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R A Thomas

Publications and source records attributed to R A Thomas.

At least 19 recordsLinked to original sources
PubMed2006

Mechanical stretch has contrasting effects on mediator release from bronchial epithelial cells, with a rho-kinase-dependent component to the mechanotransduction pathway.

INTRODUCTION: In vivo, the airway epithelium stretches and relaxes with each respiratory cycle, but little is known about the effect this pattern of elongation and relaxation has on bronchial epithelial cells. We have used a model of cell deformation to measure the effect of stretch on inflammatory cytokine release by the BEAS 2B cell line, and to examine the method of mechanotransduction in these cells. METHODS: BEAS 2B cells were cyclically stretched using the Flexercell system. IL-8 and RANTES protein and RNA levels were measured after different elongations, rates and duration of stretch. An inhibitor of Rho (Ras Homologous)-associated kinases was used, to assess the effect of blocking downstream of integrin signalling. Immunofluorescent staining of paxillin was used to study the effect of stretch on the distribution of focal contacts and the organisation of the actin cytoskeleton. RESULTS: IL-8 release by BEAS 2B cells was increased by cytokine stimulation and stretch, whereas RANTES levels in the cell supernatant decreased after stretch in a dose-, time- and rate-dependent manner. Thirty percent elongation at 20 cycles/min for 24h increased IL-8 levels by over 100% (P < 0.01). Blocking rho kinase using Y-27632 inhibited the effect of stretch on IL-8 release by the BEAS 2B cells. Immunofluorescent staining demonstrated that stretch caused dramatic disassembly of focal adhesions and resulted in the redistribution of paxillin to the peri-nuclear region. CONCLUSION: This study demonstrates a marked effect of stretch on bronchial epithelial cell function. We propose that stretch modulates epithelial cell function via the activation of rho kinases. The observation that stretch promotes focal adhesion disassembly suggests a mechanism whereby focal adhesion turnover (coordination of assembly and disassembly) is essential for mechanotransduction in bronchial epithelial cells.

Amides↗
PubMed2001

Pharmacological evidence for complex and multiple site interaction of CXCR4 with SDF-1alpha: implications for development of selective CXCR4 antagonists.

The C-X-C chemokine SDF-1 and its receptor CXCR4, mediate a pivotal role in the pathophysiology of HIV-1 infection and vascular inflammatory diseases. In this study, we investigated the pharmacological properties of SDF-1alpha interaction with CXCR4 in human leukemia cell lines. Our data, based on [125I]-SDF-1alpha radioligand binding, SDF-1alpha-induced [35S]-GTPgammaS binding and use of specific CXCR4 antagonist AMD3100 reveals the complex nature of SDF-1alpha-CXCR4 interaction. Firstly, homologous competition with cold SDF-1alpha revealed a bimodal ligand displacement curve and secondly, although AMD3100 inhibited both SDF-1alpha-mediated chemotaxis (IC(50)=4.7 nM) and [35S]-GTPgammaS binding (IC(50)=7.4 nM) with high affinity, it was intriguingly up to 3000-fold less potent (IC(50)=15.2 microM) in the radioligand binding assay. These results provide pharmacological evidence for the recently described two-site model for SDF-1alpha-CXCR4 interaction. Accordingly, inhibition of SDF-1alpha binding to one of the receptor sites is sufficient to antagonize function, without causing its complete displacement from the receptor. Furthermore, these findings have important implications in the development and evaluation of CXCR4-selective small molecule antagonists for therapeutic use.

Benzylamines↗
PubMed2001

NASA/American Cancer Society High-Resolution Flow Cytometry Project - III. Multiparametric analysis of DNA content and electronic nuclear volume in human solid tumors.

BACKGROUND: The NASA/American Cancer Society (ACS) flow cytometer can simultaneously measure electronic nuclear volume (ENV) and DNA content of nuclei. The preceding articles in this volume ("NASA/American Cancer Society High-Resolution Flow Cytometer Project-I") described the schematics, performance, and procedures used for the preparation of nuclei for analysis on this unit. In the present article, we describe the analysis of selected human tumors using the ratio of ENV/DNA content (nuclear packing efficiency [NPE]). METHODS: Tumor specimens (frozen) were minced with scalpels and stained with 1-10 microg/ml of 4',6-diamidino-2-phenylindole (DAPI) dihydrochloride at pH 6.0-7.2. Trout erythrocytes were used as internal standards. Data on ENV and DNA content were collected in list mode files. Propidium iodide-stained nuclei, analyzed on a Coulter XL cytometer, were used for comparison. RESULTS: Simultaneous measurement of ENV and DNA makes it possible to discriminate between hypodiploid or hyperdiploid tumor cells, as well as to differentiate between near-diploid aneuploid and diploid cells on the basis of their increased ENV. The NPE ratio is a valuable parameter for the detection of small quantities of tumor cells, separating overlapping diploid and aneuploid populations for cell cycle analysis and characterizing the level of differentiation in some tumors. CONCLUSION: NPE analysis provides unique measuring capabilities for the study of human solid tumors by flow cytometry.

American Cancer Society↗
PubMed2001

NASA/American Cancer Society High-Resolution Flow Cytometry Project-I.

BACKGROUND: The NASA/American Cancer Society (ACS) flow cytometer can simultaneously analyze the electronic nuclear volume (ENV) and DNA content of cells. This study describes the schematics, resolution, reproducibility, and sensitivity of biological standards analyzed on this unit. METHODS: Calibrated beads and biological standards (lymphocytes, trout erythrocytes [TRBC], calf thymocytes, and tumor cells) were analyzed for ENV versus DNA content. Parallel data (forward scatter versus DNA) from a conventional flow cytometer were obtained. RESULTS: ENV linearity studies yielded an R value of 0.999. TRBC had a coefficient of variation (CV) of 1.18 +/- 0.13. DNA indexes as low as 1.02 were detectable. DNA content of lymphocytes from 42 females was 1.9% greater than that for 60 males, with a noninstrumental variability in total DNA content of 0.5%. The ENV/DNA ratio was constant in 15 normal human tissue samples, but differed in the four animal species tested. The ENV/DNA ratio for a hypodiploid breast carcinoma was 2.3 times greater than that for normal breast tissue. CONCLUSIONS: The high-resolution ENV versus DNA analyses are highly reliable, sensitive, and can be used for the detection of near-diploid tumor cells that are difficult to identify with conventional cytometers. ENV/DNA ratio may be a useful parameter for detection of aneuploid populations.

American Cancer Society↗
PubMed2001

NASA/American Cancer Society High-Resolution Flow Cytometry Project - II. Effect of pH and DAPI concentration on dual parametric analysis of DNA/DAPI fluorescence and electronic nuclear volume.

BACKGROUND: In the present paper, we describe the effect of 4', 6-diamidino-2-phenylindole (DAPI) dihydrochloride concentration and pH on the resolution of DNA distribution histograms generated by dual-parametric simultaneous analysis of DNA content and electronic nuclear volume (ENV). METHODS: Nuclei from tissue culture cell lines and frozen human solid tumors were isolated in nuclear isolation media containing different concentrations of DAPI, at various pH levels, and analyzed on a NASA/American Cancer Society (ACS) flow cytometer. Samples stained with propidium iodide/hypotonic citrate and analyzed in a Coulter XL flow cytometer were used for comparison. RESULTS: Nuclei stained with DAPI concentration of 1-3 microg/ml, pH 6.0, gave the best resolution for the detection of the near-diploid and near-tetraploid populations. Simultaneous use of ENV and DAPI/DNA fluorescence under these conditions identified subpopulations that otherwise could not be detected by DNA analysis alone. CONCLUSIONS: Staining at 1-3 microg/ml DAPI, pH 6.0, was optimal for the detection of aneuploid populations, especially the near-diploid and/or near-tetraploid populations in human tumors.

American Cancer Society↗
PubMed2001

Comparison of conversion factors for x-ray beams from a Philips SL15 and the NPL linear accelerator.

The National Physical Laboratory provides a megavoltage photon calibration service for secondary standard dosemeter systems in terms of absorbed dose to water using a graphite calorimeter. It is therefore necessary to evaluate factors to convert absorbed dose calibrations from graphite to water for all energies provided by the calibration service. A portable graphite calorimeter is currently being developed at the NPL for measuring absorbed dose in the radiotherapy clinic (McEwen and Duane 2000 Phys. Med. Biol. 45 3675-91) and so factors are now required to be able to convert absorbed dose calibrations from graphite to water in the clinical beam. The factors used to convert absorbed dose calibrations from graphite to water which are currently in use at the NPL were determined in previous work by Burns and Dale (1990 NPL Report RSA (EXT) 7) for all photon energies provided by the high-energy x-ray calibration service. Nutbrown et al (2000 NPL Report CIRM 37) have since re-evaluated these conversion factors using two methods. In this paper the factors to convert absorbed dose calibrations from graphite to water for two clinical beams from a Philips SL15 LINAC (6 and 10 MV) that were determined using both methods are presented and compared with values for the NPL heavily filtered beams. The results from the measurements made on the clinical machines using both methods agree within 1sigma uncertainty. The weighted average of these results agrees to within 1sigma uncertainty with results given by Burns and Dale and Nutbrown et al. The uncertainty in the determination of the graphite to water conversion factors at the 1sigma level is estimated to be 0.4%.

Calibration↗
PubMed2001

Latency-associated nuclear antigen encoded by Kaposi's sarcoma-associated herpesvirus interacts with Tat and activates the long terminal repeat of human immunodeficiency virus type 1 in human cells.

The latency-associated nuclear antigen (LANA) is constitutively expressed in cells infected with the Kaposi's sarcoma (KS) herpesvirus (KSHV), also referred to as human herpesvirus 8. KSHV is tightly associated with body cavity-based lymphomas (BCBLs) in immunocompromised patients infected with human immunodeficiency virus (HIV). LANA, encoded by open reading frame 73 of KSHV, is one of a small subset of proteins expressed during latent infection and was shown to be important in tethering the viral episome to host chromosomes. Additionally, it has been shown that LANA can function as a regulator of transcription. However, its role in the progression of disease is still being elucidated. Since KS is one of the most common AIDS-associated cancers in the United States and BCBLs appear predominantly in AIDS patients, we examined whether LANA is able to regulate the HIV type 1 (HIV-1) long terminal repeat (LTR). Using luciferase-based transient transfection assays, we found that LANA was able to transactivate the HIV-1 LTR in the human B-cell line BJAB, human monocytic cell line U937, and the human embryonic kidney fibroblast cell line 293T. Moreover, we observed that the virus-encoded HIV transactivator protein Tat cooperated with LANA in activation of the LTR in a dose-response fashion with increasing amounts of LANA. Surprisingly, LANA alone was sufficient to transactivate the HIV-1 LTR in BJAB cells. In similar assays using a HIV-1 LTR construct with the core enhancer elements deleted; the activity of LANA was diminished but not abolished, indicating a mechanism which involves the cooperation of the core enhancer elements and downstream elements which include Tat. Furthermore, transient transfection of an infectious clone of HIV with LANA demonstrated effects similar to those seen in the reporter assays based on Western blot analysis of HIV Gag polypeptide p24. Interestingly, we also demonstrated that the carboxy terminus of LANA associates with Tat in cells and in vitro. These experiments suggest a role for LANA in activating the HIV-1 LTR through association with cellular molecules targeting the core enhancer elements and Tat and may have important consequences in increasing the levels of HIV in infected individuals and, hence, the disease state.

Antigens, Viral↗
PubMed2000

Endothelin receptor subtype distribution predisposes coronary arteries to damage.

Several vasoactive drugs that lower blood pressure and increase heart rate induce regional cardiotoxicity in the dog, most frequently of right coronary arteries and right atrium. The basis for this selective damage is thought to result from local changes in vascular tone and blood flow. Administration of an endothelin receptor antagonist (ETRA, SB 209670) to dogs induced damage most frequent and severe in the right coronary artery and right atrium. Because site predisposition may correlate with distribution of vasoactive receptors, the objectives of this study were to map endothelin (ET) receptor distribution and density within regions of dog heart using both gene (mRNA) and protein expression endpoints for dog ET(A) and ET(B) receptors, and, additionally, correlate ET receptor subtype density with regional cardiac blood flow. A 10- to 15-mmHg reduction in mean arterial pressure with a concomitant increase in heart rate (10-20%), a six- and twofold increase in regional blood flow to the right and left atrium, respectively, and acute hemorrhage, medial necrosis, and inflammation were observed in the right coronary arteries and arteries of the right atrium after ETRA infusion for 5 days. Radioligand protein binding to quantify both ET receptors in normal dog heart indicated a twofold greater density of ET receptors in atrial regions versus ventricular regions. Importantly, ET receptor density in coronary arteries was markedly (about five- to sixfold) increased above that in atrial or ventricular tissues. ET receptor subtype characterization indicated ET(B) receptors were three times more prevalent in right coronary arteries compared to left coronary arteries and in situ hybridization confirmed localization of ET(B) in vascular smooth muscle. ET(A) receptor density was comparable in right and left coronary arteries. Quantitative real-time polymerase chain reaction for ET(A) and ET(B) receptor mRNA transcripts supported the site prevalence for message distribution. Consequently, the composite of protein and message expression profiles for ET(A) and ET(B) receptors indicated a disproportionate distribution of ET(B) receptors within right coronary artery of dog and this, along with functional measures of blood flow after ETRA infusion indicated a predisposition for exaggerated pharmacological responses and subsequent damage to right coronary arteries by ET and/or ETRAs.

Amino Acid Sequence↗
PubMed1999

Genetic analysis reveals ongoing HIV type 1 evolution in infected human placental trophoblast.

To provide a better understanding of the role of placenta in vertical human immunodeficiency virus (HIV) transmission, we have studied the infection of placental trophoblast in a group of 15 mother-neonate pairs. By nested PCR amplification of the C2V3 env gene region, HIV-1 has been found to infect the placenta in five cases (33%). Phylogenetic analysis of the cloned sequences showed that all recovered maternal variants were of the B subtype. Further investigation into the ancestral relationships at the nucleotide level revealed that the trophoblast sequences evolved into a quasispecies population clearly distant from that observed in the mother. As expected, the populations transmitted to the trophoblast were also found to be more homogeneous than those in the mothers when characterized on the basis of pairwise nucleotide sequence distances. With regard to the predicted biological properties, the primary amino acid structure of the V3 loop domain was consistent, with a macrophage-tropic, non-syncytium-inducing phenotype in all patients. We also attempted to determine if any of a number of selected maternal or viral factors was associated with trophoblast infection. However, none of the followed parameters, including maternal age, disease stage, antiretroviral therapy, CCR5delta32 deletion status of the infant, and viral genotype, could be associated with viral transmission. Moreover, in one pair with proven trophoblast infection, HIV was also detected in the cord blood. Taken together, our data suggest that the productive trophoblast infection by HIV-1 in vivo is a relatively frequent event that may bear direct implications for a further transplacental propagation of the virus.

Adult↗
PubMed1998

The effect of growth conditions on the biodegradation of tributyl phosphate and potential for the remediation of acid mine drainage waters by a naturally-occurring mixed microbial culture.

The biodegradation of tributyl phosphate (Bu3-P, TBP), releasing phosphate at a high enough concentration locally to precipitate uranium from solution, was demonstrated by a mixed culture consisting primarily of pseudomonads. The effect of various parameters on Bu3-P biodegradation by growing cells is described. Growth at the expense of Bu3-P as the carbon and phosphorus source occurred over a pH range from 6.5 to 8, and optimally at pH 7. Bu3-P biodegradation was optimal at 30 degrees C, reduced at 20 degrees C and negligible at 4 degrees C and 37 degrees C. Incorporation of Cu or Cd inhibited, and Ni, Co and Mn reduced its degradation. Inorganic phosphate (above 10 mM) and kerosene (up to 1 g/l) reduced Bu3-P biodegradation significantly, but nitrate had no effect. Sulphate (10-100 mM) was inhibitory. When pregrown biomass was used the fastest rates of tributyl and dibutyl phosphate biodegradation were 25 mumol h-1 mg protein-1 and 37 mumol h-1 mg protein-1 respectively. Microcarrier-immobilised biomass decontaminated uranium-bearing acid mine waste water by uranium phosphate precipitation at the expense of Bu3-P hydrolysis in the presence of 35 mM SO(4)2-. At pH 4.5, 79% of the UO2(2)+ was removed at a flow rate of 1.4 ml/h on a 7-ml.test column.

Anions↗
PubMed1998

Biodegradation of metal-EDTA complexes by an enriched microbial population.

A mixed culture utilizing EDTA as the sole carbon source was isolated from a mixed inoculum of water from the River Mersey (United Kingdom) and sludge from an industrial effluent treatment plant. Fourteen component organisms were isolated from the culture, including representatives of the genera Methylobacterium, Variovorax, Enterobacter, Aureobacterium, and Bacillus. The mixed culture biodegraded metal-EDTA complexes slowly; the biodegradability was in the order Fe > Cu > Co > Ni > Cd. By incorporation of inorganic phosphate into the medium as a precipitant ligand, heavy metals were removed in parallel to EDTA degradation. The mixed culture also utilized a number of possible EDTA degradation intermediates as carbon sources.

Biodegradation, Environmental↗
PubMed1997

The biodegradation of tributyl phosphate by naturally occurring microbial isolates.

The biodegradation of tributyl phosphate by a mixed culture of Pseudomonads was demonstrated. Growth and the rate of tributyl phosphate consumption were variable and divisible into rapid and slow rates. Rapidly growing, rapidly tributyl phosphate-utilising cultures contained a 22-24 kb DNA fragment isolated by two methods, which was not visible in the cultures growing slowly. The mixed culture gave five periods of rapid growth interspersed with periods of poor growth during 7 months of weekly subculture, with the 22-24 kb DNA fragment detectable during the rapidly growing periods only. Seven Pseudomonads isolated from the culture grew at the expense of tributyl phosphate as the sole phosphorus source but spontaneously and irreversibly lost this ability after eight serial subcultures.

Biodegradation, Environmental↗
PubMed1997

A case of eosinophilic pneumonia and vasculitis induced by diflunisal.

Drug-induced pneumonitis is an uncommon complication of nonsteroidal anti-inflammatory drug administration. Herein is the first reported case of pneumonitis resulting from diflunisal therapy. The patient demonstrated clinical and biopsy evidence of systemic vasculitis. She responded dramatically to administration of systemic glucocorticoids.

Adult↗
PubMed1995

Maternal nicotine exposure: response of type II pneumocytes of neonatal rat pups.

The influence of maternal nicotine exposure during pregnancy and lactation on the Type II cells of lung tissue of one day old neonatal rat pups was investigated. The results clearly show that maternal nicotine exposure resulted in an increase in the type II cell count in the lungs of the offspring. In addition the lamellar body content of the type II cells of the nicotine exposed rat pups were significantly (P < 0.01) higher than that of the control animals. The type II cell mitochondria of lung tissue of nicotine exposed rat pups were swollen and no microvilli occurred on the alveolar surface. This clearly illustrates that nicotine interfered with type II cell integrity of tlte neonatal lung and may subsequently interfere with the normal development of the alveolar region of the lung.

Animals↗
PubMed1994

The influence of maternal nicotine exposure on the interalveolar septal status of neonatal rat lung.

The aim of this study was to determine the effect of maternal nicotine exposure (1 mg nicotine/kg body mass/day, subcutaneously) on the status of the alveolar septa of the 1 to 21 day old offspring. The data obtained showed swelling of type II and interstitial cell mitochondria. The type I:type II cell ratio decreased as a result of type II cell proliferation. The number of capillaries per unit length of septum was also significantly lower than that of control lung. Ruptured blood-air barriers also occur in the nicotine exposed lungs of rats of all age groups. The results show that maternal nicotine exposure interfered with the morphometric and morphologic characteristics of the septa of lung tissue of the offspring.

Animals↗
PubMed1994

Basal and Tat-transactivated expression from the human immunodeficiency virus type 1 long terminal repeat in human placental trophoblast rules out promoter-enhancer activation as the partial block to viral replication.

We have analysed the capacity of the trophoblast-derived malignant cell lines BeWo, JAR and JEG-3, and primary cultures of highly purified trophoblast cells to support the basal and Tat-mediated trans-activation-enhanced transcriptional activity of two distinct human immunodeficiency virus type 1 (HIV-1) isolates. Kinetic studies based on expression of long terminal repeat (LTR)-chloramphenicol acetyltransferase (CAT) constructs revealed that LTRs of both the prototype strain 3B and the highly cytopathic Zairean variant NDK were activated significantly in all target cells. Overall, the strongest activation was observed in primary trophoblasts. A novel modification of quantitative PCR was used to normalize LTR expression for transfection efficiency, enabling the calculation of specific expression rates in terms of muU CAT enzyme per fmol of transfected DNA. Using the latter criterion we determined that LTRs of both viruses were activated in decreasing order from trophoblasts to JAR, JEG-3 and BeWo cells; furthermore, the expression of HIV-1 3B LTR always significantly surpassed that of HIV-1 NDK. The effects of trans-activation on either of the LTRs, when assayed in cotransfection assays with various amounts of HIV-1 NDK-Tat expression vector, increased in a dose-dependent fashion and were comparable in a particular neoplastic cell line. Furthermore, the cell-specific LTR activity patterns did not correspond to the abundance of transcription factors binding specifically to the viral NF kappa B and SP1 motifs. Unlike SP1-binding proteins which were relatively abundant, substantially smaller amounts of proteins with NF kappa B specificity were found in all cells. Despite this apparent deficit in NF kappa B activity, trophoblasts supported a high basal activity of both LTRs. These data indicate that an insufficiency of basal or Tat-trans-activated LTR activity cannot account for the low level of HIV-1 replication in this important cell type.

Base Sequence↗
PubMed1994

Diabetes mellitus, advances and their implications for aerospace medicine.

The authors describe a case of Type 1 diabetes mellitus (insulin dependent) in a 34-year-old fighter pilot, which included a 15-month remission ("honeymoon period"). The pathogenesis, characteristics, diagnosis, evaluation, and the aeromedical implications of Type 1 diabetes are discussed. The use of C-peptide values in accessing beta cell function is also discussed. The risk of poorly controlled diabetes, diabetic ketoacidosis, and visual acuity fluctuations were major reasons to disqualify this individual when in remission. The importance of determining the type of diabetes for prognosis and aeromedical disposition is stressed.

Adult↗