Susan R. Hollán

Acetylcholinesterase activity of lymphocytes: an enzyme characteristic of T‐cells

Acetylcholinesterase (AchE) activity of normal human peripheral blood lymphocytes was determined by an adaptation of the colorimetric and radiometric techniques described for other cells. The enzyme activity seemed to be correlated to the number of T‐lymphocytes in the blood. To decide whether only the T‐cells possess AchE activity the lymphocytes were separated on Leucopac filter and on Percoll density gradient. B‐lymphocytes had no detectable enzyme activity, while the T‐lymphocyte fraction represented the total activity measured in the unseparated sample. The majority of AchE activity could be demonstrated in T‐lymphocytes of lower density (TLD). The role of AchE in the plasma membrane of various blood cells is not known. Nevertheless, the enzyme is a good marker of the integrity and functional state of the membrane. The difference observed in AchE activity of the lymphocyte populations seems to be suitable for using it to characterize T‐lymphocytes.

Hereditary triosephosphate isomerase (TPI) deficiency: two severely affected brothers one with and one without neurological symptoms

A 13-year-old Hungarian boy (B.J. Jr.) with congenital haemolytic anaemia (CHA) and hyperkinetic torsion dyskinesia was found to have severe triose-phosphate isomerase (TPI) deficiency. One of his two brothers (A.J.), a 23-year-old amateur wrestler, has CHA as well, but no neurological symptoms. Both have less than 10% TPI activity and a highly increased dihydroxyacetone phosphate (DHAP) level in their red blood cells. Their TPI had a slow electrophoretic mobility and was heat unstable. Both parents and a third brother are healthy heterozygous carriers of the defect. A.J. represents a unique phenotype from the point of view that all published “homozygotes” had severe neurological alterations from infancy or early childhood except one infant who died at 11 months, probably too young for neurological symptoms to be noted. In contrast to the two affected Hungarian brothers all but one “homozygote” has died before the age of 6 years. The striking difference in the clinical course of the defect between the two brothers with the same severe red blood cell enzyme deficiency may originate from unusual differences between two double heterozygous brothers resulting inter alia in different levels of TPI expression in various tissues. Significantly lower TPI activities were found in both the T- and B-cells of the propositus as compared to the respective cells of the neurologically symptom-free brother.

Functional aspects of cellular microcompartmentation in the development of neurodegeneration: Mutation induced aberrant protein-protein associations

Research in the last 10 years has revealed that the development of neurodegeneration is a multistep process during which one or few specific mutant protein species of altered conformation initiate aberrant protein-protein interactions resulting in aggregates forming plaques. This review focuses on the heteroassociations of the mutant proteins with subcellular structures, such as cytoskeleton, cell membranes or with glycolytic enzymes, which may be crucial in the initiation of neurodegeneration such as in Huntingtons disease or Alzheimers disease. Triosephosphate isomerase enzymopathy is a unique glycolytic enzyme deficiency coupled with neurodegeneration. We present data on the mutation induced misfolding process, which likely plays a crucial role in the enhanced associations of the enzyme with the truncated fragment of the isomerase, with the red cell membrane or with the microtubular network. On the basis of our recent clinical and experimental results obtained with two compound heterozygote Hungarian brothers it became obvious that the mutations alone are not sufficient to explain the development of the neurological sympthomes. This underscores the fact that the mutations alone are not enough for the development of the clinical phenotype of a disease.

Neutralizing and complement-dependent enhancing antibodies in different stages of HIV infection

Reclustering and indirect immunofluorescence assays on MT-4 cells [carrying both CD4 and complement receptor type 2 (CR2)] were used to measure neutralizing and enhancing antibodies in sera obtained from HIV-1-infected individuals. Heat-inactivated sera were tested before and after mixing 1:1 with fresh seronegative human serum. Using heated samples, neutralizing antibodies were found in 20 out of 20 and 11 out of 19 serum samples of asymptomatic and symptomatic [AIDS, AIDS-related complex (ARC)] HIV-seropositive patients, respectively. In complement-restored samples, neutralizing activity was found in eight sera of asymptomatic patients and in none of the sera of AIDS and ARC patients; enhancing activity could be detected in four and 12 sera, respectively. A significant positive correlation was observed between the titres of neutralizing antibodies measured in the complement-restored samples and the absolute number of CD4+ lymphocytes. These findings indicate that the appearance of complement-dependent enhancing antibodies coincident with the loss of neutralizing antibodies may indicate a poor prognosis in HIV infection.

Structural characterizations of hemoglobins J-Buda [α 61 (E 10) Lys→Asn] and G-pest [α 74 (EF 3) Asp→Asn]

Abstract Two new mutant hemoglobins, each with abnormal α-chains, have been found along with normal adult hemoglobin in three brothers of a Hungarian family. Preliminary identification of the abnormal chains and genetic studies have been reported earlier (Hollan, S. R., Szelenyi, J. G., Brimhall, B., Duerst, M., Jones, R. T. Koler, R. D. and Stocklen, Z. (1972) Nature 235, 47–50). The present paper reports the structural characterizations of the two abnormal α-chains found in affected members of this family and of the normal α-chain from one of the brothers who also had the two abnormal hemoglobins. One abnormal hemoglobin, named J-Buda, has a substitution of an asparaginyl residue for the lysyl residue 61 (E 10) of its α-chain. The second abnormal hemoglobin, named G-Pest, has an asparaginyl residue for the aspartyl residue 74 (EF 3) of its α-chain. The amino acid compositions of all of the other tryptic peptides of the abnormal α-chains and of all of the tryptic peptides of the normal α-chain from one of the affected brothers were identical to compositions reported for tryptic peptides of normal human α-chains. We conclude that except for the substitution of single amino acid residues in the two abnormal α-chains, the compositions and probably the sequences of amino acid residues of the four gene products from two α-chain loci are identical, at least in this Hungarian family.

Blast Colony Forming Cell-Binding Capacity of Bone Marrow Stroma from Myelodysplastic Patients

A specific stroma function can be quantitatively assessed by counting the stroma‐adherent blast cell colonies (CFU‐BL) that are formed from normal plastic nonadherent mononuclear bone marrow cells (PNAMNC) after a short‐term coincubation (“panning”) with the preformed stromal layer. In order to obtain information of stroma function in myelodysplasia (MDS), the “CFU‐BL‐binding capacity” of stroma from normal bone marrow and from patients with MDS were compared. Stromal cell cultures were established from mononuclear bone marrow cells in microplate cultures cultured with or without 10−6 M hydrocortisone. CFU‐BL‐binding capacity was studied by counting blast colonies seven days after panning, and the results were expressed as CFU‐BL/103 PNAMNC. Normal marrow stromal layers bound CFU‐BL only if they were cultured with hydrocortisone, while MDS stromal layers also bound CFU‐BL in the absence of hydrocortisone. For further studies of the function of MDS stroma, the effect of growth factors (stem cell factor [SCF], G‐CSF, interleukin 3 [IL‐3] and their combinations) on CFU‐BL binding by normal or MDS stroma has also been compared. Twenty‐hour incubation of the stromal layers with a standard dose (100 ng/ml) of various hemopoietic growth factors (IL‐3 alone or in combination with SCF, G‐CSF alone or in combination with SCF) did not have any effect on CFU‐BL binding by normal marrow stroma, but increased the CFU‐BL binding by stromal layers from MDS bone marrow. These findings suggest that although stromal microenvironment in MDS is capable of supporting hemopoiesis, bone marrow stroma from MDS patients differs in some characteristics from the normal stroma.

Electron Microscope and High Resolution Autoradiographic Studies of the Erythroblasts in Haemoglobin H Disease

Summary Electron microscope studies of bone marrow from a patient with haemoglobin H (HbH) disease have revealed the presence of highly condensed branching intracytoplasmic inclusions within a proportion of the early and late polychromatic erythroblasts and reticulocytes. Intraerythroblastic inclusions of this type have not yet been described in any other haematological disorder and may well be a specific feature of HbH disease. Electron microscope autoradiographic studies of the marrow cells from the patient with HbH disease revealed that (1) active protein synthesis continues in inclusion‐containing erythroblasts and (2) there is a rapid precipitation of a considerable proportion of the newly synthesized free β chains onto the surfaces of preformed inclusions, presumably after conversion to HbH. These electron microscope and electron microscope autoradiographic findings are quite different from the findings previously reported in homozygous β thalassaemia using the same techniques.

Molecular form of human lymphocyte membrane-bound acetylcholinesterase

The membrane-bound acetylcholinesterase (AchE) from human peripheral blood lymphocyte gives only one symmetrical peak on sucrose density gradient centrifugation in the presence of Triton X-100 detergent, with the calculated sedimentation coefficient of 6.5 S. However, this dimeric form of AchE was converted to a monomeric 3.8 S form when treated with 2-mercaptoethanol and iodoacetic acid. The results are consistent with studies which have shown by sodium dodecyl sulfate gel electrophoresis that the enzyme is built up of two identical monomers inter-linked by disulfide bond(s). Under reducing conditions, revealed a single species of 70,000 molecular weight, whereas under non-reducing conditions, another species of 140,000 molecular weight of the AchE was found. Polyacrylamide gel electrophoresis indicated a single band with AchE activity in the presence of Triton X-100. In contrast, in the absence of the same detergent multiple band pattern could be observed. These results suggest that membrane-bound AchE enzyme is present in homogenous dimeric form on human lymphocyte membrane.

Intense, reversible aggregation of intramembrane particles in non-haemolyzed human erythrocytes A freeze-fracture study

Acridine orange, a strongly cationic, membrane-penetrating dye, caused intense aggregation of intramembrane particles in non-haemolyzed human erythrocytes at 5 mM concentration. Simultaneously with the particle aggregation, large, empty, intramembrane particle-free or particle-depleted vesicles were detached from the red cells. Washing the erythrocytes after Acridine orange treatment resulted in complete disaggregation of the intramembrane particles. Less cationic acridine dyes (9-aminoacridine, 5-aminoacridine and Quinacrine) caused much less conspicuous alterations. Rivanol (ethacrine lactate), on the one hand, caused intramembrane particle aggregation in human red cells as well as ribosome aggregation in rabbit reticulocytes when it was dissolved in lactate-buffered sucrose. On the other, Rivanol dissolved in phosphate-buffered saline failed to induce these alterations. Neuraminidase treatment had no effect on the intensity of Acridine orange-induced particle aggregation, but impeded disaggregation. Our results indicate that, in contrast to previous observations, intense and reversible clustering of intramembrane particles is certainly possible in non-haemolyzed erythrocytes. The intramembrane particle aggregation may be due primarily to perturbation of the inner red cell surface by strongly cationic dyes and the presence of sialic acid residues on the outer red cell surface seems to be essential for the reversibility of the process.

Acetylcholinesterase (AchE) activity of lymphocytes in chronic lymphoid leukemia (CLL)

The specific AchE (EC 3.1.1.7) activity of lymphocytes from the peripheral blood of normal donors was determined. On Leukopak filter the isolated T lymphocytes showed activity, whereas in stepwise Percoll gradient, population TLD displayed enzyme activity. The THD and B cells were inactive. [Szelényi J. G., Bartha E. & Hollán S. R. (1982) Br. J. Haemat. 50, 241]. A mixed cell population derived from CLL patients had significantly lower enzyme activity than normal. With the progress of B-cell proliferation AchE activity decreased in parallel with the number of T cells. The sp. act. of TLD population isolated from CLL patients was the same as that of normal donors whereas their B cells were inactive.