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A Waseem

Publications and source records attributed to A Waseem.

At least 19 recordsLinked to original sources
PubMed2000

Human-Saccharomyces cerevisiae proliferating cell nuclear antigen hybrids: oligomeric structure and functional characterization using in vitro DNA replication.

The proliferating cell nuclear antigen (PCNA) is a highly conserved protein required for the assembly of the DNA polymerase delta (pol delta) holoenzyme. Because PCNAs from Saccharomyces cerevisiae and human do not complement each other using in vitro or in vivo assays, hybrids of the two proteins would help identify region(s) involved in the assembly of the pol delta holoenzyme. Two mutants of human PCNA, HU1 (D21E) and HU3 (D120E), and six hybrids of human and S. cerevisiae PCNA, HC1, HC5, CH2, CH3, CH4, and CH5, were prepared by swapping corresponding regions between the two proteins. In solution, all PCNA assembled into trimers, albeit to different extents. These PCNA variants were tested for stimulation of pol delta and in vitro replication of M13 and SV40 DNA as well as to stimulate the ATPase activity of replication factor C (RF-C). Our data suggest that in addition to the interdomain connecting loop and C terminus, an additional site in the N terminus is required for pol delta interaction. PCNA mutants and hybrids that stimulated pol delta and RF-C were deficient in M13 and SV40 DNA replication assays, indicating that PCNA-induced pol delta stimulation and RF-C-mediated loading are not sufficient for coordinated DNA synthesis at a replication fork.

Adenosine Triphosphatases↗
PubMed1999

Keratin 15 expression in stratified epithelia: downregulation in activated keratinocytes.

Keratin 15 (K15) is a type I keratin without a defined type II partner whose expression in epidermal diseases has not been investigated. In this study we have used LHK15, a monoclonal antibody raised against the last 17 amino acids of the K15 polypeptide, to show that K15 is expressed primarily in the basal keratinocytes of stratified tissues, including the fetal epidermis and fetal nail. Although K15 in normal hair follicles was virtually absent from hair bulbs, it was expressed by a subset of keratinocytes in the outer root sheath. By comparison, K14 expression was found throughout the outer root sheath of hair follicles; however, when both K14 alleles were naturally ablated, the expression of K15 was also observed throughout the outer root sheath of the follicles. Expression of K15 mRNA was assessed by in situ hybridization and corroborated the data from immunostaining. An increase in K15 mRNA and protein expression in hair follicles from the K14 ablated epidermis suggested an upregulation of the K15 gene in the absence of the K14 protein. In organotypical cultures where differentiating keratinocytes expressed markers of activated phenotype, i.e., K6 and K16, expression of K15 was undetectable. The expression of K15 mRNA and protein was also downregulated in two hyperproliferating situations, psoriasis and hypertrophic scars. Because keratinocytes in psoriasis and hypertrophic scars are activated, we conclude that K15 expression is not compatible with keratinocyte activation and the K15 gene is downregulated to maintain the activated phenotype.

Adult↗
PubMed1998

Isolation, sequence and expression of the gene encoding human keratin 13.

Keratins are a family of highly homologous proteins expressed as pairs of acidic and basic forms which make intermediate filaments in epithelial cells. Keratin 13 (K13) is the major acidic keratin, which together with K4, its basic partner, is expressed in the suprabasal layers of non-cornified stratified epithelia. The mechanism which allows mucosal-specific expression of this keratin remains unknown. To provide insight into the tissue-specific expression, we have isolated the human K13 gene by screening a chromosome 17 library with a specific K13 cRNA probe. Sequence analysis of unidirectional deletions produced by transposon Tn3 has revealed that the gene is 4601 nucleotides long and contains seven introns and eight exons. When driven by the CMV promoter, the gene produced K13 protein in MCF-7 cells, which normally do not express this protein. Two transcription-start sites were identified, the major being at 61 and the minor at 63 nucleotides upstream of ATG. The upstream sequence contained a TATA box and several other putative transcription factor binding sites. A single copy of the K13 gene was detected in the human genome by Southern hybridisation and polymerase chain reaction. K13 mRNA shows differential expression in cultured keratinocytes, and in A431 cells the RNA levels remained independent of calcium concentrations in the culture medium. Characterisation of the human K13 gene will facilitate elucidation of the molecular mechanism regulating K13 expression in mucosal tissues.

Amino Acid Sequence↗
PubMed1998

Activated keratinocytes in the epidermis of hypertrophic scars.

The etiology of hypertrophic scarring, a pathological end point of wound healing, is unknown. The scars most commonly occur when epithelialization has been delayed during, for example, the healing of deep dermal burn wounds. Hypertrophic scars are conventionally described as a dermal pathology in which the epidermis has only a passive role. In this study, the expression of keratin intermediate filament proteins and filaggrin has been investigated in the epidermis of hypertrophic scars and site-matched controls from the same patients. Hypertrophic scar epidermis was found to express the hyperproliferative keratins K6 and K16 in interfollicular epidermis in association with K17 and precocious expression of filaggrin. K16 mRNA was localized by in situ hybridization using a highly specific cRNA probe. In contrast to the immunohistochemical location of K16 protein, the K16 mRNA was found to be expressed in the basal cell layer of normal skin. In hypertrophic scars the mRNA distribution corroborated the abnormal K16 protein distribution. These results suggest the keratinocytes in hypertrophic scar epidermis have entered an alternative differentiation pathway and are expressing an activated phenotype. Activated keratinocytes are a feature of the early stages of wound healing producing growth factors that influence fibroblasts, endothelial cells, and the inflammatory response. We propose that cellular mechanisms in the pathogenesis of hypertrophic scarring are more complex than isolated dermal phenomena. The persistence of activated keratinocytes in hypertrophic scar epidermis implicates abnormal epidermal-mesenchymal interactions.

Adult↗
PubMed1997

Identification of a novel keratin epitope: evidence for association between non-helical sub-domains L12 during filament assembly.

Keratin filaments in simple epithelial cells are heteropolymers of keratin 8 (K8) and keratin 18 (K18) polypeptides. The assembly of these polypeptides into intermediate filaments is a complex multi-stage phenomenon that involves several levels of associations. These molecular associations are not very well characterized. Monoclonal antibodies (MAbs) with defined specificities can be used to probe these associations and to isolate various intermediates in the assembly pathway. Here we describe the specificity of a MAb LE65 that has been widely used in keratin expression studies. We report that the MAb LE65 does not recognize individual keratin polypeptides but it instead reacts with a complex of K8 with K18. The MAb also did not react with complexes of K8 or K18 with other keratins. By allowing the antibody to react with complexes reconstituted from keratin fragments plus the complementary keratin, we have mapped the MAb LE65 epitope on the L12 sub-domains of K18, residues 214-231, and K8, residues 234-265, which must associate together to achieve antibody binding. These results suggest that the non-helical linkers, L12, of complementary keratins associate directly during filament assembly. This would explain why microinjection of MAb LE65 has been shown to disrupt keratin filaments. Furthermore, it may also help to explain the mechanism of filament disruption in some skin blistering syndromes induced by spontaneous mutations in the L12 region.

Amino Acid Sequence↗
PubMed1997

Variable requirement for splicing signals for nucleocytoplasmic export of mRNAs.

Using in situ hybridisation to detect the intracellular localisation of mRNAs we have found that mRNAs expressed from intronless cDNAs of normally intronic genes are expressed well but largely retained in nuclei. The degree of nuclear retention is quite variable but in all cases addition of splicing signals to the expression cassette are required for efficient export of the mRNAs from nucleus to cytoplasm. In contrast mRNAs expressed from the intronless genes of hamster beta-adrenergic receptor and human serotonin receptor type 1A showed very little nuclear accumulation and strong expression in the cytoplasm independently of splicing signals. The data demonstrate a link between splicing and export and dissemble from the idea that splicing enhances mRNA expression by protecting nascent nuclear mRNAs from degradation.

Animals↗
PubMed1996

A keratin antibody recognizing a heterotypic complex: epitope mapping to complementary locations on both components of the complex.

Keratin filaments in simple epithelial cells are heteropolymers of keratin 8 (K8) and keratin 18 (K18), which can be stained by the monoclonal antibody (MAb) LE61. This antibody has been widely used to study keratin expression in normal and neoplastic tissues. In this study we have found that MAb LE61 does not react with individual keratin polypeptides either derived from natural sources or expressed as recombinant proteins in Escherichia coli. However, when K8 or K18 bound to nitrocellulose were incubated with complementary keratin they became reactive with this antibody. A mixture of K8 and K18 in solution also reacted strongly with the MAb LE61 in ELISA. These observations suggest that the antibody recognizes a discontinuous epitope on the keratin complex. The antibody also reacted with complexes of K8 and K18 with other keratins. To locate the epitope of this antibody we have expressed K8 and K18 fragments, deleted from the amino- and carboxyl-termini, as fusion proteins with glutathione S-transferase. These fragments were able to form a heterotypic complex with the complementary keratin. Binding of the MAb LE61 to these complexes mapped the two halves of the epitope on K8, between residues 353 and 367, and on K18, between residues 357 and 385. The two halves of the epitope appear to be in close association in the heterotypic complex since deletions from the amino-terminus did not influence the antibody binding. The highly conserved nature of this epitope in both type I and type II keratins could explain the MAb LE61 reactivity with complexes of K8 or K18 with other keratins.

Amino Acid Sequence↗
PubMed1993

Expression of keratin mRNAs and proteins in normal salivary epithelia and pleomorphic adenomas.

Control of keratin (K) gene expression may be important for cell differentiation in complex epithelia such as salivary gland. To investigate differences in distribution between keratin mRNAs and their respective proteins, a combined in situ hybridization (ISH) and immunohistochemical study was undertaken on nine normal salivary glands and seven pleomorphic adenomas. ISH employed riboprobes to K7, K8, K14, K18, and K19. Immunohistochemistry was performed on adjacent sections using monoclonal antibodies (MAbs) to the same keratins. Normal luminal cells showed abundant hybridization with probes for K7, K8, K18, and K19. Keratin 14 mRNA was present in basal and myoepithelial cells at a low level of expression. Proteins of their keratins were strongly stained. Neoplastic cells showed variable expression of mRNA and protein for K7, K8, K18, and K19. There was a high level of K14 mRNA but variable protein. The findings provide evidence that expression of these keratins in normal salivary epithelia is regulated transcriptionally and that in neoplasia this system is in considerable disarray.

Adenoma, Pleomorphic↗
PubMed1990

Localization of the gene for human simple epithelial keratin 18 to chromosome 12 using polymerase chain reaction.

Many human genes encoding keratin intermediate filament proteins are clustered on chromosomes 17 (the type I genes) and 12 (the type II genes). Some have not yet been localized, notably the genes for the primary embryonic keratins 8 and 18, normally expressed in simple epithelia: this is because the numerous pseudogenes for these keratins have made it difficult to identify the true functional gene in each case. Through the use of human-specific primers from within introns of the published gene sequence for human type I keratin 18, human genomic DNA has been specifically amplified using the polymerase chain reaction. A single reaction product was obtained. DNA from a characterized series of mouse-human somatic cell hybrid lines was tested for the presence of sequences able to initiate the chain reaction from these primers, and the presence or absence of this genomic DNA PCR product allowed us to assign a gene for human keratin 18 to chromosome 12 unambiguously. This differs from the location of other human type I keratins on chromosome 17 and may indicate the early divergence of the genes for stratifying cell keratins from that of simple, or embryonic, keratin 18.

Animals↗
PubMed1990

Identification and protein kinase C-dependent phosphorylation of alpha-adducin in human fibroblasts.

A protein of Mr approximately 120,000, related to the human erythrocyte membrane skeletal protein alpha-adducin, has been identified by immunological criteria in human fibroblasts. Using similar methods, beta-adducin (an Mr approximately 110,000 protein that forms a dimeric complex with alpha-adducin in the erythrocyte) is not present in fibroblasts. Subcellular distribution studies reveal that fibroblast alpha-adducin is largely associated with the particulate fraction and is most effectively solubilized from that fraction by a combination of nonionic detergent and high salt. Immunocytochemistry of quiescent fibroblasts shows that alpha-adducin is clustered in large perinuclear arrays that may correspond to vesicular structures; weak staining was also found in the sub-plasma membrane region. As in erythrocytes, the phosphorylation of fibroblast alpha-adducin is elevated on exposure of cells to phorbol esters that activate protein kinase C (PK-C). In addition, various mitogens such as serum, bradykinin and vasopressin also stimulate alpha-adducin phosphorylation by a PK-C-dependent pathway. The elevation in alpha-adducin phosphorylation is maintained for up to 30 min after mitogen addition. Peptide maps of phospho-alpha-adducin from both fibroblasts and erythrocytes after PK-C-mediated phosphorylation showed multiple phosphorylated peptides but with dissimilar migration patterns, suggesting divergence of structure around the phosphorylation sites. Adducin appears to play an important role in the regulation of spectrin-actin interactions in the red cell and may play a role in cytoskeletal function in the fibroblasts.

Calmodulin-Binding Proteins↗
PubMed1990

Embryonic simple epithelial keratins 8 and 18: chromosomal location emphasizes difference from other keratin pairs.

The keratins 8 and 18 of simple epithelia differ from stratified epithelial keratins in tissue expression and regulation. To examine the specific properties of human keratin 8, we cloned and sequenced the cDNA from a placental mRNA expression library and defined the optimum state of such clones for expression in bacterial plasmid vectors. Using the polymerase chain reaction we identified and sequenced three introns and located the single active gene for keratin 8, out of a background of 9 to 24 pseudogenes, on chromosome 12. This chromosome contains several genes for type II keratins and also the gene for keratin 18, the type I keratin that is coexpressed with keratin 8. This location of both members of a keratin pair on a single chromosome is thus far unique among the keratin genes; it is consistent with the hypothesis that keratins 8 and 18 may be closer to an ancestral keratin gene than the keratins of more highly differentiated epithelia.

Amino Acid Sequence↗
PubMed1989

Association of glyceraldehyde-3-phosphate dehydrogenase with the plasma membrane of the intact human red blood cell.

Glycolytic enzymes have been observed to associate in vitro with membranes and cytoplasmic filaments in a variety of systems, but their distribution in vivo is contested. We have therefore examined the distribution of glyceraldehyde-3-phosphate dehydrogenase (G3PD) in the intact human erythrocyte using indirect immunofluorescence and affinity-purified rabbit antibodies to G3PD. Antibody specificity was demonstrated by immunoblotting as well as immunofluorescence experiments with ghosts specifically depleted of and reconstituted with G3PD. Anti-G3PD immunolabeling experiments utilized both fixed whole cells and fixed cell suspensions infused with 2.3 M sucrose, frozen and thick-sectioned. In all experiments a two-step fixation protocol was employed which ensured that cytoplasmic hemoglobin was retained when cells were subjected to Triton X-100 permeabilization, the anti-genicity of G3PD was preserved, and antibody penetration was complete. We used mixtures of biotinylated affinity-purified antibodies to G3PD and dichlorotriazinylaminofluorescein-labeled, affinity-purified antibodies to hemoglobin, followed by rhodamine-streptavidin, in double-label experiments. In both whole and sectioned human erythrocytes, G3PD staining was predominantly membrane associated while hemoglobin staining was diffusely distributed throughout the cytoplasm. In isolated ghosts, some G3PD was tightly bound to the membrane and was resistant to elution with phosphate-buffered saline and NAD+/arsenate. However, in immunolabeled rat reticulocytes and erythrocytes G3PD was cytoplasmic. Nucleated human blood cells and platelets also exhibited cytoplasmic G3PD. In approximately 10% of the human erythrocyte population G3PD was also cytoplasmic. These cells were flatter in shape and exhibited strong cytoplasmic immunolabeling for hemoglobin which was sometimes concentrated along the cell membrane; possibly, these cells were late reticulocytes or early erythrocytes. We conclude that G3PD is preferentially associated with the plasma membrane of human erythrocytes in a specific fashion.

Animals↗
PubMed1988

Erythrocyte adducin. Comparison of the alpha- and beta-subunits and multiple-site phosphorylation by protein kinase C and cAMP-dependent protein kinase.

Two major substrates for human erythrocyte protein kinase C (PK-C) of Mr 120,000 and 110,000, previously named PKC-1 and PKC-2 [Palfrey, H. C. & Waseem, A. (1985) J. Biol. Chem. 260, 16021-16029] have been found to be identical to CaM-BP 103/97 or 'adducin', recently described by K. Gardner and V. Bennett [(1986) J. Biol. Chem. 261, 1339-1348; (1987) Nature (Lond.) 328, 359-362]. These proteins have been purified from the membrane skeleton by high-salt extraction, ion-exchange and gel filtration chromatography. The two proteins co-fractionate in a ratio of approximately 1:1 under a number of conditions suggesting that they exist as a complex. Physicochemical data indicate that the native adducin complex is probably an asymmetric heterodimer of alpha and beta subunits. Adducin binds to a calmodulin (CaM) affinity matrix in a Ca2+-dependent manner and is specifically eluted with EGTA. Fingerprinting of the iodinated peptides derived from the alpha and beta subunits using three different proteases yields 16-37% overlapping peptides, indicating limited similarity between the two polypeptides. Affinity-purified polyclonal antibodies against each protein show little or no cross-reactivity with the other, indicating that the beta subunit is not derived from the alpha subunit or vice versa. Proteins reactive with both anti-(alpha-adducin) and anti-(beta-adducin) antibodies are found in erythrocytes from rat, rabbit, pig, ferret and duck. Immunoblots of adducin after non-ionic detergent extraction of ghosts reveal that a significant fraction of the protein may associate with non-skeleton membrane components. The phosphorylation of adducin is stimulated by both phorbol esters and cAMP analogues in intact erythrocytes. Fingerprinting suggests that protein kinase C preferentially phosphorylates four distinct sites on the two proteins. Phosphopeptide maps of alpha-adducin are virtually identical to those of beta-adducin after phorbol ester stimulation of intact cells, or after PK-C-catalyzed phosphorylation of the purified protein, indicating strong local similarities in the two proteins. Such maps also suggest that cAMP-dependent protein kinase (cAMP-PK) modifies adducin at some similar and some distinct sites as those modified by PK-C. In vitro phosphorylation of isolated adducin by purified PK-C results in rapid incorporation of phosphate to a final level of approximately 1.5 mol/mol in both alpha and beta subunits.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Glycoprotein-concanavalin A interaction: role of protein conformation in the specific interaction of glycoprotein with concanavalin A.

The specific binding of two glycoproteins, ovomucoid and ovalbumin to concanavalin A was studied in 0.01 M Tris-HCl buffer, pH 7.4, containing 0.2 M NaCl and 1 mM each of magnesium acetate, calcium acetate and manganese chloride at 30 degrees C. Native ovalbumin was a better ligand than ovomucoid in its reaction with concanavalin A-sepharose 4B; in the inhibition of the lectin binding to dextran and cross-linked dextran, Sephadex G-150, and in the rate of precipitin reaction. Reduction of disulphide bonds of the two glycoproteins with 2-mercaptoethanol and subsequent alkylation with iodoacetate produced significant conformational change as evidenced by the increase in the hydrodynamic volume measured by intrinsic viscosity. The native and modified glycoproteins were indistinguishable in inhibiting the binding of dextran and Sephadex G-150 to the lectin, however, their pronase digest (or glycopeptides) were most effective inhibitors. Substantial (4-fold) decrease in the rate of precipitin reaction of the lectin with glycoprotein which was noted with conformationally altered glycoproteins suggested the importance of native protein conformation in the specific interaction of the two glycoproteins with concanavalin A.

Alkylation↗
PubMed1985

Protein kinase C in the human erythrocyte. Translocation to the plasma membrane and phosphorylation of bands 4.1 and 4.9 and other membrane proteins.

Protein kinase C (PK-C) was demonstrated in human erythrocytes using the exogenous substrate histone H1. The enzyme was dependent on the simultaneous presence of micromolar Ca2+ and phosphatidylserine. The Ca2+ requirement was reduced in the presence of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). Activity was normally recovered in cytoplasmic extracts, but treatment of intact cells with TPA (EC50 = 40 nM) prior to lysis caused a rapid translocation of activity from the cytoplasm to the membrane. Following translocation, PK-C activity in the isolated membrane was independent of Ca2+ and phosphatidylserine and could not be removed by manipulating ionic strength. Ghosts from TPA-treated cells showed a marked increase in the phosphorylation of five proteins (termed PK-C-1-5) of Mr 120,000, 110,000, 80,000, 78,000, and 49,000. Addition of purified bovine brain PK-C to ghosts from untreated cells resulted in the phosphorylation of the same five proteins. PK-C-3/4 corresponded to Band 4.1 and PK-C-5 to Band 4.9. Both proteins were isolated from ghosts and shown to be substrates for PK-C in vitro. PK-C-1 and -2 appear to be minor peripheral membrane proteins as both were released from the membrane by alkaline solutions. Incubation of 32P-prelabeled erythrocytes with TPA (EC50 = 40 nM) also resulted in a dose-dependent phosphorylation of PK-C-1-5. These results suggest that PK-C may play an important role in erythrocyte membrane function.

Amino Acids↗