Our analysis of DNA methylation levels within the major histocompatibility complex, including regulatory exonic and intronic regions associated with 90 genes in multiple tissues and individuals, reveals a bimodal distribution of methylation profiles , tissue specificity, inter-individual variation, and correlation with independent gene expression data.The Human Epigenome Project aims to identify, catalogue, and interpret genome-wide DNA methylation phenomena. Occurring naturally on cytosine bases at cytosine–guanine dinucleotides, DNA methylation is intimately involved in diverse biological processes and the aetiology of many diseases. Differentially methylated cytosines give rise to distinct profiles, thought to be specific for gene activity, tissue type, and disease state. The identification of such methylation variable positions will significantly improve our understanding of genome biology and our ability to diagnose disease. Here, we report the results of the pilot study for the Human Epigenome Project entailing the methylation analysis of the human major histocompatibility complex. This study involved the development of an integrated pipeline for high-throughput methylation analysis using bisulphite DNA sequencing, discovery of methylation variable positions, epigenotyping by matrix-assisted laser desorption/ionisation mass spectrometry, and development of an integrated public database available at  DNA is frequently modified by methylation, which can affect its function. The Human Epigenome Project aims to identify, catalog, and interpret DNA methylation throughout the genome DNA methylation is indispensable for vertebrate genome function. It is involved in diverse genomic processes such as gene regulation, chromosomal stability, and parental imprinting , and intThe sequencing of the human genome (IHGSC 2001) and creation of a whole-genome map of single nucleotide polymorphisms (SNPs)  laid theAs a prelude to the HEP, here we report the results of the HEP pilot study: DNA methylation profiling of the human major histocompatibility complex (MHC). The MHC, located on Chromosome 6 (6p21.3), is one of the most gene-dense regions in the human genome, containing genes with a high diversity of function, many of which are involved in the innate and adaptive immune systems. We chose to analyse the MHC for the pilot HEP study for three main reasons. (i) The MHC is associated with more diseases than any other region of the human genome, and therefore the generated data will be of interest to researchers with diverse biomedical interests. (ii) It is also the most polymorphic region in the genome, and therefore the data will allow study of the potential effects of the loss or gain of cytosine–guanine dinucleotide (CpG) methylation sites (due to SNPs) on gene expression and possibly other phenotypes. (iii) At the time when the HEP pilot study was initiated in 1999 , the MHChttp://www.epigenome.org), which integrates, for the first time, epigenetic information with the existing genome annotation.Using an integrated pipeline involving high-throughput bisulphite DNA sequencing, we have determined the DNA methylation levels within the vicinity of the promoter and other relevant regions, such as CpG islands and first exons and introns of 90 genes within the 3.8-Mb MHC region in multiple tissues and individuals. Our analysis reveals a bimodal distribution of methylation levels, tissue specificity, and inter-individual variation. We have also developed matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) assays for high-throughput epigenotyping of the analysed regions. Finally, we have established a publicly available database for the HEP data (For the DNA methylation profiling of the human MHC, the following regions of interest (ROIs) were chosen: (i) a potential regulatory region for each gene and (ii) the most CpG-dense region of each gene. It is well established that epigenetic modifications at regulatory regions, in particular promoters, correlate with the transcriptional state of the cognate gene (reviewed in http://www.ncbi.nlm.nih.gov/SNP). A total of 253 unique amplicons were successfully analysed  that allows easy access to the data from the pilot HEP study  or relatively hypermethylated (i.e., median methylation of amplicon 70% or greater) A and 4. p < 0.0001). Interestingly, within the upstream category we found that CpG sites located within the 5′ UTR were less likely to be methylated (median methylation of 7%) than the CpG sites located within 2 kb of the first start codon but not within the 5′ UTR (median methylation of 14%) (p < 0.0001). Within the intragenic category, we found that CpG sites located within introns (median methylation of 84%) were less likely to be methylated than CpG sites located within exons (median methylation of 89%) (p < 0.0001). Whether these significant but small differences reflect any bias for the presence of regulatory elements close to the transcriptional start site or within introns, or some other functional consequence, is currently hard to assess.Comparison of the methylation values for upstream amplicons (median methylation of 10%) versus intragenic amplicons (median methylation of 86%) revealed that upstream amplicons were more likely to be hypomethylated (Fourteen amplicons displayed significant heterogeneous methylation profiles (i.e., median methylation between 30% and 70%) see A. These insulin-like growth factor 2 (IGF2) gene, which is imprinted in all tissues except brain  and others with thymidines at those CpG sites . When these PCR products are sequenced directly, the cumulative effect of the molecular weight difference between cytosines and thymidines is that some molecules migrate faster than others during capillary electrophoresis. The sequence trace therefore contains two traces that do not perfectly overlap, resulting in erroneous estimation of methylation levels. Such sequences were excluded from further analyses.CpG islands are GC-rich regions that contain a high density of CpGs and are positioned at the 5′ ends of many human genes (reviewed in tenascin-XB (TNXB) gene, which was hypomethylated in muscle samples only. This hypomethylation correlates with the temporally regulated and tissue-specific expression of TNXB, which is abundantly expressed in connective tissues. It has been suggested that TNXB has a role in limb, muscle, and heart development (TNXB CpG island may have an important regulatory role (tissue specificity of methylation profiles is discussed in more detail below). Interestingly, the CpG island amplicon located within exon 3 of the HLA-G gene spanned a methylation boundary, being hypomethylated at the 5′ end with a sharp transition to a hypermethylated profile at the 3′ end. Overall, the results are consistent with the prevailing model of CpG islands being regions of the genome that are hypomethylated, especially when they occur upstream or within the 5′ end of the gene.In our study, CpG island amplicons situated in the upstream ROIs were always hypomethylated, whereas hypermethylated CpG island amplicons were found only in the intragenic regions. Among the intragenic CpG island amplicons, those situated at the 5′ end of the gene (i.e., overlapping exon 1, intron 1, or exon 2) were always hypomethylated. A tissue-specific methylation profile was observed for the CpG island situated within exon 3 of the elopment , and, thDNA methylation profiles are complex and dynamic, and can vary with developmental stage, tissue type, age, the alleles' parent-of-origin, and also phenotype or disease state . Another example is DOM3Z, which is ubiquitously expressed but occurs only at very low levels in the lung (DOM3Z that is hypermethylated in lung (and brain) but hypomethylated in the other tissues examined. It has also been demonstrated that the murine complement factor B utilises differential tissue-specific start sites  that varied significantly between individuals in liver . Furthermore, the mutation abrogated in vitro interaction with a nuclear factor, and this effect was phenocopied following in vitro DNA methylation of the region. Evidence for an interaction between genotype and epigenotype at the IGF2 gene in humans has also recently been reported (http://www.ncbi.nlm.nih.gov/SNP) and are annotated in the HEP database. One could postulate that the gain or loss of one or more critical CpG sites may affect the overall methylation profile of a locus and, consequently, promoter activity. Alternatively, non-CpG SNPs located within an epigenetically sensitive regulatory element could also influence the epigenetic makeup of that region. Therefore, mutations in regulatory sequences could influence epigenetic profiles, resulting in altered phenotypes.Examples of amplicons that displayed significant inter-individual variation in methylation profiles include a region overlapping the last exon in prostate A, and a in liver B. Althoureported . Of the reported ; Table 1A major aim of the HEP is to identify genomic regions at which DNA methylation profiles display statistically significant variation due to biological or environmental influences. Therefore, based on the tissue-specific and inter-individual variation in methylation profiles discussed above, we were interested in establishing high-throughput assays for epigenotyping. This involved the identification  of methylation variable positions (MVPs), which we define as CpG sites that have statistical power to discriminate between different biological samples or states. In other words, by assaying the methylation state of just a few select CpG sites within a given region, information can be inferred about the tissue source or disease state. Such a high-throughout MVP epigenotyping method was recently developed based on the GOOD assay . This reThe primary function of epigenetic modifications is to modulate gene expression: a specific combination of epigenetic modifications at regulatory elements, notably promoters and enhancers, influences the transcriptional state of a gene (reviewed in http://expression.gnf.org). This publicly available database contains whole-genome mRNA expression data obtained by p < 0.0001 for prostate and p < 0.01 for liver). The intragenic amplicons did not show any correlation between methylation and expression levels (p > 0.3 for both prostate and liver). A list of all upstream amplicons included in the analysis is given in We compared the amplicon methylation profiles with the human genome expression patterns available from the Genomics Institute of the Novartis Research Foundation Gene Expression Atlas database (p > 0.3 for both categories). Although the lung data show the same trend as the prostate and liver data, the lung hypomethylated data contained a number of outlier data points representing very high expression values  for public access to the data generated by the study. The results from the pilot study demonstrate that a significant proportion of the analysed loci within the MHC show tissue-specific methylation profiles, and inter-individual methylation differences are common. Furthermore, the tissue-specific differences in DNA methylation suggest that epigenetic mechanisms are involved in the use of alternative transcriptional start sites. We have also shown that the generated methylation data allow the identification of MVPs that can be typed with high quantitative resolution and sensitivity using MALDI-MS, providing a tool for large population-based studies and for diagnosing diseases in the future.One of the principal challenges in the post-genomic era is to provide a holistic view of genome function, a challenge which is currently being addressed by several large-scale studies of the transcriptome, proteome, metabolic networks, and haplotype maps. The HEP is therefore timely, since DNA methylation is an indispensable part of the genome's regulatory mechanisms. Here we have described the pilot study for the HEP—DNA methylation profiling of the MHC region—which is the first systematic large-scale study of methylation profiles at the sequence level within a multi-megabase region of the human genome. For this project, we developed an integrated pipeline for high-throughput methylation analysis using bisulphite DNA sequencing, MVP discovery, and epigenotyping by MALDI-MS, and created an integrated database  associated with nearly all the annotated genes (approximately 3,000) on human Chromosomes 6, 13, 20, and 22. The excellent genomic annotation available for these four chromosomes, e.g., high-quality transcript information and location of SNPs, will enable us to perform comprehensive analyses linking the epigenetic information gained from the HEP with the underlying genetic information. Samples from over 40 different individuals representing 20 tissues will be used in the study.The resulting data will generate a map that complements other large-scale efforts that are linking our knowledge of gene sequence and cellular phenotypes: studies involving DNA sequencing, SNPs, histone modifications, and transcriptome and proteomic analyses. The epigenome map will be invaluable for understanding gene regulation and the interactions between genes in normal and disease states. It will offer new explanations in well-studied areas such as cancer research, and will also provide a basis for novel approaches to research on environmental effects, nutrition, and ageing . The HEPHuman tissue samples were obtained from the National Disease Research Interchange (Philadelphia, Pennsylvania, United States) and consisted of tissue material from healthy individuals. Tissue samples included seven different tissue types (adipose, brain, breast, lung, liver, prostate, and muscle) from 32 different individuals . DNA wasBisulphite treatment of genomic DNA was performed with minor modifications to a method described previously .Taq polymerase (Qiagen), and 8 ng of bisulphite-treated genomic DNA. Water-only controls were also included in each 96-well PCR plate. The cycling conditions were 95 °C for 15 min followed by 40 cycles of 95 °C for 60 s, 55 °C for 45 s, and 72 °C for 90 s, and a final extension step of 10 min at 72 °C. The PCR amplicons for some of the genomic regions that displayed heterogeneous levels of CpG methylation were sub-cloned using the pGEM-T Easy Vector System according to the manufacturer's instructions (Promega, Madison, Wisconsin, United States). The clones were sequenced on ABI 3700 capillary sequencers  using ABI Prism Big Dye terminator V 3.1 sequencing chemistry.Primers were designed to be at least 21 bases (G + C ≥ 30%) and to contain at least two bases complementary to bisulphite-converted sequence to increase the specificity. To ensure that the primers were not biased for either hypomethylated or hypermethylated sequences, controls were performed using bisulphite-converted unmethylated or in vitro methylated target sequences. PCRs were performed in 96-well plates on MJ Research  thermocyclers in a final volume of 25 μl containing 250 μM dNTPs, 1X PCR Buffer , 10 pmol each of forward and reverse primer, 1 U PCR amplicons were purified using MultiScreen PCR plates (Millipore, Billerica, Massachusetts, United States) and sequenced directly in forward and reverse directions with the same primers used in the PCR. Sequencing was performed on ABI 3700 capillary sequencers using ABI Prism Big Dye terminator V 3.1 sequencing chemistry.http://www.genome.washington.edu/UWGC/analysistools/Swat.cfm). Using these offsets the positions of CpG dinucleotides were determined in genomic coordinates. Positional data were then loaded into an LDAS database (http://www.biodas.org) suitable for serving to applications using the distributed annotation system (DAS) XML format (http://www.epigenome.org. The data reported in the pilot HEP study are subject to the HEP's data release policy (http://www.sanger.ac.uk/PostGenomics/epigenome/drp.shtml).Quantitative methylation rates were estimated from sequence traces using the ESME software . This inL format . HEP metp <0.0001 in all cases). We therefore used the Mann-Whitney U test (a non-parametric test that compares the medians of two unpaired groups of samples that do not follow a Gaussian distribution) to perform three different comparative analyses for methylation profiles: (i) upstream versus intragenic amplicons, (ii) within the upstream amplicon category, CpG sites located within the 5′ UTR versus CpG sites not located within the 5′ UTR but still within 2 kb of the first start codon, and (iii) within the intragenic amplicon category, intronic versus exonic CpG sites.Kolmogorov-Smirnov tests confirmed that the methylation data did not follow Gaussian distribution .Click here for additional data file.Table S2(32 KB DOC).Click here for additional data file.http://www.ncbi.nlm.nih.gov/projects/LocusLink/) accession numbers for the genes and gene products discussed in this paper are C2 (LocusID 717), CYP21A2 (LocusID 1589), DOM3Z (LocusID 1797), HLA-G (LocusID 3135), IGF2 (LocusID 3481), TNXB (LocusID 7148), and tumour necrosis factor (LocusID 7124).The LocusLink (


Strongylocentrotus purpuratus, is an invertebrate model organism that is evolutionarily closer to humans than Drosophila and C. elegans, neither of which possesses vaults. Here we compare the structures of sea urchin and mammalian vaults and analyze the subcellular distribution of vaults during sea urchin embryogenesis.Vaults are intriguing ribonucleoprotein assemblies with an unknown function that are conserved among higher eukaryotes. The Pacific coast sea urchin, The sequence of the sea urchin major vault protein (MVP) was assembled from expressed sequence tags and genome traces, and the predicted protein was found to have 64% identity and 81% similarity to rat MVP. Sea urchin MVP includes seven ~50 residue repeats in the N-terminal half of the protein and a predicted coiled coil domain in the C-terminus, as does rat MVP. A cryoelectron microscopy (cryoEM) reconstruction of isolated sea urchin vaults reveals the assembly to have a barrel-shaped external structure that is nearly identical to the rat vault structure. Analysis of the molecular composition of the sea urchin vault indicates that it contains components that may be homologs of the mammalian vault RNA component (vRNA) and protein components (VPARP and TEP1). The sea urchin vault appears to have additional protein components in the molecular weight range of 14–55 kDa that might correspond to molecular contents. Confocal experiments indicate a dramatic relocalization of MVP from the cytoplasm to the nucleus during sea urchin embryogenesis.These results are suggestive of a role for the vault in delivering macromolecules to the nucleus during development. Strongylocentrotus purpuratus is an important model system in developmental biology and its genome is currently being sequenced by the Human Genome Sequencing Center at the Baylor College of Medicine under the auspices of the National Human Genome Research Institute (NHGRI). Sea urchin embryos are well suited for biochemical approaches to studying the cell biology of development, as large quantities of eggs can easily be obtained, and their fertilization initiates the synchronous development of optically transparent embryos [Drosophila and C. elegans. In addition, the echinoderm lineage leading to sea urchins diverged from chordates prior to the large scale gene duplication events that occurred early in the evolution of the vertebrates, and because of this many of the genes that are found as multiple paralogues in vertebrates have only a single homolog in sea urchins. Therefore sea urchins provide a system that avoids the problem of functional redundancies between multiple paralogues that often occurs in vertebrates, and serves as a useful comparison for assessing the significance of conserved genes and regulatory linkages within the genome.The sea urchin  embryos . Sea urcSea urchins cells, like mammalian cells, contain abundant quantities of vaults, which are ~13MDa ribonucleoprotein particles of as yet unknown function . Vaults Immunofluorescence studies in sea urchins indicate that MVP is present throughout the cytoplasm in cleavage-stage zygotes and in the nucleus in adult somatic cells . Within The present study provides a comparison of vaults from sea urchins and rats in terms of their molecular composition and MVP protein sequence, as well as by cryoEM imaging and three-dimensional reconstruction. In addition, images obtained by confocal microscopy are presented that indicate differential localization of MVP during sea urchin embryogenesis, suggesting that the highly conserved ribonucleoprotein vault might play a role during development.Vaults isolated from mammals, including rats, mice, and humans, are composed of three proteins (MVP, VPARP, and TEP1) and one or more small untranslated RNA molecules, called vRNA. The protein stoichiometry is thought to be 96 copies of MVP with 2–16 copies of VPARP and TEP1 per particle . The humDictyostelium MVPα and MVPβ vault proteins [S. purpuratus genome identified clear sequence homologs of both VPARP and TEP1 (data not shown). Purified sea urchin vaults also appear to contain vRNA, as demonstrated by the loss of a 26.5 kDa band after RNase treatment , which is apparently present as a single homolog per haploid genome. The fact that this sequence was found in several blastula-stage ESTs shows that it is expressed in the embryo. The deduced sequence of sea urchin MVP encodes a 95 kDa protein which is slightly smaller than the 96 kDa rat MVP, with 847 vs. 861 aa. These two MVP homologs exhibit 64% identity and 81% strong similarity  and trace sequences from the ity Fig. . The seaity Fig. . The ratDictyostelium  and nuclear export signals (NES's), as well as possible sumoylation sites. Although the PredictNLS server  did not et al. , indicatet al. . SequencDictyostelium and rat MVP [SpMVP gene to the major protein component of the purified sea urchin vault.Epitopes are also conserved between the rat MVP and the sea urchin MVP. Previously, we generated and purified a rabbit polyclonal antibody against the MVP that copurified with sea urchin microtubules . These a rat MVP . In this rat MVP . This seNegative-stain EM and cryoEM images of isolated sea urchin vaults reveal that they have the same overall morphology as mammalian vaults Fig. . As was In order to characterize the three dimensional structure of the sea urchin vault, a three-dimensional structure was calculated using cryoEM images and single particle reconstruction methods. Only particle images of well-formed, fully closed sea urchin vaults were selected for image processing. Refinement of the cryoEM data set produced a three-dimensional reconstruction at 33 Å resolution Fig. . The extCryoEM reconstructions of recombinant and tissue derived vaults often show small holes at the cap/barrel junction ,39. The Earlier immunofluorescence studies indicated that maternal and zygotic MVP is located in the cytoplasm, whereas adult MVP is predominantly nuclear . The subIn this study we used the sea urchin genome to deduce the amino acid sequence of the sea urchin MVP and characterized the structure of the sea urchin vault particle using cryoEM single particle reconstruction methods. It is known that multiple copies of MVP form the recognizable vault structure, as expression of rat MVP in insect cells leads to the assembly of vaults . Given tSDS-PAGE analysis has indicated that the molecular composition of sea urchin vaults is more complex than that of rat vaults. Protein bands in the range of 14 to 55 kDa are observed that might either be additional vault components or macromolecular contents that are present in high copy numbers within the sea urchin vault. One protein in this mass range (~50 kDa) cross-reacts with MVP antisera and is likely to be a breakdown product of MVP. The identities of the additional proteins will be the subject of future investigations. It is tempting to speculate that the difference in protein composition between sea urchin and rat vaults might indicate that sea urchin vaults are transporting or sequestering a larger assortment of macromolecules.Vaults were originally named because of their similarity to arched cathedral ceilings . This naA difference map analysis between cryoEM reconstructions of intact and RNase-treated rat vaults led to the localization of the vRNA to the flat portions of the rat vault caps . The recThe confocal images reveal a progressive enrichment of MVP in the nucleus and perinuclear cytoplasm during sea urchin embryogenesis. Our previous biochemical fractionation studies confirm that the MVP accumulates in the nucleus beginning at the mesenchyme blastula stage . This imWhile MVP localization studies in mammalian cells reveal a largely cytoplasmic location, the published staining pattern is consistent with there also being a small amount of nuclear MVP ,18. WhilSumoylation is a dynamic and reversible process that involves the covalent attachment of the small ubiquitin-like modifier (SUMO) protein to protein substrates. Similar to ubiquination, sumoylation requires four enzymatic steps to attach the carboxyl-terminal glycine of SUMO to the ε-amino group of lysine (reviewed in ). HoweveTaken together these results suggest that the vault may play a role in delivering macromolecules to the nucleus during embryonic development in sea urchins. A trafficking role has also been proposed for vaults in human cells based on the observations that MVP interacts with the estrogen receptor in MCF-7 breast cancer cells  and withThe results presented here suggest that sea urchin vaults contain various macromolecules in addition to the two main vault-associated proteins, VPARP and TEP1. Human MVP is known to interact specifically with the tyrosine phosphatase SHP-2, an enzyme that plays an important role in intracellular signaling . It has 7 intact vault particles. By comparison, the mature sea urchin egg contains approximately 4 × 108 ribosomes [It would seem likely that the sea urchin MVP is transported to the nucleus for a reason. Presumably cells must expend a considerable amount of energy to achieve this differential localization, as MVP is a relatively abundant protein. The MVP concentration in the unfertilized sea urchin egg is on the order of 10 μM, as determined by immunoblotting . This maibosomes . AlthougStrongylocentrotus purpuratus. Animals were spawned and the eggs were collected and washed as described [4, 4 mM EGTA, 2 mM DTT, 1 mM GTP, 0.2 mM PMSF, 10 μg/ml leupeptin and 1 μg/ml pepstatin A). The homogenate was centrifuged at 18 K rpm (~39,000 g) in a Beckman J20 rotor at 4°C for 45 min. The resulting supernatant fluids were decanted into a 500-ml Erlenmeyer flask at room temperature. A fresh aliquot of GTP was added such that the GTP concentration was increased by 1 mM. The DMSO was added in three aliquots with gentle mixing until a final concentration of 15% (v/v) was reached. Microtubule assembly was promoted by incubating the supernatant fluids in a 24°C water bath for 30–45 min. The microtubules were pelleted by centrifugation at 18K rpm (~39,000 g) in a Beckman J20 rotor at 24°C for 30 min. The supernatant fluids (H1S) were carefully removed and drop-frozen in liquid nitrogen and stored in a -80°C freezer. Microtubules in the pellet were purified by two subsequent cycles of assembly and disassembly as previously described [Vaults were purified to homogeneity from unfertilized eggs of the Pacific coast sea urchin escribed . Washed escribed  and used2, 75 mM NaCl, 1 mM DTT and 1 mM PMSF. This pooled supernatant was used as the starting point for the vault purification. This extract was treated as if it was a rat liver homogenate and all subsequent purification steps were as described for rat liver vaults, including purification on sucrose density gradients and two cesium chloride gradients [Sea urchin vaults were purified from the microtubule-depleted supernatant fluids that had been previously frozen in liquid nitrogen. Approximately 150 ml of frozen supernatant (H1S) was thawed and mixed with an equal volume of 50 mM Tris-HCL, pH 7.4, 1.5 mM MgClradients .Rat vaults were purified from liver by sucrose-density and cesium chloride gradient centrifugation as described by the Rome laboratory .et al., [Vault samples were analyzed by SDS-PAGE with the discontinuous buffer formulation of Laemmli et al.,  and staiS. purpuratus genome project [S. purpuratus ESTs, with the following NCBI accession numbers: BG784484, BG784394, CD305511, CD295832, CD295616, CD304000, CD310423, CD307400, CD305382, CD292179, CD305646, CD333690, CD309547, CD292477, and BG783194. The gap from A728 to G785 was covered by a sequence in the NCBI trace archives (gnl|ti|287010960). The assembled SpMVP sequence has been deposited in the NCBI database under accession number BK005641.Trace sequences from the  project were used project ,58 was pet al. and based on the alignment of 58 high-quality NES's [Strongy. purp., Homo sapiens, Mus musculus, Rattus norv., Danio rerio, and Xenopus laevis) and are indicated in Figure We note that the sea urchin MVP has a molecular weight of 95 kDa, calculated from the protein sequence. Previously the sea urchin MVP was referred to as a 107-kDa polypeptide based on its apparent molecular weight by SDS-PAGE . The proty NES's . The seaty NES's . Five moIsolated sea urchin vaults were applied to freshly glow-discharged formvar-coated copper grids (Electron Microscopy Sciences). Excess liquid was wicked away and the samples were briefly air dried. Samples were stained with uranyl acetate and observed using a JEOL 1200EXII transmission electron microscope.6 filament, a Gatan 626 cryotransfer holder, and a Gatan slow scan CCD camera (1024 × 1024 pixels, YAG scintillator). One hundred and forty-one cryomicrographs were collected with a nominal magnification of 45,000×, and with two different defocus values (-1.0 and -0.6 μm). The pixel size in the cryomicrographs is 4.1 Å on the molecular scale as determined by calibration with a catalase crystal.CryoEM grids were prepared using cryogenic plunge freezing methods described previously ,64. Digiet al. [The QVIEW software package was used to extract 481 individual vault particle images with a selection box size of 200 × 200 pixels . The iniet al. . The folAll of the image processing was performed on HP/Digital alpha unix workstations. The graphics representations were produced with the AVS  software package.Staged embryos were fixed in -20°C Methanol for 20 minutes on ice, then washed three times in PBS plus 0.2% Tween-20 (PBST). Embryos were blocked for 30 minutes on ice in PBST plus 5% Bovine Serum Albumin, then incubated with an appropriate dilution of an affinity purified antibody against MVP  overnighPLS directed the cryoEM and three-dimensional image processing as well as helped to draft the manuscript. MM performed the cryomicroscopy and image processing. JL provided technical assistance. CDS performed the antibody staining and confocal imaging of whole-mounted embryos. AJR assembled the SpMVP sequence and performed the sequence alignments used to calculate sequence similarities and differences. JAC directed the research pertaining to vault localization in the embryo, and drafted Figure


ATP channels consist of Kir6.2 and SUR2A subunits, but the distribution of these (and other KATP channel subunits) is poorly defined. We examined the localization of each of the KATP channel subunits in the mouse and rat heart.Electrophysiological data suggest that cardiac KATP channels.Immunohistochemistry of cardiac cryosections demonstrate Kir6.1 protein to be expressed in ventricular myocytes, as well as in the smooth muscle and endothelial cells of coronary resistance vessels. Endothelial capillaries also stained positive for Kir6.1 protein. Kir6.2 protein expression was found predominantly in ventricular myocytes and also in endothelial cells, but not in smooth muscle cells. SUR1 subunits are strongly expressed at the sarcolemmal surface of ventricular myocytes (but not in the coronary vasculature), whereas SUR2 protein was found to be localized predominantly in cardiac myocytes and coronary vessels . Immunocytochemistry of isolated ventricular myocytes shows co-localization of Kir6.2 and SUR2 proteins in a striated sarcomeric pattern, suggesting t-tubular expression of these proteins. Both Kir6.1 and SUR1 subunits were found to express strongly at the sarcolemma. The role(s) of these subunits in cardiomyocytes remain to be defined and may require a reassessment of the molecular nature of ventricular KATP channel subunit expression patterns in the heart. These results suggest distinct roles for KATP channel subunits in diverse cardiac structures.Collectively, our data demonstrate unique cellular and subcellular K ATP) channels are widely expressed in both excitable and non-excitable tissue types throughout the body. However, differences exist in the functional and pharmacological properties of various KATP channels in different tissues. This functional diversity of KATP channels is also reflected in the cardiovascular system. KATP channels are abundantly expressed in ventricular myocytes, where they are probably best characterized. These channels have a high unitary conductance, are inhibited by ATP in the micromolar range, are blocked by glibenclamide (but not tolbutamide) and opened by pinacidil (and not by diazoxide). KATP channels also exist in the coronary vasculature, where they function to maintain basal coronary blood flow [ATP channels in the coronary smooth muscle have a low unitary conductance (~30 pS) and are blocked by glibenclamide and activated by KATP channel openers and adenosine [ATP channels exist in the coronary endothelium [ATP channels in the heart, KATP channels with unique biophysical and pharmacological profiles are also believed to be expressed in the mitochondrial inner membrane [ATP-sensitive  mice and the coronary abnormalities that develop in Kir6.1 and SUR2 null mice [ATP channels of ventricular myocytes [ATP channel. The specific cellular and subcellular localization of proteins can be used to predict their function. We therefore used antibodies specific for each of the KATP channel subunits to determine their cellular and subcellular localization in the mouse and rat heart. Our results suggest distinct roles for each of the KATP channel subunits in diverse cardiac structures.K complex ,6. Two g complex . It is wull mice . Althougull mice , or the myocytes . They almyocytes -12. or eATP channel subunits in the heart (see earlier), we performed immunohistochemistry and immunocytochemistry to determine the cellular and subcellular localization of Kir6.1, Kir6.2, SUR1 and SUR2 subunits in mouse and rat ventricle. To this end, we stained frozen sections of cardiac ventricular tissue as well as cardiac myocytes enzymatically isolated from mouse and rat hearts. Where possible, we used different antibodies to the subunits to ensure that the staining pattern observed was specific.Given the reports of expression of each of the KATP channel subunit combinations  all detect a band that migrates with an apparent molecular size of 44 kDa in Western blotting of rat heart membrane fractions .Both the 76A and G-16 anti-Kir6.2 antibodies have previously been characterized and we demonstrated that they specifically detect a ~38 kDa band in Western blotting of Kir6.2 transfected cells and do not detect heterologously expressed Kir6.1 protein ,14. HereThe anti-SUR1 antibodies specifically detect SUR1 protein (170 kDa) in cell lysates of COS7L cells transiently transfected with SUR1/Kir6.2 cDNA as well as in membrane fractions obtained from mouse hearts . In the We used an immunohistochemistry approach to identify the localization of Kir6.1 protein in cryostat sections of mouse ventricles. We used three separate antibodies that produced similar results (NAF-1, CAF-1 and C-16). We also used another anti-Kir6.1 antibody (78A) but this antibody did not perform well in these assays. A typical result is shown in Fig The cylindrical shape of the punctate structures in Fig Given the high sensitivity of the NAF-1 antibody, we were able to perform a sub-cellular localization study of Kir6.1 protein in the mouse heart  gave similar results. The W62 antibody developed by us  did not Using anti-SUR1 antibodies, we observed staining predominantly in cardiac ventricular myocytes Fig . The lacATP channel subunits.Fixation and permeabilization procedures can affect the outcome of immunocytochemistry experiments. We therefore compared three different methods. Isolated cardiac myocytes were (a) fixed with paraformaldehyde and permeabilized with Triton X-100, (b) fixed and permeablized in a single step by ice-cold methanol or (c) subjected to a two-step protocol with paraformaldehyde fixation followed by methanol fixation/permeabilization. Typical results obtained with the anti-Kir6.2 76A) antibody are shown in Fig  antibodyATP channel subunits with mitochondria, we used MitoTracker Red as a mitochondrial marker. We found MitoTracker staining to dissipate during immunocytochemistry protocols and co-labeling with KATP channel antibodies was therefore not an effective strategy. Nevertheless, the pattern of MitoTracker staining that we observed shortly after fixation  we observed strong surface expression of this subunit with a smaller degree of intracellular labeling  or that staining could be blocked by preincubation of antibody with the peptide to which the antibody has been raised. Further, we used the primary antibodies at the lowest concentrations possible to eliminate possible non-specific cross-reactivity with other proteins. Our study is a comprehensive description of the cellular and subcellular expression patterns of KATP channel subunits in the heart given our stringent criteria and the panel of antibodies available to us.A significant strength of our study is that we extensively characterized the antibodies used. We performed Western blotting with membrane fractions obtained from the heart to ensure that a band of the expected size is detected. Further, we chose antibodies that showed little cross-reactivity with other proteins, as judged by the absence of non-specific bands. In as far as it was possible we used different antibodies to the same KATP channels in ventricular myocytes have been described more than two decades ago [ATP channels have been described to consist of hetero-octameric complexes of Kir6.2 and SUR2A subunits [ATP channels [ATP channels in the ventricular myocyte [ATP channels as determined by functional microscopy. Scanning ion conductance (patch clamp) microscopy data have demonstrated KATP channels to be organized in small clusters and to be anchored in the Z-grooves (t-tubular openings) of the sarcolemma [ATP channels are predominantly expressed in the t-tubular system. The implications of KATP channels present in the t-tubular system are not entirely clear. Since t-tubular ion channels may control action potential propagation into the cardiac myocyte, it may be possible for KATP channels to have a role in the spread of excitation and action potential duration, particularly during conditions of metabolic impairment when these channels are more prone to opening. A shorter action potential duration in the t-tubular system would imply less Ca2+ entry at the local control sites of SR Ca2+ release and hence reduced contractility, which may in part explain the negative inotropic effects observed with KATP channel openers [Sarcolemmal Kades ago . Cardiacsubunits ,18,19. Tchannels  and alsochannels . Our dat myocyte ,22. It i myocyte  and the rcolemma . Collect openers . HoweverATP channels can be recorded in hearts from knockout mice lacking Kir6.1 subunits [ATP channels. It may be possible for Kir6.1 subunits to have a role in the pathophysiological setting, as demonstrated by the upregulated Kir6.1 expression levels during cardiac remodeling after ischemia or hypoxia [ATP channels have not been studied in SUR-/- mice. However, SUR1 antisense oligonucleotides inhibit KATP channels in rat ventricular myocytes [ATP channel function. Our data, demonstrating that both Kir6.1 and SUR1 subunits exhibit strong sarcolemmal expression, may require a reassessment of the molecular composition of ventricular KATP channels during normal and pathophysiological conditions.We found clear expression of Kir6.1 and SUR1 subunits in cardiac ventricular myocytes. Interestingly, both of these two subunits are strongly expressed at the sarcolemmal surface, but their functions in the sarcolemma are currently not understood. Since ventricular Ksubunits , it appe hypoxia ,26. To omyocytes , suggestATP channels are expressed in the mitochondrial inner membrane and that these channels are involved in the protection of the heart afforded by ischemic preconditioning [ATP channels remains to be identified. There are almost as many reports describing the presence of Kir6.0 subunits in cardiac mitochondria [ATP channel subunits in ventricular mitochondria. However, the technique of immunocytochemistry does not have sufficient resolution to rule out the existence of KATP channel subunits in mitochondria and our data therefore do not add significantly to this debate, other than demonstrating that KATP channel subunits are not abundantly expressed in mitochondria of ventricular myocytes.The concept has evolved that Kitioning ,28. The chondria ,29,30. achondria ,32. We dATP channels have been identified in several different vascular tissues, including the coronary vasculature [ATP channels in coronary resistance vessels have also been implicated in physiologically important stimuli such as regulation of basal vascular tone, autoregulation of blood flow, hypoxia-induced coronary vasodilation, reactive hyperemia  and ischemia (reviewed in [A tight coupling exists between metabolic status in the heart and coronary blood flow. Kculature . KATP chiewed in ,34).ATP channels has been characterized less extensively than the KATP channels in cardiomyocytes. A recent study employing in situ hybridization histochemistry examined Kir6.1 and SUR2B mRNA expression in different vascular beds, including the coronary vasculature [ATP channel subunit expression in the intact coronary vasculature. We find Kir6.1 expression in blood vessels of different sizes, including large vessels such as the aorta and large arteries, but also in small resistance arterioles (as defined by their diameter of larger than 12–15 μm and the presence of a well-defined smooth muscle layer). Without using specific markers, we were not able to distinguish objectively between venules and arterioles, but we did occasionally observe vessels with a thin smooth muscle layer (possibly veins or venules) that only expressed Kir6.1 faintly . Collectively, our data using various anti-Kir6.1 antibodies generally suggest that Kir6.1 subunits are expressed in coronary arterial smooth muscle, and possibly to a lesser extent in coronary veins. In contrast, we did not observe any staining of the coronary smooth muscle with anti-Kir6.2 antibodies.Molecularly, the identity of coronary smooth muscle Kculature . Strong culature . The preATP channels in the aortic cells of the SUR2 knockout mouse [ATP channels in coronary artery smooth muscle  are comprised of Kir6.1/SUR2 subunits (most likely the SUR2B isoform).We found strong staining of smaller coronary resistance vessels with the anti-SUR2 antibodies. We did not have access to suitable SUR2 isoform-specific antibodies, but the staining most probably reflects SUR2B expression. Curiously, we failed to see strong SUR2 expression in larger coronary arteries. This result is in apparent contradiction to the description that SUR2B mRNA expression occurs in larger coronaries  and the ut mouse . ReasonsATP channels play a role in regulation of coronary blood flow is compelling. Endothelial KATP channels, for example, contribute to shear stress-induced endothelial release of the vasodilator nitric oxide in rabbit aorta [ATP channels by stimulating the release of nitric oxide from the endothelium [The evidence that endothelial Kit aorta  and may it aorta ,39. Furtothelium .ATP channels are heteromeric Kir6.1/Kir6.2 complexes in combination with SUR2B subunits [ATP channels in the endothelium may differ fundamentally from homomeric KATP channels found in other tissues. The investigation of endothelial KATP channels is currently the subject of some of our ongoing studies.In the present study, we used immunohistochemistry approaches and identified Kir6.1, Kir6.2 and SUR2 protein in the endothelium lining coronary vessels Fig.  as well subunits . Thus, tFor this type of study, the specificity of antibodies used is always a concern. To overcome this problem, we used multiple different antibodies where possible and obtained similar staining patterns. However, we only had access to a single antibody to each of the SUR subunits and consequently we have not been able to verify the specificity of these antibodies by comparing different antibodies to each other (as we have done in the case of the Kir6 subunits). Furthermore, we did not have access to antibodies to the various splice variants of SUR1 or SUR2 and our data therefore do not address the possibility of regional expression differences. A definitive study will require the use of tissues obtained from knockout animals . Viable knockout animals for each of the proteins under consideration have been generated, but we have not been able to obtain these animals  for this purpose. Therefore, although we have taken every step possible to minimize non-specificity issues, our results should be interpreted within this limitation.ATP channel subunits in the heart. We found each of the KATP channel subunits to be expressed in ventricular myocytes, but with varying expression patterns. The roles of Kir6.1 and SUR1 subunits in ventricular myocytes remain to be elucidated and may require a reassessment of the molecular nature of the cardiac KATP channel. Coronary smooth muscle expresses predominantly Kir6.1 and SUR2 subunits, whereas the coronary endothelium expresses Kir6.1, Kir6.2 and SUR2 subunits. Thus, there is wide diversity of KATP channel subunit expression within the heart which determines the functional responses of various cell types to physiological and pathophysiological demands.Our study is a comprehensive analysis of the various K2 1.8, MgCl2 1, NaH2PO4 0.33; pH adjusted to 7.4 with NaOH) containing pinacidil (100 μM) to cause maximal vasodilatation as to clear the vasculature of blood. Hearts were fixed by switching the perfusate to paraformaldehyde , pH adjusted to 7.4) for 15 minutes at room temperature. The heart was incubated in 4% paraformaldehyde overnight at 4°C. Following fixation, the tissue was incubated overnight at 4°C in 30% sucrose in PBS. The tissue was then embedded in M1 embedding matrix (Thermo Shandon, Pittsburgh, PA) and placed on dry ice until frozen. The block containing the tissue was sectioned using a cooled (-20°C) cryostat  at 15 μm thicknesses. The sections were transferred to Superfrost Plus slides (Fisher Scientific) for further processing.Adult mice were sacrificed by pentobarbital overdose and the hearts were rapidly removed. All animal experiments were approved by the institutional Animal Care Review Board. Hearts were perfused at 37°C through the aorta (Langendorff mode) with Tyrode's solution  containing 4% goat or donkey serum (depending on the secondary antibody being used) and 0.2% Triton X-100 at room temperature. The slides were incubated overnight at 4°C with primary antibodies (see below) diluted in TBS containing 0.1% serum and 0.2% Triton X-100. Double or triple immunofluorescent studies were carried out by incubating the tissue sections with more than one primary antibody at the same time. Sections were washed three times for 15 min each in TBS, and incubated with fluorescently labeled secondary antibodies (see below) for 1 h at room temperature. The samples were again washed three times for 15 min each in TBS. Sections were drained by blotting with filter paper and a drop of mounting medium (containing an anti-fade reagent) was added to the slides before mounting with a standard coverslip. The mounting medium was allowed to dry before the slides were imaged using a Leica PS2 confocal microscope equipped with an Argon 488 nm gas laser and Helium Neon lasers (543 and 633 lines). Most images were obtained using an emission pin hole of 1.1–1.6 AE with either a 20× (0.7 NA) or a 63× (1.2 NA) oil objective.2+-free Tyrode's solution (same composition, but without the addition of CaCl2). Myocytes were dispersed using collagenese  and proteinase (Sigma type XXIV). The ventricles were removed and chopped into small pieces and digested using Ca2+-free Tyrode's solution containing the same enzymes. Single dissociated myocytes were plated onto laminin (10 ug/cm3)-coated glass coverslips and incubated at 37°C for 15 min to allow attachment to the coverslips before being fixed. In some experiments, myocytes were incubated with 500 nM MitoTracker Red 580 (Molecular Probes, Eugene, OR) during this period.Single ventricular myocytes were isolated from adult rats or mice using previously described procedures. Briefly, adult animals were sacrificed and the heart was rapidly removed and perfused in Langendorff mode (at 37°C) for sequential 5 min periods with Tyrode's solution and nominally Ca2+ and Mg2+-free PBS (Invitrogen, Carlsbad, CA). Myocytes were then incubated with 0.1% Triton X-100 (in PBS) for 15 min at room temperature, which permeabilizes surface membranes as well as those of intracellular organelles (this step was omitted when fixing the cells only with methanol but was included in the two-step fixation protocol). Following washing (2 × 5 min) and blocking  the cells were incubated with primary antibodies (1 h at room temperature), washed (3 × 10 min in PBS-serum) and incubated with secondary antibodies (45 min at room temperature). Following 4 washes  coverslips were mounted and viewed using confocal microscopy.Three different fixation protocols were employed. Some myocytes were fixed in paraformaldehyde (4%) for 15 min at room temperature, whereas with others fixation and permeabilization was performed in a single step by incubation with ice-cold 100% methanol for 5 minutes at -20°C. However, in the majority of the myocytes presented in this study a two-step fixation protocol was used , in whicAs a negative control, the primary antibody was adsorbed with the peptide against which it was made (when available). Negative staining controls (not shown) also included a null control, in which the primary antibody was omitted, which tested for non-specific staining of the secondary antibody. To avoid background from secondary antibodies alone, we normally pre-blocked the tissue with 5% normal serum from the same host species as the labeled secondary antibody. We used labeled secondary antibodies that have been pre-adsorbed against mouse and human and we titrated the labeled secondary antibody to obtain a maximal signal-to-noise ratio.The coding regions of Kir6.1 and Kir6.2 (a gift from Dr. S. Seino, Kobe University Graduate School of Medicine, Japan) were subcloned into pcDNA3. HEK-293 or COS-7L cells were cultured in D-MEM (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum and 20 μg/mL gentamycin. Cells were co-transfected according to the manufacturer's recommendations (Fugene 6, Roche Applied Science, Indianapolis, IN) with Kir6.1 or Kir6.2 cDNA (obtained from Dr S. Seino), SUR1 (Dr J. Bryan, Baylor College of Medicine, Texas) or SUR2A cDNAs (a gift from Dr Seino). Cells were lysed 48 h post-transfection.Adult Sprague Dawley rats were anesthetized and the hearts were rapidly removed. Membranes were prepared essentially as described before . The proCells were solubilized in lysis buffer [25 mmol/L Tris, 150 mmol/L NaCl, 5 mmol/L EDTA, 1% (v/v) Triton X-100, 0.5% (w/v) deoxycholate, pH 7.5 supplemented with a cocktail of protease inhibitors (Sigma)]. After centrifugation (10 min at 16,000 g), an equal volume of 2 × Laemmli loading buffer was added to the lysate. Proteins were separated by 10% SDS-PAGE and transferred to Immun-blot PVDF membrane (Bio-Rad Laboratories, Hercules, CA). The membrane was blocked and incubated with primary antibody (see below). As secondary antibodies, we used HRP-conjugated anti-rabbit, anti-mouse IgG (Amersham Biosciences, Piscataway, NJ) or anti-goat IgG in TBS-Tween for 1 hour and detected using a chemiluminescent substrate .Antibodies (NAF1) were raised against a peptide corresponding to residues 20–31 of the Kir6.1 N-terminus (ENLRKPRIRDRLP). There is a high degree of sequence similarity in Kir6.1 subunits between species in this region. We also raised a Kir6.1 antibody (CAF-1) to this peptide in chickens. In each case, a C-terminal cysteine was added for conjugation purposes. Peptides were synthesized and the antibodies were generated commercially . Each of these antibodies were peptide affinity purified. There is sequence similarity (81% identity) between this peptide and the recently-identified human beta-V spectrin. We tested for possible cross-reactivity of NAF-1 with beta-V spectrin using antibodies generously provided by Dr Jon Morrow  and demonstrated that our antibodies had no cross-reactivity with beta-V spectrin  and the goat anti-Kir6.1 R-14 or C-16 C-terminal antibodies . In our hands, the 78A and R-14 antibodies failed to detect Kir6.1 protein in Western blotting of cardiac membrane fractions and did not appear to stain above background in immunohistochemistry assays (not shown).We used a goat anti-Kir6.2 G-16 antibody  and an antibody (76A) developed in Dr Tinker's laboratory against a peptide (DALTLASSRGPLRKRSC) corresponding to a peptide within the Kir6.2 C-terminus (amino acids 357–372).We used a goat anti-SUR1 C-16 antibody  developed to an epitope mapping at the C-terminus and a goat anti-SUR2 C-15 C-terminal antibody . This antibody was initially sold as a pan-SUR2 antibody and was able to detect the SUR2A protein in Western blots of cell lysates from Kir6.2/SUR2A transfected cells, although with less sensitivity compared to SUR2B-transfected cells (data not shown). Currently, the antibody with the same catalog number is sold as being specific to SUR2B. We have not tested recent batches of this antibody for isoform specificity.Other antibodies used for immunolocalization included a mouse monoclonal α-actin smooth muscle antibody preconjugated to FITC  and a rat monoclonal antibody  raised against ICAM-2 (CD102), which is a cell surface glycoprotein constitutively expressed on vascular endothelial cells.2 fragment donkey anti-rat IgG and a Cy3- or Cy5-conjugated donkey anti-goat IgG .Secondary antibodies used included Cy3-conjugated donkey anti-rabbit IgG, Cy3-conjgated donkey anti-chicken IgY, Cy5-conjugated F(ab')+ channel familyKir = Inward rectifying KSUR = Sulphonylurea receptorATP channel = ATP-sensitive K+ channelKAM, ER and JL carried out the immunohistochemistry experiments. PDC, SH and GL carried out the immunocytochemistry experiments. TN, AM, LP, HY, XT, JPG and AT participated in characterization of the antibodies. WAC, MA and TN participated in the design of the study. WAC conceived the study, participated in study design and coordination, and manuscript preparation. All authors made substantive intellectual contributions to the study, read and approved the final manuscript.NAF-1 antibody does not cross-react with beta V spectrin. COS7L cells have been transfected with Kir6.1 cDNA and cell lystates were subjected to Western blotting with anti-beta V spectrin antibodies or NAF-1 anti-Kir6.1 antibodies.Click here for file


African-American women are disproportionately affected by HIV, accounting for 60% of all cases among women in the United States. Although their race is not a precursor for HIV, the socioeconomic and cultural disparities associated with being African American may increase their risk of infection. Prior research has shown that interventions designed to reduce HIV infection among African-American women must address the life demands and social problems they encounter. The present study used a qualitative exploratory design to elicit information about strategies to prevent HIV transmission among young, low-income African-American women.Twenty five low income African American women, ages 18–29, participated in five focus groups of five women each conducted at a housing project in Houston, Texas, a large demographically diverse metropolitan area that is regarded as one of the HIV/AIDS epicenters in the United States. Each group was audiotaped, transcribed, and analyzed using theme and domain analysis.The participants revealed that they had most frequently placed themselves at risk for HIV infection through drugs and drinking and they also reported drug and alcohol use as important barriers to practicing safer sex. The women also reported that the need for money and having sex for money to buy food or drugs had placed them at risk for HIV transmission. About one-third of the participants stated that a barrier to their practicing safe sex was their belief that there was no risk based on their being in a monogamous relationship and feeling no need to use protection, but later learning that their mate was unfaithful. Other reasons given were lack of concern, being unprepared, partner's refusal to use a condom, and lack of money to buy condoms. Finally, the women stated that they were motivated to practice safe sex because of fear of contracting sexually transmitted diseases and HIV, desire not to become pregnant, and personal experience with someone who had contracted HIV.This study offers a foundation for further research that may be used to create culturally relevant HIV prevention programs for African-American women. Despite the impressive strides that have been made by behavioral scientists in developing culturally sensitive HIV intervention programs for minority populations in the United States, HIV infection continues to be a major public health problem and is increasingly affecting minority populations, persons infected through heterosexual contact, the poor and women . African1, suggesting a need for an effective HIV prevention program for these women.The main exposure categories for all women in the United States is heterosexual contact accounting for approximately 40% of all new AIDS cases among this group ,5. This Aside from heterosexual contact and injection drug use, depression, physical and sexual abuse, and lack of condom negotiation skills are some of the psychosocial determinants of HIV risk behaviors among women . Drug usWhile African-American women may not be placed at risk of HIV infection because of their race and ethnicity, St. Lawrence, Eldridge, Reitman, Little, Shelby, and Brasfield  note thaAn array of socioeconomic and cultural factors exacerbate high-risk behaviors that place African-American women at risk for HIV infection ,13, the Individual and small group behavior change techniques have long been used in HIV prevention interventions for women and have resulted in increased condom use by inner-city women in primary health care settings, mental health clinics, and among women living in economically disadvantaged neighborhoods . CommuniSeveral HIV interventions have been developed to promote condom use and enhance sexual communication skills among African American women. The most promising interventions have been programs that are based on social cognitive principles. However, Kalichman et al  notes thUsing constructs from social cognitive theory, the health belief model and theory of reasoned action, Roye & Hudson  conducteThe research presented here results from qualitative studies conducted among African-American women in Houston, Texas, to elicit information that could be used to develop an HIV prevention intervention for similar populations. The research had two overarching purposes: to examine the sociocultural contexts of sexual risk taking among African-American women and to determine how a videotape-based HIV prevention intervention could be tailored so that it is effective in preventing HIV transmission among African-American women.This study utilized a qualitative exploratory design to elicit information about strategies for preventing HIV transmission among African-American women. Twenty five low income African-American women, aged 18–29, participated in five focus groups of five women each conducted at a housing project in Houston, Texas. Houston, Texas, a large demographically diverse metropolitan area, was selected as the study site because of its distinction as a leading HIV/AIDS epicenter in the United States . The houFocus group participants were recruited by displaying posters and flyers at strategic locations in the housing complex identified by the project manager. The flyer listed the study inclusion criteria: African-American heterosexual female, aged 18 to 29, self-reported unprotected vaginal intercourse with two or more partners or injection drug using partner in the last six months, or having been diagnosed or treated for a sexually transmitted disease in the past year. The flyer listed a university telephone number that prospective participants could call to obtain additional information about the study and/or to schedule their participation in a group. A trained research assistant confirmed the caller's eligibility. The study participants were recruited using a convenience sampling approach and study participants encouraged their friends to enroll in the study. Of the 89 prospective participants who contacted the university, 42 agreed to participate. Of that number, seventeen individuals did not appear on the scheduled day and thus 25 individuals formed the study sample. The groups were conducted at the housing project's clubhouse by a facilitator, slightly older than the target group, trained in focus group methodology and having approximately seven years' experience conducting groups with low-income African-American women. The facilitator was assisted by a trained research assistant who took notes to ensure that pertinent information not captured by the audiotape was obtained and to record major points and items discussed.Participants were told that their involvement would help researchers and clinicians to learn about what they feel is important and better ways of helping the African-American community to combat HIV and AIDS. They were informed that their involvement was voluntary, that they could refuse to answer any question, and that they could cease participation at any time without penalty. Agreement was also obtained to audiotape the session. Prior to the start of each group, active written informed consent was obtained from each participant. The facilitator discussed with participants the issue of confidentiality of the information discussed at the meeting. Because some of the women were familiar with one another as a result of residency within the same housing complex, the facilitator ensured that all women were aware that what was discussed during the session should not leave the session at its conclusion. They were advised that they would receive a $25 mall gift certificate and condoms as incentives for their participation. Respondents were also advised that the tapes, which were anonymous, would be destroyed following transcription and checking. All questions as well as the informed consent were provided in English.Semi-structured and open-ended questions were used to elicit information from the participants based on our research interest in determining perceptions of AIDS as a threat to African-American communities, barriers and facilitators to safer sex practices, characteristics of past situations which have placed persons at risk for HIV infection, the role of alcohol and drugs in creating high-risk sexual situations, suggestions on ways to enhance the saliency of AIDS in the African-American community and on how HIV intervention videotapes could be produced to achieve maximum levels of interest [See Table Focus group questions were generated in three stages. The first involved conducting interviews with six key informants [four public health researchers, one pharmacist, and one nurse experienced in HIV/AIDS prevention among African Americans] to generate working hypotheses on facilitators and barriers to HIV prevention and program messages and methods. The hypotheses formulated were then tested in interviews with women similar to members of the target population to reshape the hypotheses and continued until no new information emerged. The resulting hypotheses were used to generate questions that were then field tested with members of the target population. At the end of the first group, additional changes were made as needed to the way in which questions were asked not to elicit different information, but rather to add clarity for the participants.The resulting focus group interviews from which this research is reported were conducted using a guide consisting of a written list of questions and probes. McCracken  has highData analysis was performed according to the standard grounded theory approach of Glaser and Strauss . The relThe study sample consisted of 25 women, ages 18 to 29. The mean age was 24 years. Among the 25 women in the sample, two had completed some post-secondary education and the remainder had completed eighth or ninth grade. Twenty of the women were unemployed and most of these women identified themselves as homemakers. Of the remaining five, the most frequently cited source of employment [n = 4] was the medical field [nursing or medical assistant]. Two identified themselves as HIV positive. None of the respondents had health insurance. The names used for the quotes given in this report are pseudonyms.The situational determinants of HIV risk taking and their impact on HIV/AIDS prevention behaviors and education programs were examined. The rate of HIV infection and AIDS is higher among African-American women in Houston and nationally  and AfriComprehension of the situations explicated by low-income African-American women that place them at risk for HIV is of critical importance when developing programs that address HIV risk reduction. Most women had placed themselves at risk of transmission through drug use or needle sharing and having unprotected sex. The sexual activity took place in some instances in exchange for drugs or money and to purchase basic necessities such as food for their children. Other women stated that they had been placed at risk of HIV transmission because they believed they were in monogamous relationships but later learned their partners had been unfaithful.Edith, a 27 year old volunteer, described the situations that have placed her at risk for HIV in the past year:When you are on drugs and then you are drinking, it impairs your senses and you don't use common sense or knowledge of what you are doing. You just get caught up in the moment.Mary, a 26 year old homemaker explains:Having sex period is a risk. When you can't feed your kids and you need money. When you go sleep around and have sex and they're not using condoms. Cause some of them say they don't use them and some of them say they don't want to use them. They have all kinds of excuses not to use them.Celia, a 25 year old medical worker, described how she was placed at risk of HIV infection:I married a man, not really knowing him, and he was sleeping with a lot of women, and sleeping with me unprotected. Yeah, right after we got married, he told me he wasn't going to cheat on me no more and when I found out some of the women he was cheating on me with, I knew that they always stay at the doctor cause something always be wrong with them. I was pregnant and hoping that nothing was wrong with me.Related to risk for HIV, the participants were asked to discuss why HIV is so prevalent within their [the African-American] community. Unprotected sex, lack of awareness, lack of medical access, and early sexual initiation were most frequently mentioned. When asked why HIV is spreading so rapidly in the African-American community, unprotected sex was stated most often. Lack of knowledge or awareness about prevention was also frequently stated. However, drug use was not highly ranked and was infrequently stated compared to women's beliefs that lack of access to medical care and non-priority of health as well as early sexual initiation and feelings of invincibility among young people were more significant contributors to HIV's rapid spread among African Americans.Betty, a 25 year old homemaker said:I think it's spreading around so fast because people that have AIDS just constantly have sex and they pass it on and pass it on and pass it on.Celia expressed her agreement and added:Everybody's sleeping with everybody. The women are sleeping with women; the men are sleeping with men and then they sleeping with each other. Ménage á trios or threesomes, foursomes, they popping Ecstasy pills, just partying hard, they smoking marijuana, they speed balling, they taking downers, handlebars. Uh, I don't know, just really not caring and so they're not even going to the doctor to see if anything is wrong with them. They're just continually sleeping and not stopping to take care of their own bodies, to make sure that they are okay.Kelli, a 24 year old operator stated:Well, first of all because we're uneducated. And we're just unconcerned. Like they say, you have to do what you have to do to get what you need, and if it's one of them five minute things, they ain't thinking about no condom.Kim, a 24 year old cashier said:I don't know – people feel like they can't get it. Maybe they feel like it just can't get to them. They feel like it can get to other people, but it can't get to them.Although the women related their individual risk for HIV infection to drugs and alcohol, they did not associate drug and alcohol use with the rapid spread of HIV within the overall African-American community.When asked to name situations that have prevented them from protecting themselves against HIV infection, the reasons given were much the same as when they were asked about situations that have placed them at risk for HIV infection. About one-third of the women named drug and alcohol use as responsible for them not taking needed precautions. Surprisingly, about one-third of women stated that their barrier was their belief that there was no risk based on their being in a monogamous relationship and feeling no need to use protection, but later learning that their mate was unfaithful. Other reasons given were lack of concern, being unprepared, partner's refusal to use a condom, and lack of money to buy condoms.Chaka discussed how drug and alcohol use were barriers to safe sex practices for her:Working in the club, you drink and if you intensify that with drugs, you don't' know who you're going home with. You don't know who been to your house and what they have done to you on account of you being high, on account of you being drunk or you done overindulged in either or and it's just not a good feeling.Charlie stated that she was placed at risk of contracting HIV by her spouse:I married a man not really knowing him and he was sleeping with a lot of women and then sleeping with me unprotected.Vicki, an 18 year old high school student said:Like I said, not having the money to go and buy the protection. That's going to prevent you from preventing it.In this culture, particularly among poorer uneducated women, men may play a more domineering role over women . There iThe women were motivated to practice safe sex because of fear of contracting sexually transmitted diseases and HIV, desire not to become pregnant, and personal experience with someone who had contracted HIV. Over one-third of women acknowledged that fear of contracting sexually transmitted infections including hepatitis and HIV motivated them to practice safe sex. A participant stated:Every disease that's in the book that's not curable is enough to scare my clothes on.Mary said:Syphilis, gonorrhea, HIV, herpes; that ought to want to motivate anybody to practice safe sex.Other women said that their desire not to become pregnant motivated them to practice safe sex. Sally was motivated by the need to care for her children.She stated:Because I have three children and I don't plan on dying until the Lord takes me.A smaller number of women declared that they were motivated to practice safe sex because they had seen, first hand, the effects of HIV.Kim said:Things that motivate me – you see somebody outside on the streets and you see them with it and you see the effect that it's had on them and you look at them and you say that's something that you don't want to do so it motivates you to practice safe sex.When asked to describe what should be included in a videotape aimed at prevention of HIV within the African-American community, the most prevalent response among participants was to include personal experiences of people affected by HIV and AIDS. They believed that testimonials from those infected with HIV and sensational footage of the ravaging effects HIV and AIDS have on the human body would be most effective. To create a video that addresses HIV prevention, over half of the participants recommended sensationalism to garner the audience's attention. The purpose of the video would be to show the illness, pain, rejection, medication regimen, and years of life lost among those infected.Fonda, a 24 year old GED preparer with 2 years of college said:To make them watch it, show the blood, the guts, the pus, the sores, the relationships...Sally said:Show them how the body deteriorates from having AIDS. Show them everything else in the world that they are going to miss out on if they don't take care of themselves.Another participant said:You need to let them see how these people are just in so much pain and rejection, and not having the finances and things. In order to live like the guy that's an athlete [Magic Johnson], you know to live by the medication, how people are going to other places to get it.A variety of other suggestions were given including the use of popular culture in the forms of rap and gospel music videos, productions and concerts and creating a Surgeon General's warning against unsafe sex [much like what has been done with tobacco],. Ceah, a 25 year old health care worker said:Drum them in any way you can. If they like rap, rap it to them. If they like gospel, you sing it to them. If prayer is what it takes, you pray it to them. By any means necessary, you get your message across. Try all ways.Other recommendations included developing a video in the form of a comedy-drama or a cartoon or using a campaign similar to the one utilized by Mothers Against Drunk Driving in which a person pre-HIV is shown followed by the person who has become ill post-HIV infection.The participants also offered suggestions for recruiting African Americans into prevention programs where the videotape could be shown. The majority of women suggested the use of financial incentives including air conditioning units, fans, food, and amusement park tickets. The women recommended recruiting participants within the communities in which they live, going door-to-door if needed, and having such programs take place in community settings, such as schools and neighborhood centers. Other suggestions included having family friendly events that men would want to attend, thus ensuring women and children will follow.Fonda believed that you have to give something to get people to participate. She said:You know, people want something for something; nobody wants nothing for nothing. Nothing is free in this world.Shirley, a GED student suggested community involvement:Fundraisers, cooking things, get the community involved with you as the person that wants to get these things started. Once you get these things started, once you get the community, you got it.Nilene suggested:Just get it [word] out, it has to be let out in some kind of way, as far as radio stations, TVs, billboards. People in the communities, the main office, the campus, I mean everywhere, it has to be everywhere. It can't be in one spot, it has to come out, go out.Veronica, a 25 year old youth coordinator with one year of college says:If you get the men there, the women will come. A basketball tournament, yes, they like basketball and once you get the men there, the women are gonna come. Seriously, it's bad to say it like that but you're giving something. It's a sex symbol, yes, you understand what I'm saying, but it's a way to get them out there.2.Prior research has indicated the need to develop HIV intervention programs that target the socioeconomic contexts of women at risk for infection including cognitive or social cognitive, educational and/or skills building content-specific components. Women in the present study recommended that HIV/AIDS videotaped messages should be developed that highlight the sensational effects of the disease. Contrary to research indicating this method does not work -32 and bHealth professionals may wish to learn more about the barriers these women face and work with social service providers to address the issues most salient to the women before developing patient education materials for HIV/AIDS prevention. Methods that appeal to the target audience should be devised but nursing professionals should remember that low-income African-American women are a heterogeneous group. Interventions such as videotapes should be developed to have a wide appeal yet have contextual, cultural, and gender specificity and it is important to remember that it is best to educate women within a community-based setting. Resources are needed to identify, recruit and retain African American women into HIV intervention programs.The author(s) declare that they have no competing interests.EJE, AFM and GOO conceived and designed the study. EJE, AFM, RJP jointly planned and executed the data analyses. EJE wrote the paper with assistance from AFM, RJP, GOO and NIO.


Household-level geographic information systems (GIS) database are usually constructed using the geographic positioning system (GPS). In some research settings, GPS receivers may fail to capture accurate readings due to structural barriers such as tall buildings. We faced this problem when constructing a household GIS database for research sites in Karachi, Pakistan because the sites are comprised of congested groups of multi-storied building and narrow lanes. In order to overcome this problem, we used high resolution satellite imagery (IKONOS) to extract relevant geographic information.The use of IKONOS satellite imagery allowed us to construct an accurate household GIS database, which included the size and orientation of the houses. The GIS database was then merged with health data, and spatial analysis of health was possible.The methodological issues introduced in this paper provide solutions to the technical barriers in constructing household GIS database in a heavily populated urban setting. Geographic data are increasingly being employed in health studies . By studOne way to facilitate the measurement of local variation in health outcomes is to create household-level geographic information systems (GIS) database. Household locations can be captured by using GPS  receivers ,7. HowevFaced by such challenges we explored satellite imagery in order to acquire household GIS data in urban slums in Karachi, Pakistan. This paper describes the methods used to construct the household GIS database and the technical barriers one might encounter during the construction of a database.Geographic studies have been considered as one of the research disciplines of large Vi (antigen) typhoid vaccine effectiveness trials as well as typhoid disease burden studies . The Vi The aim of the project was to construct a spatial database that includes household locations, study area boundary with administrative units, and other geographic features such as hospitals/clinics, schools, mosques, roads, lanes, and water bodies. The project area included four Karachi slums including Sultanabad, Hijrat Colony, Rehri Goth, and Sherpao Colony Figure . SultanaA commercially available map of Karachi was used as the base map for this GIS project. The base map was georeferenced with four identifiable landmarks using handheld GPS receivers with accuracy of approximately five meters. This accuracy was considered sufficient to identify the study areas, to order satellite imagery, and to conduct subsequent ground surveys. After georeferencing the map, the main geographic features such as roads, hospitals/healthcare centres, and other city landmarks were digitized and incorporated into the baseline GIS database Figure .Satellite imagery is available at different spatial, temporal, and spectral resolutions . DiffereThe satellite images were enhanced using an image processing software package (ERDAS Imagine, Atlanta, USA) to facilitate the digitization of house parcel boundaries. High precision, dual frequency GPS units (Trimble 4000 ssi) were used to capture data at several identifiable points on the images to be used as ground control points (GCPs). To transfer images into a GIS database, it must be geometrically rectified to a known coordinate system on the basis of a number of GCPs . Most ofAfter georeferencing the images were resampled, converted into JPEG files, and were imported in AutoCAD Version 14 (Cadopolis.com Inc., Canada). These processes allowed the parcel boundaries to be delineated through heads-up digitizing. The resampled satellite imagery was inserted as backdrop in AutoCAD. The image was aligned to correct its scale, translation and rotation by using two GCPs located at the corners of the image. After the alignment of the raster images in AutoCAD the house parcels were digitized. One image was used on multiple workstations to digitize different portions of the image, which were subsequently merged to form a complete area map of house parcels Figure .The georeferenced image and the household parcel maps were used during a ground survey. The survey team consisted of civil engineers skilled in drawing household parcels and other field staff who conducted the census survey. The ground survey included verification of the size and orientation of household structures, resketching of the structures where needed, and locating specific household parcels so that they could be given their unique address identification number (ID) which was created during census survey Figure . The addAfter completing the ground surveys, the maps were updated using AutoCAD, and the address IDs were added to household parcels in the database. Finally, the household parcel AutoCAD files were imported as polygons into the ArcGIS software package (ESRI Inc., USA). The process included several checks for missing households, duplicate address IDs, and misplacement of address IDs and data were corrected when an error was found. The corrected data were validated by randomly selecting several household parcels (about 2%) from different zones of the study area and verifying their position on ground. At this stage, we observed no discrepancies in the data between ground verification and the satellite based maps suggesting that the household level GIS database is highly accurate.The HGIS database is composed of spatial and non-spatial components Figure . The spaEntity relationships between data tables are shown as lines, and logical relationships between entities have parentheses around them Figure . In the In this paper, we have outlined methodological issues involved in the construction of a household GIS database using satellite-based technology in a situation where the GPS was not appropriate. To our knowledge, this approach has never been reported, but may offer greater value in constructing household GIS databases compared to that based on GPS. Our household GIS offers size and orientation of individual houses in dense urban environment. Such database can be instrumental in health and disease studies because they facilitate the integration of socioecological and environmental factors that may influence health. Future health studies may benefit by using satellite-based technology to construct household GIS databases.