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Biomedical subjects

K J Dunn

Publications and source records attributed to K J Dunn.

At least 19 recordsLinked to original sources
PubMed2000

Spatially restricted hypopigmentation associated with an Ednrbs-modifying locus on mouse chromosome 10.

We have used the varied expressivity of white spotting (hypopigmentation) observed in intrasubspecific crosses of Ednrb(s) mice (Mayer Ednrb(s)/Ednrb(s) and C3HeB/FeJ Ednrb(s)/Ednrb(s)) to analyze the effects of modifier loci on the patterning of hypopigmentation. We have confirmed that an Ednrb(s) modifier locus is present on mouse Chromosome 10. This locus is now termed k10, using the nomenclature established by Dunn in 1920. The k10(Mayer) allele is a recessive modifier that accounts for almost all of the genetic variance of dorsal hypopigmentation. Using intercross analyses we identified a second allele of this locus or a closely linked gene termed k10(C3H). The k10(C3H) allele is semidominant and is associated with the penetrance and expressivity of a white forelock phenotype similar to that seen in Waardenburg syndrome. Molecular linkage analysis was used to determine that the k10 critical interval was flanked by D10Mit10 and D10Mit162/D10Mit122 and cosegregates with mast cell growth factor (Mgf). Complementation crosses with a Mgf(Sl) allele (a 3-5-cM deletion) confirm the semidominant white forelock feature of the k10(C3H) allele and the dorsal spotting feature of K10(Mayer) allele. MgF was assessed as a candidate gene for k10(Mayer) and k10(C3H) by sequence and genomic analyses. No molecular differences were observed between the Mayer and C57BL/6J alleles of MgF; however, extensive genomic differences were observed between the C3HeB/FeJ and C57BL/6J alleles. This suggests that alteration of MgF expression in C3H mice may account for the k10(C3H) action on white forelock hypopigmentation. Crosses of Ednrb(s) with Kit(WJ-2) (the receptor for MGF)-deficient mice confirmed the hypothesis that synergistic interaction between the Endothelin and MGF signaling pathways regulates proper neural crest-derived melanocyte development in vivo.

Alleles↗
PubMed1999

Differential mechanisms of recognition and activation of interleukin-8 receptor subtypes.

We have probed an epitope sequence (His18-Pro19-Lys20-Phe21) in interleukin-8 (IL-8) by site-directed mutagenesis. This work shows that single and double Ala substitutions of His18 and Phe21 in IL-8 reduced up to 77-fold the binding affinity to IL-8 receptor subtypes A (CXCR1) and B (CXCR2) and to the Duffy antigen. These Ala mutants triggered neutrophil degranulation and induced calcium responses mediated by CXCR1 and CXCR2. Single Asp or Ser substitutions, H18D, F21D, F21S, and double substitutions, H18A/F21D, H18A/F21S, and H18D/F21D, reduced up to 431-fold the binding affinity to CXCR1, CXCR2, and the Duffy antigen. Interestingly, double mutants with charged residue substitutions failed to trigger degranulation or to induce wild-type calcium responses mediated by CXCR1. Except for the H18A and F21A mutants, all other IL-8 mutants failed to induce superoxide production in neutrophils. This study demonstrates that IL-8 recognizes and activates CXCR1, CXCR2, and the Duffy antigen by distinct mechanisms.

Antigens, CD↗
PubMed1999

The inversion of NMR log data sets with different measurement errors.

We present a composite-data processing method which simultaneously processes two or more data sets with different measurement errors. We examine the role of the noise level of the data in the singular value decomposition inversion process, the criteria for a proper cutoff, and its effect on the uncertainty of the solution. Examples of processed logs using the composite-data processing method are presented and discussed. The possible usefulness of the apparent T(1)/T(2) ratio extracted from the logs is illustrated.

Algorithms↗
PubMed1999

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

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

Amino Acid Sequence↗
PubMed1998

Binding of c-Raf1 kinase to a conserved acidic sequence within the carboxyl-terminal region of the HIV-1 Nef protein.

Nef is a membrane-associated cytoplasmic phosphoprotein that is well conserved among the different human (HIV-1 and HIV-2) and simian immunodeficiency viruses and has important roles in down-regulating the CD4 receptor and modulating T-cell signaling pathways. The ability to modulate T-cell signaling pathways suggests that Nef may physically interact with T-cell signaling proteins. In order to identify Nef binding proteins and map their site(s) of interaction, we targeted a highly conserved acidic sequence at the carboxyl-terminal region of Nef sharing striking similarity with an acidic sequence at the c-Raf1-binding site within the Ras effector region. Here, we used deletion and site-specific mutagenesis to generate mutant Nef proteins fused to bacterial glutathione S-transferase in in vitro precipitation assays and immunoblot analysis to map the specific interaction between the HIV-1LAI Nef and c-Raf1 to a conserved acidic sequence motif containing the core sequence Asp-Asp-X-X-X-Glu (position 174-179). Significantly, we demonstrate that substitution of the nonpolar glycine residue for either or both of the conserved negatively charged aspartic acid residues at positions 174 and 175 in the full-length recombinant Nef protein background completely abrogated binding of c-Raf1 in vitro. In addition, lysates from a permanent CEM T-cell line constitutively expressing the native HIV-1 Nef protein was used to coimmunoprecipitate a stable Nef-c-Raf1 complex, suggesting that molecular interactions between Nef and c-Raf1, an important downstream transducer of cell signaling through the c-Raf1-MAP kinase pathway, occur in vivo. This interaction may account for the Nef-induced perturbations of T-cell signaling and activation pathways in vitro and in vivo.

Amino Acid Sequence↗
PubMed1998

Hypocortisolaemia in a Labrador retriever.

A four-year-old Labrador retriever was presented with lethargy and exercise intolerance. Clinical examination was unremarkable. A subnormal cortisol response to adrenocorticotrophin hormone (ACTH) was demonstrated (plasma cortisol concentrations before and after administration of ACTH were both below the detection limit of the assay) but plasma aldosterone concentrations were within the normal range. Endogenous plasma ACTH concentrations were high, indicating primary adrenocortical disease. Following glucocorticoid supplementation at a replacement dose (prednisolone 0.1 mg/kg) the dog made a full clinical recovery.

Addison Disease↗
PubMed1998

Diagnostic investigations in 101 dogs with pyrexia of unknown origin.

Records from 101 dogs presented for investigation of unexplained pyrexia were reviewed. The most common diagnosis was immune-mediated disease (22 per cent of cases), with immune-mediated polyarthritis accounting for 20 per cent of all diagnoses. The frequency of positive results obtained in investigative tests was also assessed. Cytological and radiological examinations provided a high diagnostic success rate, although routine haematology and plasma biochemistry were also useful screening tests. On the basis of these results it is suggested that, in the investigation of unexplained pyrexia, a diagnosis of immune-mediated polyarthritis should be excluded before less common diagnoses are considered.

Animals↗
PubMed1997

ETS-1 induces increased expression of erythroid markers in the pluripotent erythroleukemic cell lines K562 and HEL.

Members of the ETS gene family are known to be expressed in hematopoietic tissues and cell lines, and there is increasing evidence that ETS proteins may play a role in normal hematopoietic cell development. We demonstrate that ETS-1 can contribute to the development of an erythroid phenotype in vitro. The pluripotent erythroleukemic K562 and HEL cell lines express messages for a number of ETS genes, but only c-ETS-1 levels are elevated in response to treatment with hemin or cytosine arabinofuranoside (Ara-C), agents which induce erythroid differentiation. Furthermore, ETS-1 antisense oligonucleotides inhibit hemoglobinization of cells treated with Ara-C or hemin, and K562 and HEL cells infected with retrovirus expressing the c-ETS-1 gene exhibit a significant increase in erythroid character (as indicated by benzidine staining for hemoglobin (Hb) and surface marker analysis), a dramatic increase in responsiveness to hemin or Ara-C, and a decreased rate of proliferation (20-40% of control rates). In contrast, infection with virus expressing ETS-2 or vector sequences only causes no detectable changes in the proliferation or erythroid character of either the HEL or K562 cell lines. These data indicate a role for ETS-1 in erythroid differentiation.

Cell Differentiation↗
PubMed1996

Rapid neurite formation in a human cortical neuronal cell line.

The subclone HCN-1 was derived from parental cell lines from cortical tissue of a patient with unilateral megalencephaly growth and immunochemistry staining characteristics [G. V. Ronnett et al. (1990) Human cortical neuronal cell line: establishment from a patient with unilateral megalencephaly. Science 248, 603-605]. As we and others have shown, HCN-1A cells can be induced to differentiate to a neuronal-like morphology. HCN-1A cells stain positively for neurofilament, neuron-specific enolase and the low-affinity neurotrophin receptors, p75NGFR, but not for myelin basic protein, S-100, or glial fibrillary acidic protein (GFAP). HCN-1A cells also stain positively for gamma-aminobutyric acid and glutamate. In the present study, we examine the effects of cell density on the requirements for efficient induction of differentiation of HCN-1A cells and analyze the time course of this induction and its reversion. We also characterize the changes in cytoskeletal proteins of HCN-1A cells in response to their differentiation neuronal phenotype.

3T3 Cells↗
PubMed1996

Self-diffusion in periodic porous media: a comparison of numerical simulation and eigenvalue methods.

Random walk computer simulations are an important tool in understanding magnetic resonance measurements in porous media. In this paper we focus on the description of pulsed field gradient spin echo (PGSE) experiments that measure the probability, P(R,t), that a diffusing water molecule will travel a distance R in a time t. Because PGSE simulations are often limited by statistical considerations, we will see that valuable insight can be gained by working with simple periodic geometries and comparing simulation data to the results of exact eigenvalue expansions. In this connection, our attention will be focused on (1) the wavevector, k, and time dependent magnetization, M(k, t); and (2) the normalized probability, Ps(delta R, t), that a diffusing particle will return to within delta R of the origin after time t.

Algorithms↗
PubMed1996

Magnetic field nonuniformities and NMR of protons diffusing in a porous medium.

Magnetic field inhomogeneity can arise either because of an externally applied field gradient or because of spatial variations in magnetic susceptibility. The latter are most important when the solid matrix includes paramagnetic substances and when the uniform applied field, and, consequently, also the Larmor precession frequency are very large. Both types of field inhomogeneity add extra phase shifts to the precessing spins. These phase shifts vary with time and position in a complex and random fashion as a result of the diffusive motion of the spins. We have studied these effects by performing detailed calculations for the case of a fluid filled porous medium with a periodic microstructure. Special attention was devoted to the question of whether the statistical distribution of the phase shifts encountered in a Hahn spin echo experiment or in a Carr-Purcell-Meiboom-Gill (CPMG) spin-echo train can be approximated as a Gaussian. The mean square phase shift is measured in such experiments as an enhanced relaxation rate of the precessing transverse magnetization. We determine this mean square phase shift for periodic composites from the diffusion eigenstates, which were calculated using a previously developed Fourier expansion method. The enhanced relaxation rate depends on the echo spacing time tau in a way that can be correlated with important length scales of the porous microstructure. Those correlations can be extended also to disordered microstructures, like the ones that are found in natural rocks. We compare these theoretically predicted correlations with CPMG measurements performed on protons in laboratory samples of brine saturated sandstone.

Diffusion↗
PubMed1995

Use of ACTH stimulation tests to monitor the treatment of canine hyperadrenocorticism.

The case histories of 60 dogs with hyperadrenocorticism were reviewed. Fifty-four of the dogs were treated with mitotane at a mean daily dose rate of 48.8 mg/kg (range 25.6 to 84 mg/kg) for between four and 21 days. The mean weekly maintenance dose of mitotane was 48.8 mg/kg. An adrenocorticotrophic hormone (ACTH) stimulation test was performed before the treatment began, and in 30 cases at the end of the induction course, and the response to ACTH was measured at regular intervals thereafter. Nine of the treated dogs developed complete hypoadrenocorticism during treatment and required permanent mineralocorticoid replacement therapy. Twelve of the dogs had normal responses to an ACTH stimulation test before treatment, and the diagnosis of hyperadrenocorticism was based on the result of a low-dose dexamethasone suppression test. These 12 dogs had consistently lower cortisol levels before and after stimulation with ACTH and four of them developed complete hypoadrenocorticism. In general the clinical signs were well controlled when the cortisol levels were less than 105 nmol/litre before and after the stimulation test. Dogs in which the clinical signs recurred had cortisol levels between 210 and 580 nmol/litre after the test, a level which is within the normal pretreatment range. Twenty-seven of the treated dogs died and six of these deaths were attributable directly to the disease or therapy. The median survival time of the 54 treated dogs was 30 months; eight dogs died during the first 16 weeks of treatment, and the dogs which survived this period had a median survival time of 39 months (mean 50 months).

Adrenal Cortex Function Tests↗
PubMed1995

Intracranial haemorrhage in a dobermann puppy with von Willebrand's disease.

Neurological examination of a lethargic, ataxic 12-week-old dobermann revealed decreased conscious proprioception in all its limbs. Haematological examination revealed a low platelet count. Cytological examination of a sample of cerebrospinal fluid revealed evidence of haemorrhage and chronic inflammation. The levels of von Willebrand's factor antigen were extremely low. Skull radiographs were consistent with mild hydrocephalus. Treatment resulted in little clinical improvement and the animal was euthanased. Post mortem examination of the brain revealed an internal hydrocephalus with haemorrhage into the ventricles. It was considered that the animal had suffered severe intracranial haemorrhage as a result of its low level of von Willebrand's factor antigen and that the bleeding may have been potentiated by the low platelet count.

Animals↗
PubMed1993

A phenotypic host range alteration determines RD114 virus restriction in feline embryonic cells.

We have characterized the restriction mechanism for RD114 virus replication in embryonic feline cells (FeF). By comparing growth properties of the virus in FeF cells with its behavior in a fetal feline glial cell line (G355) permissive for RD114, we showed that both cell lines were readily infectible by virus grown in permissive cells and that no significant differences in viral integration or viral RNA expression could be detected. However, analysis of viral protein expression revealed differences in viral env gene processing in the two cell types. Envelope precursor pR85 was produced, but the expected processed gp70 product was detectable only in permissive (G355) cells. An envelope product of 85 kDa was packaged into virions produced by FeF cells, while virions produced by G355 cells contained the expected RD114 gp70. While the gp85 env-containing virions were infectious for permissive G355 cells, they were unable to infect FeF cells. The block to infection by the gp85-containing particles in FeF cells could be abrogated by treatment with the glycosylation inhibitor tunicamycin. Our results indicate that restriction of RD114 virus involves a novel mechanism dependent on two factors: altered glycosylation of the envelope to a gp85 form and an altered RD114 receptor in FeF cells.

Animals↗
PubMed1990

An oncogenic chromosome 8-9 gene fusion isolated following transfection of human ovarian carcinoma cell line DNA.

Transfection of NIH3T3 cells with genomic DNA from the human ovarian adenocarcinoma tumor cell line OVCAR-3 identified ovc, a rearranged human DNA sequence which was generated during transfection and which induced both morphological transformation and tumorigenesis. A human alu repeat positive 10.5 kb EcoRI fragment present in all transformants was cloned, and two alu-free fragments of 1.8 kb and 2.2 kb were subcloned. The cloned 10.5 kb fragment is not biologically active in DNA transfection assays. Probe from the 2.2 kb fragment hybridizes to poly A + RNA from the transformants and several human tumor cell lines, including OVCAR-3. The 2.2 kb fragment maps to a site on human chromosome 9 (9p24) not known to contain oncogenic sequences, and identifies a two allele polymorphic restriction site. The 1.8 kb fragment maps to human chromosome 8. The ovc transforming sequences fail to hybridize to probes to any of 14 known oncogenes, indicating that they may represent a previously unknown human transforming gene.

Blotting, Northern↗
PubMed1990

Characterization of a human gene conferring sensitivity to infection by gibbon ape leukemia virus.

Gibbon ape leukemia virus (GALV) enters cells following interaction with a specific receptor protein. We have isolated human complementary DNAs (cDNAs) encoding a protein which, when expressed in normally uninfectable mouse NIH3T3 cells, confers on these cells specific sensitivity to infection by GALV. This was done by transfection into mouse cells of human DNA and selection of putative receptor gene transfectants using infection with a retrovirus carrying a drug resistance gene. Transfected genomic sequences were then cloned through their association with repetitive DNA, and these were used to isolate cDNA clones. The predicted 679-amino acid sequence encoded in these cDNAs is characteristic of an integral membrane protein in that multiple potential transmembrane domains are present. Searches of DNA and protein data banks failed to reveal homologies to other known sequences. It thus appears that the sequence isolated is novel and represents the human receptor for GALV. As expected from the wide host range of the virus, closely related homologues of the gene were found in several other vertebrate species tested.

Amino Acid Sequence↗