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Biomedical subjects

M Hadman

Publications and source records attributed to M Hadman.

9 recordsLinked to original sources
PubMed2004

Intracerebral xenotransplantation of GFP mouse bone marrow stromal cells in intact and stroke rat brain: graft survival and immunologic response.

The present study characterized survival and immunologic response of bone marrow stromal cells (BMSCs) following transplantation into intact and stroke brains. In the first study, intrastriatal transplantation of BMSC (60,000 in 3 microl) or vehicle was performed in normal adult Sprague-Dawley male rats that subsequently received daily cyclosporin A (CsA, 10 mg/kg, IP in 3 ml) or vehicle (olive oil, similar volume) starting on day of surgery up to 3 days posttransplantation. Animals were euthanized at 3 or 30 days posttransplantation and brains were processed either for green fluorescent protein (GFP) microscopy or flow cytometry (FACS). Both GFP epifluorescence and FACS scanning revealed GFP+ BMSCs in both groups of transplanted rats with or without CsA, although significantly increased (1.6- to 3-fold more) survival of GFP+ BMSCs was observed in the immunosuppressed animals. Further histologic examination revealed widespread dispersal of BMSCs away from the graft core accompanied by many long outgrowth processes in non-CsA-transplanted animals, whereas a very dense graft core, with cells expressing only sporadic short outgrowth processes, was observed in CsA-transplanted animals. There were no detectable GFP+ BMSCs in nontransplanted rats that received CsA or vehicle. Immunologic response via FACS analysis revealed a decreased presence of cytotoxic cells, characterized by near complete absence of CD8+ cells, and lack of activation depicted by low CD69 expression in CsA-treated transplanted animals. In contrast, elevated levels of CD8+ cells and increased activation of CD69 expression were observed in transplanted animals that received vehicle alone. CD4+ helper cells were almost nondetectable in transplanted rats that received CsA, but also only minimally elevated in transplanted rats that received vehicle. Nontransplanted rats that received either CsA or vehicle displayed very minimal detectable levels of all three lymphocyte markers. In the second study, a new set of male Sprague-Dawley rats initially received bilateral stereotaxic intrastriatal transplantation of BMSCs and 3 days after were subjected to unilateral transient occlusion of middle cerebral artery. The animals were allowed to survive for 3 days after stroke without CsA immunosuppression. Epifluorescence microscopy revealed significantly higher (5-fold more) survival of transplanted GFP+ BMSCs in the stroke striatum compared with the intact striatum. The majority of the grafts remained within the original dorsal striatal transplant site, characterized by no obvious migration in intact striatum, but with long-distance migration along the ischemic penumbra in the stroke striatum. Moreover, FACS scanning analyses revealed low levels of immunologic response of grafted BMSCs in both stroke and intact striata. These results, taken together, suggest that xenotransplantation of mouse BMSCs into adult rats is feasible. Immunosuppression therapy can enhance xenograft survival and reduce graft-induced immunologic response; however, in the acute phase posttransplantation, BMSCs can survive in intact and stroke brain, and may even exhibit long-distance migration and increased outgrowth processes without immunosuppression.

Animals↗
PubMed2000

Replacing two conserved tyrosines of the EphB2 receptor with glutamic acid prevents binding of SH2 domains without abrogating kinase activity and biological responses.

Eph receptor tyrosine kinases play key roles in pattern formation during embryonic development, but little is known about the mechanisms by which they elicit specific biological responses in cells. Here, we investigate the role of tyrosines 605 and 611 in the juxtamembrane region of EphB2, because they are conserved Eph receptor autophosphorylation sites and demonstrated binding sites for the SH2 domains of multiple signaling proteins. Mutation of tyrosines 605 and 611 to phenylalanine impaired EphB2 kinase activity, complicating analysis of their function as SH2 domain binding sites and their contribution to EphB2-mediated signaling. In contrast, mutation to the negatively charged glutamic acid disrupted SH2 domain binding without reducing EphB2 kinase activity. By using a panel of EphB2 mutants, we found that kinase activity is required for the changes in cell-matrix and cell - cell adhesion, cytoskeletal organization, and activation of mitogen-activated protein (MAP) kinases elicited by EphB2 in transiently transfected cells. Instead, the two juxtamembrane SH2 domain binding sites were dispensable for these effects. These results suggest that phosphorylation of tyrosines 605 and 611 is critical for EphB2-mediated cellular responses because it regulates EphB2 kinase activity.

3T3 Cells↗
PubMed1998

Apolipoprotein A-1 is a negative target of v-Jun overexpression.

The product of the Jun oncogene influences a variety of processes including cell proliferation and differentiation. Jun exerts its influence by binding to the promoter and enhancer regions of a number of different target genes resulting in their activation or repression. We describe here the isolation and characterization of a gene differentially downregulated upon overexpression of v-Jun but not c-Jun. DNA and amino acid homology search analysis revealed this gene to be identical to chicken apolipoprotein A-1, the major component of high density lipoprotein (HDL). The half life of apolipoprotein A-1 RNA remains constant in the presence or absence of v-Jun overexpression suggesting downregulation by v-Jun is at the level of promoter activity. Consistent with this hypothesis, apolipoprotein A-1 upstream promoter fragments active in normal and c-Jun expressing CEF are inactive in v-Jun transformed CEF. Analysis of expression of apolipoprotein A-1 in CEF overexpressing other oncogenes revealed a similar downregulation by Myc and v-Src but not c-Fos, v-Ha-Ras, c-Src or c-Ski. Our findings point to a potential regulatory affect on cholesterol metabolism by v-Jun, as a result of altered levels of apolipoprotein A-1 message expression.

Animals↗
PubMed1996

Isolation and cloning of JTAP-1: a cathepsin like gene upregulated in response to V-Jun induced cell transformation.

The oncogenic potential of Jun in chicken embryo fibroblasts (CEF) varies depending on its structure. V-Jun, which has a number of structural differences from c-Jun is highly transforming and tumorigenic. C-Jun however, is only weakly transforming and is not tumorigenic. We have used this difference in oncogenic potential between v-Jun and c-Jun to screen for downstream target genes associated with the v-Jun induced transformed phenotype. We describe here the identification, cloning and characterization of one of these genes, JTAP-1. JTAP-1 is consistently overexpressed 7 to 10-fold in CEF transformed by v-Jun compared with c-Jun overexpressing or normal CEF. This pattern of expression suggests that JTAP-1 is associated with the transformed phenotype. DNA and amino acid homology search analysis revealed that JTAP-1 shares a high degree of similarity with over 100 cysteine proteases from a variety of species and is likely the chicken homolog of cathepsin O. Analysis of expression of JTAP-1 in CEF overexpressing other oncogenes including v-Ha-ras, v-Src, c-Fos, and Myc revealed that it's overexpression is unique to v-Jun transformed cells. Thus, JTAP-1 is likely a specific target of v-Jun overexpression and not simply a consequence of cell transformation.

Amino Acid Sequence↗
PubMed1993

In vivo viral and cellular Jun complexes exhibit differential interaction with a number of in vitro generated 'AP-1- and CREB-like' target sequences.

A direct comparison of the relative DNA-binding capabilities of in vivo Jun-containing complexes derived from overexpression of the highly transforming viral Jun (VJ-1 CEF), the weakly transforming chicken cellular Jun (CJ-3 CEF) or background endogenous Jun (RCAS CEF) was assessed by gel mobility-shift assays using a synthetic oligonucleotide containing the consensus sequence TGACTCA (consensus AP-1). Chicken embryo fibroblasts (CEFs) expressing background c-Jun levels (RCAS CEF) contain almost undetectable levels of c-Jun but retain significant DNA-binding activity with two distinct complexes capable of binding specifically to the consensus AP-1 site. CEFs overexpressing either v-Jun or c-Jun contain these same two complexes and, while showing marked increases in Jun protein levels, do not exhibit any increase in DNA binding or transcriptional activation activity, suggesting that much of the overexpressed protein is inactive. Gel-shift assays performed in the presence of a Jun-specific antibody revealed a reduction in binding by both complexes, suggesting that each contains Jun or a Jun cross-reactive protein. Antibodies specific for Jun B, c-Fos, Fos B and CREB failed to interact with either complex. However, antibody specific for Fra-2 caused a slight supershift, suggesting that one or both complexes may contain Fra-2. Gel-shift competition assays with 16 'AP-1- and CREB-like' target sequences revealed that, within each cell type, the two protein complexes varied in their ability to recognize the mutant target sequences. These results clearly indicate differences in potential target recognition by each specific in vivo complex, and suggest that each may preferentially bind its own subset of target DNAs. In addition, a comparison of binding by individual complexes derived from CEFs overexpressing v-Jun and c-Jun also revealed differences in target recognition. Thus, in vivo complexes formed by overexpression of v-Jun and c-Jun vary in their ability to recognize and bind to a number of 'AP-1- and CREB-like' target sequences. This has important implications with regard to the mechanisms involved in cell transformation by v-Jun.

Animals↗
PubMed1992

Heterodimerization with c-Fos is not required for cell transformation of chicken embryo fibroblasts by Jun.

c-Jun belongs to a family of proteins that require dimerization for activity. Dimerization occurs through a leucine-rich region near the carboxy terminus called the leucine zipper. Jun can form dimeric complexes with other Jun family as well as Fos family members. The relative proportion of these different dimeric complexes is determined by the relative abundance of each family member at a particular time. Overexpression of v-Jun or c-Jun alone will lead to cell transformation of chicken embryo fibroblasts, albeit with varying efficiencies. Upon overexpression, v-Jun or c-Jun presumably becomes the predominant AP-1 component in the cell. Theoretically, this should lead to a larger proportion of homodimers than heterodimers. It is not clear what role, if any, the other Jun and Fos family proteins play during cell transformation. We have examined the ability of Jun to induce cell transformation in chicken embryo fibroblasts in the absence of interaction with other Jun or Fos family proteins. To this end, we have constructed a chicken v-Jun mutant that is incapable of heterodimerization. This was accomplished by replacing the leucine zipper region of Jun with that of the yeast transcription factor GCN4. This chimeric protein, VJ-GLZ, retains all of the DNA binding and transcriptional activation domains of v-Jun. As expected, in vitro translated VJ-GLZ was found to be incapable of forming heterodimers with c-Fos, FosB, and JunD.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

A simplified biochemistry for DNA sequencing.

A simplified method of DNA sequencing by dideoxy chain termination is developed that approaches a single-step protocol. Utilizing the sequencing advantages contributed by a thermophilic polymerase and a guanine analog, stable sequencing reaction concentrates have been obtained that readily perform the entire sequencing reaction simply by adding prepared DNA to each of the four reaction concentrates required by this method. The mechanics and dynamics of these reactions have been investigated and the capacity of these reactions to withstand normal user variation is demonstrated. This study focuses on one form of this simplified method embodied in the FASTaq DNA sequencing kit.

Base Sequence↗
PubMed1986

Effect of monensin on synthesis, post-translational processing, and secretion of dopamine beta-hydroxylase from PC12 pheochromocytoma cells.

Monensin was used to ascertain the location in the biosynthetic pathway where the 77,000-Mr membrane-bound subunit form of dopamine beta-hydroxylase is post-translationally converted to the 73,000-Mr soluble form. Treatment with low concentrations of monensin (less than or equal to 50 nM) completely depleted the cells of the norepinephrine and dopamine, had a small effect on protein synthesis, and enhanced post-translational processing of only dopamine beta-hydroxylase which was previously synthesized and presumably packaged into neurosecretory vesicles. At these low concentrations, exit from the Golgi apparatus did not appear to be blocked since stimulated secretion of a group of high molecular weight [35S]methionine-labeled proteins was not inhibited. Treatment with higher concentrations of monensin (200 nM) prevented the secretion of the [35S] methionine-labeled proteins normally released with a secretagogue, and also prevented the secretion of [3H] mannose-labeled proteins including dopamine beta-hydroxylase. Surprisingly, a group of lower molecular weight [35S]methionine-labeled proteins was now released from monensin-treated cells. Treatment with high concentrations of monensin (greater than or equal to 200 nM) appeared to block the secretory pathway prior to the packaging step, probably in the Golgi apparatus. If the proteins were packaged prior to monensin treatment, they were released upon stimulation with secretagogues. Monensin treatment (200 nM) enabled the post-translational processing of newly synthesized dopamine beta-hydroxylase, from the 77,000-Mr to the 73,000-Mr subunit form, to go to completion. The susceptibility of this 73,000-Mr subunit form to endoglycosidase H digestion was unaltered, suggesting that dopamine beta-hydroxylase from monensin-treated cells may have the same high mannose oligosaccharide content as native dopamine beta-hydroxylase. These experiments indicate that the post-translational processing of dopamine beta-hydroxylase occurs in the Golgi apparatus and may continue in immature granules prior to their acidification.

Adrenal Gland Neoplasms↗