Search PubMed⌕ Search

Biomedical subjects

D J Mitchell

Publications and source records attributed to D J Mitchell.

At least 19 recordsLinked to original sources
PubMed2002

Identification to the species level of the plant pathogens Phytophthora and Pythium by using unique sequences of the ITS1 region of ribosomal DNA as capture probes for PCR ELISA.

The ribosomal internal transcribed spacer 1 region was sequenced for 10 species of Pythium and eight species of Phytophthora. Alignment of the sequences revealed considerable sequence microheterogeneity, which was utilized to prepare a capture probe of unique sequence for each species. The capture probes were tested by PCR ELISA, combining the sensitivity and specificity of the polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). The probes were entirely species specific, enabling the detection and identification of the amplified DNA of species from individual cultures or from mixed samples of the DNAs of two different species. This approach to species identification, which provides a molecular technology to process large numbers of samples and still identify the fungi with a high level of confidence, may greatly reduce the resources and the time of highly trained specialists currently needed to identify these important species of plant pathogenic fungi.

Base Sequence↗
PubMed2002

A comparison of two injury surveillance systems within sports medicine clinics.

This study compares a comprehensive method of collecting injury data from sports medicine clinics, with a more simplified method of injury surveillance. The sports medicine injury surveillance (SMIS) project was implemented in a group of five allied sports medicine clinics in Melbourne. over two consecutive years. The injury surveillance method used in the second year (SMIS2) was a simplified version of that used in the first year (SMIS1). Methodological differences in the injury surveillance systems included form design, staff commitment and training, auditing process, financial incentives offered and employment of a project officer. Data were collected on 6479 new sports injury patients during SMIS1 and on 1682 patients during SMIS2. Comparative data from the two years of injury surveillance included patient profile (gender. age. days from injury to treatment, sport and context of injury) and injury information (site, cause and nature of injury). The SMIS2 methodology was associated with a lower sensitivity (p < 0.001) and a higher proportion of missing information (p < 0.001) than the SMIS1 methodology. There was also a significant difference in the nature and cause of injury variables (p < 0.001) between SMIS1 and SMIS2 and this was associated with coding changes. This study shows that the method of data collection influences both the proportion of missing information and the sensitivity of the system. A comprehensive method of injury surveillance will lead to a more complete data collection process. Methodological differences, however, do not appear to substantially alter conclusions about general patient characteristics, but do have some influence on the accuracy with which broad injury data are identified. Notwithstanding these comments, this study shows that injury surveillance activities can be successfully implemented in sports medicine clinics.

Ambulatory Care Facilities↗
PubMed2001

Immunomodulation of experimental autoimmune encephalomyelitis with ordered peptides based on MHC-TCR binding motifs.

T cell-mediated destruction of the myelin sheath causes inflammatory damage of the CNS in multiple sclerosis (MS). The major T and B cell responses in MS patients who are HLA-DR2 (about two-thirds of MS patients) react to a region between residues 84 and 103 of myelin basic protein (1 ). The crystal structure of HLA-DR2 complexed with myelin basic protein(84-102) confirmed that Lys(91) is the major TCR contact site, whereas Phe(90) is a major anchor to MHC and binds the hydrophobic P4 pocket (2 ). We have tested peptides containing repetitive 4-aa sequences designed to bind critical MHC pockets and to interfere with T cell activation. One such sequence, EYYKEYYKEYYK, ameliorates experimental autoimmune encephalomyelitis in Lewis rats, an animal model of MS.

Adjuvants, Immunologic↗
PubMed2001

Inactivation of C30A trimethylamine dehydrogenase by N-cyclopropyl-alpha-methylbenzylamine, 1-phenylcyclopropylamine, and phenylhydrazine.

Trimethylamine dehydrogenase (TMADH) from the bacterium Methylophilus methylotrophus (sp. W(3)A(1)) and its C30A mutant were inactivated with three known inactivators of monoamine oxidase, namely, phenylhydrazine, N-cyclopropyl-alpha-methylbenzylamine, and 1-phenylcyclopropylamine. All three compounds irreversibly inactivated both the wild-type and C30A mutant enzymes, although phenylhydrazine was 10 times more potent than N-cyclopropyl-alpha-methylbenzylamine, which was much more potent than 1-phenylcyclopropylamine. The change in the UV--visible absorption spectra upon modification indicated that the flavin was modified. In the case of the C30A mutant, the absence of a covalent attachment of the flavin to the polypeptide has permitted LC-electrospray mass spectrometry of the reaction product to be undertaken, demonstrating new mass peaks corresponding to various chemically modified forms of the flavin cofactor. In the case of N-cyclopropyl-alpha-methylbenzylamine, masses corresponding to hydroxy-FMN and hydroxyriboflavin were detected. 1-Phenylcyclopropylamine inactivation of the C30A mutant produced three modified flavins, as evidenced by the electrospray mass spectrum: hydroxy-FMN, FMN plus C(6)H(5)COCH(2)CH(2), and hydroxy-FMN plus C(6)H(5)COCH(2)CH(2). Phenylhydrazine inactivation of the C30A mutant gave at least seven different modified flavins: hydroxyriboflavin, hydroxy-FMN, two apparently isomeric compounds corresponding to hydroxy-FMN plus one phenyl group, two apparently isomeric compounds corresponding to FMN plus one phenyl group, and FMN plus two phenyl groups. Covalent flavin adduct formation appears to be the only modification because dialysis of the inactive enzyme followed by reconstitution with FMN restores the enzyme activity to that of a noninactivated control.

Alanine↗
PubMed2001

Spectrometric evidence for the flavin-1-phenylcyclopropylamine inactivator adduct with monoamine oxidase N.

1-Phenylcyclopropylamine (1-PCPA) is shown to be an inactivator of the fungal flavoenzyme monoamine oxidase (MAO) N. Inactivation results in an increase in absorbance at 410 nm and is accompanied by the concomitant loss of the flavin absorption band at 458 nm. The spectral properties of the covalent adduct formed between the flavin cofactor of MAO N and 1-PCPA are similar to those reported for the irreversible inactivation product formed with 1-PCPA and mammalian mitochondrial monoamine oxidase B [Silverman, R. B., and Zieske, P. A. (1985) Biochemistry 24, 2128-2138]. There is a hypsochromic shift of the 410 nm band upon lowering the pH to 2, indicating that an N(5)-flavin adduct formed upon inactivation. Use of the fungal enzyme, MAO N, which lacks the covalent attachment to the flavin adenine dinucleotide (FAD) cofactor present in the mammalian forms MAO A and MAO B, has allowed for the isolation and further structural identification of the flavin-inactivator adduct. The incorporation of two (13)C labels into the inactivator, [2,3-(13)C(2)]-1-PCPA, followed by analysis using on-line liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy, provided a means to explore the structure of the flavin-inactivator adduct of MAO N. The spectral evidence supports covalent attachment of the 1-PCPA inactivator to the cofactor as N(5)-3-oxo-3-phenylpropyl-FAD.

Aspergillus niger↗
PubMed2001

Simultaneous monitoring of gastric and oesophageal pH reveals limitations of conventional oesophageal pH monitoring in milk fed infants.

BACKGROUND: Monitoring oesophageal pH conventionally detects "acid reflux" (pH less than 4). The pH of the gastric contents determines whether or not reflux can be detected. AIM: To monitor gastric and oesophageal pH simultaneously in order to determine the effect of milk feeds on gastric pH and how this would influence interpretation of the oesophageal pH record. METHODS: Milk fed infants for whom oesophageal pH monitoring was requested underwent simultaneous gastric and oesophageal pH monitoring using a dual channel pH probe. RESULTS: Twenty of 24 records were technically satisfactory. Mean reflux index was 1.0%, range 0.0-4.0%. Gastric pH was less than 4 for 24.5% (range 0.6-69.1%) of the total time. The average time the gastric pH was greater than 4 after feeds was 130 minutes (range 29-212 minutes). The corrected reflux index (limited to the time the gastric pH was less than 4) was 2.6% (range 0.0-11.0%). CONCLUSION: The pH of the gastric contents may be greater than 4 for prolonged intervals, during which oesophageal pH monitoring using current criteria cannot detect reflux nor correlate it with clinical events. A low reflux index may reflect prolonged buffering of gastric acidity rather than the absence of reflux.

Animals↗
PubMed2000

The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters.

Certain proteins contain subunits that enable their active translocation across the plasma membrane into cells. In the specific case of HIV-1, this subunit is the basic domain Tat(49-57) (RKKRRQRRR). To establish the optimal structural requirements for this translocation process, and thereby to develop improved molecular transporters that could deliver agents into cells, a series of analogues of Tat(49-57) were prepared and their cellular uptake into Jurkat cells was determined by flow cytometry. All truncated and alanine-substituted analogues exhibited diminished cellular uptake, suggesting that the cationic residues of Tat(49-57) play a principal role in its uptake. Charge alone, however, is insufficient for transport as oligomers of several cationic amino acids (histidine, lysine, and ornithine) are less effective than Tat(49-57) in cellular uptake. In contrast, a 9-mer of l-arginine (R9) was 20-fold more efficient than Tat(49-57) at cellular uptake as determined by Michaelis-Menton kinetic analysis. The d-arginine oligomer (r9) exhibited an even greater uptake rate enhancement (>100-fold). Collectively, these studies suggest that the guanidinium groups of Tat(49-57) play a greater role in facilitating cellular uptake than either charge or backbone structure. Based on this analysis, we designed and synthesized a class of polyguanidine peptoid derivatives. Remarkably, the subset of peptoid analogues containing a six-methylene spacer between the guanidine head group and backbone (N-hxg), exhibited significantly enhanced cellular uptake compared to Tat(49-57) and even to r9. Overall, a transporter has been developed that is superior to Tat(49-57), protease resistant, and more readily and economically prepared.

Amino Acid Sequence↗
PubMed2000

Importance of L-selectin-dependent leukocyte-leukocyte interactions in human whole blood.

The objective of this study was to investigate whether leukocytes could be recruited by rolling leukocytes in a human whole blood model system. In all experiments, either neutrophils, whole blood, or diluted blood was perfused over immobilized E-selectin. With isolated neutrophils (2 x 10(5)/mL), the free-flowing neutrophils were captured by attached neutrophils to form secondary interactions that resulted in lines of rolling leukocytes. These secondary tethers accounted for 50% to 60% of all interactions and were eliminated by an L-selectin antibody, which also eliminated the lines of rolling leukocytes. Perfusion of whole blood or diluted blood revealed no lines of rolling leukocytes. The addition of red blood cells to isolated neutrophils either in a 1000:1 or a 10:1 ratio also inhibited lines of rolling leukocytes. Leukocytes were fluorescently labeled with rhodamine-6G so that leukocyte-leukocyte interactions could be studied in whole blood. A small number of secondary tethers (less than 20%) occurred and could be reduced by more than 80% with an L-selectin antibody. However, the overall impact on leukocyte recruitment was negligible. Similar experiments were performed using murine whole blood or isolated murine leukocytes. In the absence of red blood cells, murine leukocytes also formed lines of rolling leukocytes on E-selectin, and secondary tethers accounted for 50% of total interactions. However, when murine blood (diluted 1:5 with buffer) was perfused over E-selectin, secondary tethers accounted for only 13% of total interactions. These interactions were completely absent when blood was used from L-selectin-deficient mice. These data demonstrate for the first time that the importance of L-selectin-dependent leukocyte-leukocyte interactions is greatly reduced in whole blood and does not enhance overall recruitment of leukocytes in this physiologic milieu. (Blood. 2000;95:2954-2959)

Animals↗
PubMed2000

Polyarginine enters cells more efficiently than other polycationic homopolymers.

Homopolymers or peptides containing a high percentage of cationic amino acids have been shown to have a unique ability to cross the plasma membrane of cells, and consequently have been used to facilitate the uptake of a variety of biopolymers and small molecules. To investigate whether the polycationic character of these molecules, or some other structural feature, was the molecular basis for the effect, the ability of a variety of homopolymers to enter cells was assayed by confocal microscopy and flow cytometry. Polymers of L- or D-arginine containing six or more amino acids entered cells far more effectively than polymers of equal length composed of lysine, ornithine and histidine. Peptides of fewer than six amino acids were ineffective. The length of the arginine side-chain could be varied without significant loss of activity. These data combined with the inability of polymers of citrulline to enter cells demonstrated that the guanidine headgroup of arginine was the critical structural component responsible for the biological activity. Cellular uptake could be inhibited by preincubation of the cells with sodium azide, but not by low temperature (3 degrees C), indicating that the process was energy dependent, but did not involve endocytosis.

Biological Transport↗
PubMed1999

Is integer arithmetic fundamental to mental processing?: the mind's secret arithmetic.

Unlike the ability to acquire our native language, we struggle to learn multiplication and division. It may then come as a surprise that the mental machinery for performing lightning-fast integer arithmetic calculations could be within us all even though it cannot be readily accessed, nor do we have any idea of its primary function. We are led to this provocative hypothesis by analysing the extraordinary skills of autistic savants. In our view such individuals have privileged access to lower levels of information not normally available through introspection.

Art↗
PubMed1999

Suppressive immunization with DNA encoding a self-peptide prevents autoimmune disease: modulation of T cell costimulation.

Usually we rely on vaccination to promote an immune response to a pathogenic microbe. In this study, we demonstrate a suppressive from of vaccination, with DNA encoding a minigene for residues 139-151 of myelin proteolipid protein (PLP139-151), a pathogenic self-Ag. This suppressive vaccination attenuates a prototypic autoimmune disease, experimental autoimmune encephalomyelitis, which presents clinically with paralysis. Proliferative responses and production of the Th1 cytokines, IL-2 and IFN-gamma, were reduced in T cells responsive to PLP139-151. In the brains of mice that were successfully vaccinated, mRNA for IL-2, IL-15, and IFN-gamma were reduced. A mechanism underlying the reduction in severity and incidence of paralytic autoimmune disease and the reduction in Th1 cytokines involves altered costimulation of T cells; loading of APCs with DNA encoding PLP139-151 reduced the capacity of a T cell line reactive to PLP139-151 to proliferate even in the presence of exogenous CD28 costimulation. DNA immunization with the myelin minigene for PLP-altered expression of B7.1 (CD80), and B7.2 (CD86) on APCs in the spleen. Suppressive immunization against self-Ags encoded by DNA may be exploited to treat autoimmune diseases.

Animals↗
PubMed1998

On the mechanism of 5-enolpyruvylshikimate-3-phosphate synthase.

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form EPSP, a precursor for the aromatic amino acids. This paper examines a recent claim [Studelska, D. R., McDowell, L. M., Espe, M. P., Klug, C. A., and Schaefer, J. (1997) Biochemistry 36, 15555-15560] that the mechanism of EPSP synthase involves two covalent enzyme-intermediates, in complete contrast to a large body of literature that has already proven the involvement of a single noncovalent intermediate. The evidence in the paper of Studelska et al. is examined closely, and unequivocal proof is provided that those authors' NMR assignments to covalent structures are in error, and that in fact the species they observed were simply the product EPSP and a side-product EPSP ketal. Since those authors used rotational-echo double-resonance (REDOR) solid-state NMR to measure intermolecular and intramolecular distances in the proposed covalent intermediates, we have used REDOR to measure the same distances in enzyme-free and enzyme-bound preparations of purified EPSP, and enzyme-free preparations of purified EPSP ketal. The distance between the shikimate ring phosphorus atom and C8 in enzyme-free EPSP is 6.6 +/- 0.1 A, which lengthens to 7.4 +/- 0.1 A in the presence of the enzyme, and in enzyme-free EPSP ketal is 5.6 +/- 0.1 A. These are entirely consistent with those measured by Studelska et al., which were 7.5 +/- 0.5 A for a putative enzyme-enolpyruvyl species and 6.1 +/- 0.3 A for a putative enzyme-ketal species.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1998

Short peptide-based tolerogens without self-antigenic or pathogenic activity reverse autoimmune disease.

An immunodominant epitope of myelin basic protein (MBP), VHFFKNIVTPRTP (p87-99), is a major target of T cells in brain lesions of multiple sclerosis (MS), and this peptide can trigger experimental autoimmune encephalomyelitis (EAE). We designed truncated peptides based on this pathogenic 13-mer that are not antigenic. These short peptides reduced production of IFN-gamma and TNF-alpha in vivo. Moreover, paraplegic rats given the 7-mer FKNIVTP in soluble form showed total reversal of paralysis in 24 h. Truncated peptides that are too small to stimulate antigenic responses to pathogenic regions of myelin basic protein are nevertheless effective tolerogens and are able to anergize autoreactive T cells. Short peptide-based tolerogens, devoid of immunogenic and pathogenic potential, may be attractive for therapy of autoimmune diseases.

Animals↗
PubMed1998

Effects of sample preparation conditions on biomolecular solid-state NMR lineshapes.

Sample preparation conditions with the 46 kDa enzyme complex of 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase, shikimate-3-phosphate (S3P) and glyphosate (GLP) have been examined in an attempt to reduce linewidths in solid-state NMR spectra. The linewidths of 31P resonances associated with enzyme bound S3P and GLP in the lyophilized ternary complex have been reduced to 150 +/- 12 Hz and 125 +/- 7 Hz respectively, by a variety of methods involving additives and freezing techniques.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1998

Local L-NAME decreases blood flow and increases leukocyte adhesion via CD18.

Local inhibition of nitric oxide (NO) synthesis with L-arginine analogs such as NG-nitro-L-arginine methyl ester (L-NAME) decreased red blood cell velocity (VRBC) in capillaries and increased leukocyte adhesion in postcapillary venules in rat skeletal muscle. The goal of the present study was to determine the mechanism of this response to L-NAME. Using intravital videomicroscopy, we examined blood flow in the surface microvasculature of rat extensor digitorum longus muscle. L-NAME (30 mM in the pipette) locally applied to capillaries (300 microns from feeding arteriole) reduced VRBC [control VRBC = 244 +/- 53 (SE) microns/s; delta VRBC = -52 +/- 8%] and increased leukocyte adhesion (from 0.2 +/- 0.01 to 1.3 +/- 0.3 cells/100 microns) in control animals. Systemic pretreatment with fucoidan (selectin binder), superoxide dismutase and catalase (extracellular antioxidants), dimethylthiourea (intracellular antioxidant), or ketotifen (mast cell stabilizer) did not alter this response. Pretreatment with CL26, an anti-CD18 antibody, abolished the L-NAME response. Our results suggest that L-NAME increased leukocyte-endothelial interactions via an effect on CD11/CD18 or its ligand, intercellular adhesion molecule.

Animals↗
PubMed1997

Introduction of soluble proteins into the MHC class I pathway by conjugation to an HIV tat peptide.

Protection against most intracellular pathogens requires T cells that recognize pathogen-derived peptides in association with MHC class I molecules on the surface of infected cells. However, because exogenous proteins do not ordinarily enter the cytosol and access the MHC class I-processing pathway, protein-based vaccines that induce class I-restricted CTL responses have proved difficult to design. We have addressed this problem by conjugating proteins, such as OVA, to a short cationic peptide derived from HIV-1 tat (residues 49-57). When APC were exposed in vitro to such protein conjugates, they processed and presented the peptides in association with MHC class I molecules and stimulated CD8+ Ag-specific T cells. Moreover, Ag-specific CTLs were generated in vivo by immunizing mice with histocompatible dendritic cells that had been exposed to protein-tat conjugates.

Animals↗
PubMed1997

Comparative studies of phase-cycling schemes for multiple pi-pulse sequences.

Recently, a new phase cycling scheme was introduced by this laboratory for use in biological solid-state NMR experiments involving multiple pi-pulses with characteristics that suggested it may enhance the sensitivity of these kind of experiments (Y. Li and J. N. S. Evans, 1995, Chem. Phys. Lett. 241, 79 and Erratum, 1995, ibid. 246, 527; Y. Li and J. N. S. Evans, 1996, J. Magn. Reson. B 111, 296). The new sequence followed the supercycled concept proposed a decade ago for heteronuclear decoupling experiments. In this paper, more detailed experiments demonstrate that the claim of enhanced sensitivity was unfounded, and in fact the supercycle proposed differs little from the established XY-8 and XY-16 based supercycles.

Magnetic Resonance Spectroscopy↗