Biomedical subjects

T Mio

Publications and source records attributed to T Mio.

At least 19 recordsLinked to original sources

PubMed2006

High sensitivity C-reactive protein in asthma.

Asthma is characterised by chronic inflammation of the airways, but the relevance of high-sensitivity assays for C-reactive protein (hs-CRP), which are known to be a sensitive marker of low-grade systemic inflammation, has not been fully studied in asthma. The objective was to examine serum hs-CRP levels in patients with asthma and their relationship to clinical characteristics and degree of airway inflammation. Serum hs-CRP levels were cross-sectionally examined in steroid-naive (n = 22) and steroid-inhaling (n = 23) adult patients with asthma and healthy controls (n = 14). All were nonsmokers. Serum hs-CRP levels were significantly increased in steroid-naive patients (mean+/-sd 1.33+/-1.48 mg.L(-1)) compared with controls (0.21+/-0.30 mg.L(-1)), but not in patients on inhaled corticosteroid. Among steroid-naive patients, serum hs-CRP levels significantly negatively correlated with indices of pulmonary function (forced expiratory volume in one second/forced vital capacity and forced mid-expiratory flow) and positively with sputum eosinophil count. Among patients on inhaled corticosteroid, hs-CRP levels did not correlate with any indices. In conclusion, an increase in serum C-reactive protein levels measured by high-sensitivity assays may be associated with airflow obstruction and airway inflammation, and may serve as a surrogate marker of airway inflammation in asthma.

Adult↗

PubMed2005

Relationship of airway wall thickening to an imbalance between matrix metalloproteinase-9 and its inhibitor in asthma.

BACKGROUND: The balance between matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) may be critical in extracellular matrix remodelling, a characteristic of asthmatic airways. An excess of TIMP-1 over MMP-9 has been associated with chronic airflow obstruction but the mechanisms underlying this association remain unknown. Recent computed tomographic (CT) studies indicate that airway wall thickening is associated with chronic airflow obstruction. METHODS: Sputum levels of MMP-9, TIMP-1, and their molar ratio were examined in 26 patients with stable asthma and their relationship with pulmonary function and airway wall thickness, assessed by a validated CT technique which measured wall area corrected by body surface area (WA/BSA), the ratio of WA to outer wall area (WA%), and the absolute wall thickness corrected by radicalBSA of a segmental bronchus (T/ radicalBSA), was examined. RESULTS: Sputum MMP-9 levels were inversely correlated with WA% and TIMP-1 levels were positively correlated with WA/BSA and T/ radicalBSA. The MMP-9/TIMP-1 molar ratio was inversely correlated with WA% and T/ radicalBSA and positively correlated with post-bronchodilator values of mid-forced expiratory flow and maximum expiratory flow at the quartile of lung volume. CONCLUSION: Excess TIMP-1 may have a pathogenetic role in airway wall thickening in asthmatic patients which may result in chronic airflow obstruction.

Asthma↗

PubMed2003

Gamma radiation inhibits fibroblast-mediated collagen gel retraction.

Radiation exposure is known to impair healing in irradiated areas. Fibroblasts play a major role in the production and modification of extracellular matrix in wound repair. Since one important aspect of wound repair is the contraction of the wound, this study investigated the effects of radiation on the ability of fibroblasts to mediate collagen gel contraction in an in vitro model of wound retraction. After irradiation, the cells were detached and suspended in a solution of rat tail tendon collagen. Radiation exposure decreased retraction, and this effect was dose dependent. In order to define the mechanism of reduced gel retraction, we investigated alpha2beta1 cell surface integrin and fibronectin, which are thought to mediate contraction, and prostaglandin E2 (PGE2), which is known to inhibit this process. PGE2 release increased dose responsively following radiation. The cyclooxygenase inhibitor indomethacin could partially restore the contractile activity of irradiated fibroblasts. Fibronectin production in gel culture showed a significant decrease. In contrast, there was no decrease in alpha2beta1 integrin expression in radiated cells. In conclusion, radiation decreases fibroblast-mediated gel contraction. Increased PGE2 production and decreased fibronectin production by irradiated fibroblasts may contribute to this effect and may be in part responsible for poor healing of radiated tissue.

Animals↗

PubMed2001

Identification and characterization of the genes for N-acetylglucosamine kinase and N-acetylglucosamine-phosphate deacetylase in the pathogenic fungus Candida albicans.

Like bacteria and many fungi, the pathogenic fungus Candida albicans can utilize GlcNAc as a carbon source for growth. A cluster of six genes was identified in the C. albicans genome. One of the genes in the cluster was CaNAG1, which is responsible for GlcN6P deaminase and is therefore essential for GlcNAc-dependent growth. The other five genes were designated CaNAG2, CaNAG3, CaNAG4, CaNAG5 and CaNAG6. The mRNA levels of CaNAG1, CaNAG2 and CaNAG5 were significantly induced by GlcNAc, whereas those of CaNAG3, CaNAG4 and CaNAG6 were not. Neither CaNAG2 nor CaNAG5 was essential for growth, but disruption of CaNAG2 or CaNAG5 greatly retarded the growth of cells using GlcNAc as the sole carbon source. Although no homolog of CaNAG2 or CaNAG5 was found in the Saccharomyces cerevisiae genome, CaNag2p displayed sequence similarities to Escherichia coli nagA, and CaNag5p is homologous to a wide variety of hexose kinases. When expressed as a fusion protein with glutathione S-transferase (GST), CaNag5p produced GlcNAc-P from GlcNAc in the presence of ATP, whereas GST alone did not. Furthermore, the recombinant GST-CaNag2p fusion protein converted GlcNAcP, which was produced by CaNag5p, into GlcNP. These results clearly demonstrate that CaNAG2 and CaNAG5 encode GlcNAcP deacetylase and GlcNAc kinase, respectively. CaNag5p recognized glucose and mannose as substrates, whereas the recently identified human GlcNAc kinase was specific to GlcNAc. Deletion of CaNAG2 or CaNAG5 markedly, and that of CaNAG1 moderately, attenuated the virulence of C. albicans in a mouse systemic infection model. Thus, it appears that GlcNAc metabolism of C. albicans is closely associated with its virulence.

Amidohydrolases↗

PubMed2001

Influence of initial status on functional gain for Japanese patients with first cerebral hemorrhage.

It is important to identify in advance patients who will achieve the greatest functional gains from rehabilitation therapy, as specialist rehabilitation resources are still scarce in Japan. The purpose of this study was to determine whether functional score at admission influences the functional change (functional score at discharge minus functional score at admission) after inpatient rehabilitation for first cerebral hemorrhage. One hundred and ninety-three patients with cerebral hemorrhage were enrolled in this study. They were assessed using the Functional Independence Measure (FIM) at admission and discharge and underwent inpatient rehabilitation treatment. Patients were stratified into 3 groups according to their FIM total scores on admission as follows: (1) < or = 36 (severely affected patient group); (2) 37-72 (moderately affected patient group); and (3) >73 (mildly affected patient group). Scheffe's multiple comparison test showed that patients in group 1 were significantly older (mean +/- SD = 63 +/- 10 years) than those in groups 2 (56 +/- 10 years) or 3 (53 +/- 12 years). Patients in group 2 showed significantly greater FIM gain (37 +/- 17) compared with patients in groups 3 (23 +/- 12) or 1 (27 +/- 23). The results suggest that moderately affected patients at admission will show significantly higher functional gain compared with severely or mildly affected patients. Mildly affected patients at admission had a significantly shorter length of hospital stay for rehabilitation than the other groups. There was no significant difference in onset to admission interval between the 3 groups. The functional levels of affected patients on admission, as stratified by the FIM scale, roughly predict the degree of functional gain following rehabilitation in patients with first cerebral hemorrhage. Moderately affected patients will benefit from intensive rehabilitation. This study may be useful in determining how best to prioritize rehabilitation therapy.

Adult↗

PubMed2001

Influence of admission functional status on functional change after stroke rehabilitation.

OBJECTIVE: To determine whether the admission functional score influences the functional change after stroke rehabilitation. DESIGN: Two hundred forty-three patients who had received the Functional Independence Measure (FIM) assessment at admission and at discharge were enrolled in the study. The patients were stratified into three groups according to their FIM total scores at admission, i.e., < or =36, 37 to 72, and > or =73. RESULTS: The Scheffé's multiple comparison test showed that patients with FIM total scores of > or =73 at admission were significantly younger (58 +/- 11 [SD] yr) than those who had scores of 37 to 72 (64 +/- 11 yr) or < or =36 (66 +/- 12 yr). Patients with FIM total scores of 37 to 72 at admission showed significantly higher FIM gain (37 +/- 15) compared with those patients who had scores of > or =73 (20 +/- 10) or < or =36 (29 +/- 23). CONCLUSION: The functional levels of affected patients at admission stratified by the FIM scale roughly predict the degree of functional gain after rehabilitation in survivors with a first episode of ischemic stroke. Moderately affected patients will benefit from intensive rehabilitation. These findings may be useful for rehabilitation triage.

Aged↗

PubMed2001

Bradykinin augments fibroblast-mediated contraction of released collagen gels.

Bradykinin is a multifunctional mediator of inflammation believed to have a role in asthma, a disorder associated with remodeling of extracellular connective tissue. Using contraction of collagen gels as an in vitro model of wound contraction, we assessed the effects of bradykinin tissue on remodeling. Human fetal lung fibroblasts were embedded in type I collagen gels and cultured for 5 days. After release, the floating gels were cultured in the presence of bradykinin. Bradykinin significantly stimulated contraction in a concentration- and time-dependent manner. Coincubation with phosphoramidon augmented the effect of 10(-9) and 10(-8) M bradykinin. A B2 receptor antagonist attenuated the effect of bradykinin, whereas a B1 receptor antagonist had no effect, suggesting that the effect is mediated by the B2 receptor. An inhibitor of intracellular Ca2+ mobilization abolished the response; addition of EGTA to the culture medium attenuated the contraction of control gels but did not modulate the response to bradykinin. In contrast, the phospholipase C inhibitor U-73122 and the protein kinase C inhibitors staurosporine and GF-109203X attenuated the responses. These data suggest that by augmenting the contractility of fibroblasts, bradykinin may have an important role in remodeling of extracellular matrix that may result in tissue dysfunction in chronic inflammatory diseases, such as asthma.

Animals↗

PubMed2001

Cytotoxic effects of cigarette smoke extract on an alveolar type II cell-derived cell line.

Injury of the alveolar epithelium by cigarette smoke is presumed to be an important process in the pathogenesis of smoking-related pulmonary diseases. We investigated the cytotoxic effects of cigarette smoke extract (CSE) on an alveolar type II cell-derived cell line (A549). CSE caused apoptosis at concentrations of 5% or less and necrosis at 10% or more. When CSE was exposed to air before application to A549 cells, the cytotoxic effects were attenuated. CSE caused cell death without direct contact with the cells. Acrolein and hydrogen peroxide, two major volatile factors in cigarette smoke, caused cell death in a similar manner. Aldehyde dehydrogenase, a scavenger of aldehydes, and N-acetylcysteine, a scavenger of oxidants and aldehydes, completely inhibited CSE-induced apoptosis. CSE and acrolein increased intracellular oxidant activity. In conclusion, apoptosis of alveolar epithelial cells may be one of the mechanisms of lung injury induced by cigarette smoking. This cytotoxic effect might be due to an interaction between aldehydes and oxidants present in CSE or formed in CSE-exposed cells.

Aldehydes↗

PubMed2001

Fibrogenic and inflammatory cytokines modulate mRNA expressions of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-3 in type II pneumocytes.

BACKGROUND: Imbalance between proteinases and their inhibitors released from alveolar type II pneumocytes may cause development of inflammatory lung diseases. OBJECTIVES AND METHODS: We examined mRNA expressions of matrix metalloproteinase-3 (MMP-3) and tissue inhibitor of metalloproteinase-3 (TIMP-3) in a cell line (A549) and in primary culture of normal adult human type II pneumocytes using reverse transcription-competitive polymerase chain reaction. RESULTS: Interleukin-1beta (IL-1beta) and transforming growth factor-beta1 (TGF-beta1) increased MMP-3 and TIMP-3 expressions in A549 cells in a time- and concentration-dependent manner. IL-1beta mainly augmented MMP-3 expression, while TGF-beta1 mainly augmented TIMP-3 expression. Dexamethasone attenuated both IL-1beta- and TGF-beta1-stimulated expressions of MMP-3 and TIMP-3. Interleukin-10 had no significant effect. Hepatocyte growth factor alone had no effect on constitutive MMP-3 expression or TIMP-3 expression, but it augmented TGF-beta1-stimulated MMP-3 expression. The constitutive expressions were higher in normal type II pneumocytes than in A549 cells, but the regulations were similar. CONCLUSIONS: These data indicated that the matrix degradation is enhanced by IL-1beta and suppressed by TGF-beta1 via regulations in the balance between MMP-3 and TIMP-3. Further, these regulations were shown to be modulated by glucocorticoids and growth factors.

Adult↗

PubMed2001

[Relationship between changes in skin lipids by ultraviolet exposure and the cell toxicity].

The oxidative decomposition of skin lipids by UV exposure and its cell toxicity were studied in vivo and in vitro, using guinea pigs, to investigate the role of oxidative damage in cell membranes and mitochondria in nuclear genome DNA damage resulting in skin cell death by ultraviolet (UV) exposure. Two new methods were developed for this research: selective methylation by trimethylsilyldiazomethane (TMSCHN2) of free fatty acids in crude skin lipids for gas chromatography-mass spectrometry (GC-MS) analysis, and the improvement of the thiobarbituric acid (TBA) reaction for lipid peroxide analysis, which was obtained by adding 0.01% BHT and 1 mM EDTA, an antioxidant additive, into the reaction system described by Ohkawa. The following findings were noted: 1. Using an optical microscope, the infiltration of inflammatory cells such as neutrophils, lymphocytes and monocytes was noted after skin tissue was exposed to UV. 2. The increase of lipid peroxide in exposed areas was small. In the epidermis the increase was about 2 times higher than the non-exposed areas. 3. The fragments of lipid were generated in accordance with the increase of free fatty acids (C16:0, C18:1,:2, C18:0) in the surface of the exposed skin by GC-MS precise assay, and the level of 7-dehydrocholesterol was decreased. 4. The skin homogenate received peroxidation by ultraviolet more easily than living skin, and its peroxidation was inhibited with fat-soluble antioxidative agents such as flavonoids, BHT, BHA, and vitamin E and the metal chelating agent such as Fenton reaction inhibitor as expected; however, it was promoted by water soluble antioxidative agents such as glutathione and vitamin C, which are useful to the human body. 5. Steroids and nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin and aspirin, had no inhibitory effect on lipid peroxidation by ultraviolet as properties of chemicals.

Animals↗

PubMed2001

Significance of increase in lipocortin-1 gene transcripts in the blood monocytes of patients with pulmonary sarcoidosis.

BACKGROUND AND AIM OF THE WORK: Lipocortin-1 (also known as annexin-1) is upregulated by corticosteroids and plays a prominent part in many of their anti-inflammatory actions. In our previou investigation, lipocortin-1 gene expression in blood monocytes was higher in sarcoidosis patients tha in healthy subjects or IPF patients. In this study, we examined the amounts of lipocortin-1 gene tran scripts [at baseline and after stimulation by synthetic adrenocorticotrophic hormone (ACTH)] in the blood monocytes of sarcoidosis patients, and then we analyzed the relationships between these amount and several clinical indexes. METHODS: Serum and blood monocyte samples were prepared from 21 sar coidosis patients before and after ACTH stimulation. The amounts of lipocortin-1 and beta-actin gene tran scripts obtained from monocytes were quantified by RT-PCR. The patients were prospectively followe for at least 2.5 years. RESULTS: 1. Serum cortisol was increased in all patients after synthetic ACTH in jection. 2. Lipocortin-1 gene transcripts tended to increase in parallel with increases in serum cortisol but the relationship was not significant. 3. The amounts of lipocortin-1 gene transcripts induced by syn thetic ACTH were related to two indexes of disease activity/extension: the presence of parenchymal lesions (p = 0.018), and the presence of extrathoracic lesions (p = 0.043). 4. After 3 years of follow-up the patients with higher basal amounts of lipocortin-1 gene transcripts in monocytes showed improve ment of sACE activity and FEV1/FVC%. CONCLUSION: The amounts of lipocortin-1 gene transcripts induced by synthetic ACTH were related to the presence of parenchymal lesions, while their basa amounts correlated with either improvement of sACE activity or stabilization of FEV1/FVC%.

Adrenocorticotropic Hormone↗

PubMed2000

Functional cloning and mutational analysis of the human cDNA for phosphoacetylglucosamine mutase: identification of the amino acid residues essential for the catalysis.

In Saccharomyces cerevisiae, phosphoacetylglucosamine mutase is encoded by an essential gene called AGM1. The human AGM1 cDNA (HsAGM1) and the Candida albicans AGM1 gene (CaAGM1) were functionally cloned and characterized by using an S. cerevisiae strain in which the endogenous phosphoacetylglucosamine mutase was depleted. When expressed in Escherichia coli as fusion proteins with glutathione S-transferase, both HsAgm1 and CaAgm1 proteins displayed phosphoacetylglucosamine mutase activities, demonstrating that they indeed specify phosphoacetylglucosamine mutase. Sequence comparison of HsAgm1p with several hexose-phosphate mutases yielded three domains that are highly conserved among phosphoacetylglucosamine mutases and phosphoglucomutases of divergent organisms. Mutations of the conserved amino acids found in these domains, which were designated region I, II, and III, respectively, demonstrated that alanine substitutions for Ser(64) and His(65) in region I, and for Asp(276), Asp(278), and Arg(281) in region II of HsAgm1p severely diminished the enzyme activity and the ability to rescue the S. cerevisiae agm1Delta null mutant. Conservative mutations of His(65) and Asp(276) restored detectable activities, whereas those of Ser(64), Asp(278), and Arg(281) did not. These results indicate that Ser(64), Asp(278), and Arg(281) of HsAgm1p are residues essential for the catalysis. Because Ser(64) corresponds to the phosphorylating serine in the E. coli phosphoglucosamine mutase, it is likely that the activation of HsAgm1p also requires phosphorylation on Ser(64). Furthermore, alanine substitution for Arg(496) in region III significantly increased the K(m) value for N-acetylglucosamine-6-phosphate, demonstrating that Arg(496) serves as a binding site for N-acetylglucosamine-6-phosphate.

Amino Acid Sequence↗

PubMed2000

Nilvadipine protects low-density lipoprotein cholesterol from in vivo oxidation in hypertensive patients with risk factors for atherosclerosis.

OBJECTIVE: Nilvadipine, a calcium antagonist, has been shown to have antioxidant activity in vitro, but its effect on in vivo oxidation has not been assessed. The aim of this study was to investigate the antioxidant effect of this agent in vivo. The ratios of 7-keto cholestadien to cholesterol are believed to be an available marker of lipid peroxidation. Using these ratios, we have assessed the antioxidant effect of nilvadipine on low-density lipoprotein (LDL) in hypertensive patients with high risk of atherosclerosis. The risk factors of atherosclerosis may involve oxidation of LDL. METHODS: Fifteen healthy subjects (seven females and eight males aged 35-72 years, mean +/- SD = 55.3 +/-13.8 years) and fifteen hypertensive patients (seven females and eight males aged 45-80 years, mean +/- SD = 60.2 +/- 11.8 years) were recruited. Patients were treated orally with nilvadipine (4 mg b.i.d.) for 4 weeks. Cholesterol oxidation levels of LDL in patients before and after nilvadipine therapy and healthy subjects were studied. RESULTS: The ratios of 7-keto cholestadien to cholesterol in LDL of hypertensive patients before and 4 weeks after nilvadipine treatment and in healthy subjects were 6.5 +/- 1.6% (mean +/- SD), 3.8 +/- 1.2%, and 0.2 +/- 0.1%, respectively. There were significantly (P < 0.001) increased levels of cholesterol oxidation in LDL in patients with hypertension both before and after nilvadipine treatment compared with healthy subjects. However, there was a significantly (P < 0.001) decreased level of cholesterol oxidation in LDL in patients after nilvadipine treatment compared with patients before nilvadipine treatment. CONCLUSION: Our data showed that nilvadipine may protect LDL cholesterol from in vivo oxidation in hypertensive patients with high risk of atherosclerosis.

Adult↗

PubMed2000

Comparative analysis of plasma and erythrocyte 7-ketocholesterol as a marker for oxidative stress in patients with diabetes mellitus.

OBJECTIVES: To reveal increased lipid peroxidation in diabetics by quantification of cholesterol oxidation products (COPs) not only in plasma, but also in erythrocytes. DESIGN AND METHODS: We quantified 7-ketocholesterol (7-kCho) by gas chromatography-mass spectrometry as a surrogate measure for COPs. These assays were performed on both plasma and erythrocytes in 20 control subjects and 20 treated patients with relatively poorly controlled Type 2 diabetes. RESULTS: Both plasma and erythrocyte 7-kCho levels in diabetics were significantly higher than those in control subjects. Although neither plasma nor erythrocyte 7-kCho levels were associated with markers for glucose tolerance in diabetics, a negative correlation of serum HDL-cholesterol levels with erythrocyte, but not plasma, 7-kCho levels was found. CONCLUSION: Increased oxidative stress in diabetics affects oxidation of cholesterol. Assays of COPs not only in plasma, but also in erythrocytes, may yield complementary information in lipid peroxidation.

Aged↗

PubMed2000

Dicarboxylic acids as markers of fatty acid peroxidation in diabetes.

Increased urinary excretion of dicarboxylic acids (DAs) has been well known in patients with diabetic ketoacidosis (DKA). It was known that small amounts of such DAs were also detected in urine from healthy humans. Upon chemical, radiation-induced or enzymatic oxidation, cis-polyunsaturated fatty acids (PUFA) have previously been shown to generate saturated short- and medium-chain length DAs. In diabetes, it was confirmed that the imbalance between the generation of free radicals and antioxidant defense systems increases oxidative stress and leads to the damage of lipid, which contains PUFA. Some peroxidation products of PUFA, such as malondialdehyde and conjugated diene, are generally known to be elevated in patients with diabetes. The present study was undertaken to determine if urinary excretion of DAs is elevated in diabetic patients without DKA. Urine samples from ten non-ketoacidotic patients with type 2 diabetes and ten healthy subjects were examined for DAs by combined gas chromatography and mass spectrometry with selected ion monitoring. The diabetic subjects had significantly (Psebacic acid. Being stable and easily detectable compounds, DAs may be considered potential markers of oxidative attack on PUFA in diabetes.

Adult↗

PubMed2000

Effects of interferons alpha and gamma on cytokine production and phenotypic pattern of human bronchial epithelial cells.

Human bronchial epithelial cells are involved in airway immune mechanisms through secretion of cytokines and through cell-cell contacts with immunocompetent cells. The aim of our study was to assess the ability of interferon (IFN) alpha and gamma alone and in combination to modulate human bronchial epithelial cell (HBECs) release of the inflammatory cytokines IL-8 and IL-6 and fibronectin and to induce the surface expression of HLA-DR and ICAM-1 molecules involved in immune interactions with other cells. HBECs spontaneously secreted a limited amount of IL-8, which was significantly increased by IFN gamma. IFN alpha inhibited IFN gamma stimulated IL-8 secretion in a concentration-dependent manner. Further, IFN gamma induced IL-6 and fibronectin secretion, and this was also inhibited by IFN alpha. The expression of HLA-DR antigens was significantly increased by IFN gamma and partially inhibited by co-stimulation with IFN alpha. In contrast, IFN gamma also induced ICAM-1 expression by HBECs but co-stimulation with IFN alpha had no significant effect on the expression of this surface antigen. IFN alpha modulation of HBEC functions does not seem to be restricted to IFN gamma stimulation since either stimulatory or inhibitory effects of INF alpha on IL-8 production have been found in pilot experiments using IL-1 beta, TNF alpha, and TGF beta as stimuli. In summary, IFN-gamma induces a number of responses in HBECs including increased secretion of IL-6, IL-8 and fibronectin and increased expression of HLA-DR and ICAM-1. IFN alpha can inhibit all these except expression of ICAM-1 which is unaffected. IFN alpha can also interact with other inflammatory cytokines, but whether the effects are inhibitory or augmentive depends on the cytokines.

Bronchi↗

PubMed2000

Tetracycline-regulatable system to tightly control gene expression in the pathogenic fungus Candida albicans.

Conventional tools for elucidating gene function are relatively scarce in Candida albicans, the most prevalent human fungal pathogen. To this end, we developed a convenient system to control gene expression in C. albicans by the tetracycline-regulatable (TR) promoters. When the sea pansy Renilla reniformis luciferase gene (RLUC1) was placed under the control of this system, doxycycline (DOX) inhibited the luciferase activity almost completely. In the absence of DOX, the RLUC1 gene was induced to express luciferase at a level 400- to 1,000-fold higher than that in the presence of DOX. The same results were obtained in hypha-forming cells. The replacement of N-myristoyltransferase or translation elongation factor 3 promoters with TR promoters conferred a DOX-dependent growth defect in culture media. Furthermore, all the mice infected with these mutants, which are still virulent, survived following DOX administration. Consistently, we observed that the number of these mutant cells recovered from the mouse kidneys was significantly reduced following DOX administration. Thus, this system is useful for investigating gene functions, since this system is able to function in both in vitro and in vivo settings.

Animals↗

PubMed2000

Glucocorticoid-induced contractility and F-actin content of human lung fibroblasts in three-dimensional culture.

Fibroblast contractility plays a useful role in the wound healing process but contributes to architectural distortion in the lungs. Glucocorticoids (GCs) have been reported to reduce dermal fibroblast contractility, which may result in delaying wound healing, but the effects on lung fibroblasts are unknown. In this study, we examined how human lung fibroblast contractility is altered in the presence of GCs. Lung fibroblast cell lines (n = 5) were established from normal parts of surgically resected lung tissue. The effects of GCs on contractility were investigated with a type I collagen gel contraction assay. Filamentous actin (F-actin) content was detected by confocal microscopy and measured with a fluorescent phalloidin binding assay. GCs augmented fibroblast contraction in a concentration-dependent manner, with an approximate EC(50) of 1.8 x 10(-8) M, whereas other steroid derivatives had no effects. GC contractility needed de novo protein synthesis. The GC-induced increase in contractility was found to be consistent with an increase in F-actin content. In conclusion, lung fibroblast contractility was enhanced with GCs through an upregulation of lung fibroblast F-actin.

Actins↗