Biomedical subjects

X Luo

Publications and source records attributed to X Luo.

At least 19 recordsLinked to original sources

PubMed1999

Long-term follow-up of patients treated with primary radiotherapy for supradiaphragmatic Hodgkin's disease at St. Jude Children's Research Hospital.

OBJECTIVE: To assess disease control, patterns of relapse, factors predictive of relapse, and late effects of treatment, we reviewed all cases of supradiaphragmatic (SD) Hodgkin's disease (HD) treated with primary radiation therapy (RT) at our institution. METHODS: We retrospectively reviewed the disease characteristics, treatment history, and long-term outcome of the 106 patients with Stage I and II supradiaphragmatic HD who received definitive irradiation at St. Jude Children's Research Hospital between 1970 and 1995. As of the date of analysis, 95 patients are alive, with a median follow-up of 13.3 years (range, 1.9-24.2 years). RESULTS: The median age at diagnosis was 14.7 years (range, 3.7-22.7). Involved-field RT was given to 13 patients (12%), whereas 37 (35%) had mantle RT, 51 patients (48%) had subtotal nodal irradiation, and 5 (5%) had total nodal irradiation. Relapsed disease developed in 26 patients at a median of 1.8 years (range, 0.2-9.3 years). The 5- and 10-year estimated cumulative incidences of relapse were 20.9% +/- 4.0% and 25.1% +/- 4.3%, respectively. With a median dose of 36 Gy (range, 32-40), in-field failure rate was 6.2%, whereas subdiaphragmatic relapse in sites irradiated prophylactically was 1.5%. There was a trend toward an increased incidence of relapse with higher ESR (p = 0.088) and greater number of sites of disease (p = 0.087). Age, stage, histology, nodal disease > or = 6 cm, the presence of bulky mediastinal disease, and the method of staging did not affect the incidence of relapse. The pattern of failure could not be predicted based on the stage of disease, the extent of subdiaphragmatic staging, the extent of radiation therapy, or the sequence of RT fields-"ping pong" vs. sequential. Subset analysis of Stage II patients revealed significantly more relapses in clinically staged patients. Excluding Stage IA patients with high cervical disease or peripheral nodal disease, nodal extension failures were more common for patients undergoing limited-volume RT, whereas extranodal relapses were likely after STNI or TNI. The estimated 10- and 15-year cumulative incidences of second malignancies were 2.9% +/- 1.6% and 7.9% +/- 3.3%, respectively. Our patients are at increased risk of second malignancies (11-fold), and fatal cardiac (68-fold) and infectious (33-fold) complications. Overall survival at 10 years was 90.8% +/- 3.2%; event-free survival was 72.1% +/- 5.0%. CONCLUSIONS: The current analysis confirms the curative potential of RT for HD in children and adolescents. Despite successful salvage therapy, relapsed disease remained the principal cause of death in our cohort. Excess risk of septic death in asplenic patients, fatal heart disease, and second malignancies may further compromise the ultimate cure of HD in long-term survivors.

Adolescent↗

PubMed1999

Alternate coupling of receptors to Gs and Gi in pancreatic and submandibular gland cells.

Many Gs-coupled receptors can activate both cAMP and Ca2+ signaling pathways. Three mechanisms for dual activation have been proposed. One is receptor coupling to both Gs and G15 (a Gq class heterotrimeric G protein) to initiate independent signaling cascades that elevate intracellular levels of cAMP and Ca+2, respectively. The other two mechanisms involve cAMP-dependent protein kinase-mediated activation of phospholipase Cbeta either directly or by switching receptor coupling from Gs to Gi. These mechanisms were primarily inferred from studies with transfected cell lines. In native cells we found that two Gs-coupled receptors (the vasoactive intestinal peptide and beta-adrenergic receptors) in pancreatic acinar and submandibular gland duct cells, respectively, evoke a Ca2+ signal by a mechanism involving both Gs and Gi. This inference was based on the inhibitory action of antibodies specific for Galphas, Galphai, and phosphatidylinositol 4,5-bisphosphate, pertussis toxin, RGS4, a fragment of beta-adrenergic receptor kinase and inhibitors of cAMP-dependent protein kinase. By contrast, Ca2+ signaling evoked by Gs-coupled receptor agonists was not blocked by Gq class-specific antibodies and was unaffected in Galpha15 -/- knockout mice. We conclude that sequential activation of Gs and Gi, mediated by cAMP-dependent protein kinase, may represent a general mechanism in native cells for dual stimulation of signaling pathways by Gs-coupled receptors.

Animals↗

PubMed1999

Cystic fibrosis transmembrane conductance regulator regulates luminal Cl-/HCO3- exchange in mouse submandibular and pancreatic ducts.

We have demonstrated previously the regulation of Cl-/HCO3- exchange activity by the cystic fibrosis transmembrane conductance regulator (CFTR) in model systems of cells stably or transiently transfected with CFTR (Lee, M. G., Wigley, W. C., Zeng, W., Noel, L. E., Marino, C. R., Thomas, P. J., and Muallem, S. (1999) J. Biol. Chem. 274, 3414-3421). In the present work we examine the significance of this regulation in cells naturally expressing CFTR. These include the human colonic T84 cell line and the mouse submandibular gland and pancreatic ducts, tissues that express high levels of CFTR in the luminal membrane. As in heterologous expression systems, stimulation of T84 cells with forskolin increased the Cl-/HCO3- exchange activity independently of CFTR Cl- channel activity. Freshly isolated submandibular gland ducts from wild type mice showed variable Cl-/HCO3- exchange activity. Measurement of [Cl-]i revealed that this was largely the result of variable steady-state [Cl-]i. Membrane depolarization with 5 mM Ba2+ or 100 mM K+ increased and stabilized [Cl-]i. Under depolarized conditions wild type and DeltaF/DeltaF mice had comparable basal Cl-/HCO3- exchange activity. Notably, stimulation with forskolin increased Cl-/HCO3- exchange activity in submandibular gland ducts from wild type but not DeltaF/DeltaF mice. Microperfusion of the main pancreatic duct showed Cl-/HCO3- exchange activity in both the basolateral and luminal membranes. Stimulation of ducts from wild type animals with forskolin had no effect on basolateral but markedly stimulated luminal Cl-/HCO3- exchange activity. By contrast, forskolin had no effect on either basolateral or luminal Cl-/HCO3- exchange activity of ducts from DeltaF/DeltaF animals. We conclude that CFTR regulates luminal Cl-/HCO3- exchange activity in CFTR-expressing cells, and we discuss the possible physiological significance of these findings regarding cystic fibrosis.

Animals↗

PubMed1999

WT1 expression induces features of renal epithelial differentiation in mesenchymal fibroblasts.

The WT1 tumor suppressor gene, implicated in hereditofamilial and sporadic Wilms' tumor, is required for normal renal development and is up-regulated during the mesenchymal-epithelial transition. NIH3T3 fibroblasts overexpressing WT1 were less proliferative, larger in size and more firmly attached to tissue culture plastic, suggesting an alteration of their state of differentiation. These cells were studied in vivo by subcutaneous injection into nude mice. The resulting tumors exhibited epithelioid histopathology and formed desmosome-like structures. Molecular analyses of these WT1 expressing fibroblasts grown in culture and in nude mice revealed significant alterations in the expression of many kidney epithelial markers. These studies indicate that WT1 expression can initiate features of a program of epithelial differentiation consistent with a prominent role for WT1 in the mesenchymal epithelial transition that occurs during renal development. Through this work we identified a number of novel target genes for the WT1 transcription factor, including uvomorulin, integrin alpha8 and perlecan, and suggest that WTI may activate the IGF-II gene, also implicated in the development of Wilms' tumor.

3T3 Cells↗

PubMed1999

Synthesis and protein distribution of the unspliced large tenascin-C isoform in oral squamous cell carcinoma.

The inclusion or omission of the alternatively spliced region in the tenascin-C (Tn-C) mRNA gives rise to the large (Tn-C(L)) or small (Tn-C(S)) variant, respectively. Tn-C(L) is thought to be a typical component of provisional extracellular matrices (ECMs) and is expressed during tumour stroma remodelling. Tn-C(L) synthesis has been studied using RNA/RNA in situ hybridization, and Tn-C(L) protein distribution, using immunohistochemistry (clone BC-2), in 18 oral squamous cell carcinomas (OSCCs) of different grades of malignancy. While the Tn-C(L) protein was demonstrated within the whole stromal compartment regardless of grade of malignancy, the majority of the Tn-C(L) mRNA signal-bearing cells were carcinoma cells. Only a few stromal myofibroblasts were able to synthesize Tn-C(L), as revealed by alpha-smooth muscle actin double staining. In well-differentiated carcinomas (G1), the Tn-C(L) synthesizing carcinoma cells were localized as a single positive cell layer in the tumour stroma interface, particularly in invasive areas. A higher grade of malignancy (G2/G3) is associated with a significantly increased number of Tn-C(L) synthesizing carcinoma cells randomly distributed within the invading tumour areas. Double-staining experiments (Tn-C(L) mRNA ISH/BC-2 immunohistochemistry) indicate that these cells are capable of organizing and depositing a three-dimensional Tn-C(L) matrix. Even though an instructive and/or inductive role of the carcinoma cells in tumour stroma formation cannot be excluded, these results demonstrate that carcinoma cells can directly produce the ECM components of tumour stroma.

Basement Membrane↗

PubMed1999

Expression of the transmembrane protein tyrosine phosphatase RPTPalpha in human oral squamous cell carcinoma.

Little is known about the role of protein-tyrosine phosphatases (PTPs), the cellular counterparts of protein-tyrosine kinases, both for normal growth regulation and for its dysregulation in cancer. The receptor-like PTPalpha (RPTPalpha) may play a positive role in growth regulation and has been shown to be overexpressed in colon carcinoma. An RNA/RNA in situ hybridisation protocol for RPTPalpha as well as RPTPalpha immunohistochemistry was developed to evaluate RPTPalpha expression in oral squamous cell carcinomas (OSCCs) of different histological grade and to reveal the synthetically active cells and their tissue distribution. In well-differentiated OSCC (G1), RPTPalpha mRNA could be detected by in situ hybridisation exclusively in stroma cells (fibro/myofibroblasts and inflammatory cells). A higher histological grade (G2/G3) was associated with an increased number of RPTPalpha-synthesising carcinoma cells haphazardly distributed within invading tumour areas. Consistent results were obtained by immunocytochemistry. Thus, both carcinoma dedifferentiation and stroma recruitment and activation seem to be associated with an upregulation of RPTPalpha expression in OSCC. The results speak in favour of the important role of activation of stroma fibro/myofibroblasts influencing the biological behaviour of epithelial tumours and also suggest that elevated RPTPalpha expression may be a more general marker for proliferating or dedifferentiated cells.

Carcinoma, Squamous Cell↗

PubMed1999

Cardiac function and cytotoxic aldehyde production in a murine model of chronic iron-overload.

OBJECTIVES: To determine the relationship between the total chronic dose of iron administered, ex-vivo cardiac function and the concentrations of cytotoxic aldehydes in heart tissue of a murine model. METHODS: In the first experiment, 34 male B6D2F1 mice were randomized to receive intraperitoneal injections of 5, 10 or 20 mg of iron dextran for three weeks, or a placebo control. The mice were subsequently randomized to undergo ex-vivo assessment of cardiac function. In the second experiment, free radical generation, quantified by the presence of 20 separate cytotoxic aldehydes, was assessed in heart tissue of 40 mice that were randomized to receive chronic treatment with various concentrations of iron dextran (100 mg to 300 mg total chronic dose administered), placebo treatment with saline, or no treatment at all (baseline). RESULTS: Iron-loaded groups displayed dose-dependent depressions of heart rate, systolic pressure, developed pressure, coronary pressure, -dP/dt and +dP/dt, and increases in diastolic pressure. Monotonic dose-dependent increases in total heart aldehydes were observed in the iron-treated groups (r-0.97, p < 0.0001), whereas no significant differences were observed between baseline or time-placebo control groups. CONCLUSIONS: While no single mechanism is likely to account for the complex pathophysiology of iron-induced heart failure, our findings show that chronic iron-loading in a murine model results in dose-dependent alterations to cardiac function; and results in free radical mediated damage to the heart, as measured by excess concentrations of cytotoxic aldehyde-derived peroxidation products. This is the first description of the effects of excess iron on cardiac function assessed by an ex-vivo Langendorff technique in a murine model of chronic iron-overload.

Aldehydes↗

PubMed1999

Retrograde transvenous perfusion.

Serious consequences of stroke dictate that new approaches to the treatment of stroke be investigated. We have developed a method for perfusing the patient's own arterial blood retrograde through the venous system to ischemic brain tissue. This treatment has proven beneficial in preventing and reversing serious injury in the laboratory and in a small clinical trial. The laboratory investigation has also demonstrated that this therapy, retrograde transvenous neuroperfusion, can be coupled with hypothermia to potentially increase its benefit. History, experimental development, and the clinical trial are reviewed in this article.

Adult↗

PubMed1999

L-carnitine attenuates doxorubicin-induced lipid peroxidation in rats.

Doxorubicin (DOX) was administered intraperitoneally to rats in six equal, 2.5 mg/kg doses over a 2-week period with or without L-carnitine. Injury was monitored by echocardiography, release of myosin light chain-1 (MLC-1), and by measurement of aldehydic lipid peroxidation products. General observation revealed that DOX alone caused more ascites than DOX plus L-carnitine. Animals sacrificed 2 h after the sixth dose had significantly higher aldehyde concentrations than 2 h after a single dose of DOX. Aldehydes in plasma and heart remained elevated for 3 weeks after the final dose of DOX, whereas L-carnitine prevented or attenuated the DOX-induced increases in lipid peroxidation. The increase in MLC-1 2 h after the sixth dose of DOX was greater than after a single dose, suggesting cumulative damage. Echocardiography did not detect either early injury or the protective effects of L-carnitine. These data indicate that lipid peroxidation following DOX occurs early, and parallels the cumulative characteristics of DOX-induced cardiotoxicity. The protective effects of L-carnitine may be due to improved cardiac energy metabolism and reduced lipid peroxidation.

Aldehydes↗

PubMed1999

H2O2 mediates O2 toxicity in cultured fetal rat distal lung epithelial cells.

It is unknown which of the reactive oxygen species is primarily responsible for the cytotoxicity of 95% O2 for rat distal fetal lung epithelial cells in vitro. Incubation of cells with 25 U/ml polyethylene glycol (PEG)-conjugated SOD and 50 U/ml PEG-catalase, but not PEG-SOD or SOD mimics alone, significantly reduced 95% O2-mediated cytotoxicity. Liposome-entrapped catalase, without SOD, also significantly reduced 95% O2-mediated cytotoxicity. Increased formation of lipid hydroperoxides, as assessed by the formation of 8-isoprostane and aldehydes, was attenuated by both 100 microM Trolox, a vitamin E analogue, and by 5 microM U74389G, an amino steroid. Trolox, but not U74389G, prevented an increase in cell-derived H2O2, hydroxyl radical and 95% O2-mediated cytotoxicity. An increase in hydroxyl radical formation, but not cell death, observed in 95% O2, was prevented by 0.1 microM phenanthrolene, a cell permeant iron chelator. DNA extracts of rat distal fetal lung epithelial cells maintained under serum-free conditions had an electrophoretic pattern consistent with some degree of apoptosis. However, no increase in laddering was seen with exposure to 95% O2. These data are consistent with hydrogen peroxide, but not lipid hydroperoxides or hydroxyl radical, being a critical effector of O2-mediated necrotic cell death in distal lung epithelial cells.

Analysis of Variance↗

PubMed1999

Two novel azadirachtin derivatives from azadirachta indica

Two novel compounds, the first 29-oxymethylene azadirachtin analogue, 29-oxymethylene-11- demethoxycarbonyl-11alpha-hydroxyazadirachtin (azadirachtin M) (1) and 22, 23-dihydro-23alpha-hydroxy-3-tigloyl-11-deoxyazadirachtinin (azadirachtin N) (2), together with known compound 11-epi-azadirachtin H were isolated from a methanolic extract of the seed kernels of Azadirachta indica. The structures of 1 and 2 were elucidated on the basis of spectral methods.

Journal Article↗

PubMed1999

LacSwitch inducible mammalian expression system in mouse Swiss 3T3 fibroblasts.

Swiss 3T3 fibroblasts were transfected with the provided plasmids of LacSwitch Inducible Mammalian Expression System (Stratagene). Stable transfectants were selected, expanded and characterised. At first, the production of CAT in these cell lines could be induced by IPTG treatment, but the inducibility was lost after a few months in culture in a reproducible manner. Further analysis revealed that the transfectants did not lose the cat gene nor the lac repressor protein. As a result, we conclude that LacSwitch Inducible Mammalian Expression System needs further modification for use in Swiss 3T3 fibroblasts.

3T3 Cells↗

PubMed1999

Synthesis and characterization of a series of novel glutamic gamma-15N-anilide dipeptides.

The preparation of a series of novel Cbz-Gln-Gly dipeptide derivatives is reported, wherein the gamma-carboxamide groups of the glutamine side chains have been modified to gamma-15N-anilides which are substituted in the para position with -NO2, -Cl, -H, -CH3, -OCH3, and -N(CH3)2. Characterization of the free anilines (p(kappa)a values and 15N NMR chemical shifts) and corresponding gamma-anilides (15N NMR chemical shifts and FTIR wavenumbers) is also reported. Correlation of these physicochemical data to Hammett substituent parameters ((sigma)para) is discussed. These novel dipeptide derivatives should prove to be generally useful for structure-function enzymology studies of gamma-glutamyl transferring enzymes.

Anilides↗

PubMed1999

Lipid A mutant Salmonella with suppressed virulence and TNFalpha induction retain tumor-targeting in vivo.

Systemically administered tumor-targeted Salmonella has been developed as an anticancer agent, although its use could be limited by the potential induction of tumor necrosis factor alpha (TNFalpha)-mediated septic shock stimulated by lipid A. Genetic modifications of tumor-targeting Salmonella that alter lipid A and increase safety must, however, retain the useful properties of this bacteria. We report here that disruption of the Salmonella msbB gene reduces TNFalpha induction and increases the LD50 of this pathogenic bacteria by 10,000-fold. Notwithstanding this enormous difference, Salmonella retains its tumor-targeting properties, exhibiting tumor accumulation ratios in excess of 1000:1 compared with normal tissues. Administration of this bacteria to mice bearing melanoma results in tumors that are less than 6% the size of tumors in untreated controls at day 18. Thus, the antitumor activity previously demonstrated using tumor-targeting Salmonella with normal lipid A is retained. Lipid modification of tumor-specific bacterial vectors provides a means for reducing septic shock and further suggests that the antitumor activity of these bacteria may be independent of TNFalpha.

Acyltransferases↗

PubMed1999

Cell-type-dependent activity of the ubiquitous transcription factor USF in cellular proliferation and transcriptional activation.

USF1 and USF2 are basic helix-loop-helix transcription factors implicated in the control of cellular proliferation. In HeLa cells, the USF proteins are transcriptionally active and their overexpression causes marked growth inhibition. In contrast, USF overexpression had essentially no effect on the proliferation of the Saos-2 osteosarcoma cell line. USF1 and USF2 also lacked transcriptional activity in Saos-2 cells when assayed by transient cotransfection with USF-dependent reporter genes. Yet, there was no difference in the expression, subcellular localization, or DNA-binding activity of the USF proteins in HeLa and Saos-2 cells. Furthermore, Gal4-USF1 and Gal4-USF2 fusion proteins activated transcription similarly in both cell lines. Mutational analysis and domain swapping experiments revealed that the small, highly conserved USF-specific region (USR) was responsible for the inactivity of USF in Saos-2 cells. In HeLa, the USR serves a dual function. It acts as an autonomous transcriptional activation domain at promoters containing an initiator element and also induces a conformational change that is required for USF activity at promoters lacking an initiator. Taken together, these results suggest a model in which the transcriptional activity of the USF proteins, and consequently their antiproliferative activity, is tightly controlled by interaction with a specialized coactivator that recognizes the conserved USR domain and, in contrast to USF, is not ubiquitous. The activity of USF is therefore context dependent, and evidence for USF DNA-binding activity in particular cells is insufficient to indicate USF function in transcriptional activation and growth control.

Amino Acid Sequence↗

PubMed1999

Biochemical pathways of caspase activation during apoptosis.

Caspase activation plays a central role in the execution of apoptosis. The key components of the biochemical pathways of caspase activation have been recently elucidated. In this review, we focus on the two most well-studied pathways of caspase activation: the cell surface death receptor pathway and the mitochondria-initiated pathway. In the cell surface death receptor pathway, activation of caspase-8 following its recruitment to the death-inducing signaling complex (DISC) is the critical event that transmits the death signal. This event is regulated at several different levels by various viral and mammalian proteins. Activated caspase-8 can activate downstream caspases by direct cleavage or indirectly by cleaving Bid and inducing cytochrome c release from the mitochondria. In the mitochondrial-initiated pathway, caspase activation is triggered by the formation of a multimeric Apaf-1/cytochrome c complex that is fully functional in recruiting and activating procaspase-9. Activated caspase-9 will then cleave and activate downstream caspases such as caspase-3, -6, and -7. This pathway is regulated at several steps, including the release of cytochrome c from the mitochondria, the binding and hydrolysis of dATP/ATP by Apaf-1, and the inhibition of caspase activation by the proteins that belong to the inhibitors of apoptosis (IAP).

Animals↗

PubMed1999

Multiple functional P2X and P2Y receptors in the luminal and basolateral membranes of pancreatic duct cells.

Purinergic receptors in the basolateral and luminal membranes of the pancreatic duct can act by a feedback mechanism to coordinate transport activity in the two membranes during ductal secretion. The goal of the present work was to identify and localize the functional P2 receptors (P2R) in the rat pancreatic duct. The lack of selective agonists and/or antagonists for any of the cloned P2R dictated the use of molecular and functional approaches to the characterization of ductal P2R. For the molecular studies, RNA was prepared from microdissected pancreatic intralobular ducts and was shown to be free of mRNA for amylase and endothelial nitric oxide synthase (markers for acinar and endothelial cells, respectively). A new procedure is described to obtain an enriched preparation of single duct cells suitable for electrophysiological studies. Localization of P2R was achieved by testing the effect of various P2R agonists on intracellular Ca(2+) concentration ([Ca(2+)](i)) of microperfused intralobular ducts. RT-PCR analysis suggested the expression of six subtypes of P2R in the pancreatic duct: three P2YR and three P2XR. Activation of Cl(-) current by various nucleotides and coupling of the receptors activated by these nucleotides to G proteins confirmed the expression of multiple P2R in duct cells. Measurement of [Ca(2+)](i) in microperfused intralobular ducts suggested the expression of P2X(1)R, P2X(4)R, probably P2X(7)R, and as yet unidentified P2YR, possibly P2Y(1)R, in the basolateral membrane. Expression of P2Y(2)R, P2Y(4)R, and P2X(7)R was found in the luminal membrane. The unprecedented expression of such a variety of P2R in one cell type, many capable of activating Cl(-) channels, suggests that these receptors may have an important role in pancreatic duct cell function.

Animals↗

PubMed1999

Effect of the 21-aminosteroid U74389G on oxygen-induced free radical production, lipid peroxidation, and inhibition of lung growth in neonatal rats.

Bronchopulmonary dysplasia is a chronic pneumopathy of preterm infants, with significant associated mortality and morbidity, for which there is no effective preventive therapy. Pulmonary O2 toxicity is thought to be a major contributor to the development of bronchopulmonary dysplasia, and antioxidant interventions hold significant promise for therapy. The relative importance of specific reactive oxygen species in the development of O2-mediated lung injury is unknown. In this study, we tested the effect of a synthetic 21-aminosteroid, U74389G, on 95% O2-induced free radical production, lipid peroxidation, and inhibition of postnatal lung growth in a neonatal rat model. Lipid peroxidation products, as measured by total 8-isoprostane and aldehydes, and hydroxyl radical formation, assessed using salicylate metabolites, in rat lungs and serum were significantly increased after exposure to 95% O2. These changes could be completely or partially attenuated by U74389G. However, U74389G did not improve the survival rate or lung wet-to-dry weight ratio. Expression of proliferating cell nuclear antigen, a marker for DNA synthesis, was examined by immunohistochemistry. Four- or 7-d-old control rat lungs had active DNA synthesis, which was inhibited by exposure to 95% O2. U74389G had a protective effect against 95% O2-mediated inhibition of DNA synthesis. Air-exposed animals treated with U74389G had a modest reduction in lung DNA synthesis, consistent with a role for hydroxyl radicals or lipid hydroperoxides as second messengers in the normal regulation of lung growth.

Animals↗