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Biomedical subjects

A Waheed

Publications and source records attributed to A Waheed.

At least 199 records · Page 11Linked to original sources
PubMed1981

Effects of murine leukemia virus infection on long-term hematopoiesis in vitro emphasized by increased survival of bone marrow cultures derived from BALB/Mo mice.

The effect of infection with Moloney murine leukemia virus (Mo-MuLV) on long-term bone marrow cultures was studied. Cultures were derived from the bone marrow of BALB/Mo mice, which carry Mo-MuLV as an endogenous virus, or from BALB/c, 129/J, or balb/c X 129/J mice that were infected with Mo-MuLV in vitro. The following parameters were tested: longevity of generation of granulocytes; biological properties of nonadherent cells in colony-forming assays for pluripotential stem cells and committed granulocyte-macrophage colony-forming unit culture, erythroid, or metachromasia-positive mast cell-basophil colony-forming cells; differentiation of nonadherent cells following cocultivation with thymic or bone marrow stromal cells; and generation of WEHI-3 dialyzed conditioned medium-dependent permanent cell lines. Granulocytes were generated for 65 weeks in BALB/Mo marrow cultures, 31 weeks for BALB/c, 22 weeks in 129/J, and 28 weeks in balb/c X 129/J cultures. Exogenous infection of BALB/c cultures with Mo-MuLV increased the longevity of hematopoiesis to 41 weeks. Granulocyte-macrophage colony-forming unit cultures were produced for 61 weeks in BALB/Mo cultures, 25 to 40 weeks in Mo-MuLV-infected cultures, and 19 to 33 weeks in uninfected control cultures. Nonadherent cells harvested from BALB/Mo marrow cultures generated cloned permanent WEHI-3 dialyzed conditioned medium-dependent, nonleukemogenic granulocyte-macrophage colony-forming unit culture cell lines at greater efficiency than did Mo-MuLV-infected or uninfected BALB/c cultures. The cell lines differentiated to mature granulocytes following cocultivation with purified marrow or thymic stromal cells. There was no detectable differentiation of nonadherent cells to lymphocytes or mast cells. Thus, Mo-MuLV does not detectably transform granulocyte progenitor cells in vitro to granulocytic leukemia. However, Mo-MuLV replication stimulates self-renewal of granulocyte progenitor cells in both primary marrow culture and in suspension culture in WEHI-3 dialyzed conditioned medium.

Animals↗
PubMed1981

Evaluation of radioimmunoassay and in vitro colony assay techniques for determination of colony-stimulating factor and inhibitory activity in murine serum and tissue.

These studies have evaluated a newly developed radioimmunoassay (RIA) for the measurement of colony-stimulating factor (CSF) in murine serum and other biologic fluids. The routine in vitro agar gel bioassay for CSF is influenced by high molecular weight serum lipoproteins and low molecular weight tissue-derived materials that are inhibitory to colony formation. Studies with normal serum revealed that in all cases, the levels of CSF detected by the RIA were equal to or greater than levels measured by the bioassay. Dose curves with varying quantities of serum had linear responses in the RIA but not the colony assay. Using Sephadex G-150 chromatography, the murine serum was separated into CSF active and CSF inhibitory peaks as determined by bioassay. Evaluation of these fractions by RIA indicated that the assay was unaffected by the serum inhibitors. Likewise, experiments with tissue lysates indicated that the RIA was not influenced by the low molecular weight tissue inhibitors. Instead, the radioimmunoassay revealed low levels of CSF that were not detectable by bioassay. These observations indicate that the RIA is superior to the bioassay for the estimation of CSF in murine serum and other biologic materials.

Agar↗
PubMed1980

Chemical characterization and substrate specificity of rabbit liver aryl sulfatase A.

Rabbit liver aryl sulfatase A (aryl-sulfate sulfohydrolase, EC 3.1.6.1) is a glycoprotein containing 4.6% carbohydrate in the form of 25 residues of mannose, seven residues of N-acetylglucosamine, and three residues of sialic acid per enzyme monomer of molecular weight 140 000. Each monomer consists of two equivalent polypeptide chains. The protein has a relatively high content of proline, glycine and leucine, and the amino acid composition of rabbit liver aryl sulfatase A is similar to that of other known liver sulfatases. Rabbit liver aryl sulfatase A catalyzes the hydrolysis of a wide variety of sulfate esters, although it appears possible that cerebroside sulfate is a physiological substrate for the enzyme because the Km is very low (0.06 mM). The turnover rate for hydrolysis of nitrocatechol sulfate or related synthetic substrates is much higher than the rate with most naturally occurring sulfate esters such as cereroside sulfate, steroid sulfates, L-tyrosine sulfate or glucose 6-sulfate. However, the turnover rate with ascorbate 2-sulfate is comparable to the rates measured using most synthetic substrates. These results are discussed in relationship to several previously described sulfatase enzymes which were claimed to have unique specificities.

Amino Acids↗
PubMed1980

Phosphorylated oligosaccharides in lysosomal enzymes: identification of alpha-N-acetylglucosamine(1)phospho(6)mannose diester groups.

In human fibroblasts, the recognition of lysosomal enzymes by cell surface receptors is mediated by mannose 6-phosphate residues located on oligosaccharides that can be cleaved by endo-beta-N-acetylglucosaminidase H. About half of these oligosaccharides, as isolated from beta-hexosaminidase and cathepsin D secreted by human skin fibroblasts, are anionic. Most of these are resistant to alkaline phosphatase. The resistance is due to alpha-N-acetylglucosamine residues linked to mannose 6-phosphate by a phosphodiester bond. The major phosphorylated oligosaccharides contain one and two and possibly three phosphate groups blocked by N-acetylglucosamine. Besides the blocked phosphate groups these oligosaccharides contain a common inner core consisting of Man alpha 1,6-(Man alpha 1,3)Man alpha 1,6(Man alpha 1,3)Man beta GlcNAc and either one or two alpha 1,2-linked mannose residues.

Acetylglucosamine↗
PubMed1980

Production of lymphocyte-activating factor (Interleukin 1) by macrophages activated with colony-stimulating factors.

Murine peritoneal exudate macrophages incubated with medium conditioned by L929 cells were stimulated to produce lymphocyte-activating factor (LAF, Interleukin 1). This stimulatory activity was partially neutralized by antiserum prepared against partially purified L cell colony-stimulating factor (CSF) and comigrated upon gel filtration with the myeloproliferative activity. LAF-inducing activity of three different L cell CSF preparations, including one purified to homogeneity, was dependent upon the concentration of CSF. A minimum of 1,000 to 3,000 units of CSF activity was required to stimulate macrophagfes to produce LAF. Concanavalin A (Con A) stimulated splenic supernatants also contained CSF and LAF-inducing activities that co-eluted upon gel filtration. LAF-inducing activities co-eluting with the two Con A CSF peaks (apparent m.w. of 25,000 and 35,000) were effective at minimum dilutions containing 1,000 to 3,000 units of CSF activity correlating in potency with L cell-derived CSF. Based on these data, it is proposed that CSF, whether of L cell or lymphoid origin, not only has myeloproliferative activities but also is capable of stimulating macrophages to produce LAF.

Animals↗
PubMed1979

Fractionation of antibodies to L-cell colony-stimulating factor by affinity chromatography.

Purified L-cell colony-stimulating factor (CSF) was coupled to cyanogen-bromide-activated Sepharose and used to selectively fractionate antibodies to this factor. With the use of a simplified two-step washing and elution technique, there was 50%--70% binding of the anti-CSF, with recovery of 60%--100% of the bound material. Both the native antiserum and purified anti-CSF fractions were inhibitory to murine granulocyte-macrophage colony formation. The purified antibodies contained only IgG and were reduced in protein concentration to 0.1% of the serum IgG values. These fractions should prove useful tools for the study of granulocyte and macrophage differentiation.

Animals↗
PubMed1979

Purification and properties of L cell-derived colony-stimulating factor.

CSF was prepared by the growth of L cells in serum-free culture medium. This conditioned medium was subjected to a six-step purification schedule which included ultrafiltration, alcohol precipitation, and separation by DEAE-cellulose, Con A-Sepharose, Sephadex G-150, and sucrose density-gradient centrifugation. The resultant CSF was 1000-fold purified with 50% to 70% recovery of the starting activity. Granulocyte and macrophage colony formation was detected with 5 x 10(-12M CSF; maximum colonies were obtained with 3 ng per marrow culture. Two major peaks of activity were obtained; one was nonadherent to Con A, whereas the other was bound and specifically eluted with alpha-methylglucoside. Both fractions contained carbohydrate residues as they stained avidly with PAS, were inactivated by periodate, and showed altered electrophoretic mobility after treatment with neuraminidase. Following iodination, each purified fraction migrated in a single band in SDS-acrylamide gels. The molecular weight was estimated as 65,000 to 70,000 daltons. Following reduction with mercaptoethanol, the CSF fractions were reduced into subunits with molecular weights of approximately 35,000. These studies confirm the glycoprotein nature and subunit composition of L cell CSF. The methods described herein are useful for the purification of both the Con A-adherent and con A-nonadherent forms of CSF.

Chemical Phenomena↗
PubMed1979

Development of a radioimmunoassay for colony stimulating factor.

Purified L-cell colony stimulating factor (CSF) and rabbit anti-CSF serum were used to devise a radioimmunoassay for this factor. The CSF was radiolabelled with the aid of lactoperoxidase and precipitated by a double antibody technique. Addition of unlabelled CSF caused a dose-related displacement of the labelled tracer. Similar results were noted with conditioned media and murine serum. The assay required only 4 days for completion as compared with 7 days for the conventional agar gel bioassay. Moreover, the radioimmunoassay proved more sensitive and accurate than the bioassay. This technique should allow further exploration of the role of CSF in granulopoiesis.

Animals↗
PubMed1978

Binding of colony stimulating factor by sterile filtration membranes.

Loss of serum-free L-cell colony stimulating factor (CSF) activity was noted when non-sterile or ethylene oxide sterilized Millipore filters were employed; autoclaved membranes showed no inactivation. The reduction in CSF did not appear to be due to release of inhibitory or inactivating substances as extensive rinsing with various solvents did not prevent loss of CSF activity. In addition, these filtrates did not inhibit the activity of standard CSF. Less pure sources of CSF, including standard serum containing L-cell CSF and human urinary concentrate, did not lose activity upon filtration. Moreover, the addition of bovine serum albumin to serum-free CSF completely prevented membrane filtration loss. The CSF loss could be prevented by treatment of the membranes with buffers contraining 0.05% polyethylene glycol (PEG). These findings show that Millipore membranes can bind significant quantities of partially purified CSF. It is important in purification studies to recognize that CSF loss can be prevented by addition of PEG to all buffer systems.

Colony-Stimulating Factors↗