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Biomedical subjects

K E Murphy

Publications and source records attributed to K E Murphy.

At least 19 recordsLinked to original sources
PubMed2006

On the certification of cadmium at trace and ultratrace levels in standard reference materials using ID ICP-MS.

Analytical methods used for the isotope dilution inductively coupled plasma mass spectrometric (ID-ICP-MS) measurement of Cd at microg kg(-1) and sub-microg kg(-1) levels are described and applied to the certification of new dietary supplement, blood, and serum Standard Reference Materials (SRMs). The materials are: SRM 3240 Ephedra sinica Stapf Aerial Parts, SRM 3241 Ephedra sinica Stapf Native Extract, SRM 3243 Ephedra-Containing Solid Oral Dosage Form, SRM 3244 Ephedra-Containing Protein Powder, SRM 966 Toxic Metals in Bovine Blood, Level 1 (L1) and Level 2 (L2), and SRM 1598a Animal Serum. The concentration of Cd in the materials ranges from 120 microg kg(-1) down to 0.03 microg kg(-1). At these levels, the factors that most influence the accuracy of the ICP-MS data are the procedure blank and spectral and nonspectral interferences. Nonspectral interference, caused by the high concentration of dissolved solids in the matrices investigated, resulted in signal suppression. Matrix separation was used to enhance signal intensity and to reduce spectral interference for the accurate determination of Cd in SRM 1598a and SRM 3244. Chromatographic separation procedures using Chelex for SRM 1598a and anion exchange for SRM 3244 were optimized to achieve the desired separation characteristics without substantially increasing the procedure blank. Sensitivity for the determination of Cd in serum was additionally enhanced through the use of desolvation nebulization. We determined that separations were not required for the accurate ICP-MS determination of Cd in SRM 3240, SRM 3241, SRM 3243, and SRM 966 L2 under optimized analysis conditions. These samples were diluted to a minimum volume and introduced to the ICP-MS via low flow (40-100 microL/min) microconcentric nebulizers. SRM 966 L1 was also analyzed directly, but results were highly variable. The ID-ICP-MS sample preparation and ratio measurement protocols described here resulted in total expanded uncertainties of less than 1% for the determination of 90.85 microg kg(-1) Cd in SRM 3240, and less than 10% total expanded uncertainty for the determination of 0.0468 microg kg(-1) Cd in SRM 1598a.

Animals↗
PubMed2004

Assignment of canine MSS1 microsatellite markers to chromosomes by linkage data.

Recent advances in mapping the canine genome have led to an increase in the number of linkage studies aimed at dissecting the genetic causes of many hereditary diseases that affect the domestic dog. The first step in developing molecular tools for a whole genome scan was the characterization of a set of microsatellite markers, termed minimal screening set 1 (MSS1), that provided an estimated coverage of 10 cM. A limiting factor in use of the MSS1 is not all of the 172 MSS1 markers have been localized to specific chromosomes. Seventy-five of the markers were positioned on a total of 15 chromosomes with the original publication of the MSS1. The localization based on linkage data of 14 additional MSS1 markers to chromosomes using CRIMAP v. 2.4 to build a linkage map of 113 MSS1 markers that were polymorphic in a kindred of Dalmatians is reported here.

Animals↗
PubMed2004

Heritability and segregation analysis of deafness in U.S. Dalmatians.

Hereditary loss of hearing affects many breeds of the domestic dog, but the Dalmatian has the highest prevalence. Approximately 30% are affected in the United States (U.S.) population. It is widely accepted that a relationship exists between deafness and pigmentation in the dog and also in other animals. While the Dalmatian exemplifies this relationship, the genetic origin and mode of inheritance of deafness in this breed are unknown. The goals of this study were to: (1) estimate the heritability of deafness in an extended kindred of U.S. Dalmatians and (2) determine, through complex segregation analysis, whether there is a major segregating locus that has a large effect on the expression of deafness. A kindred of 266 Dalmatians was assembled, of which 199 had been diagnosed using the brainstem auditory evoked response to determine auditory status. Of these, 74.4% (N = 148) had normal hearing, 18.1% (N = 36) were unilaterally deaf, and 7.5% (N = 15) were bilaterally deaf. A heritability of 0.73 was estimated considering deafness a dichotomous trait and 0.75 considering it as a trichotomous trait. Although deafness in the Dalmatian is clearly heritable, the evidence for the presence of a single major gene affecting the disorder is not persuasive.

Animals↗
PubMed2003

Digging up the canine genome--a tale to wag about.

There is incredible morphological and behavioral diversity among the hundreds of breeds of the domestic dog, CANIS FAMILIARIS. Many of these breeds have come into existence within the last few hundred years. While there are obvious phenotypic differences among breeds, there is marked interbreed genetic homogeneity. Thus, study of canine genetics and genomics is of importance to comparative genomics, evolutionary biology and study of human hereditary diseases. The most recent version of the map of the canine genome is comprised of 3,270 markers mapped to 3,021 unique positions with an average intermarker distance of approximately 1 Mb. The markers include approximately 1,600 microsatellite markers, about 1,000 gene-based markers, and almost 700 bacterial artificial chromosome-end markers. Importantly, integration of radiation hybrid and linkage maps has greatly enhanced the utility of the map. Additionally, mapping the genome has led directly to characterization of microsatellite markers ideal for whole genome linkage scans. Thus, workers are now able to exploit the canine genome for a wide variety of genetic studies. Finally, the decision to sequence the canine genome highlights the dog's evolutionary and physiologic position between the mouse and human and its importance as a model for study of mammalian genetics and human hereditary diseases.

Animals↗
PubMed2003

Identification of isoforms and RH mapping of canine KIT.

The proto-oncogene, C-KIT (KIT), encodes a tyrosine kinase receptor, and mutations in this gene are causative for several mammalian diseases, including cancer and a form of pigmentation-associated hereditary deafness. Our laboratories are interested in a form of hereditary deafness that is associated with abnormalities in pigmentation and is common in the Dalmatian. Thus, KIT is being analyzed as a candidate gene for deafness in this breed. In addition to our interest in deafness, we are involved in mapping gene loci in the canine genome. Reported here is the identification of two isoforms of canine C-kit and radiation hybrid mapping of KIT to CFA13.

Animals↗
PubMed2001

Unique subpopulations of CD56+ NK and NK-T peripheral blood lymphocytes identified by chemokine receptor expression repertoire.

CD56, an adhesion molecule closely related to neural cell adhesion molecule, is an immunophenotypic marker for several unique populations of PBLS: Although CD56(+) cells derive from multiple lymphocyte lineages, they share a role in immunosurveillance and antitumor responses. We have studied the chemokine receptor expression patterns and functional migratory responses of three distinct CD56(+) populations from human peripheral blood. NK-T cells were found to differ greatly from NK cells, and CD16(+) NK cells from CD16(-) NK cells. CD16(+) NK cells were the predominant population responding to IL-8 and fractalkine, whereas NK-T cells were the predominant population responding to the CCR5 ligand macrophage-inflammatory protein-1beta. CD16(-) NK cells were the only CD56(+) population that uniformly expressed trafficking molecules necessary for homing into secondary lymphoid organs through high endothelial venule. These findings describe a diverse population of cells that may have trafficking patterns entirely different from each other, and from other lymphocyte types.

Adult↗
PubMed2001

Expression of chemokine receptors by lung T cells from normal and asthmatic subjects.

The lung is an important tertiary lymphoid organ with constant trafficking of T cells through the lung in both health and disease. T cell migration is controlled by a combination of adhesion receptors and chemokines expressed on vascular endothelium and in the tissue, often in an organ-specific manner. This leads to selective accumulation of different T cell subsets, a process called lymphocyte homing. There is evidence for a distinct lung-homing pathway, but no specific lung-homing receptors have been described. We analyzed the chemokine receptor profile of lung T cells to determine the extent to which lung T cells shared homing pathways with other organs such as the gut and skin. In addition, we compared expression of receptors in normal and asthmatic individuals to determine whether different pathways were used in health and disease. We observed that lung T cells expressed a profile of chemokine and adhesion receptors distinct from that of gut- and skin-homing T cells although no chemokine receptor specific for the lung was found. In particular, lung T cells expressed CCR5 and CXCR3, but not CCR9 or cutaneous lymphocyte Ag, and only low levels of CCR4 and alpha(4)beta(7). No differences were observed between lung T cells from normal vs asthmatic subjects. This study provides added support for the concept of a lung-homing pathway separate from other mucosal organs such as the gut and suggests that the chemokine pathways that control T cell migration in normal homeostasis and Th2-type inflammatory responses are similar.

Antigens, Differentiation, T-Lymphocyte↗
PubMed2001

CCR7 expression and memory T cell diversity in humans.

CCR7, along with L-selectin and LFA-1, mediates homing of T cells to secondary lymphoid organs via high endothelial venules (HEV). CCR7 has also been implicated in microenvironmental positioning of lymphocytes within secondary lymphoid organs and in return of lymphocytes and dendritic cells to the lymph after passage through nonlymphoid tissues. We have generated mAbs to human CCR7, whose specificities correlate with functional migration of lymphocyte subsets to known CCR7 ligands. We find that CCR7 is expressed on the vast majority of peripheral blood T cells, including most cells that express adhesion molecules (cutaneous lymphocyte Ag alpha(4)beta(7) integrin) required for homing to nonlymphoid tissues. A subset of CD27(neg) memory CD4 T cells from human peripheral blood is greatly enriched in the CCR7(neg) population, as well as L-selectin(neg) cells, suggesting that these cells are incapable of homing to secondary lymphoid organs. Accordingly, CD27(neg) T cells are rare within tonsil, a representative secondary lymphoid organ. All resting T cells within secondary lymphoid organs express high levels of CCR7, but many activated cells lack CCR7. CCR7 loss in activated CD4 cells accompanies CXC chemokine receptor (CXCR)5 gain, suggesting that the reciprocal expression of these two receptors may contribute to differential positioning of resting vs activated cells within the organ. Lymphocytes isolated from nonlymphoid tissues (such as skin, lung, or intestine) contain many CD27(neg) cells lacking CCR7. The ratio of CD27(neg)/CCR7(neg) cells to CD27(pos)/CCR7(pos) cells varies from tissue to tissue, and may correlate with the number of cells actively engaged in Ag recognition within a given tissue.

Biomarkers↗
PubMed2001

Determination of As, Cd, Cr, and Hg in SRM 2584 (trace elements in indoor dust) by high-resolution inductively coupled plasma mass spectrometry.

Standard reference material (SRM) 2584 (Trace Elements in Indoor Dust) was developed as a reference standard for evaluating field methods and for validating laboratory and reference methods for the assessment of lead contamination and exposure. In addition to lead, the toxic trace elements As, Cd, Cr, and Hg, at approximately 17, 10, 140, and 5 microg g(-1), respectively, have been certified in the SRM. These four analytes were successfully determined by use of high-resolution inductively coupled plasma mass spectrometry (HR-ICP-MS). Isobaric interferences at masses of As and Cr were resolved by using the high resolution mode (nominal 8000) and the medium resolution mode (nominal 3000), respectively, of the instrument. The effects of a significant drift in analyte sensitivity in the course of measurement were rectified by use of internal standardization, single spike standard addition, and an optimized analysis sequence. The results were compared with those obtained by instrumental neutron activation analysis (INAA) and isotope dilution inductively coupled plasma mass spectrometry (ID-ICP-MS). The results for the quality control, SRM 2704 (Buffalo River Sediment), were in good agreement with the certified values, indicated by the uncertainty intervals of the measured values overlapping the certified intervals at 95% confidence level.

Arsenic↗
PubMed2001

Cloning, sequence analysis and radiation hybrid mapping of a mammalian KRT2p gene.

We report here on the cloning, characterization and radiation hybrid mapping of the canine basic keratin gene KRT2p. The gene spans 8.3 kb, consists of nine exons and eight introns, and is characterized by the typical features of both basic keratins and keratins in general, including glycine-rich head and tail domains, which flank an alpha-helical rod domain of approximately 310 amino acids. Comparisons of sequence and structure reveal that canine KRT2p is strikingly similar to human KRT2p. Alignment of the predicted amino acid sequences for human and dog reveals greater than 80% identity. In the rod domain, the amino acid identity exceeds 90%. We note, however, that canine KRT2p encodes a protein 21 residues longer than human K2p due to the insertion of a glycine repeat motif, GG(G)X, in the head and tail domains of the canine gene. This is the first report of the nearly complete genome sequence for KRT2p of any organism. Radiation hybrid mapping of canine KRT2p to chromosome 27 of the dog is also reported.

Amino Acid Sequence↗
PubMed2001

DNA sequence and physical mapping of the canine transglutaminase 1 gene.

The transglutaminase 1 gene (TGM1) encodes an enzyme necessary for cross-linking the structural proteins that form the cornified envelope, an essential component of the outermost layer of the skin, the stratum corneum. Reported here is the complete coding region of canine TGM1, its chromosome localization, and its map position in the integrated canine linkage-radiation hybrid map. Canine TGM1 consists of 2,448 nucleotides distributed over 15 exons. The nucleotide sequence has 90% identity to human TGM1. The deduced canine TGM1 protein is 816 amino acids long and is 92% identical to human TGM1. Using fluorescence in situ hybridization, we localized canine TGM1 to dog (Canis familiaris) chromosome 8 (CFA 8q). Canine TGM1 localized to CFA 8 on the integrated linkage-radiation hybrid map in the interval FH2149-MYH7. Characterizing the coding region of canine TGM1 is a first step in examining the role of this enzyme in normal and defective cornification in the dog.

Amino Acid Sequence↗
PubMed2000

Isotope dilution inductively coupled plasma mass spectrometry (ID ICP-MS) for the certification of lead and cadmium in environmental standard reference materials.

Lead (Pb) and cadmium (Cd) have been determined in six new environmental standard reference materials (SRMs) using isotope dilution inductively coupled plasma mass spectrometry (ID ICP-MS). The SRMs are the following: SRM 1944, New York-New Jersey Waterway Sediment, SRMs 2583 and 2584, Trace Elements in Indoor Dust, Nominal 90 mg/kg and 10,000 mg/kg Lead, respectively, SRMs 2586 and 2587, Trace Elements in Soil Containing Lead from Paint, Nominal 500 mg/kg and 3,000 mg/kg Lead, respectively, and SRM 2782, Industrial Sludge. The capabilities of ID ICP-MS for the certification of Pb and Cd in these materials are assessed. Sample preparation and ratio measurement uncertainties have been evaluated. Reproducibility and accuracy of the established procedures are demonstrated by determination of gravimetrically prepared primary standard solutions and by comparison with isotope dilution thermal ionization mass spectrometry (ID TIMS). Material heterogeneity was readily demonstrated to be the dominant source of uncertainty in the certified values.

Cadmium↗
PubMed2000

Isolation and characterization of a cDNA clone coding for a glutathione S-transferase class delta enzyme from the biting midge Culicoides variipennis sonorensis Wirth and Jones.

Culicoides variipennis sonorensis is the primary vector of bluetongue viruses in North America. Glutathione S-transferases (GSTs) are enzymes that catalyze nucleophilic substitutions, converting reactive lipophilic molecules into soluble conjugates. Increased GST activity is associated with development of insecticide resistance. Described here is the isolation of the first cDNA encoding a C. variipennis GST. The clone consists of 720 translated bases encoding a protein with a M(r) of approximately 24,800 composed of 219 amino acids. The deduced amino acid sequence is similar (64%-74%) to class Delta (previously named Theta) GSTs from the dipteran genera Musca, Drosophila, Lucilia and Anopheles. The cDNA was subcloned into pET-11b, expressed in Epicurian coli BL21 (DE3) and has a specific activity of approximately 28,000 units/mg for the substrate 1-chloro-2,4-dinitrobenzene.

Amino Acid Sequence↗
PubMed2000

Cloning and characterisation of ITGAV, the genomic sequence for human cell adhesion protein (vitronectin) receptor alpha subunit, CD51.

The integrin family of receptors serves as major receptors for extracellular matrix-mediated cell adhesion and migration, cytoskeletal organisation, cell proliferation, survival, and differentiation. The alpha-V integrins consist of a subset which share a common alpha-V subunit combined with one of five beta subunits (beta-1, 3, 5, 6, or 8). The alpha-V integrins have been implicated in a number of developmental processes, including vasculogenesis and angiogenesis, and are therapeutic targets for inhibition of angiogenesis and osteoporosis. The human cDNA for alpha-V integrin (ITGAV) consists of a 5,717-bp transcript with a coding sequence (CDS) of 3,146 bp encoding a 150-kDa mature peptide. Here we describe the gene structure of ITGAV.

Antigens, CD↗
PubMed2000

Identification and characterization of a cDNA clone encoding the heat shock protein (Hsp60) from the biting midge, Culicoides variipennis sonorensis Wirth and Jones.

A cDNA expression library constructed from Culicoides variipennis sonorensis was screened using an antibody specific for Hsp60 of Heliothis virescens. A single clone encoding the complete heat shock protein (Hsp60) of C. variipennis was identified and its 2400-bp insert was sequenced. The encoded 62-kDa protein contains 581 amino acids and includes a 26-amino acid putative mitochondrial targeting sequence at its N terminus and a GGM motif at its carboxyl terminus. Deduced amino acid sequences are highly similar (67-78%) to Hsp60 of other species, including the fruit fly, the house mouse, the Norwegian rat, the Chinese hamster, the human, a nematode, and the tobacco budworm moth. This is the initial isolation of a coding sequence for a stress-induced protein in C. variipennis.

Amino Acid Sequence↗
PubMed1999

Identification of a novel aspartic protease (Asp 2) as beta-secretase.

The Alzheimer's disease beta-amyloid peptide (Abeta) is produced by excision from the type 1 integral membrane glycoprotein amyloid precursor protein (APP) by the sequential actions of beta- and then gamma-secretases. Here we report that Asp 2, a novel transmembrane aspartic protease, has the key activities expected of beta-secretase. Transient expression of Asp 2 in cells expressing APP causes an increase in the secretion of the N-terminal fragment of APP and an increase in the cell-associated C-terminal beta-secretase APP fragment. Mutation of either of the putative catalytic aspartyl residues in Asp 2 abrogates the production of the fragments characteristic of cleavage at the beta-secretase site. The enzyme is present in normal and Alzheimer's disease (AD) brain and is also found in cell lines known to produce Abeta. Asp 2 localizes to the Golgi/endoplasmic reticulum in transfected cells and shows clear colocalization with APP in cells stably expressing the 751-amino-acid isoform of APP.

Alzheimer Disease↗
PubMed1999

Chromosomal localization of acidic and basic keratin genes of the domestic dog.

Our laboratories are interested in characterizing genes involved in the myriad of heritable diseases affecting the domestic dog, Canis lupus familiaris, and in development of detailed genetic and physical maps of the canine genome. Included in these efforts is examination of conservation of the genetic organization, structure, and function of gene families involved in diseases of the canine skin, skeleton, and eye. To that end, study of the highly conserved keratin gene family was undertaken. Keratins belong to the superfamily of intermediate filaments and are the major structural proteins of the epidermis, hair, and nail. The keratins are highly conserved throughout vertebrate evolution both at the DNA and amino acid sequence levels. Mutations in genes encoding epithelial keratins are known to cause various diseases in humans, and similar histopathological presentations have been reported in the dog. The keratins are divided into two groups, type I (acidic) and type II (basic). In the human, the genes encoding the acidic and basic keratins are clustered on Chrs 17 and 12, respectively. The same genetic arrangement is seen in the mouse with the acidic and basic keratin gene clusters found on Chrs 11 and 15, respectively. Reported here are the chromosomal localization of acidic and basic canine keratin genes as well as supportive sequence data. Fluorescence in situ hybridization (FISH) experiments with clones isolated from a canine genomic library suggest that the acidic keratin gene cluster resides on CFA9 and the basic keratin gene cluster is located on CFA27.

Amino Acid Sequence↗