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Biomedical subjects

D Roos

Publications and source records attributed to D Roos.

At least 235 records · Page 13Linked to original sources
PubMed1988

The 47-kDa protein involved in the NADPH:O2 oxidoreductase activity of human neutrophils is phosphorylated by cyclic AMP-dependent protein kinase without induction of a respiratory burst.

When human neutrophilic granulocytes are stimulated with chemoattractants or phorbol esters, these cells respond with a so-called respiratory burst: such stimuli induce the activation of a NADPH:O2 oxidoreductase, which converts oxygen into superoxide. This activation coincides with the phosphorylation of a number of proteins, amongst which a 47-kDa phosphoprotein. Neutrophils from patients with the autosomal form of chronic granulomatous disease (CGD) fail to mount a respiratory burst and concomitantly lack phosphorylation of the 47-kDa protein. We have shown this protein to be a substrate for protein kinase C. In the present paper we describe the phosphorylation of the 47-kDa phosphoprotein by cyclic AMP-dependent protein kinase. For these studies, we used neutrophil cytoplasts, i.e., neutrophils devoid of nucleus and granules, but with an intact NADPH:O2 oxidoreductase. Addition of dibutyryl cyclic AMP (Bt2cAMP) to intact human neutrophil cytoplasts resulted in an increase in protein phosphorylation. Among the phosphorylated proteins is a 47-kDa phosphoprotein. Increased protein phosphorylation was also observed upon addition of Bt2cAMP to neutrophil cytoplast lysates. In lysates of neutrophil cytoplasts from patients with the autosomal form of CGD, phosphorylation of the 47-kDa protein was absent. This finding (confirmed by analysis on two-dimensional gels) indicates that the 47-kDa phosphoprotein, relevant for the NADPH:O2 oxidoreductase, is a substrate for the cAMP-dependent protein kinase. Unlike phorbol ester-induced phosphorylation, Bt2cAMP-induced phosphorylation is not accompanied by initiation of a respiratory burst. This observation demonstrates that 47-kDa phosphoprotein phosphorylation can be uncoupled from respiratory burst activity and indicates that other modifications of the NADPH:O2 oxidoreductase are required for induction of activity.

Bucladesine↗
PubMed1988

The PI-linked receptor FcRIII is released on stimulation of neutrophils.

Human phagocytic cells express receptors for the constant (Fc) region of immunoglobulin G. Neutrophils carry Fc receptor II (FcRII; CDw32) and FcRIII (CD16) which both bind IgG-containing immune complexes, leading to phagocytosis of the complex and activation of the neutrophil. We find that patients with paroxysmal nocturnal haemoglobinuria (PNH) have only about 10% of the normal levels of FcRIII on their neutrophils, whereas the expression of FcRII is unaffected. We show that FcRIII is a phosphatidyl inositol (PI)-anchored protein in neutrophils. Analysis of FcRIII expression in cells of PNH patients, known to be deficient in PI-linked proteins, suggests FcRIII is not PI-linked in monocytes. We find that the synthesis of FcRIII in neutrophils from PNH patients appears normal, indicating that the defect lies in the PI linkage. This lipid linkage of the receptor on neutrophils suggests that its release may be important for its function, and indeed FcRIII release was observed on stimulation of neutrophils by an inflammatory bacterial peptide (f-Met-Leu-Phe), suggesting a role for FcRIII shedding in inflammatory reactions. Activation of the PNH neutrophils with IgG-coated latex beads appeared normal (although binding of dimer IgG complexes was reduced), indicating that FcRII, rather than FcRIII, is involved in neutrophil stimulation.

Hemoglobinuria, Paroxysmal↗
PubMed1988

Purified protein kinase C phosphorylates a 47-kDa protein in control neutrophil cytoplasts but not in neutrophil cytoplasts from patients with the autosomal form of chronic granulomatous disease.

The neutrophil activators phorbol 12-myristate 13-acetate (PMA), formyl-methionyl-leucyl-phenylalanine, serum-treated zymosan, and IgG-coated latex cause an increase in protein phosphorylation in human neutrophil cytoplasts, concomitantly with an increase in oxygen consumption. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, phosphorylation was apparent in many proteins, must abundantly in 42-, 47-, 50-, 60-, and 80-kDa proteins. In neutrophil cytoplasts from autosomal chronic granulomatous disease (CGD) patients that were stimulated with PMA, the phosphorylation of a 47-kDa protein is absent. The localization of this protein in PMA-activated control cytoplasts is mainly in the cytosol and, to a lower and more variable extent, in the membrane. After addition of purified protein kinase C to lysates of nonstimulated control cytoplasts, phosphorylation occurred at the 47-kDa level in both the cytosol and the membrane fraction. With lysates of autosomal CGD cytoplasts, in vitro phosphorylation of the 47-kDa protein was completely absent. After separation of cytoplast proteins on a sodium dodecyl sulfate-polyacrylamide gel and excision of the 47-kDa protein(s), phosphorylation of the isolated 47-kDa band was observed in the presence of purified protein kinase C. This reaction was again absent when autosomal CGD cytoplasts were used as starting material. Our studies have identified the 47-kDa protein in neutrophil cytoplasts as a true substrate for protein kinase C and indicate that the defect in phosphorylation at the 47-kDa level in autosomal CGD cytoplasts is due to a defective protein.

Chromosome Aberrations↗
PubMed1988

Preparation of leukocyte-poor platelet concentrates from buffy coats. III. Effect of leukocyte contamination on storage conditions.

To study the influence of contaminating leukocytes on the storage conditions of platelet concentrates (PC), various amounts of leukocytes were added to identical PC. From 12 blood donations, 12 leukocyte-poor PC were prepared and pooled. Subsequently, the pool was divided into 12 identical PC. The plasma volume of the PC was 58.6 +/- 0.6 ml, the platelet concentration was 1.01 +/- 0.04 x 10(9)/ml (mean +/- SD) and the red cell contamination did not exceed 10(7) per PC. To 4 groups of 3 PC, pooled leukocytes were added from the same 12 blood donations. The leukocyte contamination for each group of 3 PC was 0.14 +/- 0.05, 1.96 +/- 0.09, 5.53 +/- 0.98 and 13.0 +/- 0.93 x 10(6)/ml (mean +/- SD) for groups I-IV, respectively. The PC were stored for 7 days at 22 degrees C in normal polyvinylchloride bags. A significant correlation was found between increasing concentrations of leukocytes in the PC and the drop in pH (r = -0.93), glucose consumption (r = -0.91), lactic acid production (r = 0.93) and release of lactate dehydrogenase (r = 0.92) during storage of the PC. The excretion of beta-thromboglobulin, depletion of platelet adenine nucleotides, decreased ability to incorporate 3H-adenosine into metabolic nucleotides and poor morphology of the platelets were also significantly correlated with an increased number of leukocytes in the PC. These data show that high concentrations of leukocytes in PC have a significant detrimental effect on the viability of platelets during storage at 22 degrees C. We conclude that for good storage conditions of PC, the upper limit of leukocytes per PC should not exceed 10(7).

Blood Platelets↗
PubMed1988

Immunocytochemical discovery of the 22- to 23-Kd subunit of cytochrome b558 at the surface of human peripheral phagocytes.

A monoclonal antibody raised against cytochrome b558 reacted specifically with the 22- to 23-Kd protein, the small subunit of this cytochrome. Cytochemical studies showed that the epitope was located on the surfaces of human neutrophils and monocytes. The small subunit of cytochrome b558, therefore, was expressed at least in part on the outer surface of these cells.

Antibodies, Monoclonal↗
PubMed1988

Detection of carriers of the autosomal form of chronic granulomatous disease.

The NADPH:O2 oxidoreductase catalyzing the respiratory burst in activated phagocytes from healthy individuals is not operative in phagocytes from patients with chronic granulomatous disease (CGD). In a microscopic slide test using the dye nitroblue tetrazolium (NBT), carriers of X-linked CGD can be recognized by a mosaic pattern of NBT-positive and NBT-negative cells, governed by the expression of an unaffected or an affected X chromosome, respectively. Until now, it has not been possible to detect carriers of the autosomal form of CGD (other than by family studies) because all cells of these carriers stain positive in the NBT test. We have investigated whether neutrophils from carriers of autosomal CGD can be recognized by measurement of the rate of oxygen uptake upon stimulation of the cells. It was found that with the phorbol ester PMA as a stimulus, the respiratory burst is significantly lower in autosomal CGD carriers. With serum-treated zymosan as a stimulus, no difference between controls and carriers was observed. The addition of f-Met-Leu-Phe (1 microM) to PMA-activated neutrophils of control donors caused a transient increase in oxygen consumption of about 40%. Under these conditions, an increase of more than 100% was observed in neutrophils from carriers of autosomal CGD. These findings provide a simple method for the detection of carriers of the autosomal form of CGD.

Genetic Carrier Screening↗
PubMed1987

Biochemical consequences of 2'-deoxycoformycin treatment in a patient with T-cell lymphoma. Some unusual findings.

The nucleotide content of the various blood cells and the urinary excretion of purine and pyrimidine metabolites were studied in a patient with a T-cell lymphoma (early T-cell phenotype) before and during treatment with deoxycoformycin (dCF; given intravenously [iv] during 3 days, biweekly). During and after the administration of dCF, high amounts of dATP were found in the lymphoid cells and the erythrocytes (maximally, 480 pmol/10(6) lymphocytes and 5.5 nmol/10(6) erythrocytes), but not in the polymorphonuclear leukocytes. The amount of dATP in the erythrocytes, however, was significantly lower than described in the literature. During each administration of dCF, the number of blast cells in the peripheral blood rose initially, followed by a rapid decrease. After three courses, a hematologic remission was achieved and maintained for 6 weeks; then an autologous bone-marrow transplantation was performed. During the first dCF course a large amount of deoxyadenosine was found in the urine. During the second course, this excretion was much lower, but still higher than in healthy individuals. In the patient described, dCF showed a highly specific toxicity for the immature T-lymphoblast; hardly any changes were seen in the numbers of the other hematopoietic cells, both in the blood and in the bone marrow.

Child↗
PubMed1987

Aberrant ribonucleotide pattern in lymphoid cells from patients with chronic lymphocytic leukaemia or non-Hodgkin lymphoma.

The intracellular purine and pyrimidine ribonucleotide concentrations were determined in lymphoid cells from peripheral blood of 16 patients with chronic lymphocytic leukaemia (CLL) and from peripheral blood and/or lymphoid tissue of 18 patients with non-Hodgkin lymphoma (NHL). Compared to normal peripheral lymphocytes, the lymphoid cells from CLL patients contained lower, and those from NHL patients higher amounts of nucleotides. The lymphoid cells of NHL patients showed an imbalance in the nucleotide pool compared to either normal resting peripheral or proliferating tonsillar lymphocytes. The lymphoid cells of patients with CLL showed an imbalance only when compared to normal, resting peripheral lymphocytes. The abnormalities in the nucleotide pools involved decreased ratios of purine:pyrimidine, adenine:guanine and uracil:cytosine nucleotides. Lymphocytes from CLL and NHL patients contained increased amounts (relative and/or absolute) of UDP sugars, and NHL lymphocytes also showed a changed composition of the UDP sugars. Analysis of the ribonucleotides in the lymphoid cells provides useful information for the differential diagnosis of patients suspected of having CLL or NHL, and may be valuable for the design of new chemotherapeutic regimens.

Adenosine Diphosphate↗
PubMed1987

Synergism between eosinophil cationic protein and oxygen metabolites in killing of schistosomula of Schistosoma mansoni.

To study the cytotoxic reactions responsible for mediating eosinophil damage to schistosomula of Schistosoma mansoni, we have used cytoplasts (eosinophil or neutrophil vesicles devoid of granules and nuclei, with an intact oxidase in their plasma membrane) in combination with purified eosinophil cationic protein (ECP) or major basic protein (MBP) in a cytotoxicity test toward schistosomula. Suboptimal concentrations of ECP (10(-6) M) or MBP (10(-6) M) resulting in less than 10% killing were used in combination with cytoplasts. Cytoplasts alone in the presence of immune serum tested over a wide range of cytoplast:schistosomula ratios generated superoxide and hydrogen peroxide, but were unable to damage schistosomula. However, when a suboptimal ECP concentration (10(-6) M) was combined with neutroplasts or eosinoplasts, 43.9% +/- 8.5 (n = 7) and 24.7% +/- 9.8 (n = 3), respectively, of the schistosomula were killed. Oxygen metabolites were responsible for the synergism, because cytoplasts from a patient with chronic granulomatous disease were unable to act in synergy with ECP. In contrast to ECP, no synergism was found between cytoplasts and MBP (10(-6) to 2 X 10(-5)M). These results show that oxygen metabolites are important for the killing of schistosomula by lowering the concentration of ECP needed to inflict damage.

Animals↗
PubMed1987

Imbalance in the nucleotide pools of myeloid leukemia cells and HL-60 cells: correlation with cell-cycle phase, proliferation, differentiation, and transformation.

Myeloblasts from the blood of patients with chronic myeloid leukemia (CML) in a blastoid crisis were shown to have an imbalance in the ribonucleotide pools compared with normal blood neutrophils. This imbalance includes decreased ratios of purine:pyrimidine, adenine:guanine, and uracil:cytosine nucleotides as well as an increased relative concentration and a changed composition of the uridine diphosphate (UDP) sugars, with relatively more UDP-N-acetylhexosamines. Similar, more prominent deviations were found in HL-60 promyelocytic leukemia cell line cells. We have used HL-60 cells to investigate the relationships between these changes in the ribonucleotide pools and myelocyte proliferation, maturation, and/or transformation to the malignant state. When HL-60 cells were separated by elutriation centrifugation into fractions enriched in G1, S phase, or G2 + M, we found only differences in the amount of nucleotides per cell (G2 + M greater than S phase greater than G1) corresponding with the increase in cell volume but not in the qualitative composition of the nucleotides. Therefore, throughout this study, the nucleotide content of all cells was calculated per unit of cell volume. When HL-60 cells were induced to myeloid differentiation with dimethyl sulfoxide, proliferation stopped after 3 days. After 6 days, 70-90% of the cells had matured into cells capable of nitro blue tetrazolium reduction upon stimulation with phorbol myristate acetate. During the maturation process, the mean volume of the HL-60 cells decreased, and the nucleotide content and the purine:pyrimidine and adenine:guanine nucleotide ratios increased. The composition of the UDP sugars changed dramatically, with a decrease of UDP-N-acetylhexosamines and an increase of UDP-hexoses. Similar changes were detected in HL-60 cells that stopped proliferating without dimethyl sulfoxide-induced maturation, except that the UDP sugar composition showed an increase of UDP-N-acetylhexosamines and a decrease of UDP-hexoses. Careful examination of these results indicates that the decreased ratio of purine:pyrimidine nucleotides and the decreased ratio of uracil:cytosine nucleotides observed in CML myeloblasts may be regarded as specific changes caused by transformation of myelocytes to the malignant state. The increased amount of UDP-N-acetylhexosamines and total UDP sugars in the CML cells may also be connected with the transformation process. All other deviations in the nucleotide pattern of transformed myelocytes in comparison to that of mature, normal neutrophils can be explained by the state of proliferation and/or immaturity of CML myeloblasts and HL-60 cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Cycle↗
PubMed1987

Killing of schistosomula by taurine chloramine and taurine bromamine.

Eosinophils generate hypochlorous acid when stimulated with opsonized particles. The hypochlorous acid can react with beta-amino acids such as taurine to produce chloramines with a long lifetime. In the presence of bromide, eosinophils generate hypobromous acid which can react with taurine to generate taurine bromamine. As taurine is abundantly present in leukocytes, eosinophils have the potential to generate taurine chloramine or taurine bromamine. With regard to the role of eosinophils in killing schistosomula, we have shown that both taurine chloramine and taurine bromamine in physiological concentrations are able to kill the schistosomula of Schistosoma mansoni.

Animals↗