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Biomedical subjects

S John

Publications and source records attributed to S John.

At least 109 records · Page 6Linked to original sources
PubMed1997

Characterization of serum adhesive proteins that block tumor necrosis factor-mediated cell death.

Previously we have shown that TGF-beta1 protects murine L929 fibroblasts from TNF/ActD-mediated cell death by inducing the expression of an extracellular matrix TNF-resistance triggering (TRT) protein. TRT promotes TNF-resistance via activation of tyrosine and serine/threonine kinases in L929 cells. To examine the presence of TRT activity in serum (designated STRT), human sera were diluted, treated with or without PMSF and subjected to sequential ammonium sulfate precipitation (ASP). Aliquots of the ASP protein fractions were coated onto 96-well plates, followed by thorough washing. When L929 cells were seeded and cultured on the wells coated with STRT proteins, these cells resisted killing by TNF, TNF/ActD, doxorubicin and serum deprivation, but not by anti-Fas/ActD, staurosporine and ActD. STRT activity was found at the 15% ASP fraction of untreated sera, but shifted to the 20% ASP fraction of PMSF-treated sera. Two likely STRT proteins of approximately 226 and 265 kDa were found in these fractions, compared to the corresponding nonfunctional ASP fractions. Functionally, STRT was inactivated by trypsin, but not by 5 M salt, various serine and/or cysteine protease inhibitors, and antibodies against fibronectin, vitronectin, C1q, histidine-rich glycoprotein, CD44, chondroitin sulfate and hyaluronic acid. STRT failed to alter the expression of proteins involved in apoptosis such as RIP, ICH-1L, BCL-X, TIAR and IkappaBalpha, and could not induce IkappaBalpha degradation. The induced TNF-resistance could be reversed by treatment of STRT-stimulated cells with testicular hyaluronidase, as well as with tyrosine kinase inhibitors tyrophostin, lavendustin A and AG-490 (a selective inhibitor of JAK2 kinase). However, the STRT function could not be blocked by the MEK kinase inhibitor PD98059 and the NF-kappaB inhibitors curcumin and a synthetic inhibitor peptide for NF-kappaB translocation. Together, our data suggest that tyrosine kinase activation is involved in the STRT-mediated resistance to TNF and TNF/ActD in L929 cells.

Journal Article↗
PubMed1997

Biological properties of peripheral blood progenitor cells mobilized by cyclophosphamide and granulocyte colony-stimulating factor.

Patients transplanted with mobilized blood progenitor cells (PBPC) recover their neutrophil counts more rapidly than patients transplanted with bone marrow even when they receive the same dose/kg of granulocyte-macrophage colony-forming cells (CFU-GM). Here we have sought a biological explanation for this phenomenon. Most CD34-positive PBPC are quiescent (<1% in S phase) when they are collected from the bloodstream of patients treated with cyclophosphamide and granulocyte colony-stimulating factor (G-CSF), but we have shown that they are able to resume proliferation rapidly in vitro by measuring the kinetics of CFU-GM production by primitive plastic-adherent (Pdelta) cells. Also, Pdelta cells in PBPC harvests, unlike normal marrow Pdelta cells, were insensitive to cell-cycle restraint imposed by contact with marrow-derived stromal cells. We found that Pdelta cells in PBPC collections produce relatively more CFU-GM and relatively fewer BFU-E than Pdelta cells in bone marrow, indicating that granulopoiesis might occur at the expense of erythropoiesis, but we were unable to find any differences in the kinetics of granulocytic maturation between PBPC and bone marrow. Our interpretation of these findings is that transplanted PBPC rapidly enter the cell cycle and contact with stromal cells in the marrow does not reduce the proportion of progenitors participating in neutrophil production. Consequently. neutrophil recovery after PBPC infusion is more rapid than neutrophil recovery after marrow infusion. Granulopoiesis at the expense of erythropoiesis may also contribute to this effect.

Cell Cycle↗
PubMed1997

Retinoid-induced chromatin structure alterations in the retinoic acid receptor beta2 promoter.

Transcription of the retinoic acid receptor beta2 (RARbeta2) gene is induced by retinoic acid (RA) in mouse P19 embryonal carcinoma (EC) cells. Here we studied RA-induced chromatin structure alterations in the endogenous RARbeta2 promoter and in an integrated, multicopy RARbeta2 promoter in EC cells. RA markedly increased restriction site accessibility within the promoter, including a site near the RA responsive element (RARE) to which the nuclear receptor retinoid X receptor (RXR)-RAR heterodimer binds. These changes coincided with RA-induced alterations in the DNase I hypersensitivity pattern in and around the promoter. These changes became undetectable upon removal of RA, which coincided with the extinction of transcription. Analyses with receptor-selective ligands and an antagonist showed that increase in restriction site accessibility correlates with transcriptional activation, which parallels the RA-induced in vivo footprint of the promoter. Despite these changes, the micrococcal nuclease digestion profile of this promoter was not altered by RA. These results indicate that concurrent with the binding of the RXR-RAR heterodimer to the RARE, the local chromatin structure undergoes dynamic, reversible changes in and around the promoter without globally affecting the nucleosomal organization.

Animals↗
PubMed1997

Investigation of candidate disease susceptibility genes in rheumatoid arthritis: principles and strategies.

It has been estimated that a number of non-HLA susceptibility loci exist in rheumatoid arthritis (RA), each making relatively small contributions (lambda s < 2). Previous approaches for whole genome screening are unlikely to be sufficiently sensitive to detect such loci. As the pathology of RA already indicates several molecules that may be of potential importance in disease susceptibility, we propose an alternative approach, targeting candidate genes directly. Highly polymorphic dinucleotide markers within a candidate gene sequence or close to the gene can be used as markers, and the selection of the most appropriate markers is discussed. RA sibling pair families from the Arthritis and Rheumatism Council National Repository (n = 200) are used in linkage analysis studies. The data generated are analyzed using sib pair analysis methods to examine evidence of linkage. The interpretation of such results is also discussed, in particular, minimizing the possibility of type I errors, and the interpretation of negative results.

Arthritis, Rheumatoid↗
PubMed1997

The sensitivity of different analytical methods to detect disease susceptibility genes in rheumatoid arthritis sibling pair families.

Our aim was to compare the sensitivity of 4 analytical methods to detect linkage to a known disease susceptibility locus, HLA-DRB1, in 100 rheumatoid arthritis sibling pair families with incomplete parental genotype information. Genotypes for the HLA-DRB1 and HLA-A loci were analyzed using (1) identity-by-descent (IBD), considering inheritance of maternal and paternal alleles separately; (2) maximum likelihood score-IBD (MLS-IBD), which infers missing parental genotypes; (3) identity-by-state (IBS), which does not require parental genotypes; and (4) transmission disequilibrium test (TDT), which uses affected offspring with a heterozygous parent. Due to the small number of informative meoisis for HLA-DRB1, the IBD analysis was not significant for linkage (p = 0.014). HLA-A was more informative (p = 0.0002). The MLS-IBD method for HLA-DRB1 (p = 0.00004) and HLA-A (p < or = 0.00001) was significant. Using IBS both loci gave highly significant evidence of linkage, (p < < 0.00001). The TDT detected HLA-DRB1*0401 as the allele associated with RA; no HLA-A allele was associated. Thus, sib pair families with limited parental genotypes can be used to detect disease susceptibility loci, but when selecting the method of analysis the informativeness of the markers should be taken into account.

Arthritis, Rheumatoid↗
PubMed1997

Tumor necrosis factor microsatellite haplotypes are different in male and female patients with RA.

We investigated whether tumor necrosis factor (TNF) microsatellite polymorphisms are associated with sex and age at disease onset in rheumatoid arthritis (RA). A case-control study was used to compare the frequencies of TNF microsatellite alleles in 181 Caucasian RA cases and 251 controls. TNF microsatellite genotyping was performed using fluorescent based polymerase chain reaction (PCR) techniques and HLA-DR typing using PCR based sequence specific oligonucleotide probing. The association of TNF microsatellite alleles a6, b5, and d4 with RA was confirmed. These polymorphisms were more frequent in female patients. Male patients with RA with young age at onset had a different TNF microsatellite profile, TNFa2, b1, and c2 being the most frequent. TNF microsatellite polymorphisms are different in male and female patients with RA. This difference was more obvious in patients stratified according to age at onset. Sex and age at onset should be considered in studies of genetic factors in RA.

Adult↗
PubMed1997

Linkage and association studies of the natural resistance associated macrophage protein 1 (NRAMP1) locus in rheumatoid arthritis.

OBJECTIVE: To determine if there is any evidence of linkage between the natural resistance associated macrophage protein gene, NRAMP1, and rheumatoid arthritis (RA). METHODS: Two dinucleotide markers, D2S1471, a highly polymorphic marker within 160 kb of NRAMP1, and a less polymorphic marker in the promoter region of NRAMP1, have been analyzed in 115 affected sib-pair RA families (35 with 2 parents, 18 with one parent) from the Arthritis and Rheumatism Council (ARC) National Repository and an additional 85 probands from the ARC National Twin Study and 96 controls. Individuals were typed for both markers by fluorescence based semiautomated polymerase chain reaction analysis. RESULTS: No evidence of increased allele sharing in affected sib-pairs was obtained using identity by descent (IBD), identity by state, and maximum likelihood score (MLS) analysis for the whole data set. A significant increase in allele sharing was observed with marker D2S1471 (LOD 0.74; p 0.05) in the HLA discordant subgroup using IBD and MLS-IBD. No significant differences in allele frequencies were observed for any markers in the association study. CONCLUSION: In a subset of sib-pairs that shared one or zero HLA haplotypes, LOD scores suggestive of linkage were observed. This suggests a role for NRAMP1 polymorphism in a subset of patients who do not possess HLA susceptibility alleles.

Alleles↗
PubMed1996

Characterization of an NF-1/CTF family member as a functional activator of the mouse mammary tumor virus long terminal repeat 5' enhancer.

The long terminal repeat of the mouse mammary tumor virus restricts virus expression primarily to the mammary epithelium. The extreme 5' end of the long terminal repeat contains an enhancer that has been associated with tissue-specific expression of the virus. A total of six functional cis-acting elements have been identified in the enhancer. Although proteins binding to these elements have been reported, only one has been identified; this factor, mp5, is identical or closely related to the transcription factor AP-2 (Mellentin-Michelotti, J., John, S., Pennie, W. D., Williams, T., and Hager, G. L. (1994) J. Biol. Chem. 269, 31983-31990). The other factors are hitherto unidentified and poorly described. We report here the characterization of another of the six elements, previously referred to as the F3 site (Mink, S., Hartig, E., Jennewein, P., Doppler, W., and Cato, A. C. (1992) Mol. Cell Biol. 12, 4906-4918). We show that the F3 binding activity and AP-2 act synergistically to enhance mouse mammary tumor virus-directed transcription, but only in the presence of glucocorticoid hormone. The F3 element has an NF-1-like half-site, but the activity recognizing this element has binding characteristics distinct from the NF-1/CTF family as well as the rest of the CCAAT-binding proteins. We conclude that the F3 activity represents a new member of the NF-1/CTF family.

Animals↗
PubMed1996

An IL-2 response element in the human IL-2 receptor alpha chain promoter is a composite element that binds Stat5, Elf-1, HMG-I(Y) and a GATA family protein.

Expression of the human interleukin-2 (IL-2) receptor alpha chain gene is potently upregulated by its own ligand, IL-2. In this study, we characterize an essential upstream IL-2 response element that contains both consensus and non-consensus GAS motifs, two putative Ets binding sites (EBS), one of which overlaps the consensus GAS motif, and a GATA motif, which overlaps the non-consensus GAS motif. We demonstrate that although the individual components of this element do not respond to IL-2, together they form a composite element capable of conferring IL-2 responsiveness to a heterologous promoter. Multiple factors including Stat5, Elf-1, HMG-I(Y) and GATA family proteins bind to the IL-2 response element and mutation of any one of these binding sites diminishes the activity of this element. An unidentified Ets family protein binds to the EBS overlapping the consensus GAS motif and appears to negatively regulate the human IL-2R alpha promoter. Thus, IL-2-induced IL-2R alpha promoter activity requires a complex upstream element, which appears to contain binding sites for both positive and negative regulatory factors.

Base Sequence↗
PubMed1996

Cloning of human Stat5B. Reconstitution of interleukin-2-induced Stat5A and Stat5B DNA binding activity in COS-7 cells.

We have isolated a second human Stat5 cDNA, Stat5B, and demonstrated that the genes encoding both Stat5A and Stat5B are located at chromosome 17q11.2. Both genes were constitutively transcribed in peripheral blood lymphocytes. By using specific antisera, we demonstrated that both Stat5A and Stat5B are activated by interleukin-2 (IL-2) in peripheral blood lymphocytes, natural killer-like YT leukemia cells, and human T cell lymphotropic virus type I-transformed MT-2 T cells. In COS-7 cells, which constitutively express the Janus family tyrosine kinase Jak1, reconstitution of IL-2-induced Stat5A and Stat5B DNA binding activities was dependent on the coexpression of Jak3 along with the IL-2 receptor beta chain and the common cytokine receptor gamma-chain. This IL-2-induced Stat5 activation was dependent on the presence of either of two tyrosines (Tyr-392 or Tyr-510) in the IL-2 receptor beta chain, indicating that either of these two tyrosines can serve as a docking site. Moreover, we demonstrated that human Stat5 activation is also dependent on Tyr-694 in Stat5A and Tyr-699 in Stat5B, indicating that these tyrosines are required for dimerization. The COS-7 reconstitution system described herein provides a valuable assay for further elucidation of the IL-2-activated JAK-STAT pathway.

Amino Acid Sequence↗
PubMed1996

The effect of kidney size on cadaveric renal allograft outcome.

Chronic rejection is the commonest cause of long-term renal allograft loss. Though immunologic factors are thought dominant in its pathogenesis, nonimmunologic factors, in particular, hyperfiltration damage related to reduced renal mass, have also been proposed as factors in the causation of chronic allograft rejection. We assessed the influence of renal size on graft survival and function in all cyclosporine-treated cadaver donor adult renal allograft recipients engrafted at a single center between June 1989 and July 1994, whose grafts functioned for > or = to 3 months (n=169). Patients were divided into 4 groups based on the ratio of kidney volume to recipient body surface area (volume/BSA) (ml/m2), and outcome in groups compared by methods including Cox's proportional hazards and Kaplan-Meier analysis. No significant differences between groups existed for serum creatinine levels, presence of significant proteinuria, or 1- and 5-year graft survival. There was no correlation between volume/BSA and either serum creatinine or degree of proteinuria at 3, 6, 12, 36, and 60 months posttransplant. Volume/BSA was similar in patients with good or poor renal function (58 +/-21 vs. 56 +/- 28 ml/m2), with or without significant proteinuria (57 +/- 24 vs. 60 +/- 25 ml/m2) or in patients who lost their grafts to chronic rejection compared with those with stable allograft function (64 +/- 34 vs. 59 +/- 24 ml/m2). Volume/BSA was not a predictor of graft survival on multivariate regression. We conclude that donor kidney size has no apparent effect on cadaveric renal allograft outcome in the short and intermediate-term, suggesting that close matching of donor kidney size to recipient size is not presently indicated.

Adolescent↗
PubMed1996

Importance of low affinity Elf-1 sites in the regulation of lymphoid-specific inducible gene expression.

Elf-1 is an Ets family transcription factor that regulates a number of inducible lymphoid-specific genes, including those encoding interleukin 3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), and the IL-2 receptor (IL-2R) alpha chain. A minimal oligonucleotide spanning the IL-2R alpha Elf-1 site (-97/-84) bound Elf-1 poorly, but binding activity markedly increased when this oligonucleotide was multimerized or flanking sequences were added. This result is consistent with the requirement of accessory proteins for efficient Elf-1 binding, as has been demonstrated for the GM-CSF and IL-3 promoters. A binding site selection analysis revealed the optimal Elf-1 consensus motif to be A(A/t)(C/a)CCGGAAGT(A/S), which is similar to the consensus motif for the related Drosophila E74 protein. This minimal high affinity site could bind Elf-1 and functioned as a stronger transcription element than the -97/-84 IL-2R alpha oligonucleotide when cloned upstream of a heterologous promoter. In contrast, in the context of the IL-2R alpha promoter, conversion of the naturally occurring low affinity Elf-1 site to an optimal site decreased inducible activation of a reporter construct in Jurkat cells. This finding may be explained by the observation that another Ets family protein, ER GB/Fli-1, can efficiently bind only to the optimal site, and in this context, interferes with Elf-1 binding. Therefore, high affinity Elf-1 sites may lack sufficient binding specificity, whereas naturally occurring low affinity sites presumably favor the association of Elf-1 in the context of accessory proteins. These findings offer an explanation for the lack of optimal sites in any of the known Elf-1-regulated genes.

Animals↗
PubMed1996

Molecular evidence for a common leukaemic progenitor in acute mixed lymphoid and myeloid leukaemia.

De novo acute leukaemia presenting with a mixed lymphoid and myeloid leukaemic population has rarely been described. We have used the consensus Ig heavy chain primers and DNA isolated from these two distinct populations of cells in polymerase chain reactions. We demonstrated that both populations of cells exhibited Ig heavy chain gene rearrangements. Cloning and subsequent DNA sequencing of the amplified products showed a common V-D-J junctional nucleotide sequence. This work therefore provides the first evidence that the leukaemic cells in de novo acute leukaemia with a mixed lymphoid and myeloid population are derived from a common progenitor clone and may offer an explanation for the poor prognosis in these patients.

Adult↗