Against the ATS statement on human embryonic stem cell research.
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Blastocyst↗
Biomedical subjects
Charles F Thomas
Publications and source records attributed to Charles F Thomas.
Cloning and characterization of CYP51 from Mycobacterium avium.
Mycobacterium avium complex (MAC) causes chronic lung disease in immunocompetent people and disseminated infection in patients with AIDS. MAC is intrinsically resistant to many conventional antimycobacterial agents, it develops drug resistance rapidly to macrolide antibiotics, and patients with MAC infection experience frequent relapses or the inability to completely eradicate the infection with current treatment. Treatment regimens are prolonged and complicated by drug toxicity or intolerances. We sought to identify biochemical pathways in MAC that can serve as targets for novel antimycobacterial treatment. The cytochrome P450 enzyme, CYP51, catalyzes an essential early step in sterol metabolism, removing a methyl group from lanosterol in animals and fungi, or from obtusifoliol in plants. Azoles inhibit CYP51 function, leading to an accumulation of methylated sterol precursors. This perturbation of normal sterol metabolism compromises cell membrane integrity, resulting in growth inhibition or cell death. We have cloned and characterized a CYP51 from MAC that functions as a lanosterol 14alpha-demethylase. We show the direct interactions of azoles with purified MAC-CYP51 by absorbance and electron paramagnetic resonance spectroscopy, and determine the minimum inhibitory concentrations (MICs) of econazole, ketoconazole, itraconazole, fluconazole, and voriconazole against MAC. Furthermore, we demonstrate that econazole has a MIC of 4 mug/ml and a minimum bacteriocidal concentration of 4 mug/ml, whereas ketoconazole has a MIC of 8 mug/ml and a minimum bacteriocidal concentration of 16 mug/ml. Itraconazole, voriconazole, and fluconazole did not inhibit MAC growth to any significant extent.
Anti-Infective Agents↗
Wavelet compression of three-dimensional time-lapse biological image data.
The use of multifocal-plane, time-lapse recordings of living specimens has allowed investigators to visualize dynamic events both within ensembles of cells and individual cells. Recordings of such four-dimensional (4D) data from digital optical sectioning microscopy produce very large data sets. We describe a wavelet-based data compression algorithm that capitalizes on the inherent redunancies within multidimensional data to achieve higher compression levels than can be obtained from single images. The algorithm will permit remote users to roam through large 4D data sets using communication channels of modest bandwidth at high speed. This will allow animation to be used as a powerful aid to visualizing dynamic changes in three-dimensional structures.
Algorithms↗
Microscopy of multiple visual receptor types in Drosophila.
PURPOSE: To take advantage of specialized microscopic methods and transgenic stocks, to understand the properties of each rhodopsin now that Drosophila's six rhodopsins (Rh1-Rh6) have been isolated. METHODS: The visual pigment containing organelles, the rhabdomeres, were imaged in live flies with the pseudopupil in standard and confocal microscopes. Five transgenic Drosophila strains in which Rh2-Rh6 replaced the native Rh1 in R1-6 receptors were compared with normal controls (Rh1 in R1-6) for two lines of work: (1) autofluorescence of rhodopsin; and (2) imaging rhodopsin. Other transgenic Drosophila in which the Rh1, Rh3, and Rh4 promoters drive the green fluorescent protein (GFP) reporter were used for other purposes, especially distinguishing the R7/8 types. RESULTS: We show, for the first time, that visual pigment appears pink in white light, especially for Rh1 and Rh6. While showing that rhodopsin-metarhodopsin conversions were understood by their respective wavelengths, we discovered that, for Rh6, rhodopsin and metarhodopsin could not be spectrally separated. Relative fluorescent emission, Rh1=Rh5>Rh6>Rh2>Rh4>Rh3, was of little value in explaining differences between bright and dim autofluorescence in R7. Rather, analysis of GFP driven by Rh3 and Rh4 promoters show that the rhabdomeres with bright autofluorescence are the ones that contain Rh4. CONCLUSIONS: Careful imaging provides a useful approach to analyzing Drosophila rhodopsins. Amid a considerable body of microscopic data, we identify the sources of bright and dim R7 rhabdomeres, and we demonstrate the unique properties of Rh6.
Animals↗
Expression analysis of PCSTE3, a putative pheromone receptor from the lung pathogenic fungus Pneumocystis carinii.
The fungal pathogen Pneumocystis carinii remains the most prevalent opportunistic infection in patients infected with HIV. Fungal pheromone receptors are seven transmembrane domain G-protein-coupled receptors which are expressed on specific mating types, and have ligand-binding extracellular domains for specific pheromones from cells of the opposite mating type. We have cloned and characterized PCSTE3 from P. carinii, which encodes a seven transmembrane domain protein orthologous to the Saccharomyces cerevisiae pheromone receptor Ste3. We detect PCSTE3 by indirect immunofluorescence using antibodies designed to extracellular domains of the receptor in yeast expressing the protein. Using a downstream Fus1-lacZ reporter gene, we determined that PCSTE3 does not recognize a- or alpha-factor pheromones as ligands for the receptor. We isolated P. carinii life cycle stages and examined PCSTE3 expression by immunofluorescence microscopy and flow cytometry, and found PCSTE3 expression exclusively on a population of trophic forms. PCSTE3 receptor expression was not found on cysts.
Amino Acid Sequence↗
Pneumocystis pneumonia.
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AIDS-Related Opportunistic Infections↗
Biochemical requirements for PCBCK1 kinase activity, the Pneumocystis carinii MEKK involved in cell wall integrity.
Fungal cell wall assembly is a complicated process involving multiple enzymes and coordinated signaling pathways. The cell wall integrity MAPK pathway acts to stabilize the fungal cell wall during conditions of elevated temperature by regulation of glucan synthesis. The upstream kinase, BCK1, is a critical component of this pathway. Pneumonia is a significant cause of death from the fungal opportunistic pathogen Pneumocystis in immunocompromised states, especially with HIV infection. We have previously shown that PCBCK1 functions in the cell wall integrity pathway in yeast as a functional protein kinase. Kinases have specific requirements for enzymatic function which have not been investigated in fungi. Here we examine the biochemical requirements for PCBCK1 kinase activity expressed in Saccharomyces cerevisiae bck1Delta yeast. PCBCK1 requires 10 mM MgCl(2), pH 6, temperature 30 degrees C, and 10 microM ATP for kinase activity. Interference of the Pneumocystis cell wall integrity pathway is an attractive target for drug development since glucan synthesis machinery is not present in humans.
Adenosine Triphosphate↗
Advances in the treatment of rheumatic interstitial lung disease.
PURPOSE OF REVIEW: Interstitial lung disease frequently complicates the rheumatic diseases. The purpose of this review is to outline recent advances and current concepts regarding the management of these interstitial lung diseases. RECENT FINDINGS: Several histologic lesions cause interstitial lung disease in rheumatic diseases, including nonspecific interstitial pneumonia, usual interstitial pneumonia, organizing pneumonia, lymphocytic interstitial pneumonia, desquamative interstitial pneumonia, and acute interstitial pneumonia. Although the relative frequency of occurrence of these histopathologic lesions is not definitively established, it seems that nonspecific interstitial pneumonia accounts for a large proportion of rheumatic disease-associated interstitial lung diseases. Although usual interstitial pneumonia generally responds poorly to corticosteroid therapy, other forms of interstitial pneumonia are often steroid responsive and have a more favorable long-term prognosis. Pulmonary hypertension is increasingly recognized as a complication of these interstitial lung diseases. Treatment of pulmonary hypertension in these patients provides clinical benefit and may suppress pulmonary inflammation and fibrosis. Lung transplantation is a treatment option for selected patients with severe pulmonary involvement and limited life expectancy. SUMMARY: Interstitial lung disease is common in the rheumatic diseases, may be caused by a variety of lesions that respond differently to treatment, and may lead to the development of pulmonary hypertension. Whether the prognosis of interstitial lung disease associated with rheumatic disease is similar to that associated with the idiopathic interstitial pneumonias is not known. Treatment of these interstitial lung diseases should take into account the specific histologic lesion, the activity of the underlying rheumatic disease, and associated pulmonary hypertension, if present. The diagnosis of a rheumatic disease is no longer an absolute contraindication to lung transplantation.
Humans↗
Complementation and characterization of the Pneumocystis carinii MAPK, PCM.
Mitogen-activated protein kinase (MAPK) pathways transfer environmental signals into intracellular events such as proliferation and differentiation. Fungi utilize a specific pheromone-induced MAPK pathway to regulate conjugation, formation of an ascus, and entry into meiosis. We have previously identified a MAPK, PCM, from the fungal opportunist Pneumocystis, responsible for causing severe pneumonia in patients with AIDS. In order to gain insight into the function of PCM, we expressed it in Saccharomyces cerevisiae deficient in pheromone signaling and tested activation and inhibition of this MAPK pathway. PCM restored pheromone signaling in S. cerevisiae fus3Delta kss1Delta mutants with alpha-factor pheromone (six-fold increase) and was not activated by osmotic stress. Signaling through this pathway decreased 2.5-fold with 10 microM U0126, and was unaffected with SB203580. We evaluated the conditions for native PCM kinase activity isolated from Pneumocystis carinii organisms and found that 0.1 mM MgCl2, pH 6.5, temperature 30-35 degrees C, and 10 microM ATP were optimal. The activity of PCM is significantly elevated in P. carinii trophic forms compared to cysts, implicating a role for PCM in the life cycle transition of P. carinii from trophic forms to cysts.
Butadienes↗
Pneumocystis carinii BCK1 functions in a mitogen-activated protein kinase cascade regulating fungal cell-wall assembly.
Pneumocystis pneumonia remains the most common AIDS-defining opportunistic infection in people with HIV. The process by which Pneumocystis carinii constructs its cell wall is not well known, although recent studies reveal that molecules such as beta-1-3-glucan synthetase (GSC1) and environmental pH-responsive genes such as PHR1 are important for cell-wall integrity. In closely related fungi, a specific mitogen-activated protein kinase (MAPK) cascade regulates cell-wall assembly in response to elevated temperature. The upstream mitogen-activated protein kinase kinase kinase (MAPKKK, or MEKK), BCK1, is an essential component in this pathway for maintaining cell-wall integrity and preventing fungal cell lysis. We have identified a P. carinii MEKK gene and have expressed it in Saccharomyces cerevisiae to gain insights into its function. The P. carinii MEKK, PCBCK1, corrects the temperature-sensitive cell lysis defect of bck1Delta yeast. Further, at elevated temperature PCBCK1 restored the signaling defect in bck1Delta yeast to maintain expression of the temperature-inducible beta-1-3-glucan synthetase gene, FKS2. PCBCK1, as a functional kinase, is capable of autophosphorylation and substrate phosphorylation. Since glucan machinery is not present in mammals, a better understanding of this pathway in P. carinii might aid in the development of novel medications which interfere with the integrity of the Pneumocystis cell wall.
Base Sequence↗
Pneumocystis carinii STE11, an HMG-box protein, is phosphorylated by the mitogen activated protein kinase PCM.
A pheromone-induced mitogen activated protein kinase (MAPK) pathway controls mating in fungi by regulating gene transcription. In the opportunistic fungus Pneumocystis carinii, we have identified a protein containing a high-mobility group (HMG) motif which is homologous to the transcriptional activators STE11 of Schizosaccharomyces pombe and STE12 of Saccharomyces cerevisiae. In fungi, this transcriptional activator functions in sexual development, filamentous growth, and pathogenicity. The fungal pheromone-activated MAPK phosphorylates the transcriptional activator to allow binding to pheromone-response elements in the promoter regions of certain genes. We have previously identified a P. carinii MAPK, PCM, which has significant homology to fungal MAPKs involved in mating. As an initial step in understanding the downstream molecules which interact with the PCM kinase, we have cloned a STE11 homologue in P. carinii. PCSTE11 has an open-reading frame of 1.5 kb which encodes a protein of 501 amino acids with a molecular weight of 56 kDa. Greatest homology was to S. pombe STE11 (52%). We have expressed a His-tag fusion of PCSTE11 and purified the protein with nickel affinity resin. PCM phosphorylates the purified protein indicating that PCSTE11 is associated with the MAPK cascade in P. carinii.
Amino Acid Sequence↗
Characterization of a lanosterol 14 alpha-demethylase from Pneumocystis carinii.
Pneumocystis carinii (PC) causes severe pneumonia in immunocompromised patients. PC is intrinsically resistant to treatment with azole antifungal medications. The enzyme lanosterol 14 alpha-demethylase (Erg11) is the target for azole antifungals. We cloned PCERG11 and compared its sequence to Erg11 proteins present in azole-resistant organisms, and performed chromosomal and Northern blot analysis for PCERG11. Of 13 potential sites which could confer resistance to azoles, two were identical to azole-resistant Candida. By site-directed mutagenesis we changed these two sites in PCERG11 to those present in azole-sensitive Candida to generate PCERG11-SDM (E113D, T125K). We tested the susceptibility of ERG11 deletion strains of Saccharomyces cerevisiae (SC) expressing PCERG11, PCERG11-SDM, and wild-type SCERG11 to three azole antifungals: fluconazole, itraconazole, and voriconazole. PCERG11 required a 2.2-fold higher dose of voriconazole and 3.5-fold higher dose of fluconazole than SCERG11 for a 50% reduction in growth. No difference was observed in the sensitivity to itraconazole. PCERG11-SDM has increased sensitivity to fluconazole and voriconazole, but not itraconazole. We believe that the molecular structure of the lanosterol 14 alpha-demethylase encoded by PCERG11 confers inherent resistance to azole antifungals and plays an integral part in the overall resistance of this PC to azole therapy.
Amino Acid Sequence↗
Inflammation and infection in exacerbations of chronic obstructive pulmonary disease.
Chronic obstructive pulmonary disease (COPD) is a common disorder with symptoms of chronic cough and progressive dyspnea caused by chronic bronchitis or emphysema. Acute exacerbations of COPD contribute to the accelerated decline in lung function characteristic of this disease and are associated with significant cost, morbidity, and mortality for patients. Controversy exists as to whether exacerbations are caused primarily by inflammation, infection, or perhaps a combination of both conditions. Advances in the pathogenesis of COPD have shed light on the role of inflammation in this condition and highlighted the differences in the inflammatory response present in COPD compared with asthma. Infectious agents often are suspected as causing acute exacerbations of COPD, and antibiotics are frequently prescribed empirically to patients. We review the evidence for an inflammatory and infectious etiology for exacerbations of COPD and compare and contrast how each impacts on this disease.
Humans↗
Complementation and in vivo biochemical characterization of the Pneumocystis carinii MAPK.
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Cloning, Molecular↗
Pneumocystis carinii BCK1 complements the Saccharomyces cerevisiae cell wall integrity pathway.
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Amino Acid Sequence↗
Lung epithelial cells and extracellular matrix components induce expression of Pneumocystis carinii STE20, a gene complementing the mating and pseudohyphal growth defects of STE20 mutant yeast.
Pneumocystis carinii causes severe pneumonia in immunocompromised hosts. The binding of P. carinii to alveolar epithelial cells and extracellular matrix constituents such as fibronectin and vitronectin is a central feature of infection, which initiates proliferation of the organism. Herein, we demonstrate that P. carinii binding to lung cells specifically alters the gene expression of the organism, regulating fungal growth. Subtractive hybridization was performed to isolate P. carinii genes expressed following binding to mammalian extracellular matrix constituents. P. carinii STE20 (PCSTE20), a gene participating in mating and pseudohyphal growth of other fungi, was identified following adherence to the extracellular matrix constituents fibronectin, vitronectin, collagen, and lung epithelial cells. The expression of PCSTE20 and a related P. carinii mitogen-activated protein kinase (MAPK) kinase gene, also implicated in signaling of mating, were both specifically upregulated by binding to matrix protein. The expression of general cyclin-dependent kinases and other MAPKs not involved in mating pathways were not altered by organism binding. PCSTE20 expression was also strongly enhanced following organism attachment to A549 lung epithelial cells. When expressed in a Saccharomyces cerevisiae ste20Delta mutant, PCSTE20 suppressed defects in both mating and pseudohyphal growth. These findings are consistent with the observed proliferation and filopodial extension of Pneumocystis organisms adherent to the epithelium in the lungs of immunocompromised hosts. PCSTE20 expression appears to represent a significant component in the regulation of the life cycle of this intractable opportunistic pathogen.
Amino Acid Sequence↗
Clinical response of rheumatoid arthritis-associated pulmonary fibrosis to tumor necrosis factor-alpha inhibition.
Treatment options for patients with pulmonary fibrosis associated with rheumatoid disease are limited. We report a case of a 71-year-old man with a 3-year history of seropositive rheumatoid arthritis (RA) referred to the pulmonary clinic because of progressive pulmonary symptoms associated with radiographic fibrosis that was progressive in spite of corticosteroid treatment. In an attempt to control his articular symptoms and alter the course of his pulmonary fibrosis, treatment with IV infusion of the tumor necrosis factor (TNF)-alpha inhibitor infliximab was initiated. Following 1 year of therapy with this agent, the patient reported sustained improvement in dyspnea, cough, and exercise tolerance, in addition to improvement in joint symptoms. Stabilization of pulmonary function was indicated by repeat pulmonary function test findings. This report suggests that inhibition of TNF-alpha may be of significant benefit to patients with fibrosing lung conditions in the setting of RA.
Aged↗