Biomedical subjects

J Nalbantoglu

Publications and source records attributed to J Nalbantoglu.

At least 55 records · Page 3Linked to original sources

PubMed1996

Alzheimer's beta-amyloid precursor protein is expressed on the surface of immediately ex vivo brain cells: a flow cytometric study.

Beta-amyloid precursor protein (beta APP) is ubiquitously expressed, but deposition of the beta APP proteolytic fragment A beta is virtually restricted to the brain, suggesting cell-specific processing of this molecule. Our laboratory has investigated expression of beta APP in mechanically dissociated, unfixed, immediately ex vivo cells from various mouse and rat organs by flow cytometry. Epitopes of predicted extracellular domains of beta APP recognized by the N-terminal 22C11 monoclonal antibody (mAb) and the juxtamembrane 4G8 mAb were not detectable on the surface of lymphoid cells, hepatocytes, or kidney cells. In contrast, surface 22C11 and 4G8 beta APP immunoreactivity was abundant on intact (propidium iodide-excluding) dissociated brain cells. The predicted C-terminal intracellular beta APP determinant recognized by the mAb Jonas was not detectable on the surface of intact brain cells, but was present in ethanol-permeabilized cells, consistent with a transmembrane configuration of beta APP in brain cells. Trypsinization of intact brain cells abolished cell surface immunoreactivity for 22C11, which was then reestablished by short-term culture. Augmentation of 22C11 and 4G8 surface immunoreactivity occurred when brain cells were cultured short-term in phenylarsine oxide, a general endocytosis inhibitor. By double staining protocols of brain cells with mAbs directed against beta APP ectodomain epitopes and the neuronal surface proteins Thy-1 or neural cell adhesion molecule (NCAM), we observed that all Thy-1+ and NCAM+ cells (approximately 50%) were immunoreactive for surface beta APP, but that some beta APP+ cells (approximately 20%) were negative for these neuronal markers. Our data suggest that neurons and a subpopulation of other brain cells, unlike peripheral cells, can support beta APP as a type 1 intrinsic membrane molecule with an intact ectodomain, and that beta APP surface abundance is regulated by an equilibrium between membranes vesicle insertion and endocytotic internalization. Transmembrane beta APP holoprotein may be a critical determinant of brain-predominant processing of beta APP to A beta, and may participate in a receptor/transducer function unique to brain cells.

Alzheimer Disease↗

PubMed1996

Impairment of force generation after adenovirus-mediated gene transfer to muscle is alleviated by adenoviral gene inactivation and host CD8+ T cell deficiency.

Recombinant adenovirus vectors (AdV) hold promise as a means of delivering therapeutic genes to muscle in diseases such as Duchenne muscular dystrophy (DMD). However, we have previously shown that the use of AdV is hampered by the development of reduced force-generating capacity, which occurs within 1 week and is progressive up to at least 1 month after AdV delivery in immune-competent animals. Determinations of muscle force production provide a sensitive and clinically important measure of potential adverse effects of AdV-mediated gene transfer on muscle cell function. In the present study, we investigated the role of AdV-related gene expression and host T lymphocyte responses in the genesis of muscle dysfunction following AdV injection of muscle. We report that UV-irradiation of AdV particles, which reduced AdV transcriptional activity without impairing infectivity (as confirmed by in situ polymerase chain reaction), significantly reversed early (4 days post-injection) AdV-induced contractile impairment in immune-competent mice as well as in mice lacking effective CD8+ T cell activity. The superimposed additional reduction in force-generating capacity normally found between 4 and 30 days post-AdV delivery in immune-competent mice, along with the associated loss of transgene (beta-galactosidase) expression, was largely abrogated by the absence of an intact CD8+ T lymphocyte response. Furthermore, short-term administration of a neutralizing antibody against CD4+ T cells significantly prolonged transgene expression and showed a trend toward mitigation of AdV-induced reductions in force-generating capacity. Cellular infiltration and humoral immune responses against the vector and transgene product were also blunted to varying degrees in the setting of CD8+ or CD4+ T cell deficiency. We conclude that AdV-related gene expression has an early negative (probably toxic) effect on muscle cell function that is independent of CD8+ T cell-mediated immunity. In contrast, further progression of contractile impairment and the accompanying loss of transgene expression from AdV-injected muscle are largely dependent upon the activity of CD8+ T cells. These results have implications for the design of future generation vectors and the potential need for immunosuppressive therapy after AdV-mediated gene transfer to muscle.

Adenoviridae↗

PubMed1996

The principles of gene therapy for the nervous system.

Research pertaining to gene transfer into cells of the nervous system is one of the fastest growing fields in neuroscience. An important application of gene transfer is gene therapy, which is based on introducing therapeutic genes into cells of the nervous system by ex vivo or in vivo techniques. With the eventual development of efficient and safe vectors, therapeutic genes, under the control of a suitable promoter, can be targeted to the appropriate neurons or glial cells. Gene therapy is not only applicable to the treatment of genetic diseases of the nervous system and the control of malignant neoplasia, but it also has therapeutic potential for acquired degenerative encephalopathies (Alzheimer's disease, Parkinson's disease), as well as for promoting neuronal survival and regeneration in various pathological states.

Animals↗

PubMed1996

The helix-loop-helix transcription factor USF interacts with the basal promoter of human amyloid precursor protein.

Nuclear factors from HeLa, PC12, NG108-15 and SK-N-SH cell lines recognized an oligonucleotide (-56 to -37: APP-E1) containing an E box (CANNTG) which had previously been characterized as a DNase I-protected sequence in the basal promoter of the human amyloid precursor protein (APP) gene. Binding to APP-E1 was competed with an oligonucleotide encompassing the recognition site of the transcription factor USF. Antibodies directed against USF interacted with the APP-E1-protein complex and in vitro synthesized USF could bind APP-E1. Co-expression of USF cDNA transactivated a human APP-reporter gene construct. These results suggest that USF may play a role in the expression of the APP gene.

Amyloid beta-Protein Precursor↗

PubMed1996

Expression of tumor necrosis factor alpha (TNF alpha) and interleukin 6 (IL-6) mRNA in adult human astrocytes: comparison with adult microglia and fetal astrocytes.

Astrocytes and microglia are cell populations which are implicated as being capable of regulating and effecting immune responses within the central nervous system (CNS). These functions are postulated to be mediated at least in part by production of soluble protein molecules termed cytokines. In this study, we utilized dissociated cultures of glial cells prepared from adult and fetal CNS tissue and a combined in situ hybridization-immunocytochemical technique in order to compare expression of TNF alpha and IL-6 mRNA between adult and fetal astrocytes and between adult astrocytes and microglia. Our results, using digoxygenin-labeled riboprobes, indicate that in contrast to fetal astrocytes only rare adult astrocytes express TNF alpha and IL-6 transcripts under our serum-supplemented basal culture conditions. Activation with LPS and IFN gamma increased the proportion of adult astrocytes expressing detectable TNF alpha and IL-6 mRNA signals; however, the proportion was significantly less than for microglia contained in the same cultures. These results suggest that microglia rather than astrocytes are more likely to be sources of these cytokines within the adult human CNS. Further studies of cytokine expression by glial cells will need to consider both the age and species of the glial cells used.

Adult↗

PubMed1996

Transient immunosuppression by FK506 permits a sustained high-level dystrophin expression after adenovirus-mediated dystrophin minigene transfer to skeletal muscles of adult dystrophic (mdx) mice.

Adenovirus (AV)-mediated gene transfer into skeletal muscles of adult immune-competent animals has been limited by the fact that a cell-mediated immune attack of the host against transduced muscle fibers prevented efficient long-term transgene expression. More recently, various immunomodulating strategies have been shown to improve the longevity of transgene expression after AV-mediated gene transfer. In this study we treated adult dystrophic (mdx) mice with daily subcutaneous injections of the immunosuppressive drug FK506 (tacrolimus) over 5, 10, 30 and 60 days after AV-mediated dystrophin gene transfer and compared the transduction level with saline-injected mdx controls. We show that daily FK506 treatment after AV-mediated dystrophin gene transfer into adult mdx muscle results in the maintenance of the initial transgene expression for at least 2 months, even when FK506 treatment was discontinued after 1 month. This is in keeping with the marked reduction of inflammatory infiltrates and the reduced activation level (inducible nitric oxide synthase) of macrophages in adenoviral recombinant (AVR)-injected muscles of FK506-treated animals. Moreover, we find that FK506 efficiently suppresses the humoral immune response against both the vector proteins and the transgene protein product (dystrophin). Furthermore, we demonstrate that continuous FK506 treatment over 30 days significantly improves the efficiency of gene transfer when the same vector is readministered to an animal which had been transduced 20 days earlier. In conclusion, the data suggest that sensitization by the initial antigenic load of the AVR application plays a pivotal role in triggering the humoral and cellular immune response of the host, which can be significantly counteracted by relatively short-term immunosuppressive treatment. These findings have important implications for the design of future human trials for gene replacement therapy in Duchenne muscular dystrophy.

Adenoviridae↗

PubMed1995

Apolipoprotein E4 allele as a predictor of cholinergic deficits and treatment outcome in Alzheimer disease.

Apolipoprotein E (apoE) is critical in the modulation of cholesterol and phospholipid transport between cells of different types. Human apoE is a polymorphic protein with three common alleles, APO epsilon 2, APO epsilon 3, and APO epsilon 4. ApoE4 is associated with sporadic and late-onset familial Alzheimer disease (AD). Gene dose was shown to have an effect on risk of developing AD, age of onset, accumulation of senile plaques in the brain, and reduction of choline acetyltransferase (ChAT) activity in the hippocampus of AD subjects. To characterize the possible impact of the apoE4 allele on cholinergic markers in AD, we examined the effect of apoE4 allele copy number on pre- and postsynaptic markers of cholinergic activity. ApoE4 allele copy number showed an inverse relationship with residual brain ChAT activity and nicotinic receptor binding sites in both the hippocampal formation and the temporal cortex of AD subjects. AD cases lacking the apoE4 allele showed ChAT activities close or within age-matched normal control values. The effect of the apoE4 allele on cholinomimetic drug responsiveness was assessed next in a group (n = 40) of AD patients who completed a double-blind, 30-week clinical trial of the cholinesterase inhibitor tacrine. Results showed that > 80% of apoE4-negative AD patients showed marked improvement after 30 weeks as measured by the AD assessment scale (ADAS), whereas 60% of apoE4 carriers had ADAS scores that were worse compared to baseline. These results strongly support the concept that apoE4 plays a crucial role in the cholinergic dysfunction associated with AD and may be a prognostic indicator of poor response to therapy with acetylcholinesterase inhibitors in AD patients.

Age of Onset↗

PubMed1995

Polymerase chain reaction quantification of lymphoid amyloid precursor protein mRNAs in Alzheimer's disease and Down's syndrome.

Amyloid beta-protein, the major constituent of Alzheimer's disease (AD) brain amyloid deposits, is encoded by several alternatively spliced amyloid precursor protein (APP) mRNAs. The well-established associated in Down's syndrome (DS) between APP overproduction and premature development of AD, as well as the recent demonstration of an increase in APP transcripts from lymphoblastoid cells of familial AD cases, suggest aberrant transcriptional regulation of some genes in AD. We assayed steady-state expression of the APP gene transcripts in peripheral blood mononuclear cells (PBMC) of AD and DS patients using quantitative polymerase chain reaction of reverse-transcribed mRNAs, and we compared their levels of PBMC APP expression with those of young and age-matched healthy controls. Results indicate APP mRNAs were of comparable abundance in PBMC obtained from 9 AD patients, 7 young controls and 12 age-matched controls. These data suggest regulation of APP mRNAs is normal in AD and DS PBMC.

Adult↗

PubMed1995

Neutral endopeptidase can hydrolyze beta-amyloid(1-40) but shows no effect on beta-amyloid precursor protein metabolism.

High performance liquid chromatographic analyses of incubations of beta-amyloid(1-40) with neutral endopeptidase revealed at least nine product peaks, indicating that neutral endopeptidase can cleave beta-amyloid at multiple sites. Mass spectroscopic analysis of hydrolyzed beta-amyloid identified at least five cleavage sites, between residues Glu3-Phe4, Gly9-Trp10, Phe19-Phe20, Ala30-Ile31, and Gly33-Leu34. In contrast, amyloid precursor protein metabolism in Neuro2A cells was unaffected by the expression of recombinant neutral endopeptidase in the same cells or by the addition of a secreted form of neutral endopeptidase to spent Neuro2A cell media.

Amino Acid Sequence↗

PubMed1995

Use of tissue culture models to study environmental-genetic interactions relevant to neurodegenerative diseases.

1. Clonal cell lines, primary cultured neurones and transgenic animals expressing mutant genes linked to familial forms of neurodegenerative diseases provide models in which to examine the interaction between expression of a predisposing gene and exposure to neurotoxic chemicals. Methods of establishing these models are reviewed. 2. Mutations in the gene encoding Cu/Zn-superoxide dismutase (SOD-1) have been identified in cases of familial amyotrophic lateral sclerosis linked to chromosome 21. We report that in clonal lines of PC12 cells, the cytotoxicity of a glutathione-depleting epoxide, styrene oxide, varied with SOD activity in a manner similar to that previously demonstrated for redox cycling chemicals. These preliminary data suggest that either low or high SOD-1 activities may be associated with greater toxicity of a variety of neurotoxic chemicals and their metabolites.

Amyotrophic Lateral Sclerosis↗

PubMed1994

p53 gene mutation and mdm2 gene amplification are uncommon in medulloblastoma.

Loss of heterozygosity is common for the short arm of chromosome 17 in medulloblastomas, and putative medulloblastoma suppressor loci have been localized to 17p13. The colocalization of the p53 tumor suppressor gene to 17p13 raises the possibility that its mutant alleles may play a role in the malignant transformation of "medulloblasts." Mutations and deletions of the p53 gene have been described in many tumor types and in the germline of some individuals with the Li-Fraumeni syndrome, but reports on the status of the p53 and mdm2 (a gene coding for a p53-associated protein reportedly amplified in human sarcomas) genes in medulloblastomas are few and an indication of their roles, if any, in the etiology of this important childhood tumor has yet to emerge. Here we have analyzed polymerase chain reaction-amplified products of exons 4-9 (95% of reported p53 mutations occur within this region) of the p53 gene in 9 medulloblastomas for potential mutations using the technique of single strand conformation polymorphism analysis and DNA sequencing. We found only one mutation, an A-T to T-A transversion involving the second base of codon 285 and resulting in the substitution of valine for glutamic acid, amplification of the mdm2 gene could be detected in zero of eight of these tumors. These findings suggest that genetic events associated with the inactivation of p53 gene occur in only a minor subset of medulloblastomas.

Base Sequence↗

PubMed1994

Expression of the intermediate filament-associated protein related to beta-amyloid precursor protein is developmentally regulated in cultured cells.

It was previously reported that a monoclonal antibody to beta-amyloid precursor protein (mab22C11; Boehringer Mannheim, Indianapolis, IN) labels an intermediate filament-associated protein (beta APP-IFAP) in cultured human skin fibroblasts (Dooley et al.: J Neurosci Res 33:60-67, 1992). The time course of its expression and association with different classes of intermediate filaments has been assessed in neurons, Schwann cells, and astrocytes in dissociated cultures of murine brain and spinal cord-dorsal root ganglia; in primary cultures of human muscle; and in the epithelial cell line PtK1. beta APP-IFAP was expressed in all non-neuronal cell types examined. Mab22C11 immunoreactivity was minimal or absent following dissociation or subculture, but gradually increased with time. In fibroblasts, myoblasts, and epithelial cells, the distribution eventually resembled that of vimentin. With the exception of glial fibrillary acidic protein (GFAP), beta APP-IFAP was not associated with the intermediate filament proteins characteristically found in differentiated cells, i.e., desmin, the cytokeratins, and neurofilament proteins. No labeling of neurons by mab22C11 was observed at any stage of in vitro maturation. In sections of Alzheimer's brain, the antibody labeled a subpopulation of reactive astrocytes. It is suggested that beta APP-IFAP may be the product of a member of the beta APP multigene family expressed developmentally in non-neuronal cells.

Amyloid beta-Protein Precursor↗

PubMed1994

Predictive value of apolipoprotein E genotyping in Alzheimer's disease: results of an autopsy series and an analysis of several combined studies.

Apoliprotein E (apoE) is associated with Alzheimer's neurofibrillary tangles and beta-amyloid protein in senile plaques. Recent studies have shown an increased frequency of the epsilon 4 allele of the apoE gene in familial and sporadic cases of Alzheimer's disease (AD). In the present case control study, we have determined the apoE genotype by allele-specific extension of 113 postmortem cases of sporadic AD and 77 control brains shown to be free of AD neuropathological features and then calculated the frequency of the various allelic forms of apoE (epsilon 2, epsilon 3, epsilon 4). The odds ratio associating epsilon 4 with AD was 15.5 (95% confidence interval [CI] 6.2-38.5), and the population attributable risk was 0.53. We have also combined the results of our study and several others to calculate these same parameters in a larger population (570 controls and 961 AD subjects); the odds ratio for this larger group was 6.2 (95% CI 4.9-7.8) and the population attributable risk was 0.57. These results further substantiate and strengthen the association between the epsilon 4 allele of apoE gene and AD. We have also used these results to investigate the usefulness of the determination of epsilon 4 carrier status in the diagnosis of AD.

Aged↗

PubMed1994

Amyloid precursor protein gene expression in neural cell lines: influence of DNA cytosine methylation.

Transcription of the gene encoding amyloid precursor protein (APP) varies in a cell-specific and developmentally regulated manner. The 5' region of this gene possesses a high frequency of CpG dinucleotides as well as copies of a GC-rich sequence, a potential trans factor binding element. These findings raise the possibility that DNA cytosine methylation could participate in the regulation of APP gene expression. We examined APP mRNA/18S rRNA ratio in three neural cell lines (N18TG2, SN6, SN17) cultured in 5-azacytidine (5-AZA), an inhibitor of maintenance methylase which results in loss of cytosine methylation in proliferating cells. Culture in 5-AZA globally reduced methylation in genomic DNA as assessed by an increase in HpaII restriction sites, reduced cytosine methylation in the APP gene as assessed by Southern blotting of HpaII digests, and increased APP mRNA steady state abundance in all studied cell lines. Cell lines re-acquired APP gene methylation 48 h after removal of 5-AZA from media. These results indicate that in vitro alteration of DNA methylation can affect APP gene expression, and suggest that the APP gene in neuronal cell lines may be rapidly inactivated in vitro, perhaps to neutralize its potential toxicity.

Amyloid beta-Protein Precursor↗

PubMed1994

Conspicuous accumulation of a single-stranded DNA binding protein in skeletal muscle fibers in inclusion body myositis.

In muscle biopsies from patients with inclusion body myositis (IBM), multiple sites were found in many muscle fibers that bound single-stranded but not double-stranded DNA without sequence specificity, as exemplified by several different cDNA probes. This activity was attributable to a protein, because it was abolished by proteases but not by RNAse. Most of the sites of binding were myonuclei, whereas some were rimmed vacuoles, which probably result from nuclear breakdown. No comparable binding was seen in 27 control biopsies. A number of human and viral single-stranded DNA binding proteins exist but our data does not identify the protein responsible for DNA binding in IBM. Our findings reinforce the supposition that nuclear damage plays a basic role in the pathogenesis of IBM.

Antibodies, Monoclonal↗

PubMed1993

Amyloid precursor protein in peripheral mononuclear cells is up-regulated with cell activation.

The deposition of beta/A4 protein in extraneural organs of patients with Alzheimer disease suggests that this peptide may in part be derived from a peripheral precursor. We studied expression of amyloid precursor protein (APP) in PBMC. APP expression was detectable in resting PBMC by northern blot analysis, immunoblotting studies, and immunohistochemistry. By reverse transcription-polymerase chain reaction, the 751 and 770 APP transcripts containing the Kunitz protease inhibitor (KPI) domain were approximately 10-fold more abundant than the 695 transcript lacking the KPI domain. Activation of PBMC with the lectin PHA-P was associated with an increase in apparent intracellular APP content by cytofluorometry, and an increase in the proportion of the 695 APP transcript lacking the KPI domain. We conclude that resting and activated PBMC express APP and could contribute to a circulating pool of this protein. In addition, PBMC APP is up-regulated with mitogenic stimulation and may participate in the regulation of activation of these cells.

Alternative Splicing↗

PubMed1993

An antibody against the Alzheimer's disease amyloid precursor protein recognizes distinct conformational isoforms.

The Alzheimer's disease amyloid precursor protein (APP) consists of several isoforms, which are extensively post-translationally modified and processed. A monoclonal antibody, MAbE1, was raised against a synthetic peptide from an extracellular domain that is common to all isoforms of APP. Immunoblots and immunolocalization studies on cells of neuronal and other origins demonstrated that this antibody recognized a subclass of APP isoforms when compared to a monoclonal antibody raised against a bacterial fusion protein of APP, MAb22C11. Prominent protein bands of 71 kDa and 120 kDa were only detected on immunoblots of cell lysates and no immunoreactivity was observed in protein samples obtained from cell conditioned media. Immunofluorescence labelling with MAbE1 revealed predominantly perinuclear staining of cells of neuronal and glial origin. The data suggest that this monoclonal antibody detects distinct conformational isoforms of APP present in intracellular compartments.

Alzheimer Disease↗

PubMed1993

Expression of amyloid precursor protein in a neuronal cell line: functional activity of proximal regulatory elements.

To study cell type-specific regulation of the gene for amyloid precursor protein (APP), we have analysed the human APP promoter for transcriptional activity and interaction with nuclear proteins in the neuronal cell line NG108-15. Sequences from -203 to +104 were sufficient for promoter function and regulatory elements within this region (-128 to -63) were identified by DNase I protection experiments. These results suggest that essentially the same proximal elements are recognized by nuclear factors from both neuronal and nonneuronal cells.

Amyloid beta-Protein Precursor↗