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Paul Denny

Publications and source records attributed to Paul Denny.

14 recordsLinked to original sources
PubMed2005

Detection of picomolar levels of interleukin-8 in human saliva by SPR.

Researchers at UCLA have discovered that the levels of interleukin-8 (IL-8) protein in the saliva of healthy individuals and patients with oropharyngeal squamous cell carcinoma (OSCC) are 30 pM and 86 pM, respectively. In this study, we present the development of the first immunoassay for the quantification of picomolar IL-8 concentrations in human saliva using Biacore surface plasmon resonance (SPR) in a microfluidic channel. A sandwich assay using two monoclonal antibodies, which recognize different epitopes on the antigen (IL-8), was used. Only 13 minutes were required to determine the quantity of pure IL-8 added to just 100 microL of either buffer or saliva-based samples. The limit of detection (LOD) of this immunoassay in buffer was 2.5 pM, and the precision of the response for each concentration was <3% of the coefficient of variation. When first analyzing the saliva supernatants, non-specific binding to the surface was observed. By adding carboxymethyl dextran sodium salt (10 mg mL(-1)) to compete with the surface dextran and primary antibody for non-specific interactions, the signal to noise ratio was greatly improved. The LOD of this immunoassay in saliva was 184 pM. A minimum concentration of 250 pM of exogenous IL-8 could then be consistently detected in a salivary environment. The precision of the response for each IL-8 concentration tested was <7% of the coefficient of variation. Diagnostic sensitivity for oral cancer can be achieved by pre-concentrating the saliva samples 10 fold prior to SPR analysis, making the target levels of IL-8 300 pM for healthy individuals and 860 pM for oral cancer patients.

Animals↗
PubMed2005

Dissecting the genetic complexity of human 6p deletion syndromes by using a region-specific, phenotype-driven mouse screen.

Monosomy of the human chromosome 6p terminal region results in a variety of congenital malformations that include brain, craniofacial, and organogenesis abnormalities. To examine the genetic basis of these phenotypes, we have carried out an unbiased functional analysis of the syntenic region of the mouse genome (proximal Mmu13). A genetic screen for recessive mutations in this region recovered thirteen lines with phenotypes relevant to a variety of clinical conditions. These include two loci that cause holoprosencephaly, two that underlie anophthalmia, one of which also contributes to other craniofacial abnormalities such as microcephaly, agnathia, and palatogenesis defects, and one locus responsible for developmental heart and kidney defects. Analysis of heterozygous carriers of these mutations shows that a high proportion of these loci manifest with behavioral activity and sensorimotor deficits in the heterozygous state. This finding argues for the systematic, reciprocal phenotypic assessment of dominant and recessive mouse mutants. In addition to providing a resource of single gene mutants that model 6p-associated disorders, the work reveals unsuspected genetic complexity at this region. In particular, many of the phenotypes associated with 6p deletions can be elicited by mutation in one of a number of genes. This finding implies that phenotypes associated with contiguous gene deletion syndromes can result not only from dosage sensitivity of one gene in the region but also from the combined effect of monosomy for multiple genes that function within the same biological process.

Animals↗
PubMed2005

Regulatory variation at glypican-3 underlies a major growth QTL in mice.

The genetic basis of variation in complex traits remains poorly understood, and few genes underlying variation have been identified. Previous work identified a quantitative trait locus (QTL) responsible for much of the response to selection on growth in mice, effecting a change in body mass of approximately 20%. By fine-mapping, we have resolved the location of this QTL to a 660-kb region containing only two genes of known function, Gpc3 and Gpc4, and two other putative genes of unknown function. There are no non-synonymous polymorphisms in any of these genes, indicating that the QTL affects gene regulation. Mice carrying the high-growth QTL allele have approximately 15% lower Gpc3 mRNA expression in kidney and liver, whereas expression differences at Gpc4 are non-significant. Expression profiles of the two other genes within the region are inconsistent with a factor responsible for a general effect on growth. Polymorphisms in the 3' untranslated region of Gpc3 are strong candidates for the causal sequence variation. Gpc3 loss-of-function mutations in humans and mice cause overgrowth and developmental abnormalities. However, no deleterious side-effects were detected in our mice, indicating that genes involved in Mendelian diseases also contribute to complex trait variation. Furthermore, these findings show that small changes in gene expression can have substantial phenotypic effects.

Aging↗
PubMed2005

Direct transport across the plasma membrane of mammalian cells of Leishmania HASPB as revealed by a CHO export mutant.

Leishmania HASPB is a lipoprotein that is exported to the extracellular space from both Leishmania parasites and mammalian cells via an unconventional secretory pathway. Exported HASPB remains anchored in the outer leaflet of the plasma membrane mediated by myristate and palmitate residues covalently attached to the N-terminal SH4 domain of HASPB. HASPB targeting to the plasma membrane depends on SH4 acylation that occurs at intracellular membranes. How acylated HASPB is targeted to the plasma membrane and, in particular, the subcellular site of HASPB membrane translocation is unknown. In order to address this issue, we screened for clonal CHO mutants that are incapable of exporting HASPB. A detailed characterization of such a CHO mutant cell line revealed that the expression level of the HASPB reporter molecule is unchanged compared to CHO wild-type cells; that it is both myristoylated and palmitoylated; and that it is mainly localized to the plasma membrane as judged by confocal microscopy and subcellular fractionation. However, based on a quantitative flow cytometry assay and a biochemical biotinylation assay of surface proteins, HASPB transport to the outer leaflet of the plasma membrane is largely reduced in this mutant. From these data, we conclude that the subcellular site of HASPB membrane translocation is the plasma membrane as the reporter molecule accumulates in this location when export is blocked. Thus, these results allow us to define a two-step process of HASPB cell surface biogenesis in which SH4 acylation of HASPB firstly mediates intracellular targeting to the plasma membrane. In a second step, the plasma membrane-resident machinery, which is apparently disrupted in the CHO mutant cell line, mediates membrane translocation of HASPB. Intriguingly, the angiogenic growth factor FGF-2, another protein secreted by unconventional means, is shown to be secreted normally from the HASPB export mutant cell line. These observations demonstrate that the export machinery component defective in the export mutant cell line functions specifically in the HASPB export pathway.

Acylation↗
PubMed2004

Organization and evolution of a gene-rich region of the mouse genome: a 12.7-Mb region deleted in the Del(13)Svea36H mouse.

Del(13)Svea36H (Del36H) is a deletion of approximately 20% of mouse chromosome 13 showing conserved synteny with human chromosome 6p22.1-6p22.3/6p25. The human region is lost in some deletion syndromes and is the site of several disease loci. Heterozygous Del36H mice show numerous phenotypes and may model aspects of human genetic disease. We describe 12.7 Mb of finished, annotated sequence from Del36H. Del36H has a higher gene density than the draft mouse genome, reflecting high local densities of three gene families (vomeronasal receptors, serpins, and prolactins) which are greatly expanded relative to human. Transposable elements are concentrated near these gene families. We therefore suggest that their neighborhoods are gene factories, regions of frequent recombination in which gene duplication is more frequent. The gene families show different proportions of pseudogenes, likely reflecting different strengths of purifying selection and/or gene conversion. They are also associated with relatively low simple sequence concentrations, which vary across the region with a periodicity of approximately 5 Mb. Del36H contains numerous evolutionarily conserved regions (ECRs). Many lie in noncoding regions, are detectable in species as distant as Ciona intestinalis, and therefore are candidate regulatory sequences. This analysis will facilitate functional genomic analysis of Del36H and provides insights into mouse genome evolution.

Animals↗
PubMed2004

Interleukin 6 and interleukin 8 as potential biomarkers for oral cavity and oropharyngeal squamous cell carcinoma.

BACKGROUND: Since morbidity and mortality rates due to oral cavity and oropharyngeal squamous cell carcinoma (OSCC) have improved little in the past 30 years, early detection or prevention of this disease is likely to be most effective. Using laser-capture microdissection, we have identified the expression of 2 cellular genes that are uniquely associated with OSCC: interleukin (IL) 6 and IL-8. These cytokines may contribute to the pathogenesis of this disease, and have been linked with increased tumor growth and metastasis. OBJECTIVES: To investigate whether IL-6 and/or IL-8 could serve as informative biomarkers for OSCC in saliva and/or serum and to determine if there is a role for saliva as a diagnostic medium for OSCC. PATIENTS AND METHODS: Patients with newly diagnosed T1 or T2 oral cavity or oropharyngeal histologically confirmed squamous cell carcinoma were recruited for the study. Age and sex-matched disease-free subjects were used as controls. Using quantitative real-time polymerase chain reaction analysis and enzyme-linked immunosorbent assay, we respectively assessed the expression of IL-6 and IL-8 in serum (controls, n = 32; patients with OSCC, n = 19) and saliva (controls, n = 32; patients with OSCC, n = 32) at the messenger RNA (mRNA) and protein levels. MAIN OUTCOME MEASURES: Specificity and sensitivity of these biomarkers for OSCC and their predictive value. RESULTS: Interleukin 8 was detected at higher concentrations in saliva (P<.01) and IL-6 was detected at higher concentrations in serum of patients with OSCC (P<.01). We confirmed these results at both the mRNA and the protein levels, and the results were concordant. The concentration of IL-8 in saliva and IL-6 in serum did not appear to be associated with sex, age, or alcohol or tobacco use (P>.75). Using statistical analysis, we were able to determine the threshold value, sensitivity, and specificity of each biomarker, as well as a combination of biomarkers, for detecting OSCC. CONCLUSIONS: Our findings indicate that IL-8 in saliva and IL-6 in serum hold promise as biomarkers for OSCC. A saliva-based test could be a cost-effective adjunctive tool in the diagnosis and follow-up of patients with OSCC.

Adult↗
PubMed2004

Differentially expressed protein markers in human submandibular and sublingual secretions.

Proteome analysis of secretions from individual salivary glands is important for understanding the health of the oral cavity and pathogenesis of certain diseases. However, cross-contamination of submandibular (SM) and sublingual (SL) glandular secretions can occur. The close anatomic relationship of the SM and SL ductal orifices can lead to such contamination. Additionally, these glands may share common ducts. To insure the purity of SM/SL secretions for proteomic analysis, it is important to develop unique biomarkers which could be used to verify the integrity of the individual glandular saliva. In this study, a proteomics approach based on mass spectrometry and gel electrophoresis techniques was utilized to identify and verify a set of proteins (cystatin C, calgranulin B and MUC5B mucin), which are differentially expressed in SM/SL secretions. SM/SL fluids were obtained from nine healthy subjects. Cystatin C was found to be an SM-selective protein as it was found in all SM fluids but not detected in two SL fluids. MUC5B mucin and calgranulin B, on the other hand, were found to be SL-selective proteins. All SL samples contained MUC5B mucin, whereas MUC5B mucin was not detected in four SM samples. Eight of the SL samples contained calgranulin B; however, calgranulin B was absent in eight SM samples. This set of protein markers, especially calgranulin B, can be used to determine the purity of SM/SL samples, and therefore identify potential individuals who do not exhibit cross-contaminated SM/SL secretions, an important requirement for subsequent proteome analysis of pure SM and SL secretions.

Adult↗
PubMed2003

Candidate testis-determining gene, Maestro (Mro), encodes a novel HEAT repeat protein.

Mammalian sex determination depends on the presence or absence of SRY transcripts in the embryonic gonad. Expression of SRY initiates a pathway of gene expression resulting in testis development. Here, we describe a novel gene potentially functioning in this pathway using a cDNA microarray screen for genes exhibiting sexually dimorphic expression during murine gonad development. Maestro (Mro) transcripts are first detected in the developing male gonad before overt testis differentiation. By 12.5 days postcoitus (dpc), Mro transcription is restricted to the developing testis cords and its expression is not germ cell-dependent. No expression is observed in female gonads between 10.5 and 14.5 dpc. Maestro encodes a protein containing HEAT-like repeats that localizes to the nucleolus in cell transfection assays. Maestro maps to a region of mouse chromosome 18 containing a genetic modifier of XX sex reversal. We discuss the possible function of Maestro in light of these data.

Amino Acid Sequence↗
PubMed2003

A comprehensive transcript map of the mouse Gnas imprinted complex.

The recent publication of the FANTOM mouse transcriptome has provided a unique opportunity to study the diversity of transcripts arising from a single gene locus. We have focused on the Gnas complex, as imprinting loci themselves provide unique insights into transcriptional regulation. Thirteen full-length cDNAs from the FANTOM2 set were mapped to the Gnas locus. These represented one previously described transcript and 12 putative new transcripts. Of these, eight were found to be differentially expressed from either the maternal or paternal allele. Two clones extended Nespas in the 3' direction, providing evidence of antisense transcription spanning a 30-kb genomic region from a single allele. The transcripts were summarized into six transcriptional units, Nespas, Nesp, Gnasxl, F7, exon 1A, and Gnas. The resolution of the Gnas transcript map by the FANTOM2 clones revealed a pattern of alternate splicing. In addition to the transcripts described previously as splicing onto exon 2 of Gnas, each new sense transcript had an alternate short 3'UTR independent of Gnas. Both spliced and unspliced variants of the new imprinted sense transcripts were found. Whereas the functional significance of these alternate transcripts is not known, the availability of the FANTOM clones has provided remarkable insights into the repertoire of transcripts in the Gnas complex locus.

Animals↗
PubMed2002

Comparison of human chromosome 6p25 with mouse chromosome 13 reveals a greatly expanded ov-serpin gene repertoire in the mouse.

Ov-serpins are intracellular proteinase inhibitors implicated in the regulation of tumor progression, inflammation, and cell death. The 13 human ov-serpin genes are clustered at 6p25 (3 genes) and 18q21 (10 genes), and share common structures. We show here that a 1-Mb region on mouse chromosome 13 contains at least 15 ov-serpin genes compared with the three ov-serpin genes within 0.35 Mb at human 6p25 (SERPINB1 (MNEI), SERPINB6 (PI-6), SER-PINB9 (PI-9)). The mouse serpins have characteristics of functional inhibitors and fall into three groups on the basis of similarity to MNEI, PI-6, or PI-9. The genes map between the mouse orthologs of the Werner helicase interacting protein and NAD(P)H menadioine oxidoreductase 2 genes, in a region that contains the markers D13Mit136 and D13Mit116. They have the seven-exon structure typical of human 6p25 ov-serpin genes, with identical intron phasing. Most show restricted patterns of expression, with common sites of synthesis being the placenta and immune tissue. Compared with human, this larger mouse serpin repertoire probably reflects the need to regulate a larger proteinase repertoire arising from differing evolutionary pressures on the reproductive and immune systems.

Amino Acid Sequence↗
PubMed2002

Saliva: a fountain of opportunity.

Saliva continues to demonstrate that it is more complex than generally perceived and has more diagnostic value than is generally appreciated. This article will review some of the components and functions of saliva; discuss its promise as a diagnostic aid; review some of the problems associated with inadequate salivary function; and, it is hoped, enhance oral health care providers' appreciation of the importance of saliva in everyday clinical practice.

Diagnosis↗