An open-label, long-term evaluation of the safety, efficacy and tolerability of avanafil in male patients with mild to severe erectile dysfunction.

AIM: Determine the long-term efficacy, safety and tolerability of avanafil, a highly specific, rapidly absorbed phosphodiesterase type 5 inhibitor in male patients with mild to severe erectile dysfunction (ED), with or without diabetes. METHODS: This was a 52-week, open-label extension of two 12-week, randomised, placebo-controlled, phase 3 trials. Patients were assigned to avanafil 100 mg, but could request 200 mg (for increased efficacy; '100/200-mg' group) or 50 mg (for improved tolerability). Primary end points included percentage of sexual attempts ending in successful vaginal penetration [Sexual Encounter Profile 2 (SEP2)] and intercourse (SEP3) and erectile function domain score per the International Index of Erectile Function (IIEF-EF). RESULTS: Some 712 patients enrolled; 686 were included in the intent to treat population and contributed to the data. All primary end points showed sustained improvement. SEP2 and SEP3 success rates improved from 44% to 83% and from 13% to 68% (100-mg group) and from 43% to 79% and from 11% to 66% (100/200-mg group), respectively. Mean IIEF-EF domain scores improved from 13.6 to 22.2 (100-mg group) and from 11.9 to 22.7 (100/200-mg group). Avanafil was effective in some patients ≤ 15 min and > 6 h postdose. Sixty-five per cent (112/172) of 'nonresponders' to avanafil 100 mg responded to the 200-mg dose. The most common (≥ 2%) treatment-emergent adverse events were headache, flushing, nasopharyngitis and nasal congestion; < 3% of patients discontinued therapy because of adverse events. CONCLUSIONS: The long-term tolerability and improvement in sexual function, coupled with rapid onset, suggest that avanafil is well suited for the on-demand treatment of ED.

Identification of non-functional human VNO receptor genes provides evidence for vestigiality of the human VNO.

In mammals, the vomeronasal organ (VNO) contains chemosensory receptor cells that bind to pheromones and induce a variety of social and reproductive behaviors. It has been traditionally assumed that the human VNO (Jacobson's organ) is a vestigial structure, although recent studies have shown minor evidence for a structurally intact and possibly functional VNO. The presence and function of the human VNO remains controversial, however, as pheromones and VNO receptors have not been well characterized. In this study we screened a human Bacterial Artificial Chromosome (BAC) library with multiple primer sets designed from human cDNA sequences homologous to mouse VNO receptor genes. Utilizing these BAC sequences in addition to mouse VNO receptor sequences, we screened the High Throughput Genome Sequence (HTGS) database to find additional human putative VNO receptor genes. We report the identification of 56 BACs carrying 34 distinct putative VNO receptor gene sequences, all of which appear to be pseudogenes. Sequence analysis indicates substantial homology to mouse V1R and V2R VNO receptor families. Furthermore, chromosomal localization via FISH analysis and RH mapping reveal that the majority of the BACs are localized to telomeric and centromeric chromosomal localizations and may have arisen through duplication events. These data yield insight into the present state of pheromonal olfaction in humans and into the evolutionary history of human VNO receptors.

Comparative sequencing of a multicopy subtelomeric region containing olfactory receptor genes reveals multiple interactions between non-homologous chromosomes.

In this study, we assess the evolutionary relationships among different chromosomal copies of a subtelomeric block of sequence. This block contains homology to three olfactory receptor genes and is dispersed on at least 14 different chromosome ends in humans. It is single-copy in non-human primates. We analyzed single nucleotide polymorphisms in two 1 kb subregions and a polymorphic Alu insertion within 181 copies of this block from 12 chromosome ends and found evidence for recent interactions between the subtelomeric regions of non-homologous chromosomes. First, several sequence haplotypes are each present on multiple chromosomes, and several chromosomes each have multiple alleles with divergent haplotypes. Secondly, the observed variation clearly indicates that chromosomes 5q, 8p, 11p and/or 15q have each received the block from at least two different sources by non-homologous exchange. In addition, we observe at least one ectopic gene conversion event. Awareness of such exchange among sequences on non-homologous chromosomes is critical for accurate analysis of these complex and dynamic regions of the genome.

Transcriptional activity of multiple copies of a subtelomerically located olfactory receptor gene that is polymorphic in number and location.

We report here on the transcriptional activity of multiple copies of a subtelomerically located olfactory receptor (OR) gene, OR-A. Due to recent duplication events, both the copy number and chromosomal location of OR-A vary among humans. Sequence analyses of 180 copies of this gene, derived from 12 chromosome ends in 22 individuals, show that the main coding exon of all but one copy is an intact open reading frame with 0-5 predicted amino acid differences. We detected transcription of OR-A in both olfactory epithelium and testis tissue using RT-PCR amplification with primers designed on the basis of a computationally predicted gene structure. Two alternatively spliced forms of transcripts, one encoding an isoform with an extended N-terminus, were found in both tissues. A third transcript, derived from a second promoter, was also observed in testes. The start methionine is predicted in all transcripts to lie in an upstream exon rather than the main coding exon, as is typical for most other OR genes. By examining sequence variants among transcripts, we show that transcription of this gene occurs at multiple chromosomal locations. Our results lend credence to the idea that OR diversity could be generated in rearrangement-prone subtelomeric regions and show that polymorphism in subtelomeric regions could lead to individual-to-individual variation in the expressed repertoire of OR genes.

Feline leukaemia virus status of Australian cats with lymphosarcoma.

OBJECTIVE: To determine the FeLV status of sera and tumours from Australian cats with lymphosarcoma in relation to patient characteristics, tumour characteristics (tissue involvement, histological grade and immunophenotype), haematological and biochemical values. DESIGN: Prospective study of 107 client-owned cats with naturally-occurring lymphosarcoma. PROCEDURE: An ELISA was used to detect FeLV p27 antigen in serum specimens collected from cats with lymphosarcoma. A PCR was used to detect FeLV DNA in formalin-fixed, paraffin-embedded tissue sections containing neoplastic lymphoid cells. The PCR was designed to amplify a highly conserved region of the untranslated long terminal repeat of FeLV provirus. RESULTS: Only 2 of 107 cats (2%), for which serum samples were available, were FeLV-positive on the basis of detectable p27 antigen in serum. In contrast, 25 of 97 tumours (26%) contained FeLV DNA. Of the 86 cats for which both PCR and ELISA data were available, 19(22%) had FeLV provirus in their tumours but no detectable circulating FeLV antigen in serum, while 2 (2%) had FeLV provirus and circulating FeLV antigen. FeLV PCR-positive/ELISA-negative cats (19) differed from PCR-negative/ELISA-negative cats (65) in having fewer B-cell tumours (P = 0.06), more non B-/non T-cell tumours (P = 0.02) and comprising fewer non-Siamese/Oriental pure-bred cats (P = 0.03). CONCLUSIONS: The prevalence of FeLV antigen or provirus was considerably lower in our cohort of cats compared with studies of lymphosarcoma conducted in the Northern hemisphere. This suggests that factors other than FeLV are important in the development of lymphosarcoma in many Australian cats. No firm conclusions could be drawn concerning whether FeLV provirus contributed to the development of lymphosarcoma in PCR-positive/ELISA-negative cats.

Induction of macrophage matrix metalloproteinase biosynthesis by surfactant protein D.

Recent studies strongly suggest that surfactant protein D (SP-D) plays important roles in pulmonary host defense and the regulation of immune and inflammatory reactions in the lung. Although SP-D can bind to alveolar macrophages and can elicit their chemotaxis, relatively little is known about the direct cellular consequences of SP-D on the function of these cells. Because matrix metalloproteinases (MMPs) are synthesized in increased amounts in response to various proinflammatory stimuli, we investigated the capacity of SP-D to modulate the production of MMPs by freshly isolated human alveolar macrophages. Unexpectedly we found that recombinant rat SP-D dodecamers selectively induce the biosynthesis of collagenase-1 (MMP-1), stromelysin (MMP-3), and macrophage elastase (MMP-12) without significantly increasing the production of tumor necrosis factor alpha and interleukin-1beta. SP-D did not alter the production of these MMPs by fibroblasts. Phosphatidylinositol, a surfactant-associated ligand that interacts with the carboxyl-terminal neck and carbohydrate recognition domains of SP-D, inhibited the SP-D-dependent increase in MMP biosynthesis. A trimeric, recombinant protein consisting of only the neck and carbohydrate recognition domain did not augment metalloproteinase production, suggesting that the stimulatory effect on MMP production depends on an appropriate spatial presentation of trimeric lectin domains. Although SP-D dodecamers can selectively augment metalloproteinase activity in vitro, this effect may be competitively inhibited by tissue inhibitors of metalloproteinases or surfactant-associated ligands in vivo.

Genomic analysis of orthologous mouse and human olfactory receptor loci.

Olfactory receptor (OR) genes represent approximately 1% of genomic coding sequence in mammals, and these genes are clustered on multiple chromosomes in both the mouse and human genomes. We have taken a comparative genomics approach to identify features that may be involved in the dynamic evolution of this gene family and in the transcriptional control that results in a single OR gene expressed per olfactory neuron. We sequenced approximately 350 kb of the murine P2 OR cluster and used synteny, gene linkage, and phylogenetic analysis to identify and sequence approximately 111 kb of an orthologous cluster in the human genome. In total, 18 mouse and 8 human OR genes were identified, including 7 orthologs that appear to be functional in both species. Noncoding homology is evident between orthologs and generally is confined within the transcriptional unit. We find no evidence for common regulatory features shared among paralogs, and promoter regions generally do not contain strong promoter motifs. We discuss these observations, as well as OR clustering, in the context of evolutionary expansion and transcriptional regulation of OR repertoires.

Segmental duplications: organization and impact within the current human genome project assembly.

Segmental duplications play fundamental roles in both genomic disease and gene evolution. To understand their organization within the human genome, we have developed the computational tools and methods necessary to detect identity between long stretches of genomic sequence despite the presence of high copy repeats and large insertion-deletions. Here we present our analysis of the most recent genome assembly (January 2001) in which we focus on the global organization of these segments and the role they play in the whole-genome assembly process. Initially, we considered only large recent duplication events that fell well-below levels of draft sequencing error (alignments 90%-98% similar and > or =1 kb in length). Duplications (90%-98%; > or =1 kb) comprise 3.6% of all human sequence. These duplications show clustering and up to 10-fold enrichment within pericentromeric and subtelomeric regions. In terms of assembly, duplicated sequences were found to be over-represented in unordered and unassigned contigs indicating that duplicated sequences are difficult to assign to their proper position. To assess coverage of these regions within the genome, we selected BACs containing interchromosomal duplications and characterized their duplication pattern by FISH. Only 47% (106/224) of chromosomes positive by FISH had a corresponding chromosomal position by comparison. We present data that indicate that this is attributable to misassembly, misassignment, and/or decreased sequencing coverage within duplicated regions. Surprisingly, if we consider putative duplications >98% identity, we identify 10.6% (286 Mb) of the current assembly as paralogous. The majority of these alignments, we believe, represent unmerged overlaps within unique regions. Taken together the above data indicate that segmental duplications represent a significant impediment to accurate human genome assembly, requiring the development of specialized techniques to finish these exceptional regions of the genome. The identification and characterization of these highly duplicated regions represents an important step in the complete sequencing of a human reference genome.

Posttranslational modifications of microfibril associated glycoprotein-1 (MAGP-1).

Microfibril-associated glycoprotein-1 (MAGP-1) is a small molecular weight protein associated with extracellular matrix microfibrils. Biochemical studies have shown that MAGP-1 undergoes several posttranslational modifications that may influence its associations with other microfibrillar components. To identify the sites in the molecule where posttranslational modifications occur, we expressed MAGP-1 constructs containing various point mutations as well as front and back half truncations in CHO cells. Characterization of transiently expressed protein showed that MAGP-1 undergoes O-linked glycosylation and tyrosine sulfation at sites in its amino-terminal half. This region of the protein also served as a major amine acceptor site for transglutaminase and mediated self-assembly into high molecular weight multimers through a glutamine-rich sequence. Fine mapping of the modification sites through mutational analysis demonstrated that Gln20 is a major amine acceptor site for the transglutaminase reaction and confirmed that a canonical tyrosine sulfation consensus sequence is the site of MAGP-1 sulfation. Our results also show that O-glycosylation occurs at more than one site in the molecule.

Characterization of nonfunctional V1R-like pheromone receptor sequences in human.

The vomeronasal organ (VNO) or Jacobson's organ is responsible in terrestrial vertebrates for the sensory perception of pheromones, chemicals that elicit stereotyped behaviors among individuals of the same species. Pheromone-induced behaviors and a functional VNO have been described in a number of mammals, but the existence of this sensory system in human is still debated. Recently, two nonhomologous gene families, V1R and V2R, encoding pheromone receptors have been identified in rat. These receptors belong to the seven-transmembrane domain G-protein-coupled receptor superfamily. We sought to characterize V1R-like genes in the human genome. We have identified seven different human sequences by PCR and library screening with rodent sequences. These human sequences exhibit characteristic features of V1R receptors and show 52%-59% of amino acid sequence identity with the rat sequences. Using PCR on a monochromosomal somatic cell hybrid panel and/or FISH, we demonstrate that these V1R-like sequences are distributed on chromosomes 7, 16, 20, 13, 14, 15, 21, and 22 and possibly on additional chromosomes. One sequence hybridizes to pericentromeric locations on all the acrocentric chromosomes (13, 14, 15, 21, and 22). All of the seven V1R-like sequences analyzed show interrupted reading frames, indicating that they represent nonfunctional pseudogenes. The preponderence of pseudogenes among human V1R sequences and the striking anatomical differences between rodent and human VNO raise the possibility that humans may have lost the V1R/VNO-mediated sensory functions of rodents.

Modeling the feasibility of whole genome shotgun sequencing using a pairwise end strategy.

In pairwise end sequencing, sequences are determined from both ends of random subclones derived from a DNA target. Sufficiently similar overlapping end sequences are identified and grouped into contigs. When a clone's paired end sequences fall in different contigs, the contigs are connected together to form scaffolds. Increasingly, the goals of pairwise strategies are large and highly repetitive genomic targets. Here, we consider large-scale pairwise strategies that employ mixtures of subclone sizes. We explore the properties of scaffold formation within a hybrid theory/simulation mathematical model of a genomic target that contains many repeat families. Using this model, we evaluate problems that may arise, such as falsely linked end sequences (due either to random matches or to homologous repeats) and scaffolds that terminate without extending the full length of the target. We illustrate our model with an exploration of a strategy for sequencing the human genome. Our results show that, for a strategy that generates 10-fold sequence coverage derived from the ends of clones ranging in length from 2 to 150 kb, using an appropriate rule for detecting overlaps, we expect few false links while obtaining a single scaffold extending the length of each chromosome.

Amplification of the human dihydrofolate reductase gene via double minutes is initiated by chromosome breaks.

DNA sequence amplification is one of the most frequent manifestations of genomic instability in human tumors. We have shown previously that amplification of the dihydrofolate reductase (DHFR) gene in Chinese hamster cells is initiated by chromosome breaks, followed by bridge-breakage-fusion cycles that generate large intrachromosomal repeats; these are ultimately trimmed by an unknown process to smaller, more homogenous units manifested as homogenously staining chromosome regions (HSRs). However, in most human tumor cells, amplified DNA sequences are borne on unstable, extrachromosomal double minutes (DMs), which suggests the operation of a different amplification mechanism. In this study, we have isolated a large number of independent methotrexate-resistant human cell lines, all of which contained DHFR-bearing DMs. Surprisingly, all but one of these also had suffered partial or complete loss of one of the parental DHFR-bearing chromosomes. Cells in a few populations displayed what could be transient intermediates in the amplification process, including an initial HSR, its subsequent breakage, the appearance of DHFR-containing fragments, and, finally, DMs. Our studies suggest that HSRs and DMs both are initiated by chromosome breaks, but that cell types differ in how the extra sequences ultimately are processed and/or maintained.

The microfibrillar proteins MAGP-1 and fibrillin-1 form a ternary complex with the chondroitin sulfate proteoglycan decorin.

MAGP-1 and fibrillin-1, two protein components of extracellular microfibrils, were shown by immunoprecipitation studies to interact with the chondroitin sulfate proteoglycan decorin in the medium of cultured fetal bovine chondrocytes. Decorin interacted with each protein individually and with both proteins together to form a ternary complex. Expression of truncated fibrillin-1 proteins in Chinese hamster ovary cells localized proteoglycan binding to an amino-terminal region near the proline-rich domain. A spatially analogous fibrillin-2 truncated protein did not coprecipitate the same sulfated molecule, suggesting that chondroitin sulfate proteoglycan binding in this region is specific for fibrillin-1. An interaction between fibrillin and MAGP-1 was also observed under culture conditions that abrogated decorin secretion, suggesting that the two microfibrillar proteins can associate in the absence of the proteoglycan. Sulfation of matrix proteins is important for elastic fiber assembly because inhibition of sulfation was shown to prevent microfibrillar protein incorporation into the extracellular matrix of cultured cells.

Interaction of tropoelastin with the amino-terminal domains of fibrillin-1 and fibrillin-2 suggests a role for the fibrillins in elastic fiber assembly.

Alignment of tropoelastin molecules during the process of elastogenesis is thought to require fibrillin-containing microfibrils. In this study, we have demonstrated that amino-terminal domains of two microfibrillar proteins, fibrillin-1 and fibrillin-2, interact with tropoelastin in solid phase binding assays. The tropoelastin-binding site was localized to a region beginning at the glycine-rich and proline-rich regions of fibrillin-2 and fibrillin-1, respectively, and continuing through the second 8-cysteine domain. Characterization of the binding requirements using the fibrillin-2 construct found that a folded, secondary structure was necessary for binding. Furthermore, binding between tropoelastin and fibrillin was mediated by ionic interactions involving the lysine side chains of tropoelastin. The importance of the lysine side chains was corroborated by the finding that the fibrillin-2 construct did not bind to mature elastin, whose lysine side chains have been modified to form cross-links. Interestingly, there was no interaction between the fibrillin constructs and tropoelastin in solution phase, suggesting that binding of tropoelastin to a solid substrate exposes a cryptic binding site. These results suggest that fibrillin plays an important role in elastic fiber assembly by binding tropoelastin and perhaps facilitating side chain alignment for efficient cross-linking.

Radiation-produced chromosome aberrations: colourful clues.

Ionizing radiation produces many chromosome aberrations. A rich variety of aberration types can now be seen with the technique of chromosome painting. Apart from being important in medicine and public health, radiation-produced aberrations act as colorful molecular clues to damage-processing mechanisms and, because juxtaposition of different parts of the genome is involved, to interphase nuclear organization. Recent studies using chromosome painting have helped to identify DNA double-strand-break repair and misrepair pathways, to determine the extent of chromosome territories and motions, and to characterize different aberration patterns left behind by different kinds of radiation.

PART-1: a novel human prostate-specific, androgen-regulated gene that maps to chromosome 5q12.

Genes regulated by androgenic hormones are of critical importance for the normal physiological function of the human prostate gland, and they contribute to the development and progression of prostate carcinoma. We used cDNA microarrays containing 1500 prostate-derived cDNAs to profile transcripts regulated by androgens in prostate cancer cells. This study identified a novel gene that we have designated PART-1 (prostate androgen-regulated transcript 1), which exhibited increased expression upon exposure to androgens in the LNCaP prostate cancer cell line. Northern analysis demonstrated that PART-1 is highly expressed in the prostate gland relative to other normal human tissues and is expressed as different transcripts using at least three different polyadenylation signals. The PART-1 cDNA and putative protein are not significantly homologous to any sequences in the nonredundant public sequence databases. Cloning and analysis of the putative PART-1 promoter region identified a potential binding site for the homeobox gene PBX-la, but no consensus androgen response element or sterol-regulatory element binding sites were identified. We used a radiation hybrid panel and fluorescence in situ hybridization to map the PART-1 gene to chromosome 5q12, a region that has been suggested to harbor a prostate tumor suppressor gene. These results identify a new gene involved in the androgen receptor-regulated gene network of the human prostate that may play a role in the etiology of prostate carcinogenesis.

Characterization, chromosomal localization, and the complete 30-kb DNA sequence of the human Jagged2 (JAG2) gene.

The genomic sequence of the human Jagged2 (JAG2) gene, which encodes a ligand for the Notch receptors, was determined. The 30-kb DNA sequence spanning the JAG2 gene contains 26 exons and a putative promoter region. Several potential binding sites for transcription factors, including NF-kappab, E47, E12, E2F, Ets-1, MyoD, and OCT-1, were found in the human JAG2 promoter region. The JAG2 gene was also mapped to the chromosomal region 14q32 using fluorescence in situ hybridization.

Sharp DNA bends as landmarks of protein-binding sites on straightened DNA.

We have developed a fluorescence-based method for mapping single or multiple protein-binding sites on straightened, large-size DNA molecules (> 5 kbp). In the described method, protein-DNA complexes were straightened and immobilized on a flat surface using surface tension. A fraction of the immobilized complexes displayed a sharp DNA bend with two DNA segments extending from the apex. The presence of DNA-binding proteins at the apex was verified by atomic force microscopy. The position of protein binding relative to the ends of the DNA molecule was determined by measuring the length of two DNA segments using fluorescence microscopy. We demonstrate the potential of the fluorescence-based method to localize protein-binding sites on the DNA template and to evaluate relative binding affinity. The proposed protein-binding-site mapping technique is simple and easy to perform. Practical applications include screening for DNA-binding proteins and the localization of protein-binding sites on large segments of DNA.

Comparative mapping of the region of human chromosome 7 deleted in williams syndrome.

Williams syndrome (WS) is a complex developmental disorder resulting from the deletion of a large (approximately 1.5-2 Mb) segment of human chromosome 7q11.23. Physical mapping studies have revealed that this deleted region, which contains a number of known genes, is flanked by several large, nearly identical blocks of DNA. The presence of such highly related DNA segments in close physical proximity to one another has hampered efforts to elucidate the precise long-range organization of this segment of chromosome 7. To gain insight about the structure and evolutionary origins of this important and complex genomic region, we have constructed a fully contiguous bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) contig map encompassing the corresponding region on mouse chromosome 5. In contrast to the difficulties encountered in constructing a clone-based physical map of the human WS region, the BAC/PAC-based map of the mouse WS region was straightforward to construct, with no evidence of large duplicated segments, such as those encountered in the human WS region. To confirm this difference, representative human and mouse BACs were used as probes for performing fluorescence in situ hybridization (FISH) to metaphase and interphase chromosomes. Human BACs derived from the nonunique portion of the WS region hybridized to multiple, closely spaced regions on human chromosome 7q11.23. In contrast, corresponding mouse BACs hybridized to a single site on mouse chromosome 5. Furthermore, FISH analysis revealed the presence of duplicated segments within the WS region of various nonhuman primates (chimpanzee, gorilla, orangutan, and gibbon). Hybridization was also noted at the genomic locations corresponding to human chromosome 7p22 and 7q22 in human, chimpanzee, and gorilla, but not in the other animal species examined. Together, these results indicate that the WS region is associated with large, duplicated blocks of DNA on human chromosome 7q11.23 as well as the corresponding genomic regions of other nonhuman primates. However, such duplications are not present in the mouse.