Biomedical subjects

G A Cox

Publications and source records attributed to G A Cox.

At least 19 recordsLinked to original sources

PubMed2006

Expression of terminal differentiation proteins defines stages of mouse mammary gland development.

Immunohistochemical analysis using paraffin-embedded specimens is the method of choice to evaluate protein expression at a cellular level while preserving tissue architecture in normal and neoplastic tissues. Current knowledge of the expression of terminal differentiation markers in the mouse mammary gland relies on the evaluation of frozen tissues by use of immunofluorescence. We assessed changes in patterns of expression of terminal differentiation markers throughout the development of the mouse mammary gland in paraffin-embedded tissues. The expression of alpha-smooth muscle actin (SMA) and keratins (K) 5, 8/18, and 14 was influenced by the development stage of the mammary gland. Expression of K5 and SMA was restricted to basal cells. Keratin 14 was consistently expressed by mammary basal cells, and was detected in scattered luminal cells from 13.5 days after conception through puberty. Labeling for K8/18 of luminal cells was heterogeneous at all times. Heterogeneous expression patterns in luminal cells suggest this layer has cells with a variety of biological functions. The absence of K6 expression at any stage of the development of the mammary gland was confirmed by use of reverse transcriptase-polymerase chain reaction analysis, which indicates that this intermediate filament is not a marker of the mammary gland stem cell. Finally, consistent with results of earlier studies, keratins 1, 10, 13, and 15, and filaggrin, involucrin, and loricrin were not detected at any stage of mammary gland development.

Actins↗

PubMed2004

Measurement of the total active 8B solar neutrino flux at the Sudbury Neutrino Observatory with enhanced neutral current sensitivity.

The Sudbury Neutrino Observatory has precisely determined the total active (nu(x)) 8B solar neutrino flux without assumptions about the energy dependence of the nu(e) survival probability. The measurements were made with dissolved NaCl in heavy water to enhance the sensitivity and signature for neutral-current interactions. The flux is found to be 5.21 +/- 0.27(stat)+/-0.38(syst) x 10(6) cm(-2) s(-1), in agreement with previous measurements and standard solar models. A global analysis of these and other solar and reactor neutrino results yields Deltam(2)=7.1(+1.2)(-0.6) x 10(-5) eV(2) and theta=32.5(+2.4)(-2.3) degrees. Maximal mixing is rejected at the equivalent of 5.4 standard deviations.

Journal Article↗

PubMed2004

Constraints on nucleon decay via invisible modes from the Sudbury Neutrino Observatory.

Data from the Sudbury Neutrino Observatory have been used to constrain the lifetime for nucleon decay to "invisible" modes, such as n-->3nu. The analysis was based on a search for gamma rays from the deexcitation of the residual nucleus that would result from the disappearance of either a proton or neutron from 16O. A limit of tau(inv)>2 x 10(29) yr is obtained at 90% confidence for either neutron- or proton-decay modes. This is about an order of magnitude more stringent than previous constraints on invisible proton-decay modes and 400 times more stringent than similar neutron modes.

Journal Article↗

PubMed2002

Direct evidence for neutrino flavor transformation from neutral-current interactions in the Sudbury Neutrino Observatory.

Observations of neutral-current nu interactions on deuterium in the Sudbury Neutrino Observatory are reported. Using the neutral current (NC), elastic scattering, and charged current reactions and assuming the standard 8B shape, the nu(e) component of the 8B solar flux is phis(e) = 1.76(+0.05)(-0.05)(stat)(+0.09)(-0.09)(syst) x 10(6) cm(-2) s(-1) for a kinetic energy threshold of 5 MeV. The non-nu(e) component is phi(mu)(tau) = 3.41(+0.45)(-0.45)(stat)(+0.48)(-0.45)(syst) x 10(6) cm(-2) s(-1), 5.3sigma greater than zero, providing strong evidence for solar nu(e) flavor transformation. The total flux measured with the NC reaction is phi(NC) = 5.09(+0.44)(-0.43)(stat)(+0.46)(-0.43)(syst) x 10(6) cm(-2) s(-1), consistent with solar models.

Journal Article↗

PubMed2002

Measurement of day and night neutrino energy spectra at SNO and constraints on neutrino mixing parameters.

The Sudbury Neutrino Observatory (SNO) has measured day and night solar neutrino energy spectra and rates. For charged current events, assuming an undistorted 8B spectrum, the night minus day rate is 14.0%+/-6.3%(+1.5%)(-1.4%) of the average rate. If the total flux of active neutrinos is additionally constrained to have no asymmetry, the nu(e) asymmetry is found to be 7.0%+/-4.9%(+1.3%)(-1.2%). A global solar neutrino analysis in terms of matter-enhanced oscillations of two active flavors strongly favors the large mixing angle solution.

Journal Article↗

PubMed2001

Measurement of the rate of nu(e) + d --> p + p + e(-) interactions produced by (8)B solar neutrinos at the Sudbury Neutrino Observatory.

Solar neutrinos from (8)B decay have been detected at the Sudbury Neutrino Observatory via the charged current (CC) reaction on deuterium and the elastic scattering (ES) of electrons. The flux of nu(e)'s is measured by the CC reaction rate to be straight phi(CC)(nu(e)) = 1.75 +/- 0.07(stat)(+0.12)(-0.11)(syst) +/- 0.05(theor) x 10(6) cm(-2) s(-1). Comparison of straight phi(CC)(nu(e)) to the Super-Kamiokande Collaboration's precision value of the flux inferred from the ES reaction yields a 3.3 sigma difference, assuming the systematic uncertainties are normally distributed, providing evidence of an active non- nu(e) component in the solar flux. The total flux of active 8B neutrinos is determined to be 5.44+/-0.99 x 10(6) cm(-2) s(-1).

Journal Article↗

PubMed2000

The mouse fidgetin gene defines a new role for AAA family proteins in mammalian development.

The mouse mutation fidget arose spontaneously in a heterogeneous albino stock. This mutant mouse is characterized by a side-to-side head-shaking and circling behaviour, due to reduced or absent semicircular canals. Fidget mice also have small eyes, associated with cell-cycle delay and insufficient growth of the retinal neural epithelium, and lower penetrance skeletal abnormalities, including pelvic girdle dysgenesis, skull bone fusions and polydactyly. By positional cloning, we found the gene mutated in fidget mice, fidgetin (Fign), which encodes a new member of the 'meiotic' or subfamily-7 (SF7; ref. 7) group of ATPases associated with diverse cellular activities (AAA proteins). We also discovered two closely related mammalian genes. AAA proteins are molecular chaperones that facilitate a variety of functions, including membrane fusion, proteolysis, peroxisome biogenesis, endosome sorting and meiotic spindle formation, but functions for the SF7 AAA proteins are largely unknown. Fidgetin is the first mutant AAA protein found in a mammalian developmental mutant, thus defining a new role for these proteins in embryonic development.

Adenosine Triphosphatases↗

PubMed1999

Identification and characterization of human rhinovirus-14 3C protease deamidation isoform.

A purified recombinant human rhinovirus-14 3C protease preparation contained only approximately 50% active enzyme as titrated using specifically designed irreversible 3C protease inhibitors. Analysis of the purified 3C protein by isoelectric focusing showed differently charged 3C isoforms that had isoelectric points (pI) of 8.3 (55%) and 9.0 (45%), with the latter one being consistent with the predicted pI of the human rhinovirus-14 3C protein. Further analysis indicated that the pI 8.3 protein was the deamidated form of 3C, and it displayed approximately 10-fold reduced cleavage activity relative to the original 3C protease sample. Peptide mapping followed by sequence analysis revealed that a single asparagine, Asn-164, was deamidated to aspartic acid in the pI 8.3 isoform. Converting Asn-164 to Asp by site-directed mutagenesis resulted in a mutated 3C protease with extremely low activity, as seen with the pI 8.3 isoform, indicating a role of Asn-164 in substrate recognition and binding. In addition, the deamidated 3C protease was found to be present in vivo, and its abundance was related to the viral replication cycle. Moreover, mutant virus carrying Asp-164 showed reduced viability in infected cells. Taken together, our data suggest that 3C protein deamidation plays a role in the regulation of its enzymatic activity.

3C Viral Proteases↗

PubMed1999

Characterization of dystrophin and utrophin diversity in the mouse.

Utrophin is a 400 kDa autosomal homolog of dystrophin and a component of the submembranous cytoskeleton. While multiple dystrophin isoforms have been identified along with alternatively spliced products, to date only two different mRNA species of utrophin have been identified. To determine the degree of evolutionary conservation between dystrophin and utrophin isoforms, we have compared their expression patterns in adult mice. Northern blot analysis of multiple adult tissues confirmed that only two major sizes of transcripts are produced from each gene: 13 and 5.5 kb from utrophin and 14 and 4.8 kb from dystrophin. However, western blot analysis detected several putative short utrophin isoforms that may be homologs of the dystrophin isoforms Dp140, Dp116 and Dp71. We also identified an alternatively spliced utrophin transcript that lacks the equivalent of the alternatively spliced dystrophin exon 71. Finally, we demonstrated that the C-terminal domain of utrophin targeted to neuromuscular junctions in normal mice, but localized to the sarcolemma efficiently only in the absence of dystrophin. Our results provide further evidence for a common evolutionary origin of the utrophin and dystrophin genes.

Alternative Splicing↗

PubMed1998

Identification of the mouse neuromuscular degeneration gene and mapping of a second site suppressor allele.

The nmd mouse mutation causes progressive degeneration of spinal motor neurons and muscle atrophy. We identified the mutated gene as the putative transcriptional activator and ATPase/DNA helicase previously described as Smbp2, Rip1, Gf1, or Catf1. Mutations were found in two alleles-a single amino acid deletion in nmdJ and a splice donor mutation in nmd2J. The selective vulnerability of motor neurons is striking in view of the widespread expression of this gene, although the pattern of degeneration may reflect a specific threshold since neither allele is null. In addition, the severity of the nmd phenotype is attenuated in a semidominant fashion by a major genetic locus on chromosome (Chr) 13. The identification of the nmd gene and mapping of a major suppressor provide new opportunities for understanding mechanisms of motor neuron degeneration.

Adenosine Triphosphatases↗

PubMed1998

Enzymatic characterization of refolded human rhinovirus type 14 2A protease expressed in Escherichia coli.

Reported here is the production of recombinant human rhinovirus 14 (HRV14) 2A protease from bacterial cells transformed with a heat-inducible plasmid containing the HRV14 2A cDNA sequence. Overexpressed 2A protein partitioned into the inclusion bodies was solubilized in urea and then refolded in the presence of Zn2+ Transition metals were required for the restoration of 2A protease activity as a structural component, but appeared to be inhibitory if added exogenously once the enzyme was refolded. Based on the cleavage specificity studies, a colorimetric assay was developed for the highly purified HRV14 2A protease. A peptide with the sequence RKGDIKSY-p-nitroanilide was found to be cleaved by the 2A protease with a k(cat)/Km ratio of approximately 335 M(-1)s(-1), which allows its activity to be measured continuously with a spectrophotometer or a microplate reader.

Cysteine Endopeptidases↗

PubMed1997

A continuous colorimetric assay for rhinovirus-14 3C protease using peptide p-nitroanilides as substrates.

Human rhinovirus encoded 3C protease is an attractive target for antiviral drug development. However, lack of a convenient and selective assay for 3C protease has been a hindrance in characterization of this enzyme and evaluation of a large number of potential inhibitors. In the present study we describe development of a simple, continuous colorimetric assay for this enzyme using peptide p-nitroanilides (pNA) as substrates. Several peptides mimicking the native 3C cleavage site of HRV-14 polyprotein have been synthesized with an N-acylated p-nitroaniline at position P1' and examined as substrates for the purified 3C protease. In these peptides, amino acids downstream from the original cleavage site have all been replaced with a chromophoric p-nitroaniline moiety which is directly linked to the bond undergoing enzymatic cleavage, thereby generating a new cleavage site Gln-pNA for the enzyme. Hydrolysis of these pNA peptides by 3C at the newly formed scissile bond releases free p-nitroaniline which is yellow-colored and can be continuously monitored at a visible wavelength. Kinetic parameters of 3C protease toward these peptides have been measured and analyzed. In addition, the pNA peptides have been modeled within the active site of the 3C protease to investigate the ability of the pNA group to act as a replacement for Gly-Pro in the prime side. The selectivity and applicability of this assay and its advantages over the previously described methods have been demonstrated and discussed. Since multiple tests can be performed simultaneously in one microtiter plate, the assay is ideal for evaluation of a large number of samples.

3C Viral Proteases↗

PubMed1997

Sodium/hydrogen exchanger gene defect in slow-wave epilepsy mutant mice.

The "housekeeping" sodium/hydrogen exchanger, NHE1, mediates the electroneutral 1:1 exchange of Na+ and H+ across the plasma membrane. NHE1 is ubiquitous and is studied extensively for regulation of pHi, cell volume, and response to growth factors. We describe a spontaneous mouse mutant, slow-wave epilepsy, (swe), with a neurological syndrome including ataxia and a unique epilepsy phenotype consisting of 3/sec absence and tonic-clonic seizures. swe was fine-mapped on Chromosome 4 and identified as a null allele of Nhe1. Mutants show selective neuronal death in the cerebellum and brainstem but otherwise are healthy. This first example of a disease-causing mutation in an Nhe gene provides a new tool for studying the delicate balance of neuroexcitability and cell survival within the CNS.

Animals↗

PubMed1997

Expression and purification of recombinant rhinovirus 14 3CD proteinase and its comparison to the 3C proteinase.

Human rhinovirus (HRV) is a positive-stranded RNA virus with an open reading frame that encodes for a single polyprotein of about 3000 amino acids. The HRV polyprotein is proteolytically processed; eight of nine cleavages are catalyzed by the 3C and/or the 3CD proteinases. We have expressed and purified recombinant HRV14 3C and 3CD proteinases and investigated their substrate selectivity and inhibitor sensitivity. Expressed 3CD proteinase had the P1/P1' residues of the 3C/3D cleavage site mutated from Gln/Gly to Ala/Ala in order to prevent autocleavage. The 3CD proteinase activities were measured by utilization of native, chromogenic, and fluorogenic peptide substrates. The 3CD proteinase exhibited < or =15% activity, compared to 3C, toward peptidyl p-nitroanilide substrates which contain only the p-nitroaniline moiety in the prime side. The 3C and 3CD proteinases exhibited similar activities for both internally quenched fluorogenic and native peptides. These results suggest that the two enzymes have similar but slightly different substrate specificity, especially on their preference for prime side residues. Inhibitor sensitivities toward classical proteinase inhibitors were generally similar for both enzymes. Small peptidyl inhibitors, specifically designed and synthesized for HRV14 3C, also inhibited the 3CD proteinase. Taken together, our data indicate that the 3D domain of 3CD proteinase had some influence on substrate recognition, but did not have dramatic impact on its interaction with inhibitors.

3C Viral Proteases↗

PubMed1997

Interactions between dystrophin and the sarcolemma membrane.

Dystrophin serves as a link between the subsarcolemmal cytoskeleton and the extracellular matrix. The NH2 terminus attaches to the cytoskeleton, while the COOH terminus attaches to the dystrophin associated protein (DAP) complex, which can be separated into the dystroglycan, sarcoglycan, and syntrophin subcomplexes. While the function of each DAP is not known, the dystroglycan complex binds laminin in the extracellular matrix, and binds the dystrophin COOH terminus in vitro. The syntrophins also bind the dystrophin COOH terminus in vitro, but no evidence has been reported for an interaction between dystrophin and the sarcoglycans. Human mutations have been found in dystrophin, the sarcoglycans and laminin, all of which lead to various types of muscular dystrophy. We have been studying the dystrophin domains necessary for formation of a functional complex by generating transgenic mdx (dystrophin minus) mice expressing internally truncated dystrophins. These mice provide in vivo models to study the localization of truncated dystrophin isoforms, the association of the truncated proteins with the DAP complex, and the functional capacity of the assembled DAP complexes. Expression of a dystrophin deleted for most of the NH2-terminal domain in mdx mice leads to only a mild dystrophy, indicating that dystrophin can attach to the cytoskeleton by multiple mechanisms. Truncation of the central rod domain leads to normal DAP complex formation and almost fully prevents development of dystrophy. Deletion analysis of the COOH-terminal regions indicates that a broad cysteine-rich domain is indispensable for dystrophin function. This region coincides with the in vitro identified beta-dystroglycan binding domain. Mice lacking this latter domain express very low levels of the sarcoglycans, indicating that the sarcoglycan complex binds dystrophin via dystroglycan. All deletion constructs tested lead to normal expression of the syntrophins, indicating that syntrophin associates with the DAP complex via multiple binding partners.

Actins↗

PubMed1996

Forced expression of dystrophin deletion constructs reveals structure-function correlations.

Dystrophin plays an important role in skeletal muscle by linking the cytoskeleton and the extracellular matrix. The amino terminus of dystrophin binds to actin and possibly other components of the subsarcolemmal cytoskeleton, while the carboxy terminus associates with a group of integral and peripheral membrane proteins and glycoproteins that are collectively known as the dystrophin-associated protein (DAP) complex. We have generated transgenic/mdx mice expressing "full-length" dystrophin constructs, but with consecutive deletions within the COOH-terminal domains. These mice have enabled analysis of the interaction between dystrophin and members of the DAP complex and the effects that perturbing these associations have on the dystrophic process. Deletions within the cysteine-rich region disrupt the interaction between dystrophin and the DAP complex, leading to a severe dystrophic pathology. These deletions remove the beta-dystroglycan-binding site, which leads to a parallel loss of both beta-dystroglycan and the sarcoglycan complex from the sarcolemma. In contrast, deletion of the alternatively spliced domain and the extreme COOH terminus has no apparent effect on the function of dystrophin when expressed at normal levels. The proteins resulting from these latter two deletions supported formation of a completely normal DAP complex, and their expression was associated with normal muscle morphology in mdx mice. These data indicate that the cysteine-rich domain is critical for functional activity, presumably by mediating a direct interaction with beta-dystroglycan. However, the remainder of the COOH terminus is not required for assembly of the DAP complex.

Animals↗

PubMed1995

Human cytomegalovirus proteinase: candidate glutamic acid identified as third member of putative active-site triad.

The human cytomegalovirus (HCMV) proteinase is synthesized as a 709-amino-acid precursor that undergoes at least three autoproteolytic cleavages. The mature proteinase, called assemblin, is one of the products of autoproteolysis and is composed of the first 256 amino acids of the precursor. HCMV assemblin and its homologs in other herpes group viruses contain five highly conserved domains (CD1 through CD5). An absolutely conserved serine in CD3 has been shown by site-directed mutagenesis of the simian cytomegalovirus (SCMV) and herpes simplex virus type 1 (HSV-1) enzymes and by inhibitor affinity labeling of the HSV-1 and HCMV enzymes to be the active-site nucleophile of assemblin. An absolutely conserved histidine in CD2 has also been demonstrated by site-directed mutagenesis of the SCMV and HSV-1 enzymes to be essential for proteolytic activity and has been proposed to be a second member of the catalytic triad of this serine proteinase. We report here the use of site-directed mutagenesis to investigate the active-site amino acids of HCMV assemblin. Substitutions were made for the CD3 serine and CD2 histidine residues implicated as active-site components, and for other amino acids whose influence on enzyme activity was of interest. The mutant proteinases were tested in a transient transfection assay for their ability to cleave their natural substrate, the assembly protein precursor. Results of these experiments verified that HCMV CD3 serine (Ser-132) and CD2 histidine (His-63) are essential for proteolytic activity and identified a glutamic acid (Glu-122) within CD3 that is also essential for proteolytic activity and may be conserved among all herpesvirus assemblin homologs. We suggest that CD3 Glu-122, CD3 Ser-132, and CD2 His-63 constitute the active-site triad of this serine proteinase.

Amino Acid Sequence↗

PubMed1994

Dp71 can restore the dystrophin-associated glycoprotein complex in muscle but fails to prevent dystrophy.

Two lines of transgenic mdx mice have been generated that express a 71 kD non-muscle isoform of dystrophin (Dp71) in skeletal muscle. This isoform contains the cysteine-rich and C-terminal domains of dystrophin, but lacks the N-terminal actin-binding and central spectrin-like repeat domains. Dp71 was associated with the sarcolemma membrane, where it restored normal expression and localization of all members of the dystrophin-associated glycoprotein complex. However, the skeletal muscle pathology of the transgenic mdx mice remained severe. These results indicate that the dystrophin C terminus cannot function independently to prevent dystrophic symptoms and confirms predictions based on patient data that both the N and C-terminal domains are required for normal dystrophin function.

Animals↗