Biomedical subjects

K D Brown

Publications and source records attributed to K D Brown.

At least 19 recordsLinked to original sources

PubMed2006

Programmes, resources, and needs of HIV-prevention nongovernmental organizations (NGOs) in Africa, Central/Eastern Europe and Central Asia, Latin America and the Caribbean.

This study assessed the programmes, resources, and needs of HIV-prevention nongovernmental organizations (NGOs) in 75 countries in Africa, Central/Eastern Europe and Central Asia, Latin America and the Caribbean. Multiple databases and expert recommendations were used to identify one major HIV-prevention NGO in the capital or a large city in each country, and in-depth interviews were conducted with each NGO Director. Most NGOs are carrying out their programmes with minimal funding and few regularly employed personnel. Most are highly dependent on international donors, but reliance on small grants with short funding periods limits programme development capacity. HIV-prevention activities varied by region, with African NGOs most likely to use peer education and community awareness events; Eastern European NGOs most likely to offer needle exchange; Latin American NGOs to have resource centres and offer risk reduction programmes; and Caribbean organizations to use mass education approaches. Across regions, NGOs most often targeted the general public and youth, although specialized at-risk groups were the additional focus of attention in some regions. Limited funding, governmental indifference or opposition, AIDS stigma, and social discomfort discussing sex were often cited as barriers to new HIV-prevention programmes. NGOs are critical service providers. However, their funding, programmes, and resource capacities must be strengthened if NGOs are to realize their full potential in HIV prevention.

Africa↗

PubMed2004

HIV prevention nongovernmental organizations in Central and Eastern Europe: programs, resources and challenges.

HIV incidence is rising more rapidly in some areas of Central and Eastern Europe than anywhere else in the world. Carrying out effective HIV prevention programs requires the presence of "bridges" that can reach community populations most vulnerable to the disease. Nongovernmental organizations (NGOs) are in a natural role to conduct HIV prevention programs. The Directors of 29 HIV prevention NGOs representing almost all countries in Central and Eastern Europe participated in in-depth interviews by telephone. The broad topics of these interviews included descriptions of the three largest programs conducted by each NGO during the past six months, at-risk target populations served, major barriers faced, and funding sources that sponsored HIV prevention activities. NGO programs most often targeted injection drug users (IDUs); other stigmatized groups were less frequently served by NGOs in the sample. The most common types of prevention activities were needle exchange, HIV prevention peer education, and delivering AIDS presentations and distributing educational materials. Among the major barriers that hampered effective conduct of HIV prevention programs were a shortage of available financial resources, governmental indifference or opposition, and AIDS-related stigma. National governments rarely provided substantial funds for NGO programs, and most funding came from United Nations agencies or private foundations. The information sources reported to be most helpful in assisting NGOs in program development were sharing ideas with other NGOs, participating in conferences, and accessing information from the Internet. A number of programs reported by the NGO Directors were innovative, outstanding, and comprehensive. Five such exemplary programs are described in this article. HIV epidemics in the region are still potentially controllable. NGOs need immediate support so that they can carry out their community-based activities on a larger scale.

Communicable Disease Control↗

PubMed2001

Constitutive nuclear factor kappaB activity is required for survival of activated B cell-like diffuse large B cell lymphoma cells.

Gene expression profiling has revealed that diffuse large B cell lymphoma (DLBCL) consists of at least two distinct diseases. Patients with one DLBCL subtype, termed activated B cell-like (ABC) DLBCL, have a distinctly inferior prognosis. An untapped potential of gene expression profiling is its ability to identify pathogenic signaling pathways in cancer that are amenable to therapeutic attack. The gene expression profiles of ABC DLBCLs were notable for the high expression of target genes of the nuclear factor (NF)-kappaB transcription factors, raising the possibility that constitutive activity of the NF-kappaB pathway may contribute to the poor prognosis of these patients. Two cell line models of ABC DLBCL had high nuclear NF-kappaB DNA binding activity, constitutive IkappaB kinase (IKK) activity, and rapid IkappaB(alpha) degradation that was not seen in cell lines representing the other DLBCL subtype, germinal center B-like (GCB) DLBCL. Retroviral transduction of a super-repressor form of IkappaBalpha or dominant negative forms of IKKbeta was toxic to ABC DLBCL cells but not GCB DLBCL cells. DNA content analysis showed that NF-kappaB inhibition caused both cell death and G1-phase growth arrest. These findings establish the NF-kappaB pathway as a new molecular target for drug development in the most clinically intractable subtype of DLBCL and demonstrate that the two DLBCL subtypes defined by gene expression profiling utilize distinct pathogenetic mechanisms.

Cell Survival↗

PubMed2001

Evidence for alternate splicing within the mRNA transcript encoding the DNA damage response kinase ATR.

Proper cellular response to genotoxic insult often requires the activity of one or more members of a family of high-molecular weight protein kinases referred to as phosphatidylinositol-3 kinase (PIK)-like proteins. While catalytic activity is an indispensable part of PIK-like protein function, little is currently known about factors that control their activity and/or functions. This deficiency stems, in large part, from our lack of knowledge concerning functionally significant subdomains within the large non-catalytic domain of these proteins. We have determined that the transcript encoding the PIK-like protein ATR undergoes alternate splicing within the region of the mRNA encoding its non-catalytic domain. This conclusion is based on the sequencing of a human expressed sequence tag clone encoding a portion of the ATR cDNA, and is supported by the results of reverse transcriptase-polymerase chain reaction (RT-PCR) assays conducted on total and polyA+ RNA, as well as sequencing of cloned RT-PCR products. Cloning and sequencing of a segment of human genomic DNA indicated that this event arises from splicing of a single 192 bp exon within the ATR gene. Analysis of several human tissues indicated that alternate ATR transcripts are differentially expressed, suggesting that this region of the ATR protein may be of functional importance.

Alternative Splicing↗

PubMed2001

Differential signaling and tumor necrosis factor receptor-associated factor (TRAF) degradation mediated by CD40 and the Epstein-Barr virus oncoprotein latent membrane protein 1 (LMP1).

Latent membrane protein 1 (LMP1) plays a critical role in B cell transformation by Epstein-Barr virus (EBV) and appears to mimic a constitutively active CD40 receptor. Intracellular tumor necrosis factor (TNF) receptor-associated factor (TRAF) adapter proteins, shown to contribute to signaling by both CD40 and LMP1, were recruited by both molecules to lipid-enriched membrane rafts. However, we found that TRAFs 2 and 3 were subsequently degraded after CD40- but not LMP1-induced signaling. This degradation was proteasome-dependent and required direct TRAF binding by CD40. Using a model system designed to directly compare the signaling potency of the cytoplasmic domains of LMP1 and CD40 in B lymphocytes, we found that LMP1 more potently activates c-Jun kinase and nuclear factor kappaB and induces higher levels of several B cell effector functions than does CD40. This suggests that LMP1 utilizes a modified CD40 signaling pathway. Failure to regulate TRAFs may contribute to the enhanced capacity of LMP1 to activate B cells as well as promote B cell transformation.

Animals↗

PubMed2001

Ataxia telangiectasia mutated is essential during adult neurogenesis.

Ataxia telangiectasia (A-T) is an autosomal recessive disease characterized by normal brain development followed by progressive neurodegeneration. The gene mutated in A-T (ATM) is a serine protein kinase implicated in cell cycle regulation and DNA repair. The role of ATM in the brain and the consequences of its loss on neuronal survival remain unclear. We studied the role of ATM in adult neural progenitor cells in vivo and in vitro to define the role of ATM in dividing and postmitotic neural cells from Atm-deficient (Atm(-/-)) mice in a physiologic context. We demonstrate that ATM is an abundant protein in dividing neural progenitor cells but is markedly down-regulated as cells differentiate. In the absence of ATM, neural progenitor cells of the dentate gyrus show abnormally high rates of proliferation and genomic instability. Atm(-/-) cells in vivo, and in cell culture, show a blunted response to environmental stimuli that promote neural progenitor cell proliferation, survival, and differentiation along a neuronal lineage. This study defines a role for ATM during the process of neurogenesis, demonstrates that ATM is required for normal cell fate determination and neuronal survival both in vitro and in vivo, and points to a mechanism for neuronal cell loss in progressive neurodegenerative diseases.

Animals↗

PubMed2001

Heparin-binding epidermal-growth-factor-like growth factor gene expression is induced by scrape-wounding epithelial cell monolayers: involvement of mitogen-activated protein kinase cascades.

Peptide growth factors can promote the cell migration and proliferation that is needed to repair epithelia after mechanical or chemical injury. We report here that scrape-wounding rat intestinal epithelial (RIE-1) cell monolayers caused a rapid increase in levels of heparin-binding epidermal-growth-factor-like growth factor (HB-EGF) mRNA, with a maximal response at approx. 1 h. Hybridization in situ showed that transcript induction occurred primarily in cells at or near wound borders. The increase in HB-EGF mRNA was preceded by activation of the p42 mitogen-activated protein kinase (MAPK) in the wounded cell cultures. Moreover, the induction of HB-EGF mRNA was blocked by PD098059 and U0126, inhibitors that prevent the activation of p42/p44 MAPKs and extracellular signal-regulated protein kinase 5 (ERK5). Both p42 MAPK activation and HB-EGF mRNA induction were inhibited by genistein, indicating a requirement for an upstream tyrosine kinase activity. In contrast, neither response was affected by inhibition of phosphoinositide 3-kinase activity, down-regulation of protein kinase C, or disruption of the actin cytoskeleton with cytochalasin B. We conclude that scrape-wounding epithelial cell monolayers induces HB-EGF mRNA expression by a mechanism that most probably requires p42/p44 MAPK activation, although we cannot exclude a role for ERK5. Our results suggest a physiological role for locally synthesized HB-EGF in promoting epithelial repair after injury.

Cell Line↗

PubMed2000

Cyclic AMP inhibits agonist-induced heparin-binding EGF gene expression independently of effects on p42/p44 MAPK activation.

Heparin-binding EGF-like growth factor (HB-EGF) mRNA levels are increased up to 20-fold in RIE-1 cells by two agonists that act through distinct receptor types. We demonstrated a common requirement for p42/p44 mitogen-activated protein kinase (MAPK) in this response using the selective MAPK kinase (MEK) inhibitor, PD 098059. Agonist-mediated induction of HB-EGF mRNA was markedly suppressed in cells that had been treated with cyclic AMP-elevating agents. In contrast, the activation of p42 MAPK in response to agonists was not affected by raising cellular cyclic AMP levels. We conclude that cyclic AMP negatively regulates the HB-EGF gene, but that the inhibitory action is either independent of the p42/p44 MAPK pathway or the site of action is distal to MAPK activation.

1-Methyl-3-isobutylxanthine↗

PubMed2000

Regulation of DNA-dependent protein kinase activity by ionizing radiation-activated abl kinase is an ATM-dependent process.

Ionizing radiation (IR) treatment results in activation of the nonreceptor tyrosine kinase c-Abl because of phosphorylation by ATM. In vitro evidence indicates that DNA-dependent protein kinase (DNA-PK) can also phosphorylate and thus potentially activate Abl kinase activity in response to IR exposure. To unravel the role of ATM and DNA-PK in the activation of Abl, we assayed Abl, ATM, and DNA-PK activity in ATM- and DNA-PKcs-deficient cells after irradiation. Our results show that despite the presence of higher than normal levels of DNA-PK kinase activity, c-Abl fails to become activated after IR exposure in ATM-deficient cells. Conversely, normal activation of both ATM and c-Abl occurs in DNA-PKcs-deficient cells, indicating that ATM but not DNA-PK is required for activation of Abl in response to IR treatment. Moreover, activation of Abl kinase activity by IR correlates well with activation of ATM activity in all phases of the cell cycle. These results indicate that ATM is primarily responsible for activation of Abl in response to IR exposure in a cell cycle-independent fashion. Examination of DNA-PK activity in response to IR treatment in Abl-deficient cells expressing mutant forms of Abl or in normal cells exposed to an inhibitor of Abl suggests an in vivo role for Abl in the down-regulation of DNA-PK activity. Collectively, these results suggest a convergence of the ATM and DNA-PK pathways in the cellular response to IR through c-Abl kinase.

Animals↗

PubMed2000

Characterisation of the rat heparin-binding epidermal growth factor-like growth factor gene promoter.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) gene expression is strongly activated by a variety of extracellular stimuli, acting through the Raf/MEK/MAP kinase pathway. To study the elements that respond to this pathway, we have isolated and sequenced a fragment of the rat HB-EGF gene promoter. By transfection of a series of promoter/reporter constructs into cells, a minimal promoter element was demonstrated to lie between 448 bp upstream of the transcriptional start site and 103 bp into the first exon of the gene. However co-transfection of the promoter constructs with a plasmid directing expression of RafCAAX, an activated c-Raf-1 protein, gave a fold-stimulation of activity no greater than that seen for the parental pGL3-Basic plasmid alone. In addition, agonist stimulation of cell lines stably transfected with a HB-EGF promoter/luciferase construct produced little or no increase in reporter enzyme activity. These results suggest that the c-Raf-1 responsive elements lie outside the tested region of the rat HB-EGF gene. However, it has been reported that a c-Raf-1 responsive element is present within the equivalent region of the mouse gene. A comparison of the 5'-flanking regions of the mouse, rat and human HB-EGF genes indicated that the mouse sequence diverges abruptly from that of the other two species approximately 260 bp upstream of the transcriptional start site. PCR analysis of mouse genomic DNA suggests that this sequence divergence is due to DNA rearrangement during the cloning of the mouse gene. Additional studies are therefore required to identify Raf/MAP kinase responsive elements in the HB-EGF gene.

3T3 Cells↗

PubMed2000

Solid-phase parallel synthesis of azarene pyrrolidinones as factor Xa inhibitors.

A focused library (4 x 14) prepared from 4-aminopyridine and 4-, 5-, and 6-azoindole templates was synthesized using 14 polymer supported 4-amido-2,3,5,6-tetrafluorophenyl (TFP) sulfonate esters inputs. Several compounds were identified as factor Xa inhibitors (IC50< or =0.1 microM) helping to establish the SAR among these four series of azarene pyrrolidinones.

Anticoagulants↗

PubMed2000

Ionizing radiation exposure results in up-regulation of Ku70 via a p53/ataxia-telangiectasia-mutated protein-dependent mechanism.

Genome damaging events, such as gamma-irradiation exposure, result in the induction of pathways that activate DNA repair mechanisms, halt cell cycle progression, and/or trigger apoptosis. We have investigated the effects of gamma-irradiation on cellular levels of the Ku autoantigens. Ku70 and Ku80 have been shown to form a heterodimeric complex that can bind tightly to free DNA ends and activate the protein kinase DNA-PKcs. We have found that irradiation results in an up-regulation of cellular levels of Ku70, but not Ku80, and that this enhanced level of Ku70 accumulates within the nucleus. Further, we uncovered that the postirradiation up-regulation of Ku70 utilizes a mechanism that is dependent on both p53 and damage response protein kinase ATM (ataxia-telangiectasia-mutated); however, the activation of DNA-PK does not require Ku70 up-regulation. These findings suggest that Ku70 up-regulation provides the cell with a means of assuring either proper DNA repair or an appropriate response to DNA damage independent of DNA-PKcs activation.

Antigens, Nuclear↗

PubMed2000

Amido-(propyl and allyl)-hydroxybenzamidines: development of achiral inhibitors of factor Xa.

The design, synthesis and SAR of amido-(propyl and allyl)-hydroxybenzamidine coagulation factor Xa inhibitors is described. These achiral inhibitors are selective for fXa vis a vis structurally related serine proteases and are readily prepared in 6-7 linear steps. The most potent member 9j (fXa Ki = 0.75 nM) is selective (>1000-fold) and an effective anticoagulant in mammalian plasma.

Animals↗

PubMed2000

ATM is a cytoplasmic protein in mouse brain required to prevent lysosomal accumulation.

We previously generated a mouse model with a mutation in the murine Atm gene that recapitulates many aspects of the childhood neurodegenerative disease ataxia-telangiectasia. Atm-deficient (Atm-/-) mice show neurological defects detected by motor function tests including the rota-rod, open-field tests and hind-paw footprint analysis. However, no gross histological abnormalities have been observed consistently in the cerebellum of any line of Atm-/- mice analyzed in most laboratories. Therefore, it may be that the neurologic dysfunction found in these animals is associated with predegenerative lesions. We performed a detailed analysis of the cerebellar morphology in two independently generated lines of Atm-/- mice to determine whether there was evidence of neuronal abnormality. We found a significant increase in the number of lysosomes in Atm-/- mice in the absence of any detectable signs of neuronal degeneration or other ultrastructural anomalies. In addition, we found that the ATM protein is predominantly cytoplasmic in Purkinje cells and other neurons, in contrast to the nuclear localization of ATM protein observed in cultured cells. The cytoplasmic localization of ATM in Purkinje cells is similar to that found in human cerebellum. These findings suggest that ATM may be important as a cytoplasmic protein in neurons and that its absence leads to abnormalities of cytoplasmic organelles reflected as an increase in lysosomal numbers.

Animals↗

PubMed1999

Quantitative analysis reveals differential expression of mucin (MUC2) and intestinal trefoil factor mRNAs along the longitudinal axis of rat intestine.

MUC2 and intestinal trefoil factor (ITF) are considered to have important roles in intestinal mucosal protection and epithelial repair. In order to investigate whether these genes are co-ordinately expressed, we have used competitive reverse transcription-polymerase chain reaction assays to measure MUC2 and ITF mRNA levels in human intestinal cell lines and along the longitudinal axis of rat intestine. ITF mRNA was expressed in several intestinal cell lines. However, MUC2 mRNA was detected only in LS174T cells where it was present at approx. 25-fold lower levels than the ITF transcript. In contrast, in rat intestinal tissues, MUC2 mRNA levels were generally higher than ITF mRNA levels. The levels of both transcripts increased markedly during postnatal development. In adult rats, the expression patterns of MUC2 and ITF mRNAs along the longitudinal axis of the small intestine were similar, with lowest levels in the proximal duodenum and relatively constant levels in the other regions assayed. In contrast, the expression patterns of MUC2 and ITF in different regions of the large intestine showed a marked divergence. Our results strongly suggest that expression of the MUC2 and ITF genes is not coordinately regulated in intestinal cells.

Animals↗

PubMed1999

Synthesis, SAR and in vivo activity of novel thienopyridine sulfonamide pyrrolidinones as factor Xa inhibitors.

Thienopyridine sulfonamide pyrrolidinones were found to be potent and selective inhibitors of the coagulation cascade enzyme factor Xa. SAR studies led to several compounds that were selected for further in vivo investigation. These novel aryl binding pocket moieties represent a structural modification to a series of fXa inhibitors. Several compounds proved to be efficacious i.v. antithrombotic agents.

Animals↗

PubMed1999

Sulfonamidopyrrolidinone factor Xa inhibitors: potency and selectivity enhancements via P-1 and P-4 optimization.

Sulfonamidopyrrolidinones were previously disclosed as a selective class of factor Xa (fXa) inhibitors, culminating in the identification of RPR120844 as a potent member with efficacy in vivo. Recognizing the usefulness of the central pyrrolidinone template for the presentation of ligands to the S-1 and S-4 subsites of fXa, studies to optimize the P-1 and P-4 groups were initiated. Sulfonamidopyrrolidinones containing 4-hydroxy- and 4-aminobenzamidines were discovered to be effective inhibitors of fXa. X-ray crystallographic experiments in trypsin and molecular modeling studies suggest that our inhibitors bind by insertion of the 4-hydroxybenzamidine moiety into the S-1 subsite of the fXa active site. Of the P-4 groups examined, the pyridylthienyl sulfonamides were found to confer excellent potency and selectivity especially in combination with 4-hydroxybenzamidine. Compound 20b (RPR130737) was shown to be a potent fXa inhibitor (K(i) = 2 nM) with selectivity against structurally related serine proteinases (>1000 times). Preliminary biological evaluation demonstrates the effectiveness of this inhibitor in common assays of thrombosis in vitro (e.g. activated partial thromboplastin time) and in vivo (e.g. rat FeCl(2)-induced carotid artery thrombosis model).

Amidines↗

PubMed1999

Isolation, genomic organization, and expression analysis of Men1, the murine homolog of the MEN1 gene.

The mouse homolog of the human MEN1 gene, which is defective in a dominant familial cancer syndrome, multiple endocrine neoplasia type 1 (MEN1), has been identified and characterized. The mouse Men1 transcript contains an open reading frame encoding a protein of 611 amino acids which has 97% identity and 98% similarity to human menin. Sequence of the entire Men1 gene (9.3 kb) was assembled, revealing 10 exons, with exon 1 being non-coding; a polymorphic tetranucleotide repeat was located in the 5'- flanking region. The exon-intron organization and the size of the coding exons 2-9 were well conserved between the human and mouse genes. Fluorescence in situ hybridization localized the Men1 gene to mouse Chromosome (Chr) 19, a region known to be syntenic to human Chr 11q13, the locus for the MEN1 gene. Northern analysis indicated two messages-2.7 kb and 3.1 kb-expressed in all stages of the embryo analyzed and in all eight adult tissues tested. The larger transcript differs from the smaller by the inclusion of an unspliced intron 1. Whole-mount in situ hybridization of 10.5-day and 11.5-day embryos showed ubiquitous expression of Men1 RNA. Western analysis with antibodies raised against a conserved C-terminal peptide identified an approximately 67-kDa protein in the lysates of adult mouse brain, kidney, liver, pancreas, and spleen tissues, consistent with the size of human menin. The levels of mouse menin do not appear to fluctuate during the cell cycle.

Amino Acid Sequence↗