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At least 1,621 records · Page 90Linked to original sources
PubMed1992

Retroviral expression of recombinant p47phox protein by Epstein-Barr virus-transformed B lymphocytes from a patient with autosomal chronic granulomatous disease.

Patients with chronic granulomatous disease (CGD) have recurrent infections resulting from a failure of phagocytic cells to produce superoxide. One third of CGD patients have an autosomal gene defect resulting in absence of p47phox protein, a cytoplasmic component critical to superoxide production by phagocytic cells. cDNA encoding p47phox has been cloned and recombinant p47phox (rp47phox) restores superoxide-generating activity to a cell-free assay containing cell membranes and cytosol from p47phox-deficient CGD neutrophils. The goal of the present study was to determine the feasibility of retrovirus mediated expression of rp47phox in the HL60 and U937 human hematopoietic cell lines, and in an Epstein-Barr virus transformed B-lymphocyte cell line (EBV-BCL) derived from a p47phox-deficient CGD patient. Normal EBV-BCL contain p47phox and generate small amounts of superoxide, while this CGD EBV-BCL lacks any detectable p47phox protein. Defective amphotropic retrovirus containing p47phox sequence inserted in the LXSN vector in sense and antisense orientations were used to transduce HL60, U937, and CGD EBV-BCL. p47phox mRNA sequence was detected in cells transduced with either sense or antisense retroviral constructs while rp47phox protein was detected only with the sense construct. The amount of rp47phox protein produced within these cells was greater than the native p47phox present in uninduced HL60 or U937 cells, but substantially less than that present in normal neutrophils, induced HL60 cells, or even normal EBV-BCL. Differentiation of transduced HL60 cells and the associated production of native p47phox in response to dimethyl sulfoxide was not affected. These studies demonstrate that retrovirus constructs can be used to mediate stable expression of rp47phox protein in human hematopoietic cell lines and can restore rp47phox protein within the cytosol of p47phox-deficient EBV-BCL from patients with CGD.

B-Lymphocytes↗
PubMed1991

A dimorphic 4-bp repeat in the cystic fibrosis gene is in absolute linkage disequilibrium with the delta F508 mutation: implications for prenatal diagnosis and mutation origin.

The gene causing cystic fibrosis (CF) has been recently cloned, and the major mutation (delta F508) accounting for approximately 70% of CF chromosomes has been uncovered. We have identified at the 3' end of intron 6 in the CF gene a 4-bp tandem repeat (GATT) that exhibits interesting features. First, PCR screening of 103 normal individuals revealed that the repeat exists only in two polymorphic allelic forms, either as a hexamer or a heptamer. These two alleles are in Hardy-Weinberg equilibrium and predict a heterozygote frequency of 41% (p[seven repeats] = .71; q [six repeats] = .29). Second, the allele with six repeats was found linked to delta F508 on all 76 CF chromosomes investigated, demonstrating strong linkage disequilibrium and suggesting that delta F508 had originated on the gene bearing six repeats. Third, when the repeat alleles are linked to the DNA markers XV2c and KM19, extended haplotypes are generated. These new haplotypes become informative in situations in which prenatal diagnosis cannot be performed solely with XV2c and KM19. Since this repeat marker is located in the CF gene and would be very less likely to recombine with the gene, it can serve as a valuable DNA marker for haplotype analysis. A possible crossover, however, was identified between XV2c and KM19, transferring delta F508 to a different haplotype.

Alleles↗
PubMed1995

Importance of the third thrombospondin repeat of C6 for terminal complement complex assembly.

The anti-C6 monoclonal antibody WU 6-4 was shown to be unequivocally native restricted since it neither binds to the terminal complement complex (TCC) nor to C5b6. In addition, it was shown to inhibit TCC formation by interfering with C5b6 generation. Using the pUEX expression system and C6 cDNA the WU 6-4 epitope was mapped to the third thrombospondin repeat of C6. This protein domain may therefore contribute to the C5 binding site of C6 and be involved in terminal complement complex assembly. The presence of the epitope in rabbit C6 indicates a useful model for studying inhibition of TCC formation in vivo.

Animals↗
PubMed2005

Population structure within lineages of Wheat streak mosaic virus derived from a common founding event exhibits stochastic variation inconsistent with the deterministic quasi-species model.

Structure of Wheat streak mosaic virus (WSMV) populations derived from a common founding event and subjected to serial passage at high multiplicity of infection (MOI) was evaluated. The founding population was generated by limiting dilution inoculation. Lineages of known pedigree were sampled at passage 9 (two populations) and at passage 15, with (three populations) or without mixing (four populations) of lineages at passage 10. Polymorphism within each population was assessed by sequencing 17-21 clones containing a 1371 nt region (WSMV-Sidney 81 nts 8001-9371) encompassing the entire coat protein cistron and flanking regions. Mutation frequency averaged approximately 5.0 x 10(-4)/nt across all populations and ranged from 2.4 to 11.6 x 10(-4)/nt within populations, but did not consistently increase or decrease with the number of passages removed from the founding population. Shared substitutions (19 nonsynonymous, 10 synonymous, and 3 noncoding) occurred at 32 sites among 44 haplotypes. Only four substitutions became fixed (frequency = 100%) within a population and nearly one third (10/32) never achieved a frequency of 10% or greater in any sampled population. Shared substitutions were randomly distributed with respect to genome position, with transitions outnumbering transversions 5.4:1 and a clear bias for A to G and U to C substitutions. Haplotype composition of each population was unique with complexity of each population varying unpredictably, in that the number and frequency of haplotypes within a lineage were not correlated with number of passages removed from the founding population or whether the population was derived from a single or mixed lineage. The simplest explanation is that plant virus lineages, even those propagated at high MOI, are subject to frequent, narrow genetic bottlenecks during systemic movement that result in low effective population size and stochastic changes in population structure upon serial passage.

Base Sequence↗
PubMed1997

Human fibulin-1D: molecular cloning, expression and similarity with S1-5 protein, a new member of the fibulin gene family.

Fibulin-1 is an extracellular matrix (ECM) component of basement membranes and connective tissue elastic fibers, and a blood protein. Multiple forms of fibulin-1 that differ in their C-terminal regions are produced through the process of alternative splicing of their precursor RNA. Two transcripts of 2.4 and 2.7 kb are the predominant fibulin-1 mRNAs expressed in human tissues and cultured cells. While the 2.4 kb transcript had been shown to encode fibulin-1C, the 2.7 kb transcript did not correspond to any of the previously identified human fibulin-1 variants. Herein, we report on the isolation and sequencing of cDNA corresponding to the 2.7 kb fibulin-1 transcript which encodes a novel, alternatively spliced form of human fibulin-1 that we term the D form. The deduced amino acid sequence of the D form is identical in its first 566 residues to the three known fibulin-1 variants (fibulin-1A-C); however, it has a unique 137 amino acid-C-terminal segment encoded by the alternatively spliced portion of its transcript. RNA hybridization analysis showed that the fibulin-1D transcript is coordinately expressed with that of fibulin-1C both in tissues and in cultured cells. Using antibodies specific to the unique C-terminal segment of fibulin-1D and -1C, both proteins were found to be expressed in human placenta. Recombinant fibulin-1D generated in transfected mammalian cells displayed similar ligand-binding properties as placenta-derived fibulin-1 and recombinant fibulin-1C, and it was capable of incorporating into cultured cell ECM in the absence of other fibulin-1 forms. A comparative sequence analysis revealed that the unique C-terminal region of fibulin-1D is similar to the C-terminal regions of fibulin-1C and fibulin-2. Furthermore, the C-terminal regions of fibulin-1C, -1D and -2 are similar to the C-terminal region of a recently described protein termed S1-5. In addition to this C-terminal similarity, S1-5 also contains repeated EGF-like modules and a conserved N-terminal element, thereby leading to the conclusion that S1-5 is a third member of the fibulin gene family.

Amino Acid Sequence↗
PubMed1996

A molecular mechanics and dynamics study of alternate triple-helices involving the integrase-binding site of the HIV-1 virus and oligonucleotides having a 3'-3' internucleotide junction.

Triple helix formation by oligonucleotides can be extended beyond polypurine tracts with the help of specially designed linkers. In this paper we focus our attention on the integrase-binding site of the HIV-1 virus located on the U5 LTR end which contains two adjacent purine tracts on opposite strands. Two alternate triple helices with a 3'-3' junction in the third strand are considered: 5'-GGTTTTp3'-3'pTGTGT-5' and 5'-GGAAAAp3'-3'pAGAGA-5' The structural plausibility of these triplexes is investigated using molecular mechanics and dynamics simulations, both in vacuo and in aqua. The non-isomorphism of the triplets in the GpT steps in the first sequence, gives rise to non canonical conformations in the torsion angles, hydration appears to be crucial for this triplex. Sugar puckers are predominantly South during in vacuo simulations while they turn East in aqua. In the simulation in aqua the triplexes are shrouded by an hydration shell, however, we have not been able to detect any permanent hydrogen bond bridge between DNA and water. The solvation of ions as well as their radial distribution, appear to be relatively well behaved despite the artifacts known to be generated by the simulation procedure. The experimental feasibility of these structures is discussed.

Base Sequence↗
PubMed2006

The role of prohormone convertase-2 in hypothalamic neuropeptide processing: a quantitative neuropeptidomic study.

Prohormone convertase (PC) 1/3 and 2 are involved in the generation of neuropeptides from their precursors. A quantitative peptidomic approach was used to explore the role PC2 plays in the processing of hypothalamic peptides. In this approach, extracts from mice lacking PC2 activity and from wild-type littermates were labeled with isotopic tags, combined, fractionated on a reverse phase HPLC column, and analyzed by electrospray ionization mass spectrometry. Altogether, 53 neuropeptides or other peptides derived from secretory pathway proteins were identified and sequenced using tandem mass spectrometry. These peptides arise from 21 distinct proteins: proenkephalin, proopiomelanocortin, prodynorphin, protachykinin A and B, procholecystokinin, promelanin-concentrating hormone, proneurotensin, proneuropeptide Y, provasopressin, pronociceptin/orphanin, prothyrotropin-releasing hormone, cocaine- and amphetamine-regulated transcript, chromogranin A and B, secretogranin II, prohormone convertase 1 and 2, propeptidyl-amidating monooxygenase, and proteins designated proSAAS and VGF. Approximately one third of the peptides found in wild-type mice were not detectable in PC2 knock-out mice, and another third were present at levels ranging from 25 to 75% of wild-type levels. Comparison of the cleavage sites suggests that sequences with a Trp, Tyr and/or Pro in the P1' or P2' position, or a basic residue in the P3 position, are preferentially cleaved by PC2 and not by other enzymes present in the secretory pathway.

Amino Acid Sequence↗
PubMed2005

Comparison of activities of beta-lactam antibiotics against Streptococcus pneumoniae with recombinant penicillin-binding protein genes from a penicillin-resistant strain.

We investigated the antibacterial activities of 19 beta-lactams against three recombinant bacterial strains, in which three penicillin-binding protein genes, pbp2x, pbp1a, and pbp2b, from penicillin-resistant Streptococcus pneumoniae (PRSP), were transformed to a penicillin-susceptible strain. By the acquisition of the pbp2x gene from PRSP, the minimum inhibitory concentrations (MICs) of third-generation cephalosporins were increased more than eight fold. When the strain acquired the PRSP pbp1a gene in addition to pbp2x, the MICs of all tested beta-lactams increased 2- to 16-fold. When the strain acquired the PRSP pbp2b gene in addition to pbp2x and pbp1a, the MICs of penicillins and carbapenems increased 4- to 16-fold. However, two novel carbapenems, ME1036 and L-036, showed excellent antibacterial activities against these recombinant strains, as well as against the parent PRSP.

Amino Acid Sequence↗
PubMed1994

Multiple developmental defects in Engrailed-1 mutant mice: an early mid-hindbrain deletion and patterning defects in forelimbs and sternum.

During mouse development, the homeobox-containing gene En-1 is specifically expressed across the mid-hindbrain junction, the ventral ectoderm of the limb buds, and in regions of the hindbrain, spinal cord, somites and somite-derived tissues. To address the function of En-1 during embryogenesis, we have generated mice homozygous for a targeted deletion of the En-1 homeobox. En-1 mutant mice died shortly after birth and exhibited multiple developmental defects. In the brains of newborn mutants, most of the colliculi and cerebellum were missing and the third and fourth cranial nerves were absent. A deletion of midhindbrain tissue was observed as early as 9.5 days of embryonic development and the phenotype resembles that previously reported for Wnt-1 mutant mice. In addition, patterning of the forelimb paws and sternum was disrupted, and the 13th ribs were truncated. The results of these studies suggest a cell autonomous role for En-1 in generation and/or survival of mid-hindbrain precursor cells and also a non-cell autonomous role in signalling normal development of the limbs and possibly sternum.

Animals↗
PubMed1991

Ankyrin binds to the 15th repetitive unit of erythroid and nonerythroid beta-spectrin.

Ankyrin mediates the attachment of spectrin to transmembrane integral proteins in both erythroid and nonerythroid cells by binding to the beta-subunit of spectrin. Previous studies using enzymatic digestion, 2-nitro-5-thiocyanobenzoic acid cleavage, and rotary shadowing techniques have placed the spectrin-ankyrin binding site in the COOH-terminal third of beta-spectrin, but the precise site is not known. We have used a glutathione S-transferase prokaryotic expression system to prepare recombinant erythroid and nonerythroid beta-spectrin from cDNA encoding approximately the carboxy-terminal half of these proteins. Recombinant spectrin competed on an equimolar basis with 125I-labeled native spectrin for binding to erythrocyte membrane vesicles (IOVs), and also bound ankyrin in vitro as measured by sedimentation velocity experiments. Although full length beta-spectrin could inhibit all spectrin binding to IOVs, recombinant beta-spectrin encompassing the complete ankyrin binding domain but lacking the amino-terminal half of the molecule failed to inhibit about 25% of the binding capacity of the IOVs, suggesting that the ankyrin-independent spectrin membrane binding site must lie in the amino-terminal half of beta-spectrin. A nested set of shortened recombinants was generated by nuclease digestion of beta-spectrin cDNAs from ankyrin binding constructs. These defined the ankyrin binding domain as encompassing the 15th repeat unit in both erythroid and nonerythroid beta-spectrin, amino acid residues 1,768-1,898 in erythroid beta-spectrin. The ankyrin binding repeat unit is atypical in that it lacks the conserved tryptophan at position 45 (1,811) within the repeat and contains a nonhomologous 43 residue segment in the terminal third of the repeat. It also appears that the first 30 residues of this repeat, which are highly conserved between the erythroid and nonerythroid beta-spectrins, are critical for ankyrin binding activity. We hypothesize that ankyrin binds directly to the nonhomologous segment in the 15th repeat unit of both erythroid and nonerythroid beta-spectrin, but that this sequence must be presented in the context of a properly folded spectrin "repeat unit" structure. Future studies will identify which residues within the repeat unit are essential for activity, and which residues determine the specificity of various spectrins for different forms of ankyrin.

Amino Acid Sequence↗
PubMed1988

Structure-activity relationship of toxic-shock-syndrome toxin-1: derivation and characterization of immunologically and biologically active fragments.

Toxic-shock-syndrome toxin-1 (TSST-1), a 22-kilodalton (kDa) polypeptide, was proteolyzed by papain, generating three distinct fragments, identified as 16, 12, and 10 kDa (based on molecular masses estimated from the predicted amino acid sequence). The NH2-terminal sequence analysis of the fragments indicated that the peptide bonds between Tyr-52 and Ser-53 and between Gly-87 and Val-88 were cleaved. Functional activity, evaluated through enzyme-linked immunosorbent and inhibition assays, was demonstrated only with the 16- and 12-kDa fragments. The presence of homologous and heterologous antigenic determinants on the fragments was demonstrated by immunoblotting. In in vitro stimulation of human peripheral blood mononuclear cells, the 12-kDa fragment was significantly (P = .003) more active than the 16-kDa fragment. The former composed 75% of the latter and occupied the COOH-terminal portion of the holotoxin. The functional domains were located on two-thirds of the TSST-1 molecule, toward the COOH-terminal end, and mitogenicity apparently was separable from serological activity.

Amino Acid Sequence↗
PubMed1994

Molecular cloning of an Onchocerca volvulus extracellular Cu-Zn superoxide dismutase.

Onchocerca volvulus, a human parasitic nematode, is the third leading cause of preventable blindness worldwide. This study describes the molecular cloning of a novel superoxide dismutase (SOD) from the parasite. This putative O. volvulus extracellular SOD (OvEcSOD) is 628 nucleotides (nt) long, including a 22-nt 5' spliced leader (SL1) and a portion encoding an N-terminal hydrophobic 42-amino-acid signal peptide. The remainder of the cDNA shares 71% identity with an O. volvulus cytosolic SOD sequence and is 3 nt longer. All residues involved in metal ion binding, active site formation, folding, and dimer formation in SODs are conserved. Data indicate the OvEcSOD and O. volvulus cytosolic SOD are separate gene products and that the OvEcSOD appears to possess the characteristics of a membrane-bound or secreted enzyme which may be involved in the parasite defense against phagocyte-generated reactive oxygen species.

Amino Acid Sequence↗
PubMed1990

MHC class II-derived peptides can bind to class II molecules, including self molecules, and prevent antigen presentation.

Seven synthetic peptides corresponding to the polymorphic regions of the alpha and beta chains of the I-Ak molecule were examined for their ability to inhibit the presentation of foreign antigens to antigen-specific, I-A-restricted T cell hybridomas. Two of the peptides, representing the sequences found in the first and third polymorphic regions (PMR) of the A alpha k chain (alpha k-1 and alpha k-3) were capable of inhibiting the presentation of three different HEL-derived peptide antigens to their appropriate T cells. In addition, the alpha k-1 peptide inhibited the presentation of the OVA(323-339) immunodominant peptide to the I-Ad-restricted T cell hybridomas specific for it. Prepulsing experiments demonstrated that the PMR peptides were interacting with the APC and not with the T cell hybridomas. These observations were confirmed and extended by the demonstration that the alpha k-1 and alpha k-3 peptides blocked the direct binding of HEL(46-61) to purified I-Ak and that the alpha k-1 peptide blocked the binding of OVA(323-339) to I-Ad. The binding competition experiments suggest that the alpha k-1 peptide binds to the I-Ak molecule from which it was derived with a Kd approximately 10(-5) M, while the alpha k-3 peptide binds slightly less well. These combined data, suggesting that class II-derived peptides can bind to MHC class II molecules, including the autologous molecule from which they are derived, have important implications for the molecular basis of alloreactivity and autoreactivity. Further, they suggest a possible mechanism by which selecting elements, involving only MHC molecules, may be generated in the thymus.

Amino Acid Sequence↗
PubMed2005

Organization, alternative splicing, polymorphism, and phylogenetic position of lamprey CD45 gene.

CD45 of jawed vertebrates is a receptor-type protein tyrosine phosphatase regulating lymphocyte development and activation. To shed light on the evolution of the CD45 gene, the organization of its orthologue in the lamprey, a jawless vertebrate, was determined. Compared to its mammalian and fugu counterparts, the lamprey gene was found to be lacking several exons in the segment encoding the extracellular part of the protein. In consequence, this part contains only one instead of the two or three fibronectin type III domains typical of the mammalian molecules. The lamprey transcripts of the CD45 gene occur in several variants originating by alternative splicing, including some not observed previously in other vertebrates. Most remarkable of these are splice variants generated by the use of intra-exonic splicing signals and thus lacking one half, one third, or two thirds of an exon and yet apparently translated in the correct reading frame. The lamprey gene contains polymorphic sites not only in the segment encoding the extracellular portion but also in the segment specifying the cytoplasmic part of the molecule. Polymorphism is generated by both mutations and recombination. Some of the alleles may have persisted long enough to represent transspecies polymorphism presumably maintained by positive selection. Phylogenetic analysis suggests that ancestors of the CD45 gene may have existed before the divergence of coelomate from pseudocoelomate metazoans.

Alternative Splicing↗
PubMed2004

Organization and expression of the SLC36 cluster of amino acid transporter genes.

Three closely related genes encoding amino acid transport proteins are clustered on 5q32 in humans, and Chromosome (Chr) 11 in mice. The human SLC36A1 gene, which encodes the lysosomal amino acid transporter LYAAT1/PAT1, generates multiple alternative mRNAs, some of which encode truncated proteins. SLC36A1 is expressed in numerous tissues, whereas expression of SLC36A2, which encodes the glycine transporter tramdorinl/PAT2, is most abundant in kidney and muscle. Expression of a third gene, SLC36A3, is restricted to testis. Mouse Slc36a2 also is expressed in bone and fat tissue. Polymorphisms in human SLC36A2 exclude it as a candidate locus for a peripheral neuropathy that has been mapped to 5q31-33. SLC36A2 is a candidate gene for 5q-myelodysplastic syndrome, on the basis of its chromosomal location and its expression in bone.

Amino Acid Sequence↗
PubMed1992

A mutational hotspot in the aprt gene of Chinese hamster cells.

Early work with adenine phosphoribosyltransferase-deficient mutants of CHO cells suggested that a site existed in the third exon of this gene which was preferentially susceptible to mutation by ethyl methanesulphonate. To determine whether this was real we analysed a large collection of induced mutants, and generated a high-density mutational spectrum for this exon. In addition, 4 sites outside exon 3 were analysed by blot. 37 mutations were found in 19 available sites, six of which were at nucleotide 1365, 1 of 2 sites in the putative hotspot (P less than 0.02). One other site, 1308, also was mutated in 6 cell lines and may also be preferentially mutable.

Adenine Phosphoribosyltransferase↗
PubMed1992

Early and multifocal tumors in breast, salivary, harderian and epididymal tissues developed in MMTY-Neu transgenic mice.

Transgenic mice carrying various oncogenes driven by mammary gland specific enhancers develop mammary tumors usually arising in a stochastic way. The only exception is a mouse lineage (TG.NF) carrying an activated rat Neu oncogene driven by the murine mammary tumor virus long terminal repeat (MMTV-LTR) that gave rise to rapid and multifocal mammary tumors interpreted as a result of a single-step neoplastic transformation. The effect of the oncogene appeared to be specific for breast tissue, since salivary and Harderian glands as well as epididymis expressed high levels of Neu but only developed hyperplasia (Muller et al., Cell, (1988) 54, p. 105). Here we describe a transgenic mouse lineage for the MMTV-Neu, analysed up to third generation. Multifocal tumors involving mammary glands arose very rapidly in all females independently from pregnancy and in some males. Moreover, multifocal neoplasias occurred also in salivary and Harderian glands and in the epididymis at a very high rate. These data demonstrate that the Neu oncogene can induce tumors in all the tissues where it is expressed at high levels.

Animals↗
PubMed1998

Cloning of murine low molecular weight phosphotyrosine protein phosphatase cDNA: identification of a new isoform.

The low molecular weight phosphotyrosine protein phosphatase (LMW-PTP) is a 18 kDa cytosolic enzyme, involved in the negative regulation of cell proliferation. In different mammalian species LMW-PTPs are expressed in two molecular forms produced from a single primary transcript through an alternative splicing mechanism. In this paper we report the cloning, expression and characterization of mouse isoforms of LMW-PTPs (called m-IF1 and m-IF2), very similar to the corresponding rat and human isoenzymes. Moreover we have identified a third cDNA encoding a protein (m-IF2P) that presents three substitutions compared to m-IF2. This new isoform is still active on pNPP, although to a lower extent: this reduction is mainly due to the leucine to proline substitution in position 13, within the catalytic loop. The mRNA expression level of this isoform is comparable to those of m-IF1 and m-IF2. It is likely that a gene duplication process followed by mutations has generated this new gene.

3T3 Cells↗