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Biomedical subjects

A Waheed

Publications and source records attributed to A Waheed.

At least 181 records · Page 10Linked to original sources
PubMed1983

The effect of preincubation of bone marrow cells on the binding of colony-stimulating factor.

In previous studies, the binding of 125I-CSF to fresh murine bone marrow cells was delayed for 6 to 16 hr at 37 degrees C and only minimal binding was observed at 4 degrees C. In the present experiments, marrow cells were preincubated for varying intervals at 37 degrees C in medium devoid of CSF prior to the addition of 125I-CSF. Preincubation for 6 to 24 hr led to a progressive increase in binding sites, so that cellular uptake of radiolabeled CSF was detected within 5 min of exposure to the cells. With preincubated cells, a marked difference was noted in binding kinetics at 4 degrees and 37 degrees C. The increase in cell-bound radioactivity at 4 degrees C with time yielded a curvilinear plot with 31% of maximum uptake at 5 min and 71% at 1 hr. In contrast, maximum cell uptake occurred within 5 min at 37 degrees C, with a plateau in cell bound radioactivity thereafter. This difference appeared to result from continuous internalization of the tracer at 37 degrees C, as judged by the finding of 67% of the radioactivity in the cytosol fraction after 1 hr exposure to tracer. Only 15% was detected in this fraction after 4 degrees incubation. Moreover, a high proportion of cytosol radioactivity appeared to be degraded after binding at 37 degrees C, as judged by a decrease in molecular weight of radioactive material on SDS-acrylamide gels. Degradation of the tracer was further demonstrated by secretion of radioactive peptides into the medium after binding. Only 8% secretion was noted at 4 degrees C; however, this increased to 70% to 80% at 37 degrees C. Development of binding sites in vitro appeared to require new protein synthesis, since addition of 1 microgram/ml cycloheximide during the preincubation prevented the subsequent CSF binding. Thus CSF binding sites are generated by in vitro incubation of marrow cells. Radiolabeled CSF attaches to surface binding sites, is internalized rapidly, and is degraded into lower-molecular-weight peptides. These findings appear to explain the continued requirement of CSF for in vitro colony formation.

Ammonium Chloride↗
PubMed1983

Identification of hemopoietic cells responsive to colony-stimulating factor by autoradiography.

Binding of radiolabeled L-cell colony-stimulating factor (CSF) was studied using murine bone marrow and fetal liver cells. With 10(7) cells, saturation of binding was seen with approximately 500,000 cpm of 125I-CSF. Minimal binding was detected after one hour incubation with tracer at 37 degrees C; however, marked cellular uptake of radioactivity was noted after 24-hr exposure to CSF. As judged by autoradiographs, small numbers of myeloblasts, promyelocytes, and large mononuclear cells were labeled with 1-hr exposure to tracer. By 6 hr of incubation, 50%-70% of myeloblasts and promyelocytes and small numbers of late granulocytic cells were labeled. Virtually all myeloblasts and promyelocytes and approximately 50% of myelocytes, metamyelocytes, polymorphonuclear granulocytes, and monocytes were labeled after 24-hr exposure to the radioiodinated CSF. Label was not detected on erythroblasts, eosinophils, or megakaryocytes. Suspensions of fetal liver cells had lower uptake of radioactivity than bone marrow cells. This appeared to result from a lesser concentration of granulocytic cells in fetal liver, as labeling of individual cells was similar with both tissues. In additional experiments, CSF binding to marrow cells was assessed after 30-min exposure to tracer at 0 degrees C. Uptake of 125I-CSF exceeded that observed after 24-hr incubation at 37 degrees C. With this technique, cellular label was also confined to granulocytic and monocytic cells. These findings suggest that purified CSF reacts with and may stimulate immature and mature cells of the granulocytic and monocytic lineages.

Animals↗
PubMed1982

Biosynthesis of acid alpha-glucosidase in late-onset forms of glycogenosis type II (Pompe's disease).

Cultured human skin fibroblasts from control persons and from patients with the generalized and late-onset forms of Pompe's disease were labelled with radioactive leucine and the incorporation of radioactivity into acid alpha-glucosidase and cathepsin D was analysed by immunoprecipitation, gel electrophoresis and fluorography. When the labelling was carried out for 6-12 h in the presence of NH4Cl, the labelling of secreted alpha-glucosidase relative to that of secreted cathepsin D in fibroblasts from patients with the late-onset form of Pompe's disease was less than 15% of that in fibroblasts from control persons. However, when the fibroblasts were labelled for less than 1 h, the relative rate of incorporation of radioactivity into acid alpha-glucosidase was rather similar in the two types of fibroblasts. In fibroblasts from patients with the generalized form of Pompe's disease no incorporation of radioactivity into acid alpha-glucosidase could be detected.

Cathepsin D↗
PubMed1982

Synthesis of phosphorylated recognition marker in lysosomal enzymes is located in the cis part of Golgi apparatus.

Rat liver membranes were subjected to centrifugation in a sucrose density gradient in which the Golgi apparatus was separated into several subfractions. Two enzymes involved in the synthesis of the phosphorylated recognition marker in lysosomal enzymes, UDP-N-acetylglucosamine:lysosomal enzyme precursor N-acetylglucosamine-1-phosphotransferase and alpha-N-acetylglucosaminyl phosphodiesterase fractionated with alpha-1,2-mannosidase, a marker enzyme of cis Golgi membranes and differently from galactosyltransferase, a marker enzyme of trans Golgi membranes.

Animals↗
PubMed1982

Enhanced breakdown of arylsulfatase A in multiple sulfatase deficiency.

Multiple sulfatase deficiency (mucosulfatidosis) is a lysosomal storage disorder characterized by the decrease in activities of all known sulfatases. To measure the apparent rate of synthesis and the half-life of arylsulfatase A in multiple sulfatase deficiency, fibroblasts from patients with the disease and from controls were subjected to pulse-chase labelling with radioactive amino acids. Arylsulfatase A and cathepsin D, a lysosomal enzyme that is not affected in multiple sulfatase deficiency, were isolated from cells and media by immunoprecipitation. The labelled polypeptides were separated by polyacrylamide gel electrophoresis, visualized by fluorography and quantified by liquid scintillation counting. Using single and double isotope techniques it was found that, as compared to cathepsin D, the apparent rate of synthesis of arylsulfatase A was 2--5 times lower and the half-life 4--9-times shorter in multiple sulfatase deficiency than in control fibroblasts. In multiple sulfatase deficiency fibroblasts the rates of endocytosis and the stabilities of endocytosed arylsulfatases A isolated from human urine and bovine tests were equal to those in metachromatic leucodystrophy fibroblasts. We postulate that in normal cells a gene product exists that affects the stability of sulfatases and that multiple sulfatase deficiency is due to a mutation in this gene.

Animals↗
PubMed1982

Further improvement of the enzyme-linked antiglobulin test (ELAT) for erythrocyte antibodies.

The enzyme-linked antiglobulin test (ELAT) was found to be nine times more sensitive than the direct antiglobulin test (DAT) in detecting erythrocytes sensitized by IgG antibody in vitro. The release of hemoglobin and other interfering substances from the erythrocytes gave falsely high absorbance values which were corrected by the use of a hemolysis bland with each test. ELAT results showed good precision. With the use of the hemolysis blank, the ELAT should prove to be a useful tool for hospital blood blanks in the detection of weakly reactive allo- and autoantibodies.

Antibodies↗
PubMed1982

Impaired phosphorylation of lysosomal enzymes in fibroblasts of patients with mucolipidosis III.

The incorporation of [3H]leucine and [32P]phosphate into three lysosomal enzymes, cathepsin D, beta-hexosaminidase and arylsulfatase A by fibroblasts from six patients affected with mucolipidosis III was determined. In the mutant cells the incorporation of 32P in the enzymes was reduced by 70-97% as compared to controls. The residual phosphorylation of lysosomal enzymes is definitely higher than in fibroblasts from patients with mucolipidosis II, where apparently non-phosphorylated enzymes are formed. In mucolipidosis III the major part of the newly formed enzymes accumulated extracellularly and the cellular enzymes were recovered mainly in their processed forms. In mucolipidosis III arylsulfatase A and the processed forms of cathepsin D exhibited a heterogeneity that was not observed in controls. beta-Hexosaminidase and cathepsin D secreted by mucolipidosis III fibroblasts contained only a small amount of phosphorylated oligosaccharides with either one or two phosphate groups per oligosaccharide. As in controls the major fraction of phosphate was present as acid-labile phosphodiester resistant to alkaline phosphatase. The residual phosphorylation of lysosomal enzymes may be related to the partial intracellular retention and processing of these enzymes in fibroblasts from patients with mucolipidosis III.

Cathepsin D↗
PubMed1982

Effect of colony stimulating factor on murine macrophages. Induction of antitumor activity.

Colony stimulating factor (CSF) was assessed for its capacity to stimulate antitumor activity in macrophages. Murine peritoneal macrophages incubated with CSF for 48 h inhibited [3H[thymidine (TdR) incorporation by P815 tumor cells to approximately 20% of control values. Inhibition of CSF-stimulated macrophages was significantly greater than inhibition by unstimulated macrophages (P less than 0.001). CSF had little direct effect on the proliferation of either tumor cells or macrophages alone, indicating that the antitumor activity of CSF was mediated by macrophages. it is unlikely that impurities in the CSF preparations were responsible for the effect since CSF that had been purified to homogeneity was as active as crude preparations. Furthermore the activity of CSF on macrophages was blocked by addition of purified anti-CSF antibodies. In addition to being tumoristatic, CSF-stimulated macrophages were tumoricidal as determined by a tumor colony growth assay. Tumor cells that had been incubated with CSF-stimulated macrophages showed a significant reduction in tumor colony-forming units (P less than 0.01). Thus, in addition to its effect on hemopoietic stem cells, CSF induces certain effector functions in mature macrophages that may enhance endogenous antitumor host defenses.

Animals↗
PubMed1982

Phosphorylation of lysosomal enzymes in fibroblasts. Marked deficiency of N-acetylglucosamine-1-phosphotransferase in fibroblasts of patients with mucolipidosis III.

N-Acetylglucosamine-1-phosphotransferase activity was assayed in human skin fibroblasts using [beta-32P]UDP-N-acetylglucosamine as donor and dephosphorylated beta-N-acetyl-D-hexosaminidase as acceptor. An optimal transfer rate of N-acetylglucosamine 1-phosphate required CDP-choline and ADP in order to inhibit the breakdown of [beta-32P]UDP-N-acetylglucosamine and a combination of leupeptin and iodoacetamide to protect the transferase. The transferase required Mg2 or Mn2. Using doubly labelled UDP-N-acetylglucosamine, simultaneous transfer of N-acetyl-[6-3H]glucosamine and [32P]phosphate to endogenous acceptors was demonstrated. Membranes prepared from fibroblasts from patients with mucolipidosis III were defective in transfer of N-acetylglucosamine 1-phosphate. A residual transferase activity of less than 10% of controls was detectable in fibroblast membranes of eight patients with mucolipidosis III. In membranes from fibroblasts from patients with mucolipidosis II,N-acetylglucosamine-1-phosphotransferase activity was not detectable. Our results indicate that the primary defect in mucolipidoses II and III is a deficiency in N-acetylglucosamine-1-phosphotransferase, the residual activity being higher in mucolipidosis III than in mucolipidosis II.

Cell Membrane↗
PubMed1982

Synthesis and processing of arylsulfatase A in human skin fibroblasts.

Biosynthesis of arylsulfatase A in normal and mutant human fibroblasts was studied by growing cells in the presence of L-[4,5-3H] leucine or [2-3H] mannose, isolation of labelled arylsulfatase A by immune precipitation and visualization of electrophoretically separated polypeptide by fluorography. Arylsulfatase A was synthesized as a precursor with a mean apparent molecular mass of 62 kDa. Intracellularly the precursor was converted into a 60.5 kDa polypeptide within a chase period of 1 to 7 days. The 60.5 kDa product in polyacrylamide corresponded to one of two polypeptides present in arylsulfatase A isolated from human placenta. In fibroblasts from a patient with metachromatic leukodystrophy no immune precipitable polypeptides of arylsulfatase A were detected. In normal fibroblasts less than 10% of the precursor of arylsulfatase A was secreted into the medium, whereas in mucolipidosis II fibroblasts and in control fibroblasts grown in the presence of NH4Cl up to 90% of the precursor of arylsulfatase A, appeared in the medium and remained there without change in the apparent molecular mass for at least 7 days. Arylsulfatase A polypeptides appear to contain two carbohydrate side chains. In about 90% of the polypeptides both side chains are cleaved by endo-beta-N-acetylglucosaminidase H, whereas in the remaining chains one of the two oligosaccharides is not cleaved.

Cells, Cultured↗
PubMed1982

Observations on the binding and interaction of radioiodinated colony-stimulating factor with murine bone marrow cells in vivo.

Radioiodinated L-cell-derived colony-stimulating factor (CSF) was used to characterize the binding reaction to murine bone marrow cells. The major increment in cell-associated radioactivity occurred over 24 hr incubation at 37 degrees C, but virtually no binding was observed at 4 degrees C. The reaction was saturable with approximately 1 ng/ml of purified CSF. Unlabeled CSF prevented the binding, whereas a number of other hormones and proteins did not compete for CSF uptake. Further specificity studies showed virtually no binding to human bone marrow, which is unresponsive to this form of murine CSF. Minimal CSF uptake was noted with murine peritoneal macrophages, but virtually no binding was detected with thymic, lymph node, liver, or kidney cells. The marrow cell interaction with tracer appeared to require a new protein synthesis, as the binding was prevented by cycloheximide or puromycin. Preincubation of marrow cells in medium devoid of CSF increased the degree of binding after 1 hr exposure to the tracer. This suggests that CSF binding sites may be occupied or perhaps decreased in response to ambient levels of CSF in vivo. Approximately 70% of the bound radioactivity was detected in the cytoplasm at 24 hr. This material was partially degraded as judged by a decrease in molecular weight from approximately 62,000 to 2 peaks of approximately 32,000 and approximately 49,000, but 72% of the binding activity was retained. After plateau binding was achieved, greater than 80% of the radioactivity released into the medium was degraded into biologically inactive peptides with molecular weights less than 10,000. These findings suggest that the interaction of CSF with marrow cells is characterized by binding with subsequent internalization and metabolic degradation into portions of the molecule that are devoid of biologic activity.

Animals↗
PubMed1982

Purification of colony-stimulating factor by affinity chromatography.

Studies were undertaken to determine whether L-cell-derived colony-stimulating factor (CSF) could be purified by a single step affinity chromatographic technique. A quantity of 100 X 10(6) units of purified anti-CSF was coupled to cyanogen bromide activated Sepharose 4B; colony assays revealed complete binding of the antibodies to the gel. Three 10-liter pools of serum-free L-cell CSF were concentrated by ultrafiltration, applied to the gel, and eluted with a low pH, high molarity buffer. Recovery of CSF ranged from 68%-100% with greater than 1000-fold decreases in protein content. Specific activity of the purified CSF ranged from 2.8 to 5.9 X 10(7) U of CSF/mg protein. Following iodination, each purified pool of CSF revealed a major 63,000-dalton peak of radioactivity that comigrated with CSF activity in SDS-acrylamide gels. In addition, several smaller peaks of 50,000 and 40,000 molecular weight were also detected. Approximately two-thirds of the purified CSF was adherent to concanavalin-A with elution by a competing sugar. Electrophoretic mobility was retarded by incubation with neuraminidase. These chromatographic studies confirm the observation that CSF is a glycoprotein but also suggest variable degrees of glycosylation of the molecule. This chromatographic technique should prove useful in the rapid purification of large quantities of CSF for physiologic and biochemical characterization.

Animals↗
PubMed1981

Processing of the phosphorylated recognition marker in lysosomal enzymes. Characterization and partial purification of a microsomal alpha-N-acetylglucosaminyl phosphodiesterase.

N-Acetylglucosamine(1)phospho(6)mannose groups recently identified in lysosomal enzymes were proposed to be precursors of the recognition markers terminating with mannose 6-phosphate (Tabas, I., and Kornfeld, S. (1980) J. Biol. Chem. 225, 6633-6639; Hasilik, A., Klein, U., Waheed, A., Strecker, G., and von Figura, K. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7074-7078). To study the presumptive enzyme removing N-acetylglucosamine from the diester, an assay was developed using a radioactive oligosaccharide containing diester groups of the above structure. An alpha-N-acetylglucosaminyl phosphodiesterase cleaving this substrate in vitro was found in human placenta and in rat liver. The enzyme was solubilized from the microsomal fraction of human placenta and more than 800-fold purified with 75% yield. It is distinct from the lysosomal alpha-N-acetylglucosaminidase by the criteria of immunological cross-reactivity, substrate specificity, and heat stability. The partially purified enzyme cleaves alpha-N-acetylglucosamine phosphodiester bonds in oligosaccharides from lysosomal enzymes, in lysosomal enzymes, and in UDP-N-acetylglucosamine. We propose that the microsomal alpha-N-acetylglucosaminyl phosphodiesterase is involved in the processing of the phosphorylated recognition marker in lysosomal enzymes.

Acetylglucosaminidase↗
PubMed1981

Subcellular location of two enzymes involved in the synthesis of phosphorylated recognition markers in lysosomal enzymes.

Phosphorylated recognition markers in lysosomal enzyme appear to be synthesized by transfer of alpha-N-acetylglucosamine 1-phosphate groups to C6 hydroxyl of mannose residues in glycosylated enzyme precursors and a subsequent hydrolysis from the diester groups of the N-acetylglucosamine residues. The transfer and the diesterase activities were studied in subcellular fractions of rat liver. Both activities fractionated like the Golgi marker galactosyltransferase.

Animals↗
PubMed1981

Transfusion significance of Lewis system antibodies. Success in transfusion with crossmatch-compatible blood.

Patients with nonhemolytic Lewis antibodies in the serum were transfused on the basis of major crossmatch compatibility alone. No adverse clinical affects were noted when 33 such patients received a total of 230 transfusions. Considerable savings in technologist time and reagent cost were realized by adopting this policy. Transfusion on the basis of crossmatch compatibility alone did not necessarily result in the selection of Le(a-) and/or Le(b-) blood for patients with corresponding antibodies in the serum. Crossmatch compatibility ratio for patients with anti-Leb in the serum was much higher than expected and was profoundly affected by the ABO group of the donors.

ABO Blood-Group System↗