Characterization of lysosomal hydrolases that are elevated in Gaucher's disease.

Although Gaucher's disease occurs in three distinct forms with greatly varying degrees of severity, there is no correlation between the clinical course of the disease and levels of residual glucocerebrosidase, the fundamental enzymatic deficiency. In an effort to study secondary changes which might contribute to the pathology of Gaucher's disease, homogenates of spleen, liver, and brain tissue, as well as serum from patients with Gaucher's disease were analyzed for their content of a number of lysosomal enzymes. Extracts of 8 Gaucher spleens contained 3- to 4-fold increases in acid phosphatase activity as well as 5-to 10-fold increases in galactocerebrosidase⁵ activity. The marked elevation in galactocerebrosidase activity in Gaucher spleen was documented using various [³H]galactose labeled galactocerebrosides as substrates and with [³H]galactose labeled lactocerebroside under the “lactosylceramidase I”⁵ assay conditions established by Suzuki (Tanaka, H., and Suzuki, K., 1975, J. Biol. Chem., 250, 2324–2332) that measure galactocerebrosidase activity specifically in the presence of G_mi-ganglioside β-galactosidase. Acid phosphatase determinations using extracts of liver from a case of infantile, neuropathic Gaucher's disease revealed a 2-fold elevation in this activity, whereas brain acid phosphatase activity in this case was similar to that of control tissue. Separation of hexosaminidase A and B activities on DEAE-Sephadex columns indicated increases in both forms of the enzyme in Gaucher tissue with the major increase occurring in the hexosaminidase B component. Glucuronidase and nonspecific esterase were observed to be elevated approximately 2-fold. However, not all lysosomal enzyme activities were increased. Levels of splenic arylsulfatase A and B, α-arabinosidase, sphingomyelinase, α-mannosidase, and G_mi-ganglioside β-galactosidase activities in Gaucher spleen were unremarkable. G_mi-ganglioside β-galactosidase was measured using 4-methylumbelliferyl-β-d-galactopyranoside and [³H]galactose labeled lactocerebroside under the specific assay conditions described by Suzuki for the determination of “lactosylceramidase II” activity. Although levels of arylsulfatase A and B in Gaucher spleen were similar to those of control tissue, arylsulfatase A activity was markedly reduced (20% of control) in homogenates of brain from the case of infantile (type 2) Gaucher's disease. The metabolic and pathologic consequences of these changes in lysosomal enzymes in Gaucher's disease are discussed.     

Subcellular localization of the heat-stable glucocerebrosidase activator substance in Gaucher spleen

The spleen in Gaucher's disease contains relatively large quantities of a heat-stable activator of the glucocerebrosidase of normal human tissues (Ho, M. W., and O'Brien, J. S. (1971) Proc. Nat. Acad. Sci. USA68, 2810–2813) that has been shown to be an 11,000 molecular weight acidic glycoprotein (Peters, S. P., et al. (1977) J. Biol. Chem.252, 563–573). In an effort to determine the subcellular location of the activator, a mannitol-sucrose homogenate of fresh, unfrozen spleen obtained from a 26-year-old patient with adult, nonneuropathic (Type 1) form of Gaucher's disease was subjected to subcellular fractionation. The tissue used in these experiments exhibited a β-glucocerebrosidase deficiency (11% of control tissue characteristic of Gaucher's disease. Mitochondrial and lysosomal fractions obtained by centrifugation of the spleen homogenate at 6900 and and 20,000g, respectively, contained greater than 80% of the recovered acid phosphatase and heat-stable glucocerebrosidase activator activities. In addition, 60% of the residual glucocerebrosidase activity was recovered in the mitochondrial and lysosomal fractions. The lysosomal and mitochondrial fractions were subjected to equilibrium sucrose density gradient centrifugation. Analysis of the sucrose gradient of the crude mitochondrial fraction demonstrated the mitochondrial marker enzyme (cytochrome oxidase) banding with a specific gravity of 1.19 g/ml, whereas the heat-stable activating factor banded in an acid phosphatase-rich fraction having a specific gravity of 1.12 g/ml. Sucrose gradient analysis of the crude lysosomal fraction obtained from differential centrifugation indicated the activating factor banding with a specific gravity of 1.12 g/ml. Coincident with the activating factor was glucocerebrosidase and acid phosphatase activity. Electron microscopic examination of fractions from each of the sucrose density gradients demonstrated that the glucocerebrosidase activating factor was located in the same acid phosphatase-rich fractions that contained the characteristic Gaucher deposits. Furthermore, when Gaucher deposits were isolated and purified independently by a sucrose gradient procedure, they were found to contain high concentrations of the heat-stable glucocerebrosidase activator. The specific activity of the glucocerebrosidase activating factor was approximately 15-fold greater in the extensively purified Gaucher deposits than in the crude extract of Gaucher spleen from which the deposits were isolated. These observations indicate that the heat-stable activator is associated with the storage deposits contained in lysosomes of the Gaucher cell.     

Increased levels of sialic acid associated with a sialidase deficiency in I-cell disease (mucolipidosis II) fibroblasts.

Cultured fibroblasts from three unrelated patients with I-cell disease (mucolipidosis II) have a 3 to 4 fold increase in total sialic acid when compared to control fibroblasts. Sialic acid levels in a number of other lysosomal disorders, i.e., mucopolysaccharidosis I, II, III, VI, metachromatic leukodystrophy, G_M1 gangliosidosis, mannosidosis, Gaucher's and Sandhoff's disease are within the normal range suggesting that this is a finding specific for I-cells. Additionally, sonicates of cultured fibroblasts from controls were shown to have an acid sialidase capable of removing sialic acid from added fetuin at pH 4.2 in 0.05M acetate buffer. In contrast, I-cell fibroblasts, within the limits of the assay, lack this enzyme activity.     

Composition of the membranous deposits occurring in Gaucher's disease

We have isolated and purified low-density (1.08 g/ml) glucocerebroside-rich deposits from the spleen of a patient with Gaucher's disease. The organic phase obtained by chloroform-methanol (2:1) extraction of the deposits contains glucocerebroside, phospholipid, and free cholesterol in an approximate molar ratio of 12:3:2. The lipid-free residue accounting for approximately 12% of the dry weight of the deposits, contained protein and glycoprotein and accounts for the observation that the low-density deposits are significantly more dense than pure β-glucocerebroside (1.06 g/ml). The nonlipid residue contains galactose, hexosamine, and sialic acid and a PAS-staining glycoprotein with an approximate molecular weight of 95,000. This glycoprotein is extensively degraded by Pronase, but only when the structure of the deposits has been destroyed by organic solvent extraction. The nonlipid residue also contains several polypeptides with subunit molecular weights of 26,000–38,000.The deposits appear as a series of twisted bilayers, each 60 Å thick when prepared by freeze-etching methods. These bilayers are considered to be densely packed since staining methods visualize only the external surface.     

Substrate Compositional Variation with Tissue/Region and Gba1 Mutations in Mouse Models–Implications for Gaucher Disease

Gaucher disease results from GBA1 mutations that lead to defective acid β-glucosidase (GCase) mediated cleavage of glucosylceramide (GC) and glucosylsphingosine as well as heterogeneous manifestations in the viscera and CNS. The mutation, tissue, and age-dependent accumulations of different GC species were characterized in mice with Gba1 missense mutations alone or in combination with isolated saposin C deficiency (C*). Gba1 heteroallelism for D409V and null alleles (9V/null) led to GC excesses primarily in the visceral tissues with preferential accumulations of lung GC24∶0, but not in liver, spleen, or brain. Age-dependent increases of different GC species were observed. The combined saposin C deficiency (C*) with V394L homozygosity (4L;C*) showed major GC18∶0 degradation defects in the brain, whereas the analogous mice with D409H homozygosity and C* (9H;C*) led to all GC species accumulating in visceral tissues. Glucosylsphingosine was poorly degraded in brain by V394L and D409H GCases and in visceral tissues by D409V GCase. The neonatal lethal N370S/N370S genotype had insignificant substrate accumulations in any tissue. These results demonstrate age, organ, and mutation-specific quantitative differences in GC species and glucosylsphingosine accumulations that can have influence in the tissue/regional expression of Gaucher disease phenotypes.     

Hydrolytic and transglucosylation activities of a purified calf spleen beta-glucosidase.

Certain properties of the transglucosylitic activity and the hydrolytic activity of a purified calf spleen beta-glucosidase (beta-D-glucoside glucohydrolase EC 3.2.1.21) were investigated. There was a stimulation of both activities by sodium taurocholate and "Gaucher's factor". The K-m values for 4-methylumbelliferyl-beta-D-glucoside and glucosylceramide as donors in the transglucosylation reaction were 2 mM and 0.075 mM, respectively. The K-m for ceramide as acceptor was 0.149 mM with both of these compounds. The ability of several glucoside to act as donors was examined. The capacity to catalyze this "transglucosylation" reaction is greatly diminished in spleen tissue samples from Gaucher's patients. The enzyme possesses the capacity to hyrolyze 4-methylumbellifery-beta-D-glucoside, p-nitrophenyl-beta-D-glucoside, glucosylsphingosine, glucosylceramide and deoxycorticosterol-beta-D-glucoside. It is postulated that a single enzyme protein may be responsible for both the hydrolytic and the transglucosylitic activities.     

Purification of beta-glucosidase activities from bovine spleen by affinity chromatography

Bovine spleen β-d-glucosidase, glucosylceramide: β-d-glucosidase and glucosylsphingosine: β-d-glucosidase were purified by chromatography on a “gluconate” Sepharose column. Ten other lysosomal acid hydrolases, present in the preparation applied to the column, were absent from the glucosidase fraction.     

Effects of a protein-free diet on the DNA-synthesizing and dividing cells in the lymphoid organs of rats. Changes induced by phytohemagglutinin and cortisone invivo

Levels of ³H-thymidine-labeled lymphocytes on autoradiographs of cell smears and mitotic indices (M.I.) were determined in the popliteal lymph nodes (PLN), spleen and thymus of rats fed with a balanced or a protein deprived (PD) diet for 7 weeks. The latter diet reduced the number of labeled cells per 10³ in the spleen but not in the PLN's and the thymus. The M.I.'s were reduced 50% in the spleen and dropped drastically in the thymus. Subplantar injection of phytohemagglutinin (PHA) provoked a sharp increase in the M.I.'s of the PLN's and the spleen, and also moderately increased the labeling index in the PLN's in normal rats. In PD rats PHA no longer influenced the mitoses in the PLN's and the spleen while it considerably increased the proportion of labeled cells in the latter. These discrepancies between DNA-synthesis and mitoses in the thymus and the spleen suggest a premitotic block of the cell cycle after protein deprivation. The effects of a 5 day-cortisone treatment resembled in a large measure the changes induced by a protein-free diet including the arrest of the cell cycle. The thymic lymphocytes were more sensitive to the hormone in PD than in normal rats. On the contrary, in the PLN's and the spleen mainly cortisone-resistant cells remained alive among the DNA-synthesizing lymphocytes after protein deprivation. All these data agree with the hypothesis of an intervention of endogenous glucocorticoids in the protein deficiency-induced lymphoid involution.     

Selective Extraction and Effective Separation of Galactosylsphingosine (Psychosine) and Glucosylsphingosine from Other Glycosphingolipids in Pathological Tissue Samples

To facilitate the study of the chemical pathology of galactosylsphingosine (psychosine, GalSph) in Krabbe disease and glucosylsphingosine (GlcSph) in Gaucher disease, we have devised a facile method for the effective separation of these two glycosylsphingosines from other glycosphingolipids (GSLs) in Krabbe brain and Gaucher spleen samples. The procedure involves the use of acetone to selectively extract GalSph and GlcSph, respectively, from Krabbe brain and Gaucher spleen samples. Since acetone does not extract other GSLs except modest amounts of galactosylceramide, sulfatide, and glucosylceramide, the positively charged GalSph or GlcSph in the acetone extract can be readily separated from other GSLs by batchwise cation-exchange chromatography using a Waters Accell Plus CM Cartridge. GalSph or GlcSph enriched by this simple procedure can be readily analyzed by thin-layer chromatography or high-performance liquid chromatography.     

In vitro studies of the rabbit immune system. IV. Differential mitogen responses of isolated T and B cells.

The mitogenic responses of separated rabbit lymphocyte populations functionally analogous to mouse T and B cells have been tested in vitro. Purified T cells were prepared by passage over nylon wool (NW) and purified B cells prepared by treatment with antithymocyte serum and complement (ATS + C). ATS + C kills 70% of peripheral blood lymphocytes (PBL's) and 50% of the spleen cells while passage over NW yields 40% of the applied PBL's and 5–23% of the applied spleen cells. NW-purified T cells from the spleen or PBL's respond fully to concanavalin A (Con A) but have a reduced response to phytohemaglutinin (PHA) and little or no response to goat anti-rabbit immunoglobulin (anti-Ig). PBL's that survive ATS + C (B cells) are stimulated by anti-Ig but not by Con A or PHA. B cells purified from spleen do not respond to Con A or PHA but will respond to anti-Ig under appropriate conditions. A full spleen B-cell response to anti-Ig required removal of Ig produced by the cultures that blocked anti-Ig stimulation. It is concluded that, for rabbit lymphocytes, Con A and PHA are primarily T-cell mitogens and that anti-Ig is primarily a B-cell mitogen. However, the mitogen response of unfractionated PBL or spleen cell populations indicates an overlap in reactivity. This could be due to cells sharing T and B properties, alteration of cell populations by the fractionation procedures used, or recruitment of one population in the presence of a mitogenic response of the other population.     

Changes in the homing properties of labeled lymphoid cells caused by solid tumor growth

The effect of a progressively growing fibrosarcoma upon the distribution of ⁵¹Cr-labeled cells from the lymph nodes, spleen, thymus, bone marrow and Peyer's Patches was measured in tumor-bearing recipient mice. Tumor presence caused a uniform depression of migration of labeled cells to the bone marrow. In most cases increased homing of cells to the spleen was also observed. Labeled cells prepared from lymph nodes and Peyer's Patches were generally unaffected by the presence of a growing tumor. Migration of labeled cells from tumor bearing donors into normal syngeneic recipients suggests depletion or incapacitation of parts of the T-cell population of the spleen. These results emphasize the important relationship between splenic function and tumor progression.     

Substrate specificity of Gaucher spleen phosphoprotein phosphatase

The spleen in Gaucher's disease contains elevated levels of two distinct acid phosphatases. One of the isoenzymes, a tartrate-resistant type 5 acid phosphatase which we have designated SP_II acid phosphatase, possesses considerable phosphoprotein phosphatase activity. The enzyme dephosphorylates phosvitin and casein at specific rates (V) of 38.6 and 45.0 units/mg, respectively. The dephosphorylation of the oligophosphoproteins as well as various fragments of phosvitin, histories, and monophosphopeptides was studied kinetically. Positive cooperativity (Hill coefficient = 1.3–2.0) was observed for the dephosphorylation of phosvitin and casein as well as for the dephosphorylation of fragments of phosvitin which contained as few as two vicinal phosphoserine residues. In contrast, the hydrolysis of phosphomonoesters such as o-phosphorylserine or various monophosphopeptides exhibited typical Michaelis-Menten kinetics. Cooperativity appears to depend upon the substrate rather than the enzyme. The cooperativity of dephosphorylation was not affected by altering the secondary structure of phosvitin from a random to β conformation or by acetylation of the protein; however, acetylated phosvitin was dephosphorylated more rapidly (V = 50.8 units/mg) than native phosvitin indicating that the very basic phosphatase enzyme (pI = 8.5) prefers more acidic phosphoproteins as substrates rather than basic proteins such as histone (V= 0.0013 unit/mg). A monophosphohexa-peptide (V = 0.47 unit/mg) and monophosphoheptapeptide (V = 0.18 unit/mg) proved to be much poorer substrates than phosvitin, and monophosphoproteins such as glycogen phosphorylase, phosphorylase kinase, and glycogen synthase were not dephosphorylated by the enzyme. Although the phosphatase is active on monophosphopeptides and the presence of flanking amino acids considerably decreases the K_m of the enzyme for the phosphoserine residue (up to 100-fold), the enzyme appears to prefer peptide or protein substrates that contain two or more phosphoserine residues in close proximity. Finally, previous results showing the spleen phosphatase to be composed of 16,000- and 20,000-dalton subunits were apparently due to proteolysis during isolation since when 1.0 mm phenylmethylsulfonyl fluoride was included in the isolation media, the enzyme appeared as a single 35,000-dalton species when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.     

Acetylcholine and choline levels in post-mortem human brain tissue: Preliminary observations in Alzheimer's disease

Choline and acetylcholine were measured in necropsy brain tissue (temporal cortex and caudate nucleus) obtained from elderly, mentally normal hospital cases and established cases of Alzheimer's disease. ACh levels were as expected, extremely low in all cases; in cases with Alzheimer's disease, the ACh level was lower in the temporal cortex but not changed in the caudate compared with normal cases (matched for ages and post-mortem sampling delays). The level of choline in Alzheimer's disease was not significantly different from the normal in either brain region. The choline levels in the human material were, however, substantially and significantly lower than those obtained from young adult rat cerebral cortex which was cooled after death according to the post-mortem temperature decline in the human cadaver.     

B-Lymphocyte differentiation in lethally irradiated and reconstituted mice. II. Recovery of humoral immune responsiveness.

The recovery of humoral immune responsiveness was studied in lethally irradiated, fetal liver-reconstituted mice. By means of both membrane fluorescence and antibody formation to sheep red blood cells (SRBC) as a functional assay, the rate of recovery of the compartments of B and T lymphocytes was determined in various lymphoid organs. The recovery of the immunoglobulin-positive (B) cell compartment after irradiation and reconstitution started in the spleen. This organ was also found to be the first in which the recovery of the B-cell population was completed. The interval between the recovery of the B-cell population in the spleen and that in the other organs tested was found to increase when the irradiated mice were reconstituted with spleen colony cells instead of fetal liver cells. This proved to be caused by the number and nature of the reconstituting hemopoietic stem cells. The immunoglobulin-positive (B) cells were found to appear before SRBC-reactive B cells could be demonstrated in spleen, lymph nodes, and Peyer's patches. The appearance of T lymphocytes in the various lymphoid organs required even more time. By means of cell transfer experiments, a sequential appearance of the precursors of anti-SRBC IgM-, IgG-, and IgA-plaque-forming cells could be demonstrated in spleen, bone marrow, lymph nodes, and Peyer's patches.     

Decreased graft versus host reaction after intrahepatic lymphoid tissue implantation.

The ability of the liver to reduce the intensity of the graft versus host (GVH) reaction has been investigated in F₁ hybrid rats implanted with parental lymph nodes, spleen, thymus or Peyer's patches. Intrahepatic and intrarenal tissue implantations were compared using classical GVH criteria. The intrahepatic implantation of tissues well known to induce a GVH reaction suppressed the mortality observed after intrarenal implantation whereas the number of paravascular infiltrates (PVI) in the liver was increased. These results confirm the importance of portal drainage in organ transplantation and suggest a new site of implantation for lymphoid cells. These observations are compatible with the presence in the liver of blocking complexes and/or the existence of a splenohepatic suppressor axis.     

Tissue Iron Distribution Assessed by MRI in Patients with Iron Loading Anemias

Bone marrow, spleen, liver and kidney proton transverse relaxation rates (R2), together with cardiac R2* from patients with sickle cell disease (SCD), paroxysmal nocturnal hemoglobinuria (PNH) and non-transfusion dependent thalassemia (NTDT) have been compared with a control group. Increased liver and bone marrow R2 values for the three groups of patients in comparison with the controls have been found. SCD and PNH patients also present an increased spleen R2 in comparison with the controls. The simultaneous measurement of R2 values for several tissue types by magnetic resonance imaging (MRI) has allowed the identification of iron distribution patterns in diseases associated with iron imbalance. Preferential liver iron loading is found in the highly transfused SCD patients, while the low transfused ones present a preferential iron loading of the spleen. Similar to the highly transfused SCD group, PNH patients preferentially accumulate iron in the liver. A reduced spleen iron accumulation in comparison with the liver and bone marrow loading has been found in NTDT patients, presumably related to the differential increased intestinal iron absorption. The correlation between serum ferritin and tissue R2 is moderate to good for the liver, spleen and bone marrow in SCD and PNH patients. However, serum ferritin does not correlate with NTDT liver R2, spleen R2 or heart R2*. As opposed to serum ferritin measurements, tissue R2 values are a more direct measurement of each tissue’s iron loading. This kind of determination will allow a better understanding of the different patterns of tissue iron biodistribution in diseases predisposed to tissue iron accumulation.     

Mössbauer spectroscopy of the iron cores in human liver ferritin, ferritin in normal human spleen and ferritin in spleen from patient with primary myelofibrosis: preliminary results of comparative analysis

Comparative study of human liver ferritin and spleen tissues from healthy human and patient with primary myelofibrosis was carried out using Mössbauer spectroscopy with a high velocity resolution at 295 and 90 K and with a low velocity resolution at 20 K. The results obtained demonstrated that the iron content in patient’s spleen in the form of iron storage proteins was about ten times larger than that in normal tissue. However, in the case of patient with primary myelofibrosis the magnetic anisotropy energy barrier differed from that in normal case and, probably, the iron core size was supposed to be slightly larger than that in both normal spleen tissue and normal human liver ferritin in contrast to well-known data for iron overload in patients with thalassemia accompanied by the iron-core size increase. Therefore, the iron overload in the case of patient with primary myelofibrosis may be related to increase in the ferritin content mainly. It was also found that Mössbauer hyperfine parameters for normal and patient’s spleen and normal human liver ferritin demonstrated some small differences related, probably, to some small structural variations in the ferritin iron cores of patient’s spleen.     

Novel Procedure for Measuring Psychosine Derivatives by an HPLC Method

We developed a sensitive and simple method to determine galactosylsphingosine and glucosylsphingosine as a 4-fluoro-7-nitrobenzofurazan autofluorescent compound, using HPLC equipped with a Showdex sugar column. Amounts of galactosylsphingosine were successfully measured in the picomole range. This novel procedure is more stable and simpler than the previous method using o-phthalaldehyde. It was applied to tissues from the twitcher mouse, an animal model of human globoid cell leukodystrophy. The amount of galactosylsphingosine was 34-102 micrograms/kg of wet tissues in control cerebrum and cerebellum, whereas in twitcher mice the range was 2,251-4,228 micrograms/kg of wet tissues. The psychosine concentration was also increased in the liver and kidney of twitcher mice, respectively, 1,513 micrograms and 1,106 micrograms/kg of wet tissue (normal liver, 125 micrograms; normal kidney, 74 micrograms/kg of wet tissue). This novel procedure is useful for the pathochemical evaluation of lysosphingolipids in various sphingolipidoses as well as in other neuropathological and cellular conditions.     

Further evidence for non-T-cell regulation of delayed hypersensitivity in the guinea pig.

The nature of the suppressor activity in the spleens of guinea pigs immunized with dinitrophenyl-bovine γ-globulin in Freund's incomplete adjuvant was investigated. An anti-T-cell serum was prepared in rabbits and, after extensive absorption, showed specific killing for T-lymphocytes. After treatment with this antiserum and complement, spleen cells from animals immunized with the antigen in Freund's complete adjuvant showed marked reduction in ability to transfer sensitivity to normal recipients. However, when immune spleen cells, treated in the same way, were transferred into antigen immunized animals which had been pretreated with cyclophosphamide, the suppressor activity was unaltered. These results confirm earlier impressions that the regulation of delayed hypersensitivity reactions in the guinea pig is normally mediated by non-T-cells.