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Biomedical subjects

A Waheed

Publications and source records attributed to A Waheed.

At least 19 recordsLinked to original sources
PubMed1992

Carbonic anhydrase IV on brain capillary endothelial cells: a marker associated with the blood-brain barrier.

Carbonic anhydrase (CA) activity plays an important role in controlling cerebrospinal fluid production and also influences neuroexcitation and susceptibility to seizures. Until recently, CA II was the only CA demonstrated in brain. Its distribution is limited to the epithelial cells of the choroid plexus and to the myelin-forming cells, the oligodendrocytes. In this report, we present immunoblots, using an antibody raised to CA IV from rat lung, that show that CA IV is also present in rat and mouse brain. Results of immunohistochemistry and immunoelectron microscopy on sections from rat and mouse brain are presented that show the distribution of CA IV to be quite distinct from that of CA II. CA IV is expressed on and is limited to the luminal surface of endothelial cells of cerebral capillaries. These results establish CA IV as a cytochemical marker associated with the blood-brain barrier and suggest an important role for CA IV in CO2 and HCO3- homeostasis in brain.

Animals↗
PubMed1992

Rat skeletal muscle membrane associated carbonic anhydrase is 39-kDa, glycosylated, GPI-anchored CA IV.

Sarcolemmal membrane vesicle preparations from white and red muscles of rat were found to contain a carbonic anhydrase which was indistinguishable from carbonic anhydrase IV from rat lung. This isozyme appears to account for all of the carbonic anhydrase activity in the sarcolemmal vesicle preparations. Digestion of 39-kDa CA IV with endoglycosidase F reduced the Mr to 36 kDa, suggesting that it contains one N-linked oligosaccharide. Treatment of sarcolemmal vesicles with phosphatidylinositol-specific phospholipase C released all of the activity, indicating that the enzyme is anchored to membranes by a phosphatidylinositol-glycan linkage. White muscle sarcoplasmic reticulum vesicles also contain a small amount of 39-kDa CA IV-type enzyme. A 52-kDa polypeptide in sarcoplasmic reticulum membranes cross-reacts with anti-human CA II and anti-rat CA II antisera, but does not bind to the sulfonamide affinity column. This cross-reacting polypeptide has no detectable CA activity.

Animals↗
PubMed1992

Human carbonic anhydrase IV: cDNA cloning, sequence comparison, and expression in COS cell membranes.

We have isolated a full-length cDNA for human carbonic anhydrase IV (CA IV) from a lambda gt10 human kidney cDNA library. The 1105-base-pair (bp) cDNA contains a 47-bp 5' untranslated region, a 936-bp open reading frame, and a 122-bp 3' untranslated region. The deduced amino acid sequence is colinear with the N-terminal sequence and the sequence of several tryptic peptides of human lung CA IV. It includes an 18-amino acid signal sequence, a 260-amino acid region that shows 30-36% similarity with the 29-kDa cytoplasmic CAs (CA I, CA II, and CA III), and an additional 27-amino acid C-terminal sequence that ends in a 21-amino acid hydrophobic domain. Of the 17 "active site" residues that are highly conserved in other human CAs, 16 are also present in CA IV. Expression of the cDNA in COS cells produced a 35-kDa enzyme that was membrane associated, resistant to inactivation by SDS, contained no carbohydrate, and reacted on Western blots with antiserum to the 35-kDa CA IV from human lung. Treatment of membranes from transfected COS cells with phosphatidylinositol-specific phospholipase C released 20-30% of the expressed enzyme from membranes, indicating that at least 20-30% of the expressed enzyme was anchored to membranes by a glycosyl-phosphatidylinositol linkage.

Amino Acid Sequence↗
PubMed1992

Membrane-associated carbonic anhydrase from rat lung. Purification, characterization, tissue distribution, and comparison with carbonic anhydrase IVs of other mammals.

Carbonic anhydrase (CA) IV was purified to homogeneity from rat lung microsomal and plasma membranes. The single N-terminal amino acid sequence showed 55% similarity to that reported for human CA IV. A monospecific antibody to the 39-kDa rat enzyme that cross-reacts on Western blots with CA IVs from other mammalian species was produced in rabbits. Digestion of rat lung enzyme with endoglycosidase (peptide-N-glycosidase F) reduced the Mr to 36,000, suggesting that rat CA contains one N-linked oligosaccharide chain. All of eight additional mammalian CA IVs that were examined also contained oligosaccharide chains, as evidenced by reduction in Mr from 52,000 (cow, sheep, and rabbit), 42,000 (pig, guinea pig, and dog), and 39,000 (mouse and hamster) to 36,000 after treatment of the respective lung microsomal membranes with peptide-N-glycosidase F. The 36-kDa human enzyme showed no change in molecular mass with this treatment. Thus, the human CA IV is the exceptional one in lacking carbohydrate. Rat lung CA IV was found to be relatively resistant to sodium dodecyl sulfate and to be anchored to membranes by a phosphatidylinositol-glycan linkage; both properties were found to be shared by other mammalian CA IVs. Western blot analysis indicated distribution of CA IV in rat tissues other than kidney and lung where it was previously known to be present. CA IV was particularly abundant in rat brain, muscle, heart, and liver, all locations where the CA IV enzyme was not known to be present previously. None was detected in rat skin or spleen.

Amino Acid Sequence↗
PubMed1992

Multipotent marrow stromal cell line is able to induce hematopoiesis in vivo.

Several murine marrow stromal cells were established from murine bone marrow cultures. Stromal cell lines transfected with a tumor-inducing polyoma virus middle T antigen (MTAg) were inoculated into nude mice subcutaneously. KUSA-MTAg cells, one of these cell lines, led to the rapid local development of bone marrow consisting of trilineage hematopoietic cells and bone; other cell lines produced spindle cell sarcoma or hemangiosarcoma. These results suggested that a single stromal cell line, KUSA-MTAg cells, may induce hematopoietic stem cells or early progenitors of three lineages of hematopoietic cells in vivo. Interestingly, untransfected KUSA cells expressed three new mesenchymal phenotypes, osteocytes, adipocytes, and myotubes, after treatment with 5-azacytidine.

Adipose Tissue↗
PubMed1991

Localization of carbonic anhydrase IV in a specific capillary bed of the human eye.

Carbonic anhydrase (CA) activity plays an important role in controlling aqueous humor production in the eye and in regulating intraocular pressure. Prior studies identified the soluble isozymes CA II and CA I in the human eye and also suggested a distinct membrane-associated CA. We used an antibody to CA IV, the membrane-anchored isozyme from human lung, to study CA IV in eye tissues and to compare its distribution with that of CA II. We found intense immunostaining for CA IV associated with endothelial cells of one specific uveal capillary bed, the choriocapillaris. CA IV was not detected in endothelial cells of the contiguous capillaries of the iris or in endothelial cells of other vessels. Immunoreactivity for CA IV was also intense in epithelial and fiber cells of the lens but was not detectable in the neuroretina, the ciliary process (except for capillaries), and the cornea, all sites where immunostaining with anti-CA II antibody was intense. These studies indicate that the membrane-associated CA in human eye, which was suspected from histochemical studies, is CA IV. Defining the physiological role of this ocular isozyme remains a challenge.

Adult↗
PubMed1991

Glycosylation of the Mr 46,000 mannose 6-phosphate receptor. Effect on ligand binding, stability, and conformation.

Using site-directed mutagenesis the N-glycosylation sites of the Mr 46,000 mannose 6-phosphate receptor (MPR 46) were identified as asparagine residues 57, 83, 107, and 113. The two outer asparagines carry high mannose-type and the two inner asparagines carry complex-type oligosaccharides. The glycosylation mutants were analyzed for stability, binding activity, and subcellular distribution. Replacing asparagine 57, 83, or 107 by threonine decreased only the stability of the receptor. Replacing asparagine 113 by threonine decreased the stability and binding activity. Deletion of three or all four N-glycosylation sites led in addition to an accumulation of the mutant receptors in endoplasmic reticulum-like structures. Nonglycosylated MPR 46 synthesized in the presence of tunicamycin, thus preserving the asparagine residues, had a normal stability and high affinity binding. The decreased stability and binding activity of the receptor mutants is therefore due to the exchange of asparagine residues rather than to the loss of N-linked oligosaccharides. The nonglycosylated receptor, however, displayed a decreased conformational stability after solubilization as a single cycle of freezing and thawing reduced the binding activity to one-third of the control. Simultaneously, the receptor lost its quaternary structure. It is concluded from these results that the N-glycosylation of the receptor is required for the stability of a high affinity conformation, but not for the binding itself or the intracellular stability.

Cell Compartmentation↗
PubMed1991

Mr 46,000 mannose 6-phosphate receptor. The role of histidine and arginine residues for binding of ligand.

The chemical modification of histidine and arginine residues results in a loss of binding of the Mr 46,000 mannose 6-phosphate receptor (MPR 46) to a phosphomannan affinity matrix (Stein, M., Meyer, J. E., Hasilik, A., and von Figura, K. (1987) Biol. Chem. Hoppe-Seyler 368, 927-936). Reversal of the modification or presence of mannose 6-phosphate during the modification partially restores or protects the binding activity, indicating that histidine and arginine residues contribute to the mannose 6-phosphate binding site. The 5 histidine and 8 arginine residues within the luminal domain of MPR 46, which contains the ligand binding site, were exchanged by site-directed mutagenesis. Only the conservative replacement of His-131 and Arg-137 by serine and lysine, respectively, results in a loss of binding activity without affecting other properties of the receptor such as the presence of intramolecular disulfide bonds, immunoreactivity, processing of N-linked oligosaccharides, formation of dimers, intracellular distribution, and surface expression. Conservative replacement of other histidine and arginine residues did not affect the binding activity. Nonconservative replacement of several arginine residues reduced binding activity and immunoreactivity, indicating that the loss of a positive charge at these positions alters the folding of MPR 46. We conclude from these results that His-131 and Arg-137 are essential for binding of ligands by MPR 46.

Arginine↗
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↗
PubMed1977

Purification and properties of the urea amidolyase from Candida utilis.

Urea amidolyase was purified to homogeneity from extracts of Candida utilis. The purification involves protamine sulfate precipitation, ammonium sulfate precipitation, polyethylene glycol precipitation, Sepharose 6B gel filtration, DEAE-cellulose column chromatography, and hydroxylapatite column chromatography. The final preparation is pure as judged by disc-gel electrophoresis. The molecular weight of urea amidolyase, as determined by gel filtration and disc-gel electrophoresis, is between 500,000 and 520,000. Treatment with sodium dodecyl sulfate results in two peptides with molecular weights of 70,000 and 170,000. The urea carboxylase and allophanate hydrolase activities of urea amidolyase may be distinguished from one another on the basis of (a) the effect of the stabilizers, urea and glycerol, (b) the effect of storage pH on activity, and (c) selective inhibition by sulfhydryl reagents.

Candida↗