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Michael R Knowles

Publications and source records attributed to Michael R Knowles.

32 records · Page 2Linked to original sources
PubMed2004

A proteomic investigation of drug-induced steatosis in rat liver.

A significant problem faced by pharmaceutical companies today is the failure of lead compounds in the later stages of development due to unexpected toxicities. We have used two-dimensional differential in-gel electrophoresis and mass spectrometry to identify a proteomic signature associated with hepatocellular steatosis in rats after dosing with a compound in preclinical development. Liver toxicity was monitored over a 5 day dosing regime using blood biochemical parameter measurements and histopathological analysis. As early as 6 h postdosing, livers showed hepatocellular vacuolation, which increased in extent and severity over the course of the study. Alterations in plasma glucose, alanine aminotransferase, and aspartate aminotransferase were not detected until the third day of dosing and changed in magnitude up to the final day. The proteomic changes were observed at the earliest time point, and many of these could be associated with known toxicological mechanisms involved in liver steatosis. This included up-regulation of pyruvate dehydrogenase, phenylalanine hydroxylase, and 2-oxoisovalerate dehydrogenase, which are involved in acetyl-CoA production, and down-regulation of sulfite oxidase, which could play a role in triglyceride accumulation. In addition, down-regulation of the chaperone-like protein, glucose-regulated protein 78, was consistent with the decreased expression of the secretory proteins serum paraoxonase, serum albumin, and peroxiredoxin IV. The correlation of these protein changes with the clinical and histological data and their occurrence before the onset of the biochemical changes suggest that they could serve as predictive biomarkers of compounds with a propensity to induce liver steatosis.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed2003

Primary ciliary dyskinesia: diagnostic and phenotypic features.

Primary ciliary dyskinesia (PCD) is a genetic disease characterized by abnormalities in ciliary structure/function. We hypothesized that the major clinical and biologic phenotypic markers of the disease could be evaluated by studying a cohort of subjects suspected of having PCD. Of 110 subjects evaluated, PCD was diagnosed in 78 subjects using a combination of compatible clinical features coupled with tests of ciliary ultrastructure and function. Chronic rhinitis/sinusitis (n = 78; 100%), recurrent otitis media (n = 74; 95%), neonatal respiratory symptoms (n = 57; 73%), and situs inversus (n = 43; 55%) are strong phenotypic markers of the disease. Mucoid Pseudomonas aeruginosa (n = 12; 15%) and nontuberculous mycobacteria (n = 8; 10%) were present in older (> 30 years) patients with PCD. All subjects had defects in ciliary structure, 66% in the outer dynein arm. Nasal nitric oxide production was very low in PCD (nl/minute; 19 +/- 17 vs. 376 +/- 124 in normal control subjects). Rigorous clinical and ciliary phenotyping and measures of nasal nitric oxide are useful for the diagnosis of PCD. An increased awareness of the clinical presentation and diagnostic criteria for PCD will help lead to better diagnosis and care for this orphan disease.

Adult↗
PubMed2003

Investigation of the possible role of a novel gene, DPCD, in primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is an autosomal recessive disease caused by mutations that affect the proper function of cilia. Recently, deletion of DNA polymerase lambda (Poll) in mice produced a phenotype characteristic of PCD (Kobayashi et al., 2002, Mol. Cell. Biol. 22:2769-2776). Because it is unclear how a mutation in a DNA polymerase would result in a specific defect in axonemes, the targeting construct was examined further. Analysis of the genomic region surrounding the Poll gene revealed an uncharacterized gene, named Dpcd, that is predicted to be transcribed from the opposite strand relative to Poll. The deletion of Poll would also remove the first exon of Dpcd. Because it is possible that the PCD phenotype observed is due to the absence of either gene, the expression of these genes during ciliogenesis of human airway epithelial cells was examined. Northern analysis demonstrated that DPCD expression increases during ciliated cell differentiation; the expression of POLL decreases. To examine directly whether DPCD is mutated in cases of human PCD, the complete coding sequence of DPCD was sequenced from 51 unrelated PCD patients. No disease-causing mutations were confirmed; however, one variant could not be excluded. Therefore, DPCD remains a novel candidate gene for PCD.

Bronchi↗
PubMed2003

Multiplex proteomic analysis by two-dimensional differential in-gel electrophoresis.

This paper describes the use of fluorescence two-dimensional differential in-gel electrophoresis in a multiplex analysis of two distinct proteomes. As a model system, cerebral cortex tissues were analyzed from neurokinin1 receptor knockout (NK(1)R-/-) and wild type (NK(1)R+/+) mice in an attempt to identify molecular pathways involved in the function of this protein. Paired NK(1)R-/- and NK(1)R+/+ samples were labeled with fluorescent Cy3 and Cy5 dyes and electrophoresed on the same two-dimensional gels. Scanning the gels at wavelengths specific for each dye revealed the two different proteomes which were overlaid and the differences in abundance of specific protein spots were determined by the Amersham Biosciences DeCyder Differential In-gel Analysis software. A Cy2-labeled sample pool was co-electrophoresed with all Cy3- and Cy5-labeled sample pairs as an internal standard providing a link for inter-gel comparisons and for more robust statistical analysis of the data. Eight spots were found to be upregulated and two downregulated in the NK(1)R-/- mice compared to NK(1)R+/+ controls. Matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass fingerprinting was used to identify the proteins. The results illustrate the power of this multiplex proteomics technology and illustrate how proteomics can be used to understand gene function.

Animals↗
PubMed2002

Nontuberculous mycobacteria. I: multicenter prevalence study in cystic fibrosis.

Nontuberculous mycobacteria (NTM) are potential respiratory pathogens in cystic fibrosis (CF). To assess the species-specific prevalence and risk factors for acquisition, we conducted a prospective, cross-sectional study of the prevalence of NTM and clinical features of patients at 21 U.S. centers. Almost 10% of patients with CF who were 10 years or older were included (n = 986). The overall prevalence of NTM in sputum was 13.0% (range by center, 7-24%). Mycobacterium avium complex (72%) and Mycobacterium abscessus (16%) were the most common species. When compared with patients with CF without NTM, culture-positive subjects were older (26 vs. 22 years, p < 0.001), had a higher FEV1 (60 vs. 54%, p < 0.01), higher frequency of Staphylococcus aureus (43 vs. 31%, p < 0.01), and lower frequency of Pseudomonas aeruginosa (71 vs. 82%, p < 0.01). Molecular typing revealed that almost all patients within each center had unique NTM strains. In summary, NTM are common in patients with CF, but neither person-to-person nor nosocomial acquisition explained the high prevalence. Older age was the most significant predictor for isolation of NTM. The clinical significance of NTM in CF is incompletely defined, but patients with these organisms should be monitored with repeat cultures.

Adolescent↗
PubMed2002

Nontuberculous mycobacteria. II: nested-cohort study of impact on cystic fibrosis lung disease.

The prevalence of nontuberculous mycobacteria (NTM) is high (approximately 13%) in sputum of patients with cystic fibrosis (CF), but the impact on lung disease is unknown. We followed 60 incident NTM-positive and 99 culture-negative patients with CF for 15 months and assessed clinical impact of NTM by FEV1 and high-resolution computed tomography (HRCT) of the chest. Mycobacterium avium complex was seen in 75% of NTM-positive subjects. The annual rate of decline in FEV1 was not different among control versus NTM-positive subjects who did not, or did, meet American Thoracic Society microbiologic criteria for NTM disease (3 +/- 1, 3 +/- 2, and 5 +/- 2%, respectively). More subjects with three or more positive cultures for NTM had two or more characteristic findings on entry HRCT (60%, 9/15) as compared with subjects with two positive cultures or less (32%) or negative cultures (19%; p < 0.02). All subjects with three or more positive cultures and exit HRCTs (n = 6) showed progression of HRCT findings, whereas only 17% of subjects with two positive cultures or less had progression (p = 0.0006). In summary, no significant short-term effect on FEV1 was detected in patients with multiple positive NTM cultures, but an abnormal HRCT was predictive of progression. Patients with CF and multiple positive NTM cultures, characteristic HRCT findings, and progression of HRCT changes should be monitored closely and considered for antimycobacterial therapy.

Adult↗
PubMed2002

Increasing concentration of inhaled saline with or without amiloride: effect on mucociliary clearance in normal subjects.

Mucociliary clearance is determined by ciliary activity and rheology of airway surface liquid. To test the hypothesis that mucociliary clearance would increase after inhalation of an osmotically active agent that would increase the volume of airway surface liquid, we measured mucociliary clearance in 16 normal subjects after inhalation of varying tonicities of saline alone, and after pretreatment with a Na+ channel blocker (amiloride). Subjects inhaled vehicle (0.12% saline) or amiloride, followed by inhalation of 0.12, 0.9, or 7% saline. Subsequently, mucociliary clearance rates were measured by gamma scintigraphy of inhaled 99mTc Fe2O3. Mucociliary clearance of whole and peripheral lung was increased (approximately twofold) after inhalation of increasing concentrations of saline (p < 0.04). Pretreatment with amiloride increased mucociliary clearance rates (approximately twofold) after inhalation of 0.12 and 0.9% saline (p < 0.05), but not 7% saline. The rates of mucociliary clearance by pretreatment with amiloride and 7% saline alone (approximately 1.4% per minute) approached the rapid mucociliary clearance rates (approximately 2.0% per minute) reported in systemic pseudohypoaldosteronism, which has loss-of-function mutations of the epithelial Na+ channel and an increased volume of airway surface liquid. We conclude that maneuvers that increase the volume of airway surface liquid are associated with increased rates of mucociliary clearance in normal subjects.

Administration, Inhalation↗
PubMed2002

Alterations of stress related proteins in genetically altered mice revealed by two-dimensional differential in-gel electrophoresis analysis.

Transgenic, knockout and knockin mice are useful tools for linking specific genes with behaviour and other complex biological processes. However, complications arising due to compensatory changes, genetic background differences and other factors could lead to difficulty in interpreting the resulting changes in phenotype. We have used fluorescence two-dimensional differential in-gel electrophoresis in combination with matrix-assisted laser desorption/ionization-time of flight mass fingerprinting to investigate the possibility that distinct genetic alterations can lead to common protein expression changes in genetically modified mice. Brain proteomes were compared from two transgenic mouse strains (Tg2576 x TgPS1 and Tg2576), two knockout mouse strains (5-HT(7)R -/- and GABA(A)Ralpha5 -/-) and one knockin mouse strain (GABA(A)Ralpha1-H101R). Both of the transgenic models showed an isoform change in the heat shock 70 related protein, mortalin. The knockout and knockin models showed similar changes in mortalin expression along with an alteration of the anti-oxidant protein 2. The observed proteomic alterations indicate that stress-responsive protein pathways may be altered artefactually in all of the mouse models used in this study and highlights an area where caution is needed in interpreting proteomic changes in genetically modified mice.

Animals↗
PubMed2002

In vivo airway surface liquid Cl- analysis with solid-state electrodes.

The pathogenesis of cystic fibrosis (CF) airways disease remains controversial. Hypotheses that link mutations in CFTR and defects in ion transport to CF lung disease predict that alterations in airway surface liquid (ASL) isotonic volume, or ion composition, are critically important. ASL [Cl-] is pivotal in discriminating between these hypotheses, but there is no consensus on this value given the difficulty in measuring [Cl-] in the "thin" ASL (approximately 30 microm) in vivo. Consequently, a miniaturized solid-state electrode with a shallow depth of immersion was constructed to measure ASL [Cl-] in vivo. In initial experiments, the electrode measured [Cl-] in physiologic salt solutions, small volume (7.6 microl) test solutions, and in in vitro cell culture models, with > or =93% accuracy. Based on discrepancies in reported values and/or absence of data, ASL Cl- measurements were made in the following airway regions and species. First, ASL [Cl-] was measured in normal human nasal cavity and averaged 117.3 +/- 11.2 mM (n = 6). Second, ASL [Cl-] measured in large airway (tracheobronchial) regions were as follows: rabbit trachea and bronchus = 114.3 +/- 1.8 mM; (n = 6) and 126.9 +/- 1.7 mM; (n = 3), respectively; mouse trachea = 112.8 +/- 4.2 mM (n = 13); and monkey bronchus = 112.3 +/- 10.9 mM (n = 3). Third, Cl- measurements were made in small (1-2 mm) diameter airways of the rabbit (108.3 +/- 7.1 mM, n = 5) and monkey (128.5 +/- 6.8 mM, n = 3). The measured [Cl-], in excess of 100 mM throughout all airway regions tested in multiple species, is consistent with the isotonic volume hypothesis to describe ASL physiology.

Adult↗
PubMedDate not supplied

The I148T CFTR allele occurs on multiple haplotypes: a complex allele is associated with cystic fibrosis.

PURPOSE: To determine whether intragenic changes modulate the cystic fibrosis (CF) phenotype in individuals who are positive for the I148T allele. METHODS: The genes from individuals who carried at least one copy of the I148T allele were analyzed for additional changes that may be acting as genetic modifiers. RESULTS: Seven of eight individuals with a known or suspected diagnosis of CF who carried I148T in combination with a severe CF mutation also carried 3199del6. Eight apparently healthy adult individuals who were compound heterozygous for I148T and a severe CF mutation or homozygous for I148T did not carry the deletion ( = 0.0014). The I148T allele occurs on at least three haplotypes: an IVS-8 9T background, a 7T background, or a 9T + 3199del6 background. The 3199del6 allele was not identified in 386 non-CF chromosomes. CONCLUSIONS: It is concluded that I148T occurs on at least three haplotypes and the complex allele I148T + 9T + 3199del6 is associated with a classic CF phenotype.

Adolescent↗