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Biomedical subjects

M McGrogan

Publications and source records attributed to M McGrogan.

At least 19 recordsLinked to original sources
PubMed2002

hNT neurons express an immunosuppressive protein that blocks T-lymphocyte proliferation and interleukin-2 production.

Ntera2/D1 cells had an A1 B8 Bw6 Cw7 DR3 DR52 major histocompatibility complex (MHC) genotype. Its neuronal derivative, hNT neurons, expressed A1 B8 Bw6 MHC class I molecules, but did not activate, and its hNT supernatant suppressed allogeneic mixed lymphocyte cultures (MLC) >98% (p<0.01), phytohemagglutinin (PHA)-activated T-cell proliferation >87% (p<0.01), even 48 h after stimulation, suppressed phorbol 12-myristate 13-acetate (PMA)/ionomycin-induced T-cell proliferation >99% (p<0.001), and reduced interleukin-2 (IL-2) production (p<0.01), while maintaining T cells in a quiescent G(0)/G(1) state without lowering their viability. This immunosuppressive activity was attributed to a 40-100-kDa anionic hNT protein with an isoelectric point of 4.8.

Brain Tissue Transplantation↗
PubMed2000

Transplantation of cultured human neuronal cells for patients with stroke.

Transplantation of cultured neuronal cells is safe in animal models and improves motor and cognitive deficits in rats with stroke. The authors studied the safety and feasibility of human neuronal cellular transplantation in patients with basal ganglia stroke and fixed motor deficits, including 12 patients (aged 44 to 75 years) with an infarct 6 months to 6 years previously (stable for at least 2 months). Serial evaluations (12 to 18 months) showed no adverse cell-related serologic or imaging-defined effects. The total European Stroke Scale score improved in six patients (3 to 10 points), with a mean improvement 2.9 points in all patients (p = 0. 046). Six of 11 PET scans at 6 months showed improved fluorodeoxyglucose uptake at the implant site. Neuronal transplantation is feasible in patients with motor infarction.

Adult↗
PubMed1995

Expression, purification, and characterization of the recombinant proform of eosinophil granule major basic protein.

The cDNA for the highly toxic eosinophil granule major basic protein (MBP) encodes a 25-kDa acidic precursor (proMBP) that is processed to form the 14-kDa mature MBP. To characterize the biochemical and biological properties of proMBP, and compare these to the known properties of MBP, we expressed recombinant proMBP in Chinese hamster ovary cells and purified the secreted form from supernatants. We developed a mAb specific for proMBP, J163-15E10, and by using a proMBP-specific RIA we found that recombinant proMBP was expressed quite efficiently at levels between 10 and 100 mg/l. By SDS-PAGE and immunoblotting analyses of bulk Chinese hamster ovary supernatants, recombinant proMBP was electrophoretically heterogeneous with an apparent molecular mass ranging from 3 x 10(4) to 1 x 10(5) daltons. Despite difficulties encountered because of the extreme molecular heterogeneity of the proform, two methods for purification of a predominant 33-kDa form of recombinant proMBP are presented. Glycosylation analysis of purified 33-kDa proMBP indicated that approximately 5 kDa is likely accounted for by the addition of one glycosaminoglycan group, three O-linked, and one N-linked complex type carbohydrate groups. Functional studies of purified recombinant proMBP were also conducted. Using amounts of proMBP determined to be optimal for MBP activity, it was shown that proMBP not only lacked the ability to inhibit protein synthesis in K562 cells, but it also lacked the ability to stimulate basophil histamine release or generate neutrophil superoxide anion release. Furthermore, proMBP inhibited in a dose-responsive manner the basophil histamine release and superoxide anion generation stimulated by MBP. The development of a mAb and RIA specific for proMBP will now make it possible to analyze biologic fluids for the presence of this protein, especially in pregnancy, when proMBP is increased.

Amino Acid Sequence↗
PubMed1994

Bactericidal/permeability-increasing protein and lipopolysaccharide (LPS)-binding protein. LPS binding properties and effects on LPS-mediated cell activation.

We have previously shown that human bactericidal/permeability-increasing protein (BPI) is able to inhibit serum-dependent lipopolysaccharide (LPS)-mediated activation of human monocytes and neutrophils in vitro, and to counteract the lethal effects of LPS challenge in vivo. Lipopolysaccharide-binding protein (LBP) is a serum protein which participates in LPS-mediated activation of cells (Tobias, P. S., Mathison, J., Mintz, D., Lee, J. D., Kravchenko, V., Kato, K., Pugin, J., and Ulevitch, R. J. (1992) Am. J. Respir. Cell. Mol. Biol. 7, 239-245). We have proposed that BPI functions in a negative feedback loop which opposes this activation (Marra, M. N., Wilde, C. G., Collins, M. S., Snable, J. L., Thornton, M. B., and Scott, R. W. (1992) J. Immunol. 148, 532-537). We have now cloned and expressed recombinant forms of human BPI and LBP. Here we demonstrate that purified recombinant human LBP can replace the serum requirement for both LPS binding to human monocytes and LPS-mediated secretion of tumor necrosis factor alpha from these cells. These activities of LBP are inhibited by a neutralizing anti-CD14 monoclonal antibody. We further demonstrate that purified recombinant human BPI can inhibit LBP-mediated LPS binding to cells and their subsequent activation. Comparison of the LPS binding properties of BPI and LBP in enzyme-linked immunosorbent type assays and in the Limulus amebocyte lysate assay suggest that BPI has a stronger affinity for LPS than does LBP. Direct competition between BPI and LBP for LPS may explain the inhibition by BPI of the proinflammatory effects of LBP in the presence of LPS.

Acute-Phase Proteins↗
PubMed1994

The molecular biology of production cell lines.

The emergence of a wide variety of biological expression systems for the large-scale production of therapeutic proteins has shifted the focus from vectors to host organisms. Although expression systems now span bacteria, fungi, plants, insects, and mammalian cells, the vast majority of recombinant-derived biopharmaceuticals at the present time have been produced in Escherichia coli and in mammalian cells. This promises to change as the economic benefits of the newer systems permit the development of a new generation of proteins heretofore considered unfeasible for commercial development. Despite the impressive results which have been observed for many of the newer systems, there are many commercial considerations which suggest that E. coli and CHO cell expression systems may continue to dominate the manufacture of biopharmaceuticals for a long time to come.

Animals↗
PubMed1993

Protease nexin 1 is expressed in the human placenta.

Protease nexin 1 (PN1), a serine protease inhibitor that inactivates thrombin, urokinase, and plasmin, is produced abundantly in cultures of human fibroblasts and rat and human glioma cells. The major sites of PN1 synthesis in vivo and the specific physiological function(s) of this serpin are unknown. Using Northern blot analysis and a full-length PN1 cDNA probe we demonstrated the presence of PN1 mRNA in human term placentas. In situ hybridization of placental tissue with a PN1 riboprobe showed that PN1 mRNA is present throughout the placenta and is also abundant in the placental membranes. Immunohistochemical analysis with an anti-PN1 antibody showed co-localization of PN1 and its mRNA within the placenta.

Amyloid beta-Protein Precursor↗
PubMed1991

Protease specificity and heparin binding and activation of recombinant protease nexin I.

Structural and functional properties of alpha-protease nexin I (alpha-PNI) expressed in Chinese hamster ovary cells were studied. All three cysteines were in the reduced form, showing that the potential disulfide bridge between residues Cys117 and Cys131 was not formed. Heparin association rate enhancements were from ka = 8.3 x 10(5) to 0.7-1.6 x 10(9) M-1 s-1 for the interaction of PNI with thrombin, from ka = 5.1 x 10(3) to 3.5 x 10(5) M-1 s-1 for interaction with Factor Xa, and from ka = 2.2 x 10(6) to 1.0 x 10(7) M-1 s-1 for interaction with trypsin; there was no rate enhancement of the plasmin interaction (ka = 1.0 x 10(5) M-1 s-1). The minimal heparin pentasaccharide had no effect on these interactions. Cleavage of the reactive center loop of PNI by three different proteases gave the typical stressed to relaxed change in thermal stability, but unlike with antithrombin III, there was no loss of heparin affinity. A similar difference from antithrombin was that PNI-thrombin complexes retained normal heparin affinity. These results are compatible with a role for protease nexin I as a cell-associated thrombin inhibitor that remains bound to the cell surface even after complexing with the protease, as compared with the role of antithrombin III as a circulating inhibitor of thrombin that becomes activated on binding to the microvasculature and is released on complex formation.

Amino Acid Sequence↗
PubMed1989

Molecular cloning, characterization, and expression of a human 14-kDa lectin.

Full length cDNAs coding for a 14-kDa beta-galactoside binding lectin have been isolated from HL-60 cells and human placenta. Oligonucleotide probes based on a pentapeptide present in several partial sequences of homologous human lectins were used to screen a lambda GT10 HL-60 cDNA library. The HL-60 cDNA clones that were isolated were used to design a synthetic primer representing the 3'-untranslated region of the HL-60 lectin. This primer was then used to synthesize a lambda GT10 human placenta cDNA library, and restriction fragments of the HL-60 cDNA clones were used to screen the library. The cDNA clones for both HL-60 and placenta lectin had identical sequences with short 5'- and 3'-untranslated regions and coded for a 135-amino acid protein which lacks a hydrophobic signal peptide sequence. Biochemical data show that, despite the presence of a possible N-linked glycosylation site, the protein is not glycosylated. Northern and Southern blot analyses indicate that the 14-kDa lectin is encoded for by a single gene. The lectin cDNA was expressed in Escherichia coli and biologically active protein was purified from cell lysates by affinity chromatography.

Amino Acid Sequence↗
PubMed1988

Isolation of a complementary DNA clone encoding a precursor to human eosinophil major basic protein.

A 14-kD protein was purified from human PMNs and its NH2-terminal sequence was determined. Comparison of a portion of the NH2-terminal sequence of this protein to the recently reported NH2-terminal sequence of eosinophil major basic protein (MBP) showed them to be identical. To aid further characterization of the structural and functional properties of this molecule, we isolated from an HL-60 cDNA library a single class of cDNA clones whose sequence matched exactly the NH2-terminal amino acid sequence of the 14-kD polypeptide. Northern analysis of HL-60 cells suggests that MBP is constitutively expressed in HL-60 cells and is highly transcribed from a single copy gene. The sequence of the full-length cDNA clones predicts that MBP is synthesized as a 23-kD precursor form (pro-MBP) which is subsequently cleaved to release the mature 14-kD MBP. The putative pro-MBP has a predicted pI of 6.0, but both the charged and the hydrophobic residues are asymmetrically distributed, creating a bipolar molecule. The NH2-terminal half has a predicted pI of 3.7 and is hydrophilic, while the COOH-terminal half (corresponding to mature MBP) has a predicted pI of 11.1 and is hydrophobic.

Base Sequence↗
PubMed1988

cDNA cloning and expression of murine macrophage colony-stimulating factor from L929 cells.

A 4-kilobase and a 2-kilobase cDNA clone encoding a murine macrophage colony-stimulating factor have been isolated. Except for 2 amino acid residue differences, these two clones encode the same 520 amino acid residue protein, which is preceded by a 32-amino acid residue signal peptide. The two clones, whose molecular masses correspond to the two transcripts observed in murine L929 fibroblasts, contain 3' untranslated regions that are markedly different in sequence and length. Both clones can be expressed in COS cells and the recombinant protein is active in a mouse bone marrow colony assay.

Amino Acid Sequence↗
PubMed1985

Heterogeneity at the 5' termini of mouse dihydrofolate reductase mRNAs. Evidence for multiple promoter regions.

We have determined the sequence of the 1000 base pairs of DNA preceding the coding region of the mouse dihydrofolate reductase gene. 700 base pairs upstream of the translation start codon (position + 1) is the sequence CAACT, separated by 50 base pairs from the sequence TAATAA; these sequences resemble controlling elements responsible for accurate and efficient transcription initiation in a variety of eukaryotic genes. The region between -244 and -101 consists of a 3-fold tandem repeat of a 48-base pair sequence. S1 mapping results indicate that the 5' termini of the multiple dihydrofolate reductase mRNAs are heterogeneous, with the major terminus at -115 and with minor termini in the regions of -275 and -450. In addition, a portion of the 5'-ward region of the gene from -543 to -405 is represented differentially in some, but not all, mouse dihydrofolate reductase mRNAs. The above findings are consistent with the presence of two transcription promoter elements in the 5' end of the mouse dihydrofolate reductase gene. This has been substantiated by inserting portions of the 5'-ward 1000 base pairs of the gene into modular dihydrofolate reductase plasmids. When such constructs are transfected into Chinese hamster ovary cells lacking dihydrofolate reductase, function can be restored with equal transfection frequency when sequences surrounding the 5' CAACT-TAATAA region at -700 or a complete 48-base pair repeat from the region of -148 to -101 are present in the plasmid construct.

Animals↗
PubMed1984

Biological activity of recombinant human interleukin-2 produced in Escherichia coli.

The gene for interleukin-2 was isolated from the Jurkat cell line and from normal peripheral blood lymphocytes and, when inserted in Escherichia coli, was expressed at high concentrations. This interleukin-2 was purified to apparent homogeneity and tested for biological activity in a variety of assays in vitro and in vivo. The recombinant lymphokine supports the growth of murine and human interleukin-2 dependent cell lines, enhances the generation of murine and human cytolytic cells in vitro, and generates lymphokine activated killer cells from murine and human lymphocytes. It has a serum half-life of 2 to 3 minutes in the mouse and significantly enhances the generation of cytolytic cells in vivo after alloimmunization. No functional differences between native and the recombinant interleukin-2 molecules have been detected.

Animals↗
PubMed1982

Nucleotide sequence surrounding multiple polyadenylation sites in the mouse dihydrofolate reductase gene.

We have previously reported the presence of four dihydrofolate reductase messenger RNAs differing in the length of 3' untranslated regions in murine cells (Setzer, D. R., McGrogan, M., Nunberg, J. H., and Schimke, R. T. (1980) Cell 22, 361-370). We have now mapped the 3' ends of these RNAs more precisely and have demonstrated colinearity between their shared sequences. Analysis of three larger dihydrofolate reductase RNAs has shown that these RNA species contain very long 3' noncoding regions, bringing the total number of dihydrofolate reductase RNAs to seven, ranging in length from 750 to 5600 nucleotides. We have determined the nucleotide sequence at and surrounding the polyadenylation sites of the four smaller RNAs. We find no striking structures in this sequence that might constitute multiple polyadenylation signals, but conclude that the putative polyadenylation signal AAUAAA is not required for polyadenylation of at least three of the four dihydrofolate reductase messengers.

Animals↗
PubMed1980

Size heterogeneity in the 3' end of dihydrofolate reductase messenger RNAs in mouse cells.

We have examined in detail the RNA coding for dihydrofolate reductase (DHFR) in methotrexate-resistant mouse cells. We find four distinct DHFR messengers, ranging in size from 750 to 1600 nucleotides. All four are polyadenylated and polysomal and can be translated in vitro to produce a 21,000 dalton protein co-migrating with purified dihydrofolate reductase on SDS polyacrylamide gels. The major difference in these RNAs is the length of 3' untranslated regions, varying from about 80 nucleotides in the smallest mRNA to about 930 nucleotides in the largest. The RNAs are also present in methotrexate-sensitive murine cells and mouse liver. Multiple DHFR RNAs are found in the poly(A)+ RNA of methotrexate-resistant Chinese hamster ovary cells but are of different molecular weights than the mouse messengers. We discuss these results in terms of the function of 3' untranslated regions of eucaryotic mRNAs and the possible origin and significance of multiple messenger RNAs for a single protein.

Animals↗
PubMed1979

Purification of specific adenovirus 2 RNAs by preparative hybridization and selective thermal elution.

A method is described for the preparation isolation of highly purified adenovirus RNA species. Cytoplasmic RNAs from cells infected with adenovirus 2 were selected by hybridization to viral DNA fragments bound to nitrocellulose membranes. A series of washes at elevated temperatures (50-70 degrees) determined conditions at which the true hybrids were stable but non-specific RNA was removed. This temperature has been found to correlate with the base composition of the DNA fragment. After washing at this predetermined temperature, the specific RNA was eluted at 85 degrees. The purity of the eluted RNA was greater than 95% as determined by size, sequence specificity, and template activity in an in vitro protein synthesizing system. The method described should be generally useful for purification of specific RNAs.

Adenoviridae↗
PubMed1978

Two regions of the adenovirus 2 genome specify families of late polysomal RNAs containing common sequences.

Late cytoplasmic RNAs specified by two regions of the adenovirus 2 genome (39.3--51.8 and 70.7--83.4 map units) were analyzed by size fractionation of poly(A)-[3H]RNA and subsequent hybridization to DNA fragments. Both regions encode RNAs whose sequence content exceeds the coding capacity of the region. These multiple transcripts are likely to function as mRNAs, because they are present on polyribosomes. The DNA segment 39.3--51.8 specifies 27S, 22S, and 18S RNAs. The genome sites specifying these three size classes were determined by hybridizations with seven different DNA fragments from this region of the genome. The 3' termini of all three size classes are specified by sequences near a common site, position 50.1. The 27S RNA includes sequences beginning near 39.3, the 22S RNA contains sequences from 41.0, and the 18S RNA includes sequences from approximately 45.3 on the unit genome. A second family of four RNAs is transcribed from 70.7--83.4. These 28S, 22S, 18S, and 16S RNAs have a relationship similar to the RNAs transcribed from 39.3--51.8. Sequences near the 5' ends of these four size classes are specified by different genome sites. However, the 3' termini of all four size classes were localized near map position 80.4. The synthesis of families of RNA may allow the translation of multiple polypeptides from a genome segment that has only one terminator site for mRNA synthesis.

Adenoviruses, Human↗