Biomedical subjects

A Waheed

Publications and source records attributed to A Waheed.

At least 127 records · Page 7Linked to original sources

PubMed1988

Mannose 6-phosphate/insulin like growth factor II receptor: the two types of ligands bind simultaneously to one receptor at different sites.

Pentamannose 6-phosphate/trilysine substituted aprotinin (PMP-lys-aprotinin) and insulin like growth factor II (IGF II) were used as affinity ligands for the mannose 6-phosphate (M6P) and IGF II binding sites of the M6P/IGF II receptor. Both ligands were cross linked to intact receptor and tryptic fragments of the receptor. The pattern of receptor fragments with M6P and IGF II binding sites differed indicating that the two binding sites are located on different segments of the receptor. The receptor was incubated with [125I]IGF II and pentamannose 6-phosphate substituted bovine serum albumin (PMP-BSA). From these mixtures [125I]IGF II receptor complexes could be precipitated with antibodies against the PMP-BSA indicating that the M6P/IGF II receptor can bind simultaneously IGF II and M6P-containing ligands.

Affinity Labels↗

PubMed1988

Mannose 6-phosphate/insulin-like growth factor II receptor: distinct binding sites for mannose 6-phosphate and insulin-like growth factor II.

Pentamannosyl phosphate substituted bovine serum albumin (PMP-BSA) and insulin like growth factor II (IGF II) bind specifically to immobilized mannose 6-phosphate/insulin like growth factor II receptor. An excess of IGF II inhibited binding of PMP-BSA by less than or equal to 20%, and an excess of PMP-BSA inhibited binding of IGF II by less than or equal to 10%. Polyclonal antibodies against the receptor purified from human liver inhibited preferentially the binding of PMP-BSA, and a monocloncal antibody 2C2 inhibited only the binding of IGF II to the receptor. Similar results were obtained for binding of PMP-BSA and IGF II to human skin fibroblasts. These results suggest that the binding sites for mannose 6-phosphate and IGF II reside in different portions of the receptor.

Binding Sites↗

PubMed1988

Probable metachromatic leukodystrophy/pseudodeficiency compound heterozygote at the arylsulfatase A locus with neurological and psychiatric symptomatology.

Metachromatic leukodystrophy (MLD) is an autosomal recessive progressive demyelination disorder caused by the deficiency of arylsulfatase A (ASA). However, there exist individuals with low ASA activity without clinical symptoms. This state is described as ASA pseudodeficiency (PD). A number of patients with low ASA activity and various neuropsychiatric symptoms have been observed. It is controversial to what extent low ASA activity predisposes for neurological and/or psychiatric symptomatology. Therefore, persons with low ASA activity who were collected from a large-scale screening among neuropsychiatric patients and healthy controls are presently being extensively evaluated using biochemical, genetic, and clinical methods. Here we present a female patient, who had been first hospitalized with the diagnosis encephalomyelitis disseminata. Her ASA activity determined in fibroblast extracts is intermediate between adult MLD and PD. Sulfatide degradation in cultured fibroblasts is diminished. The subunit pattern obtained after SDS-polyacrylamide gel electrophoresis and immunoblotting was determined in the index patient and 2 sibs. It is compatible with a compound genotype ASA-/ASAp in the index case. It appears probable that in this patient low ASA activity leads to the accumulation of sulfatide and either causes the appearance of neuropsychiatric symptoms or at least contributes to the demyelination process.

Adult↗

PubMed1988

Human lysosomal acid phosphatase: cloning, expression and chromosomal assignment.

A 2112-bp cDNA clone (lambda CT29) encoding the entire sequence of the human lysosomal acid phosphatase (EC 3.1.3.2) was isolated from a lambda gt11 human placenta cDNA library. The cDNA hybridized with a 2.3-kb mRNA from human liver and HL-60 promyelocytes. The gene for lysosomal acid phosphatase was localized to human chromosome 11. The cDNA includes a 12-bp 5' non-coding region, an open reading frame of 1269 bp and an 831-bp 3' non-coding region with a putative polyadenylation signal 25 bp upstream of a 3' poly(A) tract. The deduced amino acid sequence reveals a putative signal sequence of 30 amino acids followed by a sequence of 393 amino acids that contains eight potential glycosylation sites and a hydrophobic region, which could function as a transmembrane domain. A 60% homology between the known 23 N-terminal amino acid residues of human prostatic acid phosphatase and the N-terminal sequence of lysosomal acid phosphatase suggests an evolutionary link between these two phosphatases. Insertion of the cDNA into the expression vector pSVL yielded a construct that encoded enzymatically active acid phosphatase in transfected monkey COS cells.

Acid Phosphatase↗

PubMed1988

Human lysosomal acid phosphatase is transported as a transmembrane protein to lysosomes in transfected baby hamster kidney cells.

BHK cells transfected with human lysosomal acid phosphatase (LAP) cDNA (CT29) expressed 70-fold higher enzyme activities of acid phosphatase than non-transfected BHK cells. The CT29-LAP was synthesized in BHK cells as a heterogeneously glycosylated precursor that was tightly membrane associated. Transfer to the trans-Golgi was associated with a small increase in size (approximately 7 kd) and partial processing of the oligosaccharides to complex type structures. CT29-LAP was transferred into lysosomes as shown by subcellular fractionation, immunofluorescence and immunoelectron microscopy. Lack of mannose-6-phosphate residues suggested that transport does not involve mannose-6-phosphate receptors. Part of the membrane-associated CT29-LAP was processed to a soluble form. The mechanism that converts CT29-LAP into a soluble form was sensitive to NH4Cl, and reduced the size of the polypeptide by 7 kd. In vitro translation of CT29-derived cRNA in the presence of microsomal membranes yielded a CT29-LAP precursor that is protected from proteinase K except for a small peptide of approximately 2 kd. In combination with the sequence data available for LAP, these observations suggest that CT29-LAP is synthesized and transported to lysosomes as a transmembrane protein. In the lysosomes, CT29-LAP is released from the membrane by proteolytic cleavage, which removes a C-terminal peptide including the transmembrane domain and the cytosolic tail of 18 amino acids.

Acid Phosphatase↗

PubMed1988

Characterization of two clones isolated from the TC-1 murine marrow stromal cell line: growth factor and retrovirus production and physical support of hemopoiesis.

We previously reported the isolation of an adherent murine marrow cell line termed TC-1, and the initial characterization of two subclones derived from this line. In this study we report a further characterization of two subclones from the non-cloned TC-1 cell line. One subclone, TC-1-C-3, consisted of large, slow-growing syncytial polypoid cells that grew to relatively low saturation densities, did not form colonies in soft agar and showed desmosome-like junctions. The other subclone, TC-1-C-11, consisted of smaller, rapidly growing fibroblast-like diploid cells which showed anchorage-independent growth in soft agar. Both these subclones produced growth factors which stimulated giant macrophage colonies in soft agar culture in vitro, but only the TC-1-C-3 subclone produced a retrovirus, whose source was most likely the endogenous ecotropic Emv-2 provirus present in chromosomal DNA in C57BL mice. This retrovirus from the TC-1-C-3 subclone did not appear capable of transforming TC-1-C-11 cells. Together, these data suggest that TC-1-C-3 cells have a special capacity for supporting hemopoiesis. The question of whether the mechanism of this support relates to an intrinsic property of the cell or is possibly related to retrovirus production remains unanswered.

Animals↗

PubMed1988

Effect of pH on binding and dissociation of colony-stimulating factor.

125I-labeled colony-stimulating factor (CSF) binds to granulocytic and monocytic cells in the bone marrow in an irreversible manner. Addition of a 1000-fold excess of unlabeled CSF does not displace the bound material. The present studies showed that brief exposures to pH 2.7-5.0 caused a marked release of the bound material. Such treatments were nontoxic to the marrow cells as judged by trypan blue dye exclusion, assay of colony-forming cells, and by analysis of rebinding of fresh 125I-CSF to the acid-treated cells. The CSF released from marrow cells by low pH revealed two peaks of radioactivity on SDS-acrylamide gel. The first peak (67,500 Da) corresponded to native CSF; a second peak of 53,500 Da was observed. Despite this apparent mild degradation of CSF, the released material showed greater binding to marrow and greater precipitation by anti-CSF than the native 125I-CSF. Further studies showed that acid treatment of marrow cells led to stabilization of the CSF receptors. Pretreatment at pH 4.0 led to retention of binding sites after conversion of marrow cultures to pH 7.5 and incubation at 22-37 degrees C. In contrast, cells that were not exposed to low pH lost receptors rapidly at these temperatures. The extent of preservation of the binding sites was related to the duration of acid exposure. These studies indicate that CSF is retained on the cell surface after binding at 0 degree C and that the CSF can be eluted by acid conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Enhancement of release from MHC class II antigen-positive monocytes of hematopoietic colony stimulating factors CSF-1 and G-CSF by recombinant human tumor necrosis factor-alpha: synergism with recombinant human interferon-gamma.

The influence of purified recombinant human tumor necrosis factor-alpha (rhuTNF-alpha) was assessed alone and in combination with purified recombinant human interferon gamma (rhuIFN-gamma) for its effects on enhancing release from human monocytes of activities that stimulate colony formation by granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells. RhuTNF-alpha or rhuIFN-gamma enhanced release of colony stimulating factors (CSFs), which were determined by a combination of human and mouse colony assays, morphological assessment of colony types and neutralization studies with anti-human macrophage CSF (CSF-1) and anti-human granulocyte (G)-CSF to be CSF-1 and G-CSF. The activity in the uninduced and induced monocyte conditioned media (CM) for CFU-GM-type colonies and clusters was attributed to the presence of both CSF-1 and G-CSF, while the activity in the monocyte CM for BFU-E and CFU-GEMM colonies was attributed to the presence of G-CSF. Monocytes were separated by two-color fluorescence using a dye laser flow cytometry system with cells labeled with anti-leu M3 conjugated with fluorescein isothiocyanate and anti-HLA-DR conjugated with phycoerythrin. While "constitutive" release of CSFs from monocytes was apparent from both the leu M3+, HLA-DR+ and the leu M3+, HLA-DR- (low density or negative DR) fractions, enhanced release of CSFs in response to rhuTNF-alpha or rhuIFN-gamma was confined to the leu M3+, HLA-DR+ population of cells. RhuTNF-alpha and rhuIFN-gamma synergized to enhance release of CSFs such that low concentrations of each molecule, which were inactive when used alone, were active when the two molecules were used together. These studies suggest a role, at least in vitro, for TNF-alpha and IFN-gamma in the release of CSFs from cells of the mononuclear phagocytic lineage.

Animals↗

PubMed1988

Targeting of phosphomannosyl-deficient arylsulfatase A to lysosomes of I-cell fibroblasts.

Fibroblasts from I-cell disease, a genetically-determined lysosomal storage disease, are shown to contain large amounts of phase-dense lysosomes. These lysosomes accumulated acridine orange and were specifically labeled with antibodies to arylsulfatase A. In normal skin fibroblasts the number of arylsulfatase-containing lysosomes was considerably lower. By immunocytochemistry, metabolic labeling and enzyme assay, the arylsulfatase A in I-cell fibroblasts was shown to be synthesized, stored and secreted at a level that was several-fold higher than that present in heterozygous I-cell or normal fibroblasts. Arylsulfatase A in I-cell fibroblasts differed from arylsulfatase in normal fibroblasts by the absence of endoglycosidase H-sensitive phosphorylated oligosaccharides. These findings indicate that arylsulfatase A in I-cells is targeted to lysosomes by a mechanism that does not appear to involve the phosphorylated mannose marker.

Cell Line↗

PubMed1988

Arylsulfatase A from human tissues contains an endo-beta-N-acetylglucosaminidase F-resistant oligosaccharide.

Homogeneous arylsulfatase A from human placenta, liver and urine contains two nonidentical subunits of 59 and 54 kDa. The two subunits are immunologically identical. The relative amount of low molecular weight subunits is only 20-30% of the total enzyme protein. Treatment of the enzyme under various conditions with endo-beta-N-acetylglucosaminidase F results in a decrease in the apparent molecular weight of both subunits by 1-2 kDa. a value that corresponds to the loss of a single N-linked oligosaccharide. However, as judged by carbohydrate staining, endo-beta-N-acetylglucosaminidase F does not remove all carbohydrate from the subunits or from glycopeptides of arylsulfatase A. In contrast, human prostatic acid phosphatase, a glycoprotein with a high content of mannose, hybrid and complex oligosaccharides is completely deglycosylated under identical experimental conditions. Several attempts to deglycosylate arylsulfatase A by chemical methods were unsuccessful due to poor recovery of the protein. From the present studies we conclude that arylsulfatase A contains an endo-beta-N-acetylglucosaminidase F resistant, perhaps O-linked carbohydrate.

Acetylglucosaminidase↗

PubMed1987

Demonstration of prostatic-type acid phosphatase in non-lysosomal granules in the crypt epithelium of the human duodenum.

Human prostatic-type of acid phosphatase has been demonstrated by biochemical methods to be expressed in a number of cells and tissues in addition to the prostate gland. However, the function of this activity is unknown, nor has the enzyme been convincingly localized at the cellular level in any non-prostatic tissues. Using biochemical and immunocytochemical methods, we demonstrate that human intestinal epithelium contains both a lysosomal and prostatic type of acid phosphatase. The prostatic-type enzyme is present only in the epithelium of the crypts and to a lesser extent in the transitional zone at the base of the villi, in contrast to the widely-distributed lysosomal type. The prostatic enzyme is contained in granules that do not react with anti-lysosomal acid phosphatase and are probably secretory in nature.

Acid Phosphatase↗

PubMed1987

Cloning and tissue-specific expression of mouse macrophage colony-stimulating factor mRNA.

Macrophage colony-stimulating factor (CSF-1) stimulates the production of macrophages from bone marrow progenitor cells. We have identified a cDNA clone for murine CSF-1 by antibody screening of a mouse L-cell cDNA library in the expression vector lambda gt11. A screen of about 150,000 recombinant plaques yielded 6 clones that reacted well with an antibody raised against denatured and reduced mouse L-cell CSF-1. These clones were further screened with synthetic oligonucleotides based on the amino-terminal amino acid sequence of CSF-1. One clone, which hybridized to the oligonucleotides, was sequenced and found to contain a single open reading frame. This encompassed 68 amino acids of the mature protein, including the entire amino-terminal sequence we previously reported. This is preceded by what appears to be a 31 amino acid signal peptide. Blot analysis showed that this cDNA hybridizes to a major mRNA species of about 4.5 kilobases (kb) as well as several smaller, less abundant mRNA species (3.8, 2.3, and 1.4 kb) present in mouse L cells. A similar pattern of hybridization was observed with mRNA from a human pancreatic carcinoma cell line that produces CSF-1. Striking differences in the qualitative and quantitative expression of mRNA species for CSF-1 were observed in various mouse tissues. Liver expressed primarily a 1.4-kb species, heart and lung expressed primarily a 4.5-kb species, brain expressed high levels of both the 4.5-kb and 1.4-kb species, and intestine lacked detectable CSF-1 transcripts. Southern blot analysis suggests that the CSF-1 gene is present as a single copy in the mouse haploid genome and that it is not rearranged or amplified in L cells.

Amino Acid Sequence↗

PubMed1987

Comparative effects in vivo of recombinant murine interleukin 3, natural murine colony-stimulating factor-1, and recombinant murine granulocyte-macrophage colony-stimulating factor on myelopoiesis in mice.

Purified murine colony-stimulating factors (CSF) recombinant interleukin 3 (IL-3), natural CSF-1, and recombinant granulocyte-macrophage (GM) CSF were assessed in vivo for their effects on BDF1 mouse bone marrow and spleen granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells in untreated mice and in mice pretreated with purified iron-saturated human lactoferrin (LF). The CSF and LF preparations did not contain detectable endotoxin (less than 0.1 ng). Mice pretreated with LF were more sensitive to the effects of CSF. In mice pretreated with LF, 2,000 U IL-3 or 20,000 U CSF-1 significantly enhanced the cycling status and absolute numbers of all progenitors, whereas 20,000 U GM-CSF significantly increased the cycling status of CFU-GM and CFU-GEMM, but had no effect on cycling of BFU-E or on numbers of any of the progenitors. The effects of CSF in mice pretreated with LF were not mimicked by 0.1-100 ng E. coli lipopolysaccharide.

Animals↗

PubMed1987

Interactions between purified murine colony-stimulating factors (natural CSF-1, recombinant GM-CSF, and recombinant IL-3) on the in vitro proliferation of purified murine granulocyte-macrophage progenitor cells.

Purified preparations of natural CSF-1 (nCSF-1), recombinant GM-CSF (rGM-CSF), and recombinant IL-3 (rIL-3), alone and in combination, were investigated for their proliferative effects on highly enriched murine granulocyte-macrophage progenitor cells (CFU-GM). These CFU-GM had cloning efficiencies of 62%-95% in the presence of 10% (vol/vol) pokeweed mitogen-stimulated spleen cell-conditioned medium, and contained few, if any (less than or equal to 3%), contaminating morphologically recognizable monocytes or lymphocytes. The combination of low concentrations of nCSF-1 plus rIL-3, or nCSF-1 plus rGM-CSF, increased colony number greater than additively compared to the sum of colony formation with each factor alone, whereas total aggregate (colony plus cluster) number increased additively. At plateau concentrations, the previous CSF combinations increased colony number additively. Colony size was increased when nCSF-1 plus either rGM-CSF or rIL-3 were added simultaneously at either low or plateau concentrations, when compared to the size of colonies with any of the CSFs alone. Addition of rGM-CSF plus rIL-3 demonstrated no cooperative proliferative effect on either colony number or size. It is likely that these effects are mediated at the progenitor cell level and do not require accessory cell participation.

Animals↗

PubMed1987

Multilineage synergistic activity produced by a murine adherent marrow cell line.

We reported previously that a cell line (TC-1) derived from adherent marrow cells produced colony-stimulating factor 1 (CSF-1) and a separate activity that acts synergistically with CSF-1 to stimulate giant macrophage colonies. We now report that an activity in TC-1 conditioned media (CM) separate from CSF-1 also synergizes multilineage colony formation by pure interleukin 3 (IL 3) and a crude source of granulocyte-macrophage colony-stimulating activity (GM-CSA) (murine lung-conditioned media). IL 3-induced megakaryocyte colony formation is also synergized. The CSF-1-dependent synergistic activity is not blocked by antibodies to IL 3 and is characterized as a nondialyzable (mol wt cutoff 3,000), heat-stable (56 degrees C, 30') activity that binds to DE-52 cellulose under conditions in which IL 3 does not. This material has an apparent mol wt of approximately 200,000 by Sephadex G100 chromatography, and the bulk of it binds to Concanavalin A (Con A) and elutes off with alpha-methyl mannoside, indicating that it is a glycoprotein. As reported separately, these purified active fractions also have a pre-B cell-inducing activity. In addition, a non-IL 3 activity stimulates proliferation of the factor-dependent cell lines FDC-P1 and DA-1. These data indicate that an adherent marrow cell line produces a growth factor(s) that synergizes with IL 3, GM-CSA, and CSF-1 and induces pre-B cell formation. This may be an important regulator of early multilineage lymphohemopoiesis.

Animals↗

PubMed1987

Role of colony-stimulating factor in myelopoiesis in murine long-term bone marrow cultures.

Weekly medium change or midweek feeding of long-term bone marrow cultures (LTMBCs) results in a significant increase in total myeloid cell production. Proliferative myeloid cells peak 48 hours after feeding, and nonproliferative myeloid cells reach maximum levels at 72 hours. This increase in myelopoiesis is invariably preceded by a significant elevation in biologically and immunologically measurable colony-stimulating factor (CSF) in the supernatants of LTBMC. The level peaks 24 hours after medium change, then gradually returns to basal values. The decrease in CSF relates to its consumption by generating myeloid precursors because no fluctuation in the levels occur in cultures without active myelopoiesis. No significant inhibitors or promoters of CSF were detected. When highly purified L cell CSF, CSF in lung-conditioned medium, or CSF concentrated from LTBMC supernatant is added to cultures, an identical increase in myelopoiesis occurs. Anti-CSF antiserum, added to culture at the time of medium change, totally neutralizes supernatant CSF levels but does not affect myelopoiesis. These findings suggest a potential regulatory role for CSF in myelopoiesis in LTBMC. CSF appears to function within the microenvironment through a mechanism involving cell:cell interactions or by causing the production of other substances that stimulate myelopoiesis. Because exogenous CSF stimulates myelopoiesis, it is likely that it too can react either directly or through microenvironmental cells to stimulate primitive myeloid cells to divide.

Animals↗

PubMed1987

The effects of purified recombinant murine interleukin-3 and/or purified natural murine CSF-1 in vivo on the proliferation of murine high- and low-proliferative potential colony-forming cells: demonstration of in vivo synergism.

Purified natural murine L cell (macrophage) colony-stimulating factor (nCSF-1) and purified recombinant murine interleukin-3 (rIL-3) were administered to normal or lactoferrin-pretreated mice 20 to 24 hours before sacrifice. rIL-3 and nCSF-1 administered separately increased the percentage of macrophage high-proliferative potential colony-forming cells (HPP-CFC) and low-proliferative potential colony-forming cells (LPP-CFC) in active cell cycle. Endotoxin was not detected in the samples of nCSF-1 or rIL-3 with the Limulus lysate test, and the in vitro and in vivo hematopoietic stimulatory effects of both molecules were abolished or markedly reduced by 30 minutes' treatment at 100 degrees C, which demonstrates that the effects noted in vivo were not due to endotoxin. Combinations of low concentrations of rIL-3 and nCSF-1, which by themselves were inactive, increased the percentage of HPP-CFC and LPP-CFC in active cell cycle in a synergistic fashion. No significant change in the number of HPP-CFC or LPP-CFC per femur or femoral nucleated cellularity was observed. Thus, rIL-3 and nCSF-1 can synergize to effect the proliferation of the same cell populations in vivo.

Animals↗

PubMed1987

Identification of the hematopoietic growth factors elaborated by bone marrow stromal cells using antibody neutralization analysis.

These studies were undertaken to characterize the subclasses of hematopoietic growth factors produced by stromal cells in long-term murine bone marrow cultures. Exposure of these cultures to extremely high doses of irradiation (500 Gy), followed by endotoxin stimulation, permitted detection and characterization of various growth factor activities in the unconcentrated conditioned medium. To determine the nature of these activities, neutralization studies were performed using antisera against the following subclasses of purified colony-stimulating factors (CSFs): purified L-cell CSF-1, recombinant granulocyte-macrophage CSF (rGM-CSF), and recombinant interleukin 3 (rIL3). The antiserum against CSF-1 consistently abrogated 100% of the CSF bioactivity in irradiated stromal cell-conditioned medium (CM) but was only capable of neutralizing 62%-91% of the bioactivity in endotoxin-stimulated, irradiated stromal cell-CM. Antisera against rGM-CSF and rIL3 demonstrated variable effects. When the antisera were used in combinations, only the mixture of anti-CSF-1 + anti-GM-CSF resulted in 100% neutralization of the activities in endotoxin-stimulated, irradiated stromal cell-CM. This CM stimulated the IL3/GM-CSF-responsive cell line FDC-P1 but not the IL3-responsive (GM-CSF-unresponsive) cell line 32D cl-23. The FDC-P1 growth-promoting activity was inhibited only by the antiserum against GM-CSF and not by antiserum against IL3. These experiments indicate that stromal cells from long-term bone marrow cultures can produce and release CSF-1 and GM-CSF while the production of IL3 in this system, if there is any, could not be demonstrated.

Animals↗