Biomedical subjects

Michael A Johnson

Publications and source records attributed to Michael A Johnson.

12 recordsLinked to original sources

PubMed2006

Dopamine release is severely compromised in the R6/2 mouse model of Huntington's disease.

Recently, alterations in dopamine signaling have been implicated in Huntington's disease. In this work, dopamine release and uptake was measured in striatal slices from the R6/2 transgenic mouse model of Huntington's disease using fast-scan cyclic voltammetry at carbon-fiber microelectrodes. Dopamine release in brain slices from 6-week-old R6/2 mice is substantially reduced (53% of wild type), while dopamine uptake is unaffected. In agreement with this, R6/2 mice injected with the dopamine uptake inhibitor cocaine exhibited a blunted motor activity response (54% of wild type). At 10 weeks of age, an even more dramatic motor activity decrease in response to cocaine injection (21% of wild type) was observed. Moreover, the pre-drug activity of 10-week-old R6/2 mice was significantly reduced (by 37%) compared with 6-week-old R6/2 mice. Striatal dopamine release decreased with age, indicating that progressive alterations in dopaminergic pathways may affect motor activity. The inhibition constants of cocaine and methamphetamine (METH) determined in brain slices differed little between genotype or age group, suggesting that the decreased responses to cocaine and METH arise from compromised dopamine release rather than differences in uptake or drug action. Collectively, these data demonstrate (i) a reduction in the ability of dopamine terminals to release dopamine and (ii) the importance of this attenuation of release on the motor symptoms of Huntington's disease.

Age Factors↗

PubMed2006

S-nitrosothiol depletion in amyotrophic lateral sclerosis.

Recent data suggest that either excessive or deficient levels of protein S-nitrosylation may contribute to disease. Disruption of S-nitrosothiol (SNO) homeostasis may result not only from altered nitric oxide (NO) synthase activity but also from alterations in the activity of denitrosylases that remove NO groups. A subset of patients with familial amyotrophic lateral sclerosis (ALS) have mutations in superoxide dismutase 1 (SOD1) that increase the denitrosylase activity of SOD1. Here, we show that the increased denitrosylase activity of SOD1 mutants leads to an aberrant decrease in intracellular protein and peptide S-nitrosylation in cell and animal models of ALS. Deficient S-nitrosylation is particularly prominent in the mitochondria of cells expressing SOD1 mutants. Our results suggest that SNO depletion disrupts the function and/or subcellular localization of proteins that are regulated by S-nitrosylation such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and thereby contributes to ALS pathogenesis. Repletion of intracellular SNO levels with SNO donor compounds rescues cells from mutant SOD1-induced death. These results suggest that aberrant depletion of intracellular SNOs contributes to motor neuron death in ALS, and raises the possibility that deficient S-nitrosylation is a general mechanism of disease pathogenesis. SNO donor compounds may provide new therapeutic options for diseases such as ALS that are associated with deficient S-nitrosylation.

Active Transport, Cell Nucleus↗

PubMed2006

Maximal voluntary hyperpnoea increases blood lactate concentration during exercise.

Ventilatory work during heavy endurance exercise has not been thought to influence systemic lactate concentration. We evaluated the effect of maximal isocapnic volitional hyperpnoea upon arterialised venous blood lactate concentration ([lac-]B) during leg cycling exercise at maximum lactate steady state (MLSS). Seven healthy males performed a lactate minimum test to estimate MLSS, which was then resolved using separate 30 min constant power tests (MLSS=207+/-8 W, mean +/- SEM). Thereafter, a 30 min control trial at MLSS was performed. In a further experimental trial, the control trial was mimicked except that from 20 to 28 min maximal isocapnic volitional hyperpnoea was superimposed on exercise. Over 20-28 min minute ventilation, oxygen uptake, and heart rate during the control and experimental trials were 87.3+/-2.4 and 168.3+/-7.0 l min(-1) (P<0.01), the latter being comparable to that achieved in the maximal phase of the lactate minimum test (171.9+/-6.8 l min(-1)), 3.46+/-0.20 and 3.83 +/- 0.20 l min(-1) (P<0.01), and 158.5+/-2.7 and 166.8+/-2.7 beats min(-1) (P<0.05), respectively. From 20 to 30 min of the experimental trial [lac-]B increased from 3.7+/-0.2 to 4.7+/-0.3 mmol l(-1) (P<0.05). The partial pressure of carbon dioxide in arterialised venous blood increased approximately 3 mmHg during volitional hyperpnoea, which may have attenuated the [lac-]B increase. These results show that, during heavy exercise, respiratory muscle work may affect [lac-]B. We speculate that the changes observed were related to the altered lactate turnover in respiratory muscles, locomotor muscles, or both.

Adult↗

PubMed2006

Sequence and function of canine herpesvirus alpha-transinducing factor and its interaction with an immediate early promoter.

The sequence of the alpha-transinducing factor (alpha-TIF) of canine herpesvirus (CHV-l) was determined. Alignment of the predicted CHV-1 alpha-TIF amino acid sequence with other alpha-TIF homologues reveals a core region of similarity with divergent amino and carboxyl termini. Analysis of the CHV-1 infected cell protein 4 promoter region identified a region containing nine copies of a 52 bp repeat that showed significant up-regulation of transcription by alpha-TIF. This region contained an imperfect 'TAATGARAT' motif, the binding site for herpes simplex virus 1 alpha-TIF, with an imperfect Oct-1 binding site immediately following. The infectious laryngotracheitis virus alpha-TIF was also shown to up-regulate transcription through this region of the promoter. Transfection of CHV-1 genomic DNA failed to yield infectious virus in canine kidney cell lines. Co-transfection of genomic DNA and an alpha-TIF expression plasmid resulted in virus plaques, indicating a potential essential role for alpha-TIF in CHV-1 infection.

Animals↗

PubMed2005

Porcine adenovirus as a delivery system for swine vaccines and immunotherapeutics.

Porcine adenovirus (PAdV) has many qualities which make it an ideal choice for use as a delivery vector in swine. It is a low grade pathogen, present almost world-wide in a number of serotypes varying in their virulence and tissue tropism, which may allow for serotype specific vaccine targeting. PAdV is species specific having only been isolated from swine, reducing the possibility of its spread to other animals or man following administration. When engineered to contain a foreign gene, recombinant PAdV (rPAdV) can be grown to high titres in tissue culture cells making it cheap to produce. Knowledge of the complete nucleotide sequence of the PAdV genome has enabled rationally directed insertions of foreign genes which remain stably inserted in the genome and can be expressed at high levels following delivery to the target host. Importantly, recombinant PAdV can be administered by injection or by the oral route in feed or drinking water. We have delivered a range of antigens and immunomodulatory molecules to commercially available pigs using rPAdV and found it to be a very effective delivery system. Significantly, recombinant PAdV serotype 3 is highly effective as a delivery vehicle even when administered in the face of high levels of artificially induced serotype specific neutralising antibody to the vector.

Adenoviridae Infections↗

PubMed2004

Accelerated CuZn-SOD-mediated oxidation and reduction in the presence of hydrogen peroxide.

Copper, zinc-superoxide dismutase (CuZn-SOD) is a cytosolic, antioxidant enzyme that scavenges potentially damaging superoxide radical (()O(2)(-)). Under the proper conditions, CuZn-SOD also catalyzes the oxidation and reduction of certain small molecules. Here, we demonstrate that increased exposure to hydrogen peroxide (H(2)O(2)), a by-product of the ()O(2)(-) scavenging reaction, dramatically increases the ability of CuZn-SOD to oxidize melatonin and reduce S-nitrosoglutathione (GSNO). After a 15min in vitro incubation with CuZn-SOD and 1mM H(2)O(2), 76% of the melatonin was oxidized, compared to 52% with 0.25mM H(2)O(2), and just 9% without H(2)O(2). Pre-incubation with 1mM H(2)O(2) resulted in a 100% increase in the rate of GSNO breakdown by CuZn-SOD in the presence of glutathione (GSH) compared to untreated CuZn-SOD. Collectively, these data suggest that even small increases in intracellular H(2)O(2) levels may result in the oxidation and/or reduction of small molecules critical for proper cellular function.

Binding Sites↗

PubMed2004

Resolving neurotransmitters detected by fast-scan cyclic voltammetry.

Carbon-fiber microelectrodes are frequently used as chemical sensors in biological preparations. In this work, we evaluated the ability of cyclic voltammograms recorded at fast-scan rates to resolve neurochemicals when analyzed by principal component regression. A calibration set of 30 cyclic voltammograms was constructed from 9 different substances at a variety of concentrations. The set was reduced by principal component analysis, and it was found that 99.5% of the variance in the data could be captured with five principal components. This set was used to evaluate cyclic voltammograms obtained with one or two compounds present in solution. In most cases, satisfactory predictions of the identity and concentration of analytes were obtained. Chemical dynamics were also resolved from a set of fast-scan cyclic voltammograms obtained with the electrode implanted in a region of a brain slice that contains dopaminergic terminals. Following stimulation, principal component regression of the data resolved the changes in dopamine and pH that were evoked. In a second test of the method, vesicular release was measured from adrenal medullary cells and the data were evaluated with a calibration set composed of epinephrine and norepinephrine. Cells that secreted one or the other were identified. Overall, the results show that principal component regression with appropriate calibration data allows resolution of substances that give overlapping cyclic voltammograms.

Animals↗

PubMed2003

A recombinant fowl adenovirus expressing the S1 gene of infectious bronchitis virus protects against challenge with infectious bronchitis virus.

The spike peplomer S1 subunit sequence from avian infectious bronchitis virus (IBV) Vic S strain was expressed in a plasmid under the control of the fowl adenovirus (FAV) major late promoter (MLP). Two recombinants were constructed in FAV serotype 8 (FAV 8) by inserting the expression cassette between the SnaBI and XbaI restriction enzyme sites (clone DA3) or between the SpeI sites (clone CA6-20). Expression of the S1 gene in the recombinants was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) by 20h post-infection. Commercial broiler chickens were orally vaccinated at day 0 or day 6 post-hatch and challenged at day 35 post-hatch. FAV antibody ELISA confirmed that maternal antibody directed against inclusion body hepatitis (serotype 8) had decayed in control birds and that FAV specific serum IgG responses were produced in vaccinated birds at the time of challenge. Further, an S1 specific antibody response was detected prior to challenge. Birds were challenged with either Vic S (serotype B) or N1/62 (serotype C) strains of IBV. The tracheas of challenged birds were analyzed by RT-PCR and re-isolation of virus. In birds vaccinated at day 6, 90-100% protection at the trachea was induced against either homologous or heterologous challenge. The construction of a recombinant FAV expressing S1 of IBV demonstrates the potential of an alternative vaccination strategy against IBV.

Administration, Oral↗

PubMed2003

Protection of pigs against 'in contact' challenge with classical swine fever following oral or subcutaneous vaccination with a recombinant porcine adenovirus.

A recombinant porcine adenovirus expressing the classical swine fever virus (CSFV) gp55 gene (rPAdV-gp55) was administered to commercially available outbred pigs via the subcutaneous or oral route and their susceptibility to 'in contact' challenge with classical swine fever determined. Animals vaccinated subcutaneously with a single dose of recombinant vaccine and challenged by 'in contact' exposure were protected from disease, whereas pigs given an equivalent single oral dose did not survive challenge. However, pigs given two oral doses of rPAdV-gp55, 22 days apart, were completely protected from disease. In addition, two doses of rPAdV-gp55 given subcutaneously was shown to boost CSFV neutralising antibody compared with a single dose, but neither a single dose nor two doses given orally induced detectable neutralising antibody responses.

Adenoviruses, Porcine↗

PubMed2003

Characterization of a protective monoclonal antibody recognizing Staphylococcus aureus MSCRAMM protein clumping factor A.

The Staphylococcus aureus MSCRAMM (microbial surface components recognizing adhesive matrix molecules) protein clumping factor A (ClfA) has been shown to be a critical virulence factor in several experimental models of infection. This report describes the generation, characterization, and in vivo evaluation of a murine monoclonal antibody (MAb) against ClfA. Flow cytometric analysis revealed that MAb 12-9 recognized ClfA protein expressed by all of the clinical S. aureus strains obtained from a variety of sources. In assays measuring whole-cell S. aureus binding to human fibrinogen, MAb 12-9 inhibited S. aureus binding by over 90% and displaced up to 35% of the previously adherent S. aureus bacteria. Furthermore, a single infusion of MAb 12-9 was protective against an intravenous challenge with a methicillin-resistant strain of S. aureus in a murine sepsis model (P < 0.0001). These data suggest that anti-ClfA MAb 12-9 should be further investigated as a novel immunotherapy for the treatment and prevention of life-threatening S. aureus infections.

Adhesins, Bacterial↗

PubMed2002

Biological activity of the tryprostatins and their diastereomers on human carcinoma cell lines.

Tryprostatin A 1 and B 2 are indole alkaloid-based fungal products that act in the G2/M phase of the cell cycle. Tryprostatin A and B as well as their two enantiomers and four diastereomers have been synthesized via a common strategy. As a measure of cytotoxicity, these eight stereoisomers were assayed for their growth inhibitory properties in human breast, prostate, and lung cancer cell lines. The ability of the tryprostatins and the tryprostatin stereoisomers to induce topoisomerase II-mediated DNA relaxation or to inhibit tubulin polymerization was also examined. Although none of the stereoisomers were significantly active in topoisomerase II- or tubulin-based assays, ds2-try B 11 was found to exhibit a cytotoxicity profile more potent than etoposide 3 in the human cancer cell lines examined. In addition, ds2-try B 11 is comprised of an L-tryptophan derivative coupled to a D-proline moiety, the latter stereochemistry of which may enhance the activity of 11 and potential analogues in vivo.

Antineoplastic Agents↗

PubMedDate not supplied

DOTA-D-Tyr(1)-octreotate: a somatostatin analogue for labeling with metal and halogen radionuclides for cancer imaging and therapy.

The goal of this study was to evaluate a somatostatin receptor ligand, DOTA-D-Tyr(1)-octreotate (DOTA-DY1-TATE), that has the chelator 1,4,7,10-tetraazacyclotetradecane-N,N',N'',N'"-tetraacetic acid (DOTA) attached to the D-Tyr(1) residue, allowing radiolabeling with both radiohalogens and radiometals. A potential advantage of having a chelator attached to the Tyr(1) residue is that halogen radiolabels may residualize or remain trapped in tumor cells rather than clear from the tumor. DOTA-DY1-TATE was synthesized by solid-phase methods and radiolabeled with (61)Cu, (64)Cu, and (125)I in high radiochemical purity and specific activity. A competitive binding assay demonstrated that (nat)Cu-DOTA-DY1-TATE and DOTA-(nat)I-DY1-TATE had comparable affinity to (nat)In-DTPA-OC in AR42J rat pancreatic tumor cells membranes. (61)Cu-DOTA-DY1-TATE had a dissociation constant (K(d)) of 176.4 pM and a receptor concentration (B(max)) of 244.4 fmol/mg. A tumor uptake of 1.515 %ID/g was determined for (64)Cu-DOTA-DY1-TATE and 0.814 %ID/g for DOTA-(125)I-DY1-TATE in AR42J tumor bearing Lewis rats at 1 h postinjection. DOTA-(125)I-DY1-TATE remained in the tumor at a higher concentration out to 4 h postinjection, suggesting that the iodine may have residualized in the tumor cells. MicroPET imaging of (64)Cu-DOTA-DY1-TATE in AR42J tumor bearing rats and SCID mice at 2 h postinjection showed significant uptake and good contrast in the thigh tumors in the rat model and in the neck and thigh tumors of the mouse. This study demonstrates that DOTA-DY1-TATE is a somatostatin analogue that can be labeled with both metal and halogen radionuclides, and its (64)Cu- and (125)I-radiolabeled compounds showed somatostatin receptor-mediated uptake in normal and tumor tissues.

Animals↗