha A to further increase the sensitivity of Daudi cells to rIFN-alpha A. At 10(-6) M, dexamethasone, prednisolone, or hydrocortisone alone had little effect on Daudi cell growth, but they greatly potentiated the antiproliferative activity of rIFN-alpha A. The synergy between rIFN-alpha A and glucocorticoids on Daudi cells was not related to the inhibitory effects of glucocorticoids on prostaglandin or leukotriene synthesis, since no synergy was observed between rIFN-alpha A and indomethacin or nordihydroguaiaretic acid. Glucocorticoids and rIFN-alpha A also had appreciable synergistic antiproliferative effects on two out of five other IFN-sensitive lymphoid cell lines. When Raji B lymphoblastoid cells, which were quite resistant to the antiproliferative effect of rIFN-alpha A, were treated with the combination of glucocorticoids and rIFN-alpha A, no significant synergistic effects were observed. The synergistic antiproliferative effects of glucocorticoids and rIFN-alpha A observed with some IFN-sensitive lymphoid cell lines in this in vitro study may have clinical relevance in the treatment of certain lymphoid malignancies that are sensitive to rIFN-alpha A therapy.     

Glycosidase and phosphatase activities in U-937 and some clones and sublines

1. The activities of nine glycosidases, lysozyme and acid and alkaline phosphatases were compared in the histiocytic lymphoma cell line U-937 and a set of clones and sublines derived from this line. 2. The patterns of the different enzyme activities and selected enzyme ratios have been used as a method to distinguish between different clones and sublines. 3. Sublines with high lysozyme levels were rich in most cell-bound glycosidases. 4. During long-term growth distinct enzyme patterns of individual lines were preserved. 5. The enzyme pattern during a cell culture growth cycle was basically stable.     

The characterization of SV40-transformed cell lines derived from mouse teratocarcinoma: growth properties and differentiated characteristics.

Mouse teratocarcinoma cells derived from embryoid bodies of 129SVsl mice were cultured in vitro to permit their differentiation. These cells were then infected with simiam virus 40 (SV40) and 31 cloned cell lines (SVTER) were derived from these cultures. All 31 SVTER cell lines contained the SV40 tumor (T) antigen and grew as permanent lines in culture. Mock-infected embryoid body cultures did not give rise to permanent cell lines. The morphology of each SVTER cell line was distinct and did not change during successive subclonings. The growth properties and tumorigenic potential of all 31 SVTER cell lines were investigated. None of these lines produced tumors in 129SVsl mice. Each cell line was tested for its ability to (1) grow in medium containing 1% serum, (2) plate on cell monolayer, and (3) form clones in methocel suspension. Only three of the SVTER cell lines were transformed with respect to all three of these criteria. Most of these cell lines were minimal transformants. The SVTER cell lines were tested for creatine phospholinase (CPK), an enzyme activity chracteristic of mouse brain and muscle tissue, and the protease, plasminogen activator (PA) which is found in embryoid bodies and several differentiated cell types. Some of the SVTER cell lines contained high levels of CPK, while others had high levels of PA and a third group of cells contained neither enzyme activity. No SVTER cell line was found with high levels of both these enzyme activities. This result suggests that mutually exclusive sets of genes are expressed in these cells as might be expected from the distinct tissue distribution of the two enzyme activities studied. These SVTER cell lines may be useful in reconstructing developmental pathways of differentiating teratomas in vitro.     

Reciprocal recombination products of VK-JK joining reactions in human lymphoid cell lines.

The recombination process that joins a VK to a JK segment of an immunoglobulin gene generates a second, reciprocal recombination product called f fragment. In this second product the regions flanking the VK and JK segments in the germline are joined in a head to head fashion. We now analysed f fragments in the human lymphoid cell lines Daudi, JI and IARC/BL41. All three f fragments contain JK1 flanks; the VK derived moiety of f Daudi and f41 could be traced back to known germline VK genes. There is a precise head to head joining of the heptanucleotide signal sequences in f Daudi and fJI while in f41 six nucleotides are present between the signal sequences. In contrast to the VK-JK recombination products, the f fragments were found to lack somatic mutations. The structures of the f fragments are discussed in the context of the VK-JK rearrangement mechanism.     

Properties of a beta-D-mannosidase from Aspergillus niger

The beta-D-mannosidase (beta-D-mannoside mannohydrolase, EC 3.2.1.25) from culture filtrate of Aspergillus niger has been purified in large amounts by fractionation with (NH4)2SO4 and DEAE-cellulose chromatography. The removal of traces of alpha-D-galactosidase was performed on a Sepharose-epsilon-aminocaproyl-galactosylamine column. The final enzyme preparation (specific activity 188 units) has no other glycosidase activity and is judged homogeneous. The enzyme has a molecular weight of 130 000 +/- 5000 and an isoelectric point of 4.7. The amino acid composition of the enzyme is characterized by high proportion of acidic amino acids and no cysteine residues and a single chain structure of the enzyme is suggested. The enzyme shows maximum activity on p-nitrophenyl-beta-D-mannopyrano-side at pH 3.5 and at 55 degrees C. The presence of 80% of beta-sheet structure in the protein and 20.8% of monosaccharides (Gal : 1.3; Man : 7; GlcNAc : 1) could explain this relative high heat stability (up to 2 h at 55 degrees C). Enzyme activity is inhibited by mannose (Ki = 7.85 mM) and the specificity is examined.     

Beta-galactosidase, phospho-beta-galactosidase and phospho-beta-glucosidase activities in lactobacilli strains isolated from human faeces

AIMS: Lactose intolerance, a serious health problem for Asians, can be solved using probiotic bacteria having high lactose hydrolysis activities. We determined the distribution of beta-galactosidase (beta-gal), phospho-beta-galactosidase (P-betagal) and phospho-beta-glucosidase (P-beta-glc) activities in species of lactic acid bacteria (LAB) isolated from human faeces to select strains for potential use in fermented dairy products, e.g. yogurt. METHODS AND RESULTS: The sugar substrates, o-nitrophenyl-beta-D- galactopyranoside 6-phosphate and o-nitrophenyl-beta-D-glucopyranoside 6-phosphate, were synthesized and used to measure respectively P-beta-gal and P-beta-glc activities. Sixty-five toluene-treated strains were examined for three lactase enzyme activities. Lactobacillus mucosae OLL2848 showed the highest beta-gal activity (107.09 U mg(-1) of protein) among the Lactobacillus strains from human faeces. Lactobacillus gasseri OLL2836 and OLL 2948 showed the highest P-beta-gal (46.58 U) and P-beta-glc (50.19 U)activity, respectively, with no beta-gal activity. CONCLUSIONS: The expression of P-beta-glc induced by lactose was characteristic of Lact. gasseri. Because this LAB is a major inhabitant of the human intestine. This enzyme is a key glycosidase involved in lactose utilization. SIGNIFICANCE AND IMPACT OF STUDY: This is the first report describing the distribution of three glycosidase activities used in lactose metabolism in LAB isolated from human faeces for possible use in functional foods.     

New aspects in the synthesis and secretion of lysozyme by cultured human monocyte cell lines

The main purpose of this study was to investigate lysozyme synthesis and secretion in three human monocyte cell lines: U-937, HL-60, and THP-1, using sensitive fluorescence-based assay of lysozyme activity. PMA and hIFN-γ were evaluated for inducing lysozyme activity. Using well-defined cell lines from the cell culture collection, no lysozyme activity could be detected in the cultured U-937 cells either with or without addition of the inducing factors. These data suggested, contrary to previous reports, that U-937 cell line cannot synthesize or secrete active lysozyme. THP-1 and HL-60 cells were proved to produce enzymatically active lysozyme in increasing amounts with the time course. PMA and hIFN-γ had no significant inducing effect on the production or the release of active lysozyme in THP-1 and HL-60 cells. We showed inhibiting effect of PMA and hIFN-γ on the lysozyme activity, particularly in HL-60 cell line.     

Selective inhibitory effect of Hu-IFN-gamma on the agarose clonability of tumor-derived lymphoid cell lines

Recombinant human interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma) were compared for their ability to influence the proliferative capacity of tumor-derived cell lines and of normal B lymphocytes infected in vitro by Epstein-Barr virus (EBV). EBV-induced B-cell proliferation was suppressed almost completely when 10(2) U/ml IFN-alpha were added to the culture medium while the same dose of IFN-gamma had significantly lower inhibitory activity. The pure IFNs differed in their ability to influence the growth of three Burkitt lymphoma-derived cell lines, Raji, Daudi, and Namalwa, depending on whether the cells were propagated in suspension or in semisolid cultures. IFN-alpha inhibited cell proliferation under both culture conditions with thresholds of sensitivity characteristics for each cell line. In contrast, IFN-gamma had no effect on the growth in suspension but it abolished the clonogenic potential of tumor cell lines in semisolid agarose. The results suggest that the two IFN types may exert their growth inhibitory activity through different mechanisms of action.     

Purification and characterization of goose type lysozyme from cassowary (Casuarius casuarius) egg white

A novel goose-type lysozyme was purified from egg white of cassowary bird (Casuarius casuarius). The purification step was composed of two fractionation steps: pH treatment steps followed by a cation exchange column chromatography. The molecular mass of the purified enzyme was estimated to be 20.8 kDa by SDS-PAGE. This enzyme was composed of 186 amino acid residues and showed similar amino acid composition to reported goose-type lysozymes. The N-terminal amino acid sequencing from transblotted protein found that this protein had no N-terminal. This enzyme showed either lytic or chitinase activities and had some different properties from those reported for goose lysozyme. The optimum pH and temperature on lytic activity of this lysozyme were pH 5 and 30 degrees C at ionic strength of 0.1, respectively. This lysozyme was stable up to 30 degrees C for lytic activity and the activity was completely abolished at 80 degrees C. The chitinase activity against glycol chitin showed dual optimum pH around 4.5 and 11. The optimum temperature for chitinase activity was at 50 degrees C and the enzyme was stable up to 40 degrees C.     

Methionine adenosyltransferase activity in cultured cells and in human tissues

We have investigated methionine adenosyltransferase activity (MAT) in extracts of a variety of normal and malignant human tissues and cultured cell lines. MAT activity assayed from 17 different cultured cell lines varied to a great extent. Ramos (human, Burkitt's lymphoma) and EL4 (mouse, T cell lymphoma) cell showed MAT activity near 300 pmol/mg per min. Daudi (human, Burkitt's lymphoma) and almost all monolayer cells had MAT activity below 100 pmol/mg per min. Human peripheral blood lymphocytes had MAT activity of 36 pmol/mg permin. The MAT activity of the cell lines can be related to doubling time: cell lines with short doubling times have much higher MAT activity than other cell lines. A large variation in MAT activity in different human tissues was observed. In autopsy samples MAT activity was highest in the brain and in the colon. Malignant tissue samples gave much higher MAT activity than normal tissues. Lung cancer (carcinoma squamocellulare pulmonis) had MAT activity of 30.7 pmol/mg per min, while in normal lung it was 2.4 pmol/mg per min.     

Glycosidase activities of the 293 and NS0 cell lines, and of an antibody-producing hybridoma cell line

We have previously demonstrated that Chinese hamster ovary (CHO) cell lysates harbor sialidase, beta-galactosidase, beta-hexosaminidase, and fucosidase activities that can accumulate extracellularly in CHO cell culture, thereby potentially leading to extracellular modification of glycoprotein oligosaccharides. The sialidase activity in CHO cell lysates was surprisingly active and stable at pH 7.5, with a half-life of 57 h at 37 degrees C.We have extended this work to determine whether 293, NS0, or hybridoma cell lysates contain similar glycosidase activities. The pH-activity profiles of beta-galactosidase and beta-hexosaminidase in lysates of these three cell lines resemble the pH-activity profiles for these enzymes in CHO cell lysate, whereas the pH-activity profiles of sialidase and fucosidase appear to be cell-type dependent. Sialidase activities were relatively stable at pH 4.5 in 293, NS0, and hybridoma cell lysates. However, the activities in 293 and NS0 cell lysates were unstable at pH 7.5, with no activity remaining after a 2-h incubation at 37 degrees C. The sialidase activity in hybridoma cell lysate was moderately stable at pH 7.5 with 30% of the activity remaining after a 2-h incubation at 37 degrees C. We conclude that the sialidase activites from 293, NS0, and hybridoma cells have characteristics similar to the vast majority of reported mammalian sialidase activities, and that these activities are markedly differant from the CHO cell sialidase activity.Finally, sialidase, beta-galactosidase, beta-hexosaminidase, and fucosidase activities were measured at pH 7 in cell-free bioreactor supernatants of the hybridoma cell line. As previously observed in CHO cell culture, all four glycosidase activities were present in the hybridoma supernatants. However, the sialidase activity in hybridoma supernatant was an order of magnitude lower than in CHO cell culture supernatant despite the fact that the hybridoma cell lysis rate was an order of magnitude higher. (c) 1994 John Wiley & Sons, Inc.     

Unspecific activation of caspases during the induction of apoptosis by didemnin B in human cell lines

Caspases have been implicated in the induction of apoptosis in most systems studied. The importance of caspases for apoptosis was further investigated using the system of didemnin B-induced apoptosis. We found that benzyloxycarbonyl-VAD-fluoromethylketone, a general caspase inhibitor, inhibits didemnin B-induced apoptosis in HL-60 and Daudi cells. Acetyl-YVAD-chloromethylketone, a caspase-1-like activity inhibitor, inhibits didemnin B-induced apoptosis in Daudi cells, whereas the caspase-3-like activity inhibitor, acetyl-DEVD-aldehyde, has no effect. Using immunoblots to investigate cleavage of caspases-1 and -3, we found that both caspases are activated in both cell lines. We showed that the caspase substrate poly(ADP-ribose)polymerase is cleaved in these cells after didemnin B treatment. In both cell lines, poly(ADP-ribose)polymerase cleavage is inhibited by benzyloxycarbonyl-VAD-fluoromethylketone and also by acetyl-YVAD-chloromethylketone in Daudi cells. These results indicate that a caspase(s) other than caspase-3 is required for didemnin B-induced apoptosis. We show that caspases may be activated during apoptosis that are not required for the progression of apoptosis.     

Bacillus cereus autolytic endoglucosaminidase active on cell wall peptidoglycan with N-unsubstituted glucosamine residues

An autolytic glycosidase from a lysozyme-resistant strain of Bacillus cereus capable of cleaving the glycosidic linkages of N-unsubstituted glucosamine in the cell wall peptidoglycan was studied. This glycosidase activity, together with N-acetylmuramyl-L-alanine amidase activity, was found in an autolytic enzyme preparation obtained from the 20,000 x g precipitate fraction by means of autolysis followed by ammonium sulfate fractionation. The major saccharide fragments resulting from digestion of the untreated, non-N-acetylated, cell wall peptidoglycan of B. cereus with the autolytic enzyme preparation were identified as N-acetylmuramyl-glucosamine and its dimer. The peptidoglycan N-acetylated with acetic anhydride could also be digested with the same enzyme preparation, giving N-acetylmuramyl-N-acetylglucosamine and its dimer as the major saccharide fragments.     

Purification and characterization of endo-beta-galactosidase from Escherichia freundii induced by hog gastric mucin

A new procedure for inducing and purifying endo-beta-galactosidase from Escherichia freundii was described. The enzyme was found to be induced with high efficiency in culture medium containing Smith-degraded hog gastric mucin, which was prepared from a commercially available starting material. Endo-beta-galactosidase was then purified by ammonium sulfate fractionation, DEAE-Sephadex chromatography, and affinity chromatography on Sepharose conjugated with the Smith-degraded mucin. The enzyme thus purified by only three steps showed no other glycosidase or protease activities and had higher specific activity compared to the previous method. This new method has a great advantage since the gastric mucin is abundantly available and the efficiency of enzyme production was high without significant induction of exoglycosidase. The hydrolysis of oligosaccharides, glycosphingolipid, and keratansulfate was studied by using this newly purified enzyme. Kinetic data indicate that hydrolyzability of these substrates is largely affected by substrate concentration, enzyme concentration and the structure of substrates. Based on these results, the specificity of E. freundii endo-beta-galactosidase was discussed.