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A Waheed

Publications and source records attributed to A Waheed.

At least 163 records · Page 9Linked to original sources
PubMed1985

Isolation and characterization of a homogeneous acid phosphatase from catfish liver.

A homogeneous, tartrate-inhibitable acid phosphatase (AcPase) was obtained from the liver of channel catfish (Ictalurus punctatus) by the use of Affi Gel-10-coupled aminohexyltartramic acid affinity chromatography. The enzyme has a molecular weight of 82,500 and is a dimer consisting of two apparently equivalent subunits with subunit weights of 35,000 +/- 3000. Amino acid composition data are presented and compared with those of mammalian acid phosphatases. Data suggest that the enzyme is a metalloacid phosphatase. Catfish liver AcPase exhibits two molecular forms with pI 5.66 and 5.37 which were separated by chromatofocusing. A spontaneous conversion of the less acidic form to a more acidic form was observed and this conversion was accompanied by a decreased sensitivity towards tartrate inhibition.

Acid Phosphatase↗
PubMed1985

Structural and immunological relationships among mammalian arylsulfatase A enzymes.

Structural and immunological properties of numerous arylsulfatase A enzymes (EC 3.1.6) were examined in order to assess the relationships among these enzymes in animals. Arylsulfatase A enzymes from all animals bind to a Concanavalin A-Sepharose column, consistent with the conclusion that they are all glycoproteins. At pH 7.5 the apparent mol. wts of the enzymes are 80-182 kDa, while at pH 4.5 the mammalian arylsulfatase A enzymes dimerize and exhibit apparent mol. wts in the range of 297-348 kDa, but the enzymes from opossum and other lower classes of animals do not aggregate at pH 4.5. The mammalian arylsulfatase A enzymes, which aggregate at pH 4.5, also bind to rabbit liver arylsulfatase A monomers immobilized on an Affi-Gel 10 matrix. The arylsulfatase A enzymes that were studied all exhibit the anomalous kinetic behavior regarded as characteristic of these enzymes. However, not all of the inactivated enzymes are reactivated by sulfate ions. Goat antiserum raised against homogeneous rabbit liver arylsulfatase A cross-reacts with all of the mammalian enzymes in Ouchterlony gel diffusion experiments, whereas the enzymes from lower classes of animals do not cross-react. Quantitative immunoprecipitation experiments demonstrate that the mammalian enzymes are very similar to each other, with greater than 60% primary sequence homology indicated, while arylsulfatase A from opossum and other lower classes of animals show only a partial immunological similarity with the mammalian enzymes. Taken together, the data suggest that the active site of the enzyme and the structural features of the protein are highly conserved during the evolution of the enzyme molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Amino-terminal amino acid sequence of murine colony-stimulating factor 1.

We report the amino acid composition, the NH2-terminal sequence, and the tryptic map by HPLC for murine L-cell colony-stimulating factor 1 (CSF-1). CSF-1 purified by affinity chromatography was S-carboxymethylated under denaturing conditions and subjected to sequence microanalysis. CSF-1 contains four or five cysteine residues per subunit and approximately 30-40% carbohydrate by weight. Three contiguous cysteine residues were located in the NH2-terminal sequence. An additional two cysteine residues were located on the tryptic map. Previous studies as well as immunoblotting studies reported here suggest that sulfhydryl bridging plays a major role in maintaining the conformation of the protein. Carbohydrate was located on two tryptic fragments. The findings of a single NH2-terminal sequence in high yield and a relatively simple tryptic map (15 major peptides) suggest that CSF-1 contains two identical subunits. The NH2-terminal sequence of CSF-1 has only limited homology to insulin or insulin-like hormones but no homology with granulocyte/macrophage-CSF or interleukin 3.

Amino Acid Sequence↗
PubMed1985

Purification of mammalian arylsulfatase A enzymes by subunit affinity chromatography.

Rabbit liver arylsulfatase A (arylsulfatase sulfohydrolase, EC 3.1.6.1) monomer was immobilized on cyanogen bromide-activated Sepharose-6MB and on Affi-Gel-10 under various experimental conditions in order to study the effects of variables in sulfatase monomer/oligomer subunit affinity chromatography. First, the number of reactive groups on activated Sepharose-6MB and Affi-Gel-10 was determined by a procedure involving spectrophotometric titration with L-tyrosine. After covalent coupling of sulfatase monomers to the gels, the enzyme binding capacities of the sulfatase subunit affinity gel matrixes were determined at pH 4.5. The maximum binding of free monomers from solution could be achieved when the Affi-Gel-10 protein monomer matrix was prepared at low degrees of covalent loading. The introduction of a batch technique for equilibration of the protein sample with the monomer affinity matrix also increased the efficiency of the subunit affinity gel in purification procedures. The effect of pH on the stability of the heterodimers formed between monomers of rabbit liver arylsulfatase A immobilized on Affi-Gel-10 and free monomers of arylsulfatase A enzymes from different tissues and organisms was studied using the batch technique. For all sulfatase A enzymes tested, the midpoint of the pH transition for subunit association was pH 6.2, suggesting that the amino acid residues involved in the dimerization are similar. The versatility of the Affi-Gel-10 monomer affinity matrix was further demonstrated by purifying 13 mammalian arylsulfatase A enzymes to homogeneity, as assessed by Sephacryl chromatography, native and SDS gel electrophoresis. The molecular weights of the homogeneous monomers and their peptide subunits were in the range of 110-180 KDa and 50-64 KDa, respectively. The amino acid compositions of these enzymes were also determined.

Animals↗
PubMed1985

Effect of Listeria monocytogenes infection on serum levels of colony-stimulating factor and number of progenitor cells in immune and nonimmune mice.

Studies were performed to determine changes in serum macrophage colony-stimulating factor (M-CSF) levels and the number of macrophage progenitor cells in bone marrow and spleens of nonimmune and immune mice infected with Listeria monocytogenes. Immunity in mice was established by infecting mice 6 weeks before use with a sublethal dose of L. monocytogenes. When challenged with 10(4) L. monocytogenes organisms, immune mice had an early (12 h) peak in M-CSF serum concentrations. Levels remained elevated for 24 h but fell towards normal by 48 h. By contrast, M-CSF levels in nonimmune mice did not rise until 24 h after challenge, remained elevated for 7 days, and returned to normal by 14 days. The number of macrophage progenitor cells in the bone marrow of immune mice rose slightly during infection, whereas the number in nonimmune mice fell significantly by days 4 and 7. Progenitor cells in spleens of immune mice more than doubled during infection; in nonimmune mice, a sixfold increase was noted. These results indicate that important parameters of monocyte production differ in immune and nonimmune mice during listeria infection and suggest a possible mechanism for differences in resistance to infection.

Animals↗
PubMed1985

Hematopoietic factor production by a cell line (TC-1) derived from adherent murine marrow cells.

An adherent cell line, termed TC-1, has been isolated from long-term liquid culture of murine marrow cells by repeated exposure of the adherent cells to 0.1% trypsin. This is an alkaline phosphatase-positive cell line showing variable staining with acid phosphatase and alpha-naphthyl acetate esterase. On electron microscopy, the cells have moderate amounts of rough endoplasmic reticulum and variable numbers of polyribosomes. Some cells contain large clusters of laked glycogen particles. Intermediate junctions are present between some cells. Conditioned medium from this cell line produced from 384 to 638 units of CSF-1 per milliliter by radioimmunoassay and a CSF-1-dependent synergistic activity, which stimulates giant macrophage colony formation of marrow cells in soft agar. The conditioned medium also stimulates 3H-TdR incorporation by marrow cells in liquid culture and induces secondary adherent cell lines. The growth factor(s) produced by the TC-1 stromal cell line may be important in the regulation of early stages of hematopoietic differentiation. Two subclones, TC-1-C-11 and TC-1-C-3, have been isolated from passage 25 of the TC-1 cells by a penicylinder separation technique. The TC-1-C-11 is phenotypically like the parent TC-1 line and produces macrophage growth factors. The TC-1-C-3 grows as an epithelioid monolayer with visible junctions among adjacent cells under phase contrast microscopy. This subclone produces retrovirus and is capable of providing anchorage support for hematopoietic stem cells. The TC-1 cell line and its subclones may provide models for the control of early stem cell proliferation and differentiation.

Agar↗
PubMed1985

Macrophage colony-stimulating factor (M-CSF) enhances the capacity of murine macrophages to secrete oxygen reduction products.

The capacity of macrophage colony-stimulating factor (M-CSF) to enhance respiratory burst activity in peritoneal macrophages was measured. Macrophages incubated for 48 hr or more with concentrated L cell-conditioned medium as a source of M-CSF released two to three times as much O2- in response to PMA as did unexposed macrophages. Stimulation was noted at concentrations of colony-stimulating activity from 0.1 to 2000 U/ml and was maximal at 10 to 100 U/ml. Purified, endotoxin-free CSF enhanced secretion to a similar degree as unpurified L cell-conditioned medium. Release of O2- by M-CSF macrophages occurred over 60 min and was triggered by opsonized zymosan as well as PMA. H2O2 release was also enhanced in macrophages exposed to both unpurified and purified M-CSF. These data indicate that M-CSF enhances the capacity of mature macrophages to release oxygen reduction products, and they are consistent with reports that CSF can stimulate the release of other secretory products.

Animals↗
PubMed1985

Effect of anti-CSF on in vivo hemopoiesis.

Studies were undertaken to determine whether colony-stimulating factor in the serum is important in the control of granulopoiesis and monocytopoiesis. Groups of mice were injected with either antiserum to colony-stimulating factor (CSF) or normal rabbit serum every 12 h for intervals of 6-7 days. Antibody treatment did not lead to a reduction in circulating granulocytes or monocytes nor a decrease in marrow cellularity. In further studies, two dose levels of purified anti-CSF were employed; rabbit IgG served as a control. Virtually no effect was observed on blood monocytes, blood granulocytes, or marrow granulocytes; however, antibody treatment did cause a decline in marrow colony-forming cells and a reciprocal increase in erythroid progenitor cells. Immunoassays showed a virtual absence of serum anti-CSF after five days, despite continued administration of the antibody. Modified immunoassays indicated that this was due to development of murine antibodies directed against anti-CSF. In further experiments, mice received the purified antibody or whole antiserum in conjunction with cortisone to reduce murine antibody formation. Despite persistent anti-CSF levels in the serum, blood granulocytes, blood monocytes, and marrow precursor cells were unaffected. These findings suggest that serum levels of CSF do not appear to be responsible for in vivo granulopoiesis or monocytopoiesis.

Animals↗
PubMed1985

Release from mouse macrophages of acidic isoferritins that suppress hematopoietic progenitor cells is induced by purified L cell colony stimulating factor and suppressed by human lactoferrin.

Purified mouse L cell colony-stimulating factor (CSF) and purified iron-saturated human lactoferrin (LF) were assessed for their effects on release of acidic isoferritin-inhibitory activity (AIFIA) from resident peritoneal and spleen macrophages of B6D2F1 mice. Constitutive release of AIFIA was dependent on the number of macrophages conditioning the culture medium. Detection of release of AIFIA required at least 10(4) macrophages/ml, and increased release was noted with increased concentrations of cells. This release was enhanced by CSF and was induced by CSF from concentrations of 10(3) macrophages/ml, from which constitutive release of AIFIA was not detected. Increased concentrations of CSF induced increased release of AIFIA. The inducing effect was removed by pretreating CSF with rabbit anti-L cell CSF serum. LF suppressed the constitutive as well as the CSF-induced release of AIFIA, but results were dependent on the relative concentrations of LF and CSF used. The suppressive effects of LF were removed by pretreating LF with goat anti-human LF. Constitutive, but not CSF-induced, release of AIFIA could be ablated by removal of Ia antigen-positive macrophages with low concentrations of monoclonal anti-Ia plus complement. Treating macrophages with higher concentrations of anti-Ia in the absence of complement blocked the LF suppression of constitutive AIFIA release but not the CSF-induction of AIFIA release. Release of AIFIA from mouse macrophages can be modulated by CSF and LF. This modulation may be of significance for the regulation of myelopoiesis.

Animals↗
PubMed1985

Studies of the human testis. XIX. Preparation of an antibody to human testosterone-oestradiol-binding globulin and its application to the study of testicular androgen-binding protein.

Human testosterone-oestradiol-binding globulin (hTeBG) was purified from pregnancy serum by ammonium sulphate precipitation, preparative flatbed electrofocusing, Concanavalin A-Sepharose affinity chromatography, Sephadex G-150 gel filtration, DEAE-Sephadex chromatography and preparative polyacrylamide gel electrophoresis. The yield was 0.3 mg of hTeBG with a specific acitivity of 1.1 nmoles DHT bound per mg. An antiserum to TeBG was raised in rabbits. Anti-hTeBG IgG was separated from rabbit TeBG by DEAE-Affi-Gel-Blue chromatography. Anti-hTeBG was titrated using protein A-Sepharose which quantitatively binds IgG and therefore bound [3H]DHT-hTeBG-anti-TeBG complexes. The androgen binding components from human testis were separated on Concanavalin A-Sepharose columns into excluded and retained fractions. The antibody bound both testis fractions with titration curves which paralleled that of TeBG, indicating that these androphilic proteins share common immunodeterminants with hTe-BG. The possibility that these testicular proteins are identical in amino acid sequence to TeBG and differ only in carbohydrate content will require further verification. Finally, these results indicate that antibodies to TeBG can be used to study human testicular androgen-binding protein.

Androgen-Binding Protein↗
PubMed1984

Structural and chemical characterization of a homogeneous peptide N-glycosidase from almond.

A peptide N-glycosidase that catalyzes the hydrolysis of N-linked oligosaccharide chains from glycopeptides and glycoproteins has been purified to homogeneity from almond emulsin and from almond meal. Purification from almond emulsin using ion-exchange chromatography, gel filtration chromatography, and preparative polyacrylamide gel electrophoresis gave an enzyme which was purified more than 700-fold and with a yield of 63%. An alternative procedure, more suitable for efficient large scale purification, used ion-exchange, affinity, and gel filtration chromatography. When purification began with almond emulsin, the enzyme was purified 1200-fold with a 37% yield, while when purification began with almond powder, the enzyme was purified 9000-fold with a yield of 45%. The homogeneous enzyme is stable at 4 degrees C for several months in 10 mM sodium acetate, pH 5.0, buffer. The peptide N-glycosidase is itself shown to be a glycoprotein consisting of a single polypeptide chain with a molecular weight of 66 800 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Circular dichroism spectra of the native molecule indicate the presence of a high (approximately 80%) alpha-helix content. The amino acid and carbohydrate contents of the enzyme are presented. When a convenient new assay with a turkey ovomucoid glycopeptide as a substrate is used, the enzyme preparation exhibits a broad pH optimum centered between pH 4 and pH 6. The enzyme is readily inactivated by SDS and guanidine hydrochloride, but it is stable in the presence of moderate concentrations of several other protein denaturants.(ABSTRACT TRUNCATED AT 250 WORDS)

Amidohydrolases↗
PubMed1984

Changes in serum colony-stimulating factor and monocytic progenitor cells during Listeria monocytogenes infection in mice.

The capacity of a host to produce and mobilize monocytes is an essential component of host defenses during the early phases of infection caused by Listeria monocytogenes. In this study, the concentrations of colony stimulating factor (CSF) and the numbers of monocyte progenitor cells (CFUm) were measured in mice during infection with L. monocytogenes. The concentration of CSF in serum increased sharply during the first 24 h of infection and remained elevated for the next 7 days. The number of CFUm in the bone marrow, however, decreased during the first 4 days after injection of L. monocytogenes. Thereafter, the number increased slowly, returning to normal on day 14. The decrease in marrow progenitor cells did not appear to result from a reduced sensitivity to CSF. In contrast to bone marrow changes, spleen progenitor cells increased greater than 400%, reaching a peak 7 days after bacterial challenge. These data indicate that monocyte production during L. monocytogenes infection is correlated with a rise in serum CSF concentration, depletion of bone marrow CFUm, and an increase in the number of spleen CFUm.

Animals↗
PubMed1984

The role of colony stimulating factor in the regulation of granulopoiesis.

It is now clear that a variety of colony stimulating factors may be responsible for growth and maturation of progenitor cells in the marrow. Four of these CSFs have been purified to homogeneity with the distinct possibility of large-scale production of these factors by molecular cloning techniques. Studies of the binding and interaction of these factors with marrow cells are now under way. In vivo experiments with diffusion chamber techniques have shown the relevance of CSF in stimulating granulopoiesis when the progenitor cells are separated from their microenvironment but only limited effects in normal mice. This suggests that extensive microenvironmental influences may be involved in CSF-mediated granulopoiesis.

Animals↗
PubMed1983

Production of colony stimulating factor in long-term bone marrow cultures.

Previous studies have shown no detectable colony-stimulating factor (CSF) in media harvested from long-term bone marrow cultures. In the present experiments supernatants from long-term cultures established in three laboratories were assayed for CSF by colony assay and by radioimmunoassay (RIA). Most samples were devoid of biologic activity but all contained CSF as judged by RIA. Biologic activity was found in the majority of samples after diafiltration to remove low molecular weight inhibitors or 5-fold concentration by ultrafiltration. Samples that remained inactive in the colony assay were subjected to gel filtration on Sephadex G-150 to remove potential high molecular weight inhibitors. Biologic activity remained lower than that by RIA in two of three samples tested. Thus, most long-term cultures appear to contain biologically active CSF but this activity is masked by various types of inhibitors. In addition some media appear to contain material that is only detected by RIA.

Animals↗
PubMed1983

Phorbol ester-stimulated murine myelopoiesis: role of colony-stimulating factors.

Tumor promoting phorbol esters, such as 12-0-tetradecanoyl-phorbol-13-acetate (TPA), stimulate colony formation in vitro by murine granulocyte-macrophage progenitors (GM-CFC) without added colony stimulating factors (CSF). To determine whether TPA induces CSF production in vitro, marrow cells were cultured for 1 to 7 days in liquid medium with or without TPA. No CSF was detected in any sample by a double antibody radioimmunoassay (sensitivity = 2 units/0.1 ml), however, colony-stimulating activity was detected in supernatant fluid from all TPA containing cultures by bioassay. This activity appeared to result from a direct effect of TPA rather than from production of CSF, as equivalent activity was found in TPA-containing medium incubated in the absence of marrow cells. Rabbit antiserum to purified L-cell CSF inhibited colony formation stimulated by L-cell CSF and WEHI-3 CSF, but had no effect on colony formation induced by TPA. Cells from long-term marrow cultures responded to TPA with colony formation, despite culture conditions and cell fractionation procedures that reduced the frequency of CSF-producing macrophages to less than 1.0%. TPA inhibited binding of radioiodinated L-cell CSF to marrow cells, especially if the cells were first exposed to TPA. These results do not support induction of CSF production as the major mechanism of phorbol ester stimulation of myelopoiesis. Phorbol esters may directly stimulate GM-CFC and/or enhance their response to CSF by a mechanism involving CSF binding sites.

Animals↗
PubMed1983

Two allelic forms of human arylsulfatase A with different numbers of asparagine-linked oligosaccharides.

The biosynthesis of arylsulfatase A in human skin fibroblasts was studied by labeling cells and isolating arylsulfatase A using immune precipitation and polyacrylamide gel electrophoresis under denaturing and reducing conditions. Arylsulfatase A was synthesized as precursor polypeptides of 62 kDa or 59.5 kDa. Cell lines synthesizing either or both polypeptides were found. The results of a family study were consistent with the assumption that the two arylsulfatase A polypeptides are of allelic nature. In various heterozygous cell lines, the two polypeptides were formed at equal or different rates. The relative rate of biosynthesis was constant for an individual cell line, suggesting that both allelic products were under separate genetic control. In a group of 21 unrelated individuals, the gene frequency of alleles for the 62- and 59.5-kDa precursor forms was 3:1. The two allelic forms of the arylsulfatase A polypeptides were converted into a 57-kDa form by endo-beta-N-acetylglucosaminidase H, an enzyme specifically removing asparagine-linked oligosaccharides of the high-mannose (and hybrid) type. The apparent difference in the number of asparagine-linked oligosaccharides suggests that the two allelic genes differ in a region coding the sequence Asn-X-Thr(Ser), which is required for attachment of asparagine-linked oligosaccharides.

Alleles↗