Neutral alpha-D-mannosidase activity in human granulocytes

The presence of neutral soluble alpha-D-mannosidase activity was shown in human granulocytes. For detection of the enzyme different methods were used: addition of stabilizing agents; sorption of acid alpha-D-mannosidase on concanavalin A-sepharose; inhibition of acid alpha-D-mannosidase; determination of neutral alpha-D-mannosidase in granulocytes of patients with inherited defect of acid alpha-D-mannosidase (mannosidosis). The specific activity of neutral alpha-D-mannosidase in granulocytes of donors calculated in nmol/min/mg of protein was near to the activity in lymphocytes. However the activity in granulocytes calculated in nmol/min/10(8) of cells was approximately 3 times lower than that in lymphocytes. The activity of neutral alpha-D-mannosidase in immature myeloid cells of a patient with chronic myeloid leukaemia was 10 times higher than in natural granulocytes of the same patient. This high activity may be in connection with the process of cell differentiation or the result of malignant transformation.     

ADP-ribosylation of nuclear proteins in normal lymphocytes and in low-grade malignant non-Hodgkin lymphoma cells

Normal lymphocytes and lymphocytes from patients with low-grade malignant non-Hodgkin lymphoma were isolated from blood by a Percoll gradient procedure. Absence of cell proliferation in both cell types was indicated by very low [3H]thymidine incorporation rates. Determination of endogenous protein-bound single ADP-ribose residues by a radioimmunoassay revealed that the leukemic cells had 2.5-times lower levels of the NH2OH-sensitive and a 4-fold lower amount of NH2OH-resistant ADP-ribose . protein conjugate subfractions, respectively, than normal lymphocytes. By contrast, "total" ADP-ribose transferase activity, as measured in homogenates or permeabilized cells in the presence of DNase, was two-times higher in leukemic cells, whereas activity determined in permeabilized cells in the absence of added DNase was practically identical in both cell types. The apparent discrepancy between ADP-ribose transferase activity and endogenous levels of protein-bound single ADP-ribose residues may be explained in part by an enzyme inhibitor present in normal human lymphocytes. NAD + NADH levels were decreased 2.5-fold in the leukemic cells. This decrease, however, does not explain the reduced levels of mono(ADP-ribose) . protein conjugates since the ratio of protein-bound single ADP-ribose residues to NAD is distinctly different in leukemic lymphocytes compared to normal lymphocytes.     

Plasma and intracellular levels of lactate dehydrogenase, phosphohexose isomerase and lysozyme activity in acute leukemia

Plasma and intracellular levels of lactate dehydrogenase (LDH), phosphohexose isomerase (PHI) and lysozyme activities were investigated in 20 patients with acute myelocytic leukemia (AML), 18 patients with acute lymphatic leukemia (ALL) and 10 patients with chronic myelocytic leukemia in blast transformation (CML/BT). Though the plasma levels of LDH and PHI in all patients with acute leukemia were elevated as compared to control persons there was no distinctive pattern which could be of use in the classification of acute leukemia. On the other hand the intracellular levels of these enzymes could be of value in classifying acute leukemia. The leukemic lymphoblasts were characterized by low levels of PHI and lysozyme as compared to leukemic myeloblasts or to normal lymphocytes (p less than 0.01). The LDH/PHI ratio is also significantly higher in leukemic lymphoblasts than in leukemic myeloblasts or in normal lymphocytes (p always less than 0.01). These characteristics might also be made use of in identifying the blasts of CML/BT als "lymphoid" or "myeloid" in corresponding cases.     

Characteristics of the cyclic nucleotide phosphodiesterases of normal and leukemic lymphocytes

The specific activity of cylic AMP phosphodiesterase and cyclic GMP phosphodiesterase of leukemic lymphocytes was 5–10-fold greater than that of purified normal lymphocytes or homogenates of spleen, thymus or lymph nodes of normal mice. This rise was demonstrable over a wide range of substrate concentrations. Both normal and leukemic lymphocytes contained a heat-stable, calcium-dependent activator of phosphodiesterase. However, the increased activity of phosphodiesterase in leukemic lymphocytes was not due to this protein activator since (a) phophodiesterase activity from these cells was not stimulated by this activator and (b) phosphodiesterase activity of leukemic lymphocytes was not inhibited by the calcium chelator, ethyleneglycol-bis-(β-aminoethylether)-N′,N′-tetraacetic acid, suggesting that the enzyme was not already maximally activated. A comparison of several other properties of phosphodiesterase from normal and leukemic lymphocytes showed that the enzymes have similar pH optima, similar stabilitis to freezing and thawing and similar sensitivities to inhibition by the phosphodiesterase inhibitors, chlorpromazine, papaverine and isobutylmethylxanthine. However, the subcellular distribution of the phosphodiesterases was different, and the phosphodiesterase of leukemic lymphocytes was significantly more resistant to heat than that of normal lymphocytes.Although no differences were found between the phosphodiesterases of normal and leukemic lymphocytes in their sensitivities to drugs, there were marked differences in drug sensitivity between the phosphodiesterase of lymphocytes and that of other tissue. For example, concentrations of chlorpromazine which inhibited phosphodiesterase of cerebrum by 70% had no effect on phosphodiesterase activity of lymphocytes. On the other hand, the papaverine-induced inhibition of phosphodiesterase was similar in lymphocytes and cerebrum.Since an optimal concentration of cyclic nucleotides is essential to maintain normal cell growth, these results suggest that the abnormal growth characteristics of leukemic lymphocytes may be explained by their high activity of phosphodiesterase. Furthermore, the qualitative and quantitative differences between the phosphodiesterases of leukemic lymphocytes and other tissues raise the possibility of selectively inhibiting the phosphodiesterase of the leukemic lymphocytes, thereby reducing their rate of growth, without affecting other tissues.     

Effects of mitogenic stimulation on lymphocyte alpha-D-mannosidases

Three types of alpha-D-mannosidase are present in human and murine lymphocytes. Their levels increased substantially when the cells were activated by T-cell mitogens, concanavalin A (Con A) and phytohaemagglutinin (PHA), and in the murine cells also by lipopolysaccharide (LPS), a B-cell mitogen. The intracellular localization of the alpha-D-mannosidases in the non-stimulated and activated murine cells was investigated by fractionation of lymphocyte lysates on colloidal silica (Percoll) and discontinuous sucrose gradients. In both types of cell, an enzyme having optimal activity at neutral pH was obtained in the cytosolic fraction and another alpha-D-mannosidase most active at an intermediate pH was obtained partly in membrane-bound form. In contrast, an acidic alpha-D-mannosidase, which was particularly elevated in the activated murine spleen cells, had a distribution in these lymphoblasts which was markedly different from that in non-stimulated lymphocytes. In the latter, the major proportion of the activity was obtained in a cytosolic fraction and the remainder in a particulate fraction of light density, whereas the enzyme in activated lymphocytes was distributed between vesicles of light and heavy density comparable with lysosomal organelles. Moreover, the acidic alpha-D-mannosidase still remained membrane bound even when cell lysates were prepared under hypotonic conditions which disrupt lysosome integrity. These results suggest that lymphocyte activation involves either stabilization of fragile lysosomes present in resting cells or de novo synthesis of lysosome-like structures. The acidic alpha-D-mannosidase present within isolated, intact lysosomes was found to be in a form, A, whereas a different form, B, was most prominent in whole-cell extracts of both types of lymphocyte.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alteration of beta-hexosaminidase activity and isoenzymes in human leukemic cells

beta-Hexosaminidase (EC 3.2.1.20; Hex) activity and isoenzyme characteristics were analyzed in human normal and leukemic leukocytes. Unseparated CLL and CML cells had a specific activity that was lower, whereas ALL and AML blasts had a higher specific activity than normal lymphocytes and granulocytes. CLL B-cells had a lower specific activity compared with that in normal non-T-lymphocytes; CLL T-cells and normal T-cells had similar activity. Isoenzyme separation was performed by chromatofocusing on PBE-94 coupled with an automated enzyme assay. When using a single linear pH elution gradient, normal leukocytes and all leukemia cells contained two forms of isoenzyme (B and A). When a double pH elution gradient was performed, an extra distinct form of Hex (I) was recorded. Hex I was present in small amounts in normal granulocytes and PHA-stimulated normal lymphocytes; isoenzyme I was found in high amounts in all leukemias tested. The activity ratios I/B and I/A, as well as the I isoenzyme profile, may facilitate differentiation between normal and leukemic cells and between lymphoblastic and myeloblastic leukemias.     

Influence of mature and immature myeloid cells on lymphocyte reactivity in chronic myelocytic leukemia

Summary Earlier studies have shown anergy in chronic myeloid leukemia (CML), and it is known that myeloid cells influence lymphocyte responses. Therefore, lymphocytes from CML patients who had received no cytostatics for 2 weeks were stimulated in 89 tests with PHA and ConA. In 39 control tests, normal lymphocytes were used.Lymphocytes from CML patients were significantly less (p<0.05) markedly stimulated than normal ones. Lymphocytes from CML patients with more than 10×10⁹ white blood cells (WBC) per liter blood were inhibited to a greater degree than those from patients with a normal WBC count.When normal lymphocytes were stimulated with PHA in the presence of mononuclear cells from the blood of CML patients (mostly leukemic myelocytes), their response was significantly (p<0.05) inhibited. Inhibition with leukemic myelocytes was significantly (p<0.05) greater than that with mature granulocytes from CML patients. The latter did not seem to have an inhibitory effect.We suggest that patients with manifest CML are anergic to some extent because leukemic myelocytes have a suppressor effect.Visiting scientist and Anna Villa Rusconi Fellow, on secondment from Institute of Medical Pathology, University of Ferrara, Italy     

Cell contact induces the synthesis of a lysosomal enzyme precursor in lymphocytes and its direct transfer to fibroblasts

The activity of a lysosomal enzyme, alpha-D-mannosidase (EC 3.2.1.24), increased markedly in normal lymphocytes when they were cultured together with fibroblasts from a patient with an inherited deficiency of this enzyme. Cell-to-cell contact was obligatory for this increase in activity, which also required new protein synthesis. The enzyme induced in the co-cultured lymphocytes was a high molecular weight form of alpha-D-mannosidase that was not detected in lymphocytes cultured alone, which had only the low molecular weight mature enzyme. It was this precursor form alone that was directly transferred to the mannosidosis fibroblasts, where it was present initially in organelles of low density. When the culture period was extended the lymphocyte precursor enzyme was transported to the heavy lysosomes in the recipient cells, and correctly processed to the functionally effective mature enzyme.     

Characterization of Low Density Lipoprotein Receptors in Freshly Isolated Leukemic Guinea Pig Lymphocytes (L2C)

Abstract

The present study shows that L₂C leukemic guinea pig lymphocytes have 10 times as many low density lipoprotein (LDL) receptors per cell as normal lymphocytes. The affinity of these receptors is higher for guinea pig LDL than for human LDL. In contrast to normal cells, in which the degradation of the receptor-bound LDL is quite efficient, the leukemic cells only degraded a small fraction of these same receptor-bound LDL. Thus, the internalization index was nearly 4 times higher in the normal cells than in the leukemic cells.

In L₂C cells, cholesterol homeostasis derived 38% of its cholesterol input from receptor-mediated degradation of LDL and 62% from cholesterol synthesis, whereas in normal cells, these fractions were 97% and 3% respectively     

Cytoagglutination and cytotoxicity of Wheat Germ Agglutinin isolectins against normal lymphocytes and cultured leukemic cell lines--relationship between structure and biological activity

The relationships between degree of lectin-cell binding, cytotoxicity and cytoagglutinating activity of three Wheat Germ Agglutinin isolectins (WGA-1, WGA-2, WGA-3) against normal lymphocytes and cultured leukemic cell lines (Jurkat, MOLT-4, Raji, Daudi, K-562) were studied. All WGA-isolectins interacted in a similar degree with normal lymphocytes, while in the case of leukemic cells, the degree of isolectin-cell binding increased in the order: WGA-1< or =WGA-3相似文献   

Quantitative parameters of the IgG binding by Fc receptors and the functional activity of blood lymphocytes in healthy cattle and cattle with chronic lympholeukemia

The binding of IgG to lymphocyte Fc receptors in the blood of healthy cattle and of cattle with chronic lympholeukemia has been studied by fluorometric techniques before and after the incubation of lymphocytes in a serum-free medium at 37 degrees C. The study has shown that changes in the intensity of binding of IgG to Fc receptors of normal and leukemic lymphocytes correlate with changes in the cell-mediated cytotoxic activity of the corresponding lymphocytes. Lower cell-mediated cytotoxic activity of leukemic lymphocytes in comparison with that of normal lymphocytes was parallelled by a lower association constant of IgG with leukemic lymphocytes Fc receptors.     

Purification and properties of major alpha-D-mannosidase in the luminal fluid of porcine epididymis.

A lysosomal type alpha-D-mannosidase was successfully purified by DEAE-Sephacel, Red-Amicon and Superdex 200 column chromatographies from porcine cauda epididymal fluid. The purified enzyme consisted of 63 and 51 kDa subunits at equimolar amounts. It cleaved alpha1-2 linked mannosyl residues and less but significantly cleaved alpha1-3 and alpha1-6 linked mannosyl residues in the high-mannose oligosaccharides. The optimal pH to hydrolyze oligosaccharide was in the acidic pH range (pH 3.5 approximately 4.0).Total alpha-D-mannosidase activities in the porcine epididymal fluid increased from proximal to distal caput epididymis, which maintained to cauda epididymis. At least two kinds of alpha-D-mannosidase (lysosomal type enzyme and 135 kDa alpha-D-mannosidase (MAN2B2)) were contained in the porcine epididymal fluid. The activity of the lysosomal type enzyme is much higher than MAN2B2 at the physiological pH.These results suggest that the lysosomal type alpha-D-mannosidase is the predominantly active enzyme in the luminal fluid of porcine epididymis and that it participates in the glycoprotein modification on the sperm surface during epididymal transit.     

Lymphocytes transfer only the lysosomal form of alpha-D-mannosidase during cell-to-cell contact

We have examined the changes in the activities of the different types of alpha-D-mannosidase when fibroblasts from patients deficient in the lysosomal form of the enzyme are cultured together with normal lymphocytes. Our results show that whereas the mannosidosis cells acquired high levels of this enzyme, the activities of both the Golgi and the endoplasmic reticulum forms of alpha-D-mannosidase remained the same as in the fibroblasts cultured alone in the absence of lymphocytes. The increase in the activity of the lysosomal enzyme in the cocultured fibroblasts was not affected by the presence of mannose 6-phosphate or alpha-methyl mannoside, inhibitors of receptor- and lectin-mediated uptake of lysosomal enzymes, respectively, but it did require cell-to-cell contact. Ion-exchange HPLC and electrophoresis in polyacrylamide gradient gels showed that the acquired enzyme had the same elution profile and molecular size as the lysosomal form of the enzyme present in the lymphocytes. Immunoprecipitation studies using antibody specific for the lymphocyte type of lysosomal alpha-D-mannosidase confirmed that the increased activity in the cocultured mannosidosis cells resulted from the acquisition of the lymphocyte enzyme. Cytochemical examination revealed, however, that the transferred lymphocyte enzyme was localized in cytoplasmic organelles in the peripheral regions of the recipient fibroblasts. These results show that lymphocytes transfer only the lysosomal form of alpha-D-mannosidase during cell-to-cell contact with mannosidosis cells.     

Studies on the production of anti-human-lymphocyte serum (ALS) in bulls]

The applicability of bulls as productive animals was considered for the preparation of anti-humans ALS. The course of immunologic response was studied by lymphoagglutination, lymphocytotoxicity, rosette inhibition, hemagglutination tests and by precipitin formation in two experimental groups immunized by different amounts of lymphocytes from peripheral blood of normal donors. The animals were found to respond well already after the second application of very small amounts of antigen (on day 0-4 times 10(7), on day 21-2 times 10(8) lymphocytes). They showed lymphoagglutination titre 1 : 512-2000, lymphocytotoxic titre being higher than 1 : 4000 and the rosette inhibition test gave a minimum titre of 1 : 65000. On the other hand, further application of a high amount of antigen (2 times 10(9), or 4 times 10(9) lymphocytes) did not lead to further increase in the titre; on the contrary - hyperimmunization resulted in a lower titre in the case of the rosette inhibition test, which is known to correlate best with the in vivo immunosuppressive activity. The hemagglutinin titre was also acceptable under the above conditions and the formation of undersirable precipitins against human serum proteins was negligible. Good response reached by a simple and economical immunization scheme speaks for the suitability of bulls for the production of ALS.     

Influence of the donors' clinical status on in vitro and in vivo tumor-cytotoxic activation of interleukin-2-exposed lymphocytes and their circulation in different organs

Summary In-vitro-generated lymphokine-activated killer (LAK) cells of BALB/c mice, bearing the syngeneic colon carcinoma C-26 for 7 days, were as efficient as those from normal mice in lysing C-26 cells whereas LAK cells from 14-day tumor-bearing and 5- and 14-day tumor-resected animals had a lower C-26 cytotoxicity. The level of C-26 lysis returned to normal values 30 days after surgery. To identify the best source of LAK cells in vivo, groups of normal mice were treated with 10⁴, 3×10⁴ or 10⁵ U/day of interleukin 2 (IL-2) for 7 days intraperitoneally (i. p.) or intravenously (i. v.) (3×10⁴ dose only). The highest lysis on C-26 was obtained from peritoneal exudate cells of mice given 3×10⁴ and 10⁵ U whereas spleen cells were lytic only when taken from mice treated with 10⁵ U IL-2. Peripheral blood lymphocytes lacked any cytotoxicity except for the group of mice which received IL-2 i. v. The kinetics of in vivo LAK activation in different organs showed a peak of anti-(C-26) lytic activity at day 5 in peritoneal exudate cells and spleen cells of mice given IL-2 for 5 days whereas administration of LAK cells alone had no effect; IL-2 plus LAK cells gave a lower peak of LAK activity as compared with IL-2 alone. A lower level of in vivo LAK activation was found in mice whose tumor was resected 5 days before; such impairment was evident even 14 days after surgery. Homing experiments were carried out with i. v. injected ⁵¹Cr-labelled LAK cells in normal or tumor-resected mice. In normal mice the highest radioactivity at 30 min was found in the lungs; liver and spleen also showed high radioactivity whereas blood had a negligible amount of radioactivity. Radioactivity declined rapidly in lungs (less than 10% after 24 h) while remaining at appreciable levels in the liver after 24 h and 48 h; spleen showed constant levels of 12%–15%. Homing of LAK cells was altered in mice receiving IL-2 i. p. for 5 days with slower and lower radioactivity peaks in the lung and higher levels in liver. In tumor-excised mice lower levels of radioactivity were found in lungs. These results show that: (a) alterations in LAK activity occur in early-tumor-resected and large-tumor-bearing animals; (b) the route of IL-2 administration is critical in LAK activation in vivo; (c) treatment with IL-2 modifies LAK homing.This study was in part supported by grant no. 87.01565.44 of the Finalized Project Oncology of CNR (Rome, Italy)     

Effect of cortisol on the ultrastructure of normal, leukemic, and cultured human lymphocytes.

The effect of cortisol on the ultrastructure of normal, leukemic, and cultured human lymphocytes during a 2-hr incubation was investigated. The presence of 10(-5) M cortisol in the incubation medium produced in normal lymphocytes a variety of alterations in cytoplasmic organelles. Mitochondria were most affected and showed evidence of irreversible deterioration (formation of myelin figures). Occasional cells demonstrated an overt rearrangement of their cytoplasmic membranes resulting in a bizarre array of parallel cisternae-like structures. More commonly, the usually underdeveloped Golgi of normal lymphocytes became very pronounced in structure. All of these alterations were produced within 2 hr of incubation, but only in normal human lymphocytes. Under identical conditions, no evidence of ultrastructural changes were produced by cortisol in either lymphocytes from chronic lymphocytic leukemic patients, or those from the RPMI 1788 cell line.     

Stimulation of sterol and DNA synthesis in leukemic blood cells by low concentrations of phytohemagglutinin

The response of leukemic cells from AKR/J mice to phytohemagglutinin (PHA) was compared with that of normal lymphocytes. PHA stimulated first cholesterol synthesis and then DNA synthesis in both lymphocytes and leukemic cells. The neoplastic cells were, however, much more sensitive to PHA, requiring less time and a lower concentration of the lectin for optimum stimulation as compared to lymphocytes. In fact, the amount of PHA which was required to activate lymphocytes to proliferate, as measured by increases in sterol and DNA synthesis, was inhibitory to leukemic cells. The basal level of cholesterol synthesis and the induction of cholesterol synthesis following PHA activation were depressed in lymphocytes and leukemic cells by treatment with 25-hydroxycholesterol and 7-ketocholesterol. These two oxygenated derivatives of cholesterol are known to be potent and specific inhibitors of sterol synthesis. Blockage of sterol synthesis by these reagents also abolished PHA-activated DNA synthesis in lymphocytes and leukemic cells. The results support the hypothesis that the synthesis of cholesterol is an important event leading to cell proliferation.     

Substrate specificities of rat kidney lysosomal and cytosolic alpha-D-mannosidases and effects of swainsonine suggest a role of the cytosolic enzyme in glycoprotein catabolism

Swainsonine is a potent inhibitor of lysosomal alpha-D-mannosidase, causes the production of hybrid glycoproteins, and is reported to produce a phenocopy of hereditary alpha-mannosidosis. We now report that the effects of swainsonine administration in the rat are different in two respects from those found in other animals thus far studied. Swainsonine caused the accumulation of oligosaccharide in kidney and urine but not in liver or brain. The accumulated oligosaccharides were mainly Man(alpha 1-3)[Man(alpha 1-6)]Man(beta 1-4)GlcNAc, Man(alpha 1-3)[Man(alpha 1-6)[Man(alpha 1-3)]Man(beta 1-4) GlcNAc, and Man(alpha 1-3)[Man(alpha 1-6)]Man(alpha 1-6)[Man(alpha 1-3)]Man(beta 1-4)GlcNAc. Analogous branched Man4 and Man5 structures are found in pig and sheep tissues, but they are N, N'-diacetylchitobiose derivatives. The substrate specificities of rat kidney lysosomal and cytosolic alpha-D-mannosidases were investigated because in one type of hereditary alpha-mannosidosis, that occurring in man, the major storage products are linear rather than branched oligosaccharides. The lysosomal enzyme showed much greater activity toward linear oligosaccharides than toward the branched oligosaccharides induced in the kidney by swainsonine. On the other hand, cytosolic alpha-D-mannosidase preferred the branched oligosaccharides, a result suggesting that this mannosidase might be inhibitable by swainsonine and that the enzyme might play a normal role in glycoprotein catabolism. Swainsonine was indeed found to inhibit this enzyme at relatively high concentrations (I50 at 100 microM swainsonine), and concentrations of this magnitude were in fact found in the cytosol of kidney of swainsonine-fed rats. The kidney cytosolic alpha-D-mannosidase levels were reduced in these rats and, more important, the accumulated oligosaccharides were present mainly in the cytosol rather than in lysosomes. These results point to possible involvement of cytosolic alpha-D-mannosidase in glycoprotein degradation in the rat.