bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcriptional activation of stress genes and cytotoxicity in human liver carcinoma cells (HepG2) exposed to 2,4,6‐trinitrotoluene, 2,4‐dinitrotoluene, and 2,6‐dinitrotoluene
Authors: Tchounwou Paul B. , Wilson Barbara A. , Ishaque Ali B. , Schneider John
Published: August 2002

The CAT‐Tox (L) assay has recently been developed and validated for detecting and quantifying the specific molecular mechanisms that underlie toxicity of various xenobotic chemicals. We performed this assay to measure the transcriptional responses associated with 2,4,6‐trinitrotoluene (TNT) and 2 of its byproducts [2,4 and 2,6–dinitotoluenes (DNTs)] to 13 different recombinant cell lines generated from human liver carcinoma cells (HepG2) by creating stable transfectants of mammalian promoter chloramphenicol acetyltransferase (CAT) gene fusions. Cytoxicity test with the parental HepG2 cells, using the MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide]‐based assay for cell viability, yielded LC50 values of 105±6 μg/mL for TNT in 1% dimethyl sulfoxide (DMSO), and >300 m;g/mL for DNTs, upon 48 h of exposure. TNT appeared to be more toxic than 2,4‐DNT, which also showed a higher toxicity compared to 2,6‐DNT. Of the 13 recombinant constructs evaluated, 8 (CYP 1A1, GST Ya, XRE, HMTIIA, c‐fos, HSP70, GADD153, and GADD45), 5 (c‐fos, HSP70, GADD153, GADD45, and GRP78), and none showed inductions to significant levels (p<0.05), for TNT, 2,4‐DNT, and 2,6‐DNT, respectively. For most constructs, the induction of stress genes was concentration‐dependent. These results show the potential for TNT and 2,4‐DNT to cause protein damage and/or perturbations of protein biosynthesis (HSP70 and GRP78), alterations in DNA sequence or its helical structure (c‐fos, GADD153, GADD45), and the potential involvement of TNT in the biotransformation process (CYP 1A1, GST Ya, XRE), and in the toxicokinetics of metal ions (HMTIIA). Within the range of concentrations tested (0–300 mg TNT or DNT/mL in 1% DMSO), no significant inductions (p>0.05) of NFKBRE, p53RE, CRE, and RARE were found. © 2001 John Wiley & Sons, Inc. Environ Toxicol 16: 209–216, 2001
A channel assignment scheme for SCM/WDM‐based personal communication network
Authors: Chao Han‐Chieh , Wang Reen‐Cheng , Hong Jenn‐Yuh
Published: August 2002

As the cover range for the radio cells becomes gradually smaller than before, the procedures for mobile terminals call setup and control become complicated due to the high handoff frequency. The broadcast nature of WDM star couplers makes the handoff scheme easier and reduces the number of reconnections. In this study, we proposed a channel allocation algorithm, called pre‐empted assigned offset (PAO) scheme, for the WDM‐ and SCM/WDM‐based PCN architectures. The main idea is to keep the offset conflict probability lower. The simulation results show that the proposed scheme outperforms those previously introduced schemes. Especially in offset conflict probability under a moderate roaming rate for WDM and even under heavy roaming situations for SCM/WDM. Copyright © 2001 John Wiley & Sons, Ltd.
Uplink–downlink design issues for next generation satellite networks
Authors: Batsios Nikolaos , Pavlidou F.‐N.
Published: August 2002

Next Generation Mobile Systems will implement one's dream of anywhere, anytime communications. Providing global coverage and mobility, satellite systems will play an important role in order for this to be achieved. The design options of satellite systems include several choices. The basic ones are the choice of orbit, processing and switching, intersatellite links, and link layers technologies. In addition to the network architecture, the protocols must be developed for efficient use of the satellite radio resources. The most important protocol design issues concentrate on Medium Access Control (MAC) techniques, routing and traffic management. In this paper some of the special features of satellite systems are considered. Emphasis is given in MAC protocols design, as well as in research programmes that led to their development. Finally, an adaptive hybrid access scheme that combines CDMA technology and random access techniques is presented. This new scheme is called CDMA/PRMA and from the results obtained it is a very promising access technique for next generation satellite systems. Copyright © 2002 John Wiley & Sons, Ltd.
Noninvasive assessment of adrenal activity associated with husbandry and behavioral factors in the North American clouded leopard population
Authors: Wielebnowski Nadja C. , Fletchall Norah , Carlstead Kathy , Busso Juan M. , Brown Janine L.
Published: August 2002

The North American clouded leopard (Neofelis nebulosa) population is far from self‐sustaining. Breeding success is poor and behavioral problems (i.e., fur‐plucking, tail‐chewing, excessive hiding or pacing, and intersexual aggression that results in mate killing) are common. This study was undertaken to investigate whether some of these problems may be indicators of chronic stress (as reflected by persistently elevated glucocorticoid levels) and whether they are associated with specific management factors. A fecal corticoid metabolite assay was validated to monitor adrenal activity in clouded leopards. Adrenocorticotropic hormone (ACTH) challenges conducted in four clouded leopards established the biological relevance of the assay system. Fecal corticoid concentrations increased 14‐fold above baseline within 24 hours after ACTH administration. Adrenal activity then was monitored in 72 (36 males; 36 females) clouded leopards (65% of the North American Species Survival Plan population) during a 6‐week period and compared to husbandry and behavior data. There was a significant (P < 0.01) gender difference in fecal corticoid concentrations, with females producing higher concentrations than males. Multiple regression analyses revealed negative associations (P < 0.01) between enclosure height, number of hours keepers spent with each animal per week, and corticoid concentrations. A positive correlation (P < 0.001) was found between the number of keepers caring for an individual and corticoid concentrations. Higher fecal corticoid concentrations (P ≤ 0.05) were measured in clouded leopards kept on public display or near potential predators compared to individuals maintained off exhibit or in the absence of predators. Individuals that performed self‐injuring behaviors also had elevated fecal corticoids (P < 0.01). Spearman‐rank correlation analysis of keeper ratings and hormone data revealed positive associations (P ≤ 0.05) between some behaviors (pace, sleep, hide, and fearful/tense) and fecal corticoid concentrations. Overall these results indicate that noninvasive fecal corticoid monitoring has enormous potential for investigating how management and behavioral problems are related to animal well‐being. If conducted under carefully controlled experimental paradigms, this technique could allow researchers and managers to identify problem areas of captive management for clouded leopards (e.g., enclosure height, keeper time) and evaluate the efficacy of strategies designed to promote animal welfare and increased reproductive success. Zoo Biol 21:77–98, 2002. © 2002 Wiley‐Liss, Inc.
A Case Study in the Specification and Analysis of Design Alternatives for a User Interface
Authors: Duke David , Fields Bob , Harrison Michael D.
Published: August 2002

There is considerable interest within the Human Computer Interaction (HCI) community in the use of media spaces to enhance awareness and interaction between workers in offices or other spatially distributed environments. In addition to the technical challenges of providing reliable and efficient audio-visual communication, there are important social questions, in particular how users are able to control access to their personal environments, and how to advise other users about their level of availability. Within AMODEUS-2
1
, an ESPRIT Basic Research Action concerned with the development, transfer and assessment of techniques for modelling human-computer interaction, a prototype media space has been analysed by various user and system oriented modelling techniques. This paper describes how formal specification can be used to express requirements on the interfaces needed to control access and availability in a media space. Beyond its obvious use in clarifying the subtle relationship between these concerns, the paper describes how the specification assists in assessing design options originating from other modelling disciplines.
Advances in Intravenous Lipid Emulsions
Authors: Carpentier Yvon A. , Dupont Isabelle E.
Published: August 2002

Over the past decade, our views have considerably evolved with respect to the metabolism of intravenous lipid emulsions and their composition. Substantial progress has been made in understanding the metabolic pathways of emulsion particles and the delivery of their various components (fatty acids and vitamins) to specific tissues or cells. Although soybean long‐chain triglycerides represent a valuable source of energy, concerns have been raised about their high content of polyunsaturated fatty acids (mainly n‐6 essential fatty acids), which may adversely affect immune functions and antioxidant status. Introduction of medium‐chain triglycerides or olive oil to lipid emulsions can largely help bypass these disadvantages. Recently, incorporation of n‐3 fatty acids in lipid preparations was suggested to have potential application in several chronic and acute diseases because of their ability to reduce inflammatory and thrombotic responses and cell sensitivity to various stimuli. Hence lipid emulsions should no longer be considered only as a means of providing energy substrates; they also modulate key metabolic functions. Such improved knowledge may lead to optimizing the metabolic care of certain patients.
Comorbidity and the Elderly Trauma Patient
Authors: McMahon Damian J. , Schwab C. William , Kauder Donald
Published: August 2002

The elderly are forming an increasingly larger proportion of the population in developed countries with increasingly active life styles. The injured elderly patient has a combination of decreased physiologic reserve and a high incidence of preexisting medical conditions that cause comparably worse outcome, complications, longer hospital stay, and high costs. Although the management of specific injuries is similar in the elderly, many benefit from an overall more aggressive approach to early resuscitation and optimization of cardiopulmonary dynamics. An awareness of the importance of preexisting medical conditions and a coordinated, directed approach to the management of the injuries and the concomitant diseases leads to the most effective care. Upon recovery from injury there is often a change of functional level that precipitates a change in social circumstance. Ethical dilemmas, both at individual and community levels, may arise more frequently in the older trauma patient population. Increased triage to a trauma center, particularly when concomitant disease is present, is justified on the basis of improving outcomes.
Benign Liver Tumors: Differential Diagnosis and Indications for Surgery
Authors: Weimann Arved , Ringe Burckhardt , Klempnauer Jürgen , Lamesch Peter , Gratz Klaus F. , Prokop Mathias , Maschek Hansjörg , Tusch Günter , Pichlmayr Rudolf
Published: August 2002

The differential diagnosis for hemangioma, focal nodular hyperplasia (FNH), and hepatocellular adenoma may be difficult. Reliable diagnosis is mandatory for the decision of whether to apply surgery or observation. Experience with long‐term observation in nonoperated patients with hemangioma and FNH is limited. A group of 437 patients from a single institution were analyzed with regard to a diagnostic algorithm, the indications for surgery, and observation. There were 238 hemangiomas, 150 cases of FNH, 44 adenomas, and 5 mixed tumors. Of the 437 patients, 173 underwent surgery; 103 with hemangioma and 54 with FNH were observed at our own institution, whereas 117 patients underwent follow‐up elsewhere or were lost. Among the operated patients with confirmed histology, a good diagnostic yield was found for a combination of ultrasonography (US), contrast (bolus)‐enhanced computed tomography (CT), and labeled red blood cell (RBC) scanning: sensitivity 85.7%, specificity 100%, positive predictive value (PPV) 100%, negative predictive value (NPV) 81.8%, and accuracy 91.3%. For FNH the combination of US and CT plus cholescintigraphy showed a sensitivity 82.1%, specificity 97.1%, PPV 95.8%, NPV 84.6%, and accuracy 90.3%. Surgical mortality was 0.6%. Observation of patients with hemangioma and FNH for a median of 32 months revealed no increase in tumor size in 80% and a decrease in fewer than 7%. There was no tumor rupture and no evidence of malignant transformation. We concluded that liver hemangioma and FNH can be differentiated from adenoma with high sensitivity, specificity, and accuracy by labeled RBC scanning and cholescintigraphy in combination with US and contrast‐enhanced CT. In the case of symptoms or an equivocal diagnosis with respect to adenoma or hepatocellular carcinoma, surgery can be performed with very low risk. Because in asymptomatic patients with observed hemangioma or FNH no increase of tumor size can be expected for many years, the indications for surgery must be carefully evaluated.
Topical Formalin for Management of Bleeding Malignant Ulcers
Authors: Adebamowo Clement A.
Published: August 2002

Hemorrhage, an infrequent but serious complication of malignant ulceration, can be difficult to manage. Although many methods exist for treatment, none is wholly satisfactory. The use of topical formalin was studied in a group of patients with bleeding from malignant ulcers, and the results show that the agent achieved control of bleeding in 86% of the patients after two applications. There were no significant complications arising from the treatment. Topical formalin is a safe, inexpensive, effective agent for control of bleeding from malignant ulcers.
Stable ergodicity and julienne quasi-conformality
Authors: Pugh Charles , Shub Michael
Published: August 2002

In this paper we dramatically expand the domain of known stably ergodic, partially hyperbolic dynamical systems. For example, all partially hyperbolic affine diffeomorphisms of compact homogeneous spaces which have the accessibility property are stably ergodic. Our main tools are the new concepts - julienne density point and julienne quasi-conformality of the stable and unstable holonomy maps. Julienne quasi-conformal holonomy maps preserve all julienne density points.
Response by coccinellids to spatial variation in cereal aphid density
Authors: Elliott N. , Kieckhefer R. W.
Published: August 2002

The objectives of this study were to determine if coccinellids adjusted their distribution within spring wheat fields in response to spatial variation cereal aphid density in the fields and to describe the patterns of cereal aphid population growth that resulted. Field experiments were completed in which the physical dimensions of patches infested with cereal aphids, cereal aphid density, and access to patches by coccinellids were varied. Aphid infestations consisted of naturally occurring densities (natural patches) and much greater densities created by supplementing patches with aphids (supplemented patches). Coccinellids were denied access to some supplemented patches (exclusion patches) but allowed unlimited access to others. Densities of adult Hippodamia convergens and Coccinella septempunctata were correlated with aphid density in patches whereas density of Coleomegilla maculata was not. Aggregation by coccinellids was independent of patch area. The realized aphid population growth rate (r) was lower in supplemented than natural patches in all four trials but was significantly lower in only one trial. The lower r in supplemented patches was not exclusively caused by coccinellid predation, and emigration of aphids from patches probably also contributed. r was significantly greater in exclusion patches than supplemented and natural patches, indicating that coccinellids markedly reduced aphid numbers in patches even when aphid density was extremely high.
Fas/Cd95 Pathway Induces Mouse Liver Regeneration and Allows for Highly Efficient Retrovirus–Mediated Gene Transfer
Authors: Guidotti Jacques-Emmanuel , Mallet Vincent O. , Parlier David , Mitchell Claudia , Fabre Monique , Jaffray Patrick , Lambert Martine , Kahn Axel , Gilgenkrantz Hélène
Published: August 2002

Stable gene transfer into hepatocytes has been proposed to compensate for genetic deficiencies that affect liver function, or to deliver diffusible factors into the circulation. This strategy can be achieved using retroviral vectors; however, cell division must occur. We describe a simple and reproductive method that enables the induction of hepatocyte replication in a controlled fashion, thus allowing an efficient in vivo retroviral liver transduction that is applicable to mouse models of human genetic disorders. The approach is based on liver susceptibility to apoptosis via the Fas/CD95 pathway. We show that, 4 days following a single Fas agonist antibody (JO2) injection, hepatocyte replication occurs, the intensity of which is correlated with the level of the induced hepatic cytolysis. This treatment enables in vivo liver transduction, and its efficiency also correlates with the level of hepatic cytolysis. When recombinant retroviral vectors were infused intravenously during the period of hepatocyte replication, 15.4% ± 1.7% of the hepatocytes were transduced, reaching up to 32.5%.
Transplantation for Primary Biliary Cirrhosis: Retrospective Analysis of 400 Patients in A Single Center
Authors: Liermann Garcia Raquel F. , Garcia Christian Evangelista , McMaster Paul , Neuberger James
Published: August 2002

Orthotopic liver transplantation (OLT) is an effective treatment for patients with advanced primary biliary cirrhosis (PBC). We have conducted a retrospective analysis of 400 consecutive patients transplanted for PBC between 1983 and 1999. Mean follow–up was 56 months. The proportion of patients grafted for PBC fell progressively, from 35% in 1990 (n = 80) to 21% in 1999 (n = 111); comparison of patients grafted in the 2 decades showed that the median age increased from 53 to 56 years and the median serum bilirubin at transplantation fell from 270 μmol/L to 132 μmol/L. The overall actuarial patient and graft survival at 1, 5, and 10 years is 83%, 78%, and 67% and 82%, 75%, and 61%, respectively. The net gain in 5–year survival compared with predicted survival in the absence of transplantation fell from 37% (range, 82%–90%) to 16% (range, 91%–99%). Multiple organ failure (16.1%) and sepsis (9.6%) were the major causes of early deaths ( <6 months). Recurrent PBC, diagnosed on allograft histology, was found in 68 (17%) patients, at a mean time of 36 months. We were unable to identify any pretransplantation donor or recipient factor, which identified those patients at risk of recurrence, although recurrence was much earlier and more frequently seen in patients receiving tacrolimus (P = .04). PBC remains a good indication for liver transplantation, with excellent survival rates. The age at transplantation increased although patients tended to be grafted earlier. Survival rates have increased although there is a reduction in the survival benefit. Recurrence may be common, but does not seem to affect medium–term graft survival.
Trends in Survival of Patients With Hepatocellular Carcinoma Between 1977 and 1996 in the United States
Authors: El-Serag Hashem B. , Mason Andrew C. , Key Charles
Published: August 2002

The recent increase in the incidence of hepatocellular cancer in the United States is thought to underlie the rising mortality of this malignancy. However, it remains unknown whether survival of patients with hepatocellular carcinoma (HCC) has changed during the same time period. Using the SEER database (Surveillance, Epidemiology, and End Results) of the National Cancer Institute, we examined the temporal changes and determinants of survival among patients with histologically proven HCC over a 20–year period. Between 1977 and 1996, 7,389 patients diagnosed with HCC were followed in the survival database of SEER. The overall 1–year relative survival rate increased from 14% (95% confidence intervals (CI): 12–16) during 1977–1981 to 23% (95% CI: 21–24) during 1992 to 1996. Between the same two time periods, less improvement was seen in the 5–year survival rates, which increased from 2% (95% CI: 1–3) to only 5% (95% CI: 4–7). The median survival increased slightly from 0.57 years during 1977 to 1981 to 0.64 years during 1992 to 1996. In general, there were no significant differences in survival between men and women or between ethnic groups. During 1987 to 1991, a small fraction (0.8%) of patients underwent radical surgery; these patients had 1–year survival of 59% (95% CI: 35–83%), and 5–year survival of 35% (95% CI: 12–58%). Similar rates were seen during 1992–1996. In conclusion, a small improvement in survival of patients with HCC was seen between 1977 and 1996. Most of this apparent benefit is restricted to the first year following cancer diagnosis, raising the possibility of lead–time bias. There were no significant differences related to gender or ethnicity.
Ezrin–Radixin–Moesin-Binding Phosphoprotein 50 Is Expressed At the Apical Membrane of Rat Liver Epithelia
Authors: Fouassier Laura , Duan ChengYing , Feranchak Andrew P. , Yun Chris C.H. , Sutherland Eileen , Simon Francis , Fitz Gregory J. , Doctor Brian R.
Published: August 2002

Ezrin–radixin–moesin (ERM)–binding phosphoprotein 50 (EBP50) and NHE3 Kinase A regulatory protein (E3KARP) are membrane–cytoskeleton linking proteins that utilize 2 PSD–95/DIg/ZO–1 (PDZ) domains and an ERM binding site to coordinate cyclic adenosine monophosphate (cAMP)–regulated ion transport in a number of distinct epithelia. ERM family members serve to anchor EBP50 and E3KARP to the actin cytoskeleton and sequester protein kinase A (PKA) to these protein complexes. In hepatocytes and cholangiocytes, the epithelial cells of the bile secretory unit, cAMP–activated PKA stimulates secretion and bile formation, but the molecular mechanisms, including the potential contribution of EBP50 and E3KARP, remain undetermined. The present studies evaluated the comparative expression and localization of EBP50 and E3KARP in rat hepatocytes and cholangiocytes. Complementary DNAs encoding rat EBP50 and E3KARP were identified by reverse transcription–polymerase chain reaction in both epithelial cell types and subsequently sequenced. Northern and Western analysis showed the presence of EBP50 messenger RNA and protein in both hepatocytes and cholangiocytes. Confocal immunofluorescence revealed EBP50 was concentrated at the apical domain of both cell types. E3KARP was also expressed in cholangiocytes but had a distinct cytoplasmic/nuclear distribution. In dominant–negative transfection studies, patch clamp analysis of Mz–ChA1 cholangiocarcinoma cells showed that expression of the PDZ1 domain of EBP50 selectively decreased the endogenous cAMP–mediated Cl secretory response. The apical expression of EBP50, presence of specific ERM proteins, and functional effects of PDZ1 expression on cholangiocyte secretion suggest EBP50 is positioned to contribute to the organization and regulation of bile secretory proteins in both hepatocytes and cholangiocytes.
Halofuginone to Prevent and Treat Thioacetamide–Induced Liver Fibrosis in Rats
Authors: Bruck Rafael , Genina Olga , Aeed Hussein , Alexiev Rosaly , Nagler Arnon , Avni Yona , Pines Mark
Published: August 2002

Hepatic fibrosis is associated with activation of hepatic stellate cells (HSC), the major source of the extracellular matrix (ECM) proteins. The predominant ECM protein synthesized by the HSC is collagen type I. We evaluated the effect of halofuginone—an inhibitor of collagen synthesis—on thioacetamide (TAA)–induced liver fibrosis in rats. In the control rats the HSC did not express smooth muscle actin, collagen type I gene, or tissue inhibitor of metalloproteinases–2 (TIMP–2), suggesting that they were in their quiescent state. When treated with TAA, the livers displayed large fibrous septa, which were populated by smooth muscle actin–positive cells expressing high levels of the collagen α1(I) gene and containing high levels of TIMP–2, all of which are characteristic of advanced fibrosis. Halofuginone given orally before fibrosis induction prevented the activation of most of the stellate cells and the remaining cells expressed low levels of collagen α1(I) gene, resulting in low levels of collagen. The level of TIMP–2 was almost the same as in the control livers. When given to rats with established fibrosis, halofuginone caused almost complete resolution of the fibrotic condition. The levels of collagen, collagen α1(I) gene expression, TIMP–2 content, and smooth muscle actin–positive cells were as in the control rats. Halofuginone inhibited the proliferation of other cell types of the fibrotic liver in vivo and inhibited collagen production and collagen α1(I) gene expression in the SV40–immortalized rat HSC–T6 cells in vitro. These results suggest that halofuginone may become an effective and novel mode of therapy in the treatment of liver fibrosis.
Increased Activity in the Biliary Con A-Binding Fraction Accounts for the Difference in Crystallization Behavior in Bile From Chilean Gallstone Patients Compared With Dutch Gallstone Patients
Authors: Miquel Juan Francisco , van der Putten Joris , Pimentel Fernando , Mok Kam S. , Groen Albert K.
Published: August 2002

Chile has one of the highest prevalences of cholesterol gallstone disease in the world. Recent data indicate that this is partly caused by genetic (Indian) factors. However, the causal factors inducing increased gallstone formation have not been elucidated. The aim of this study was to compare biliary composition and cholesterol crystallization in bile from patients of high and moderate risk areas (Chile and The Netherlands) for gallstone disease. Bile was sampled at cholecystectomy from 30 Chilean and 26 Dutch gallstone patients. The Con A-binding fraction (CABF) was extracted from fresh native bile samples by incubation with Con A–sepharose. Reconstitution of the CABF to the Con A–extracted native bile induced almost full recovery of crystallization confirming the validity of this technique. There was no difference between the two groups regarding sex and age. Chilean bile nucleated significantly faster (3.5 ± 0.6 vs. 7.9 ± 1.5 days) despite the fact that Dutch bile had a significantly higher cholesterol saturation index (CSI) (1.6 vs. 1.2, P = .01). The total lipid content was not different. Chilean bile contained more total protein (5 vs. 2.9 mg/mL, P = .008). IgG, IgM, Haptoglobin and α–1–acid glycoprotein were not different between the two groups. IgA, though, was significantly higher in the Chilean samples (0.44 vs. 0.19 mg/mL, P < .001). Extraction of CABF increased crystal observation time (COT) and decreased crystal growth in both groups. However, the effects were much more pronounced in the Chilean samples. Compared with Dutch bile, Chilean bile crystallizes much faster despite a lower CSI. Chilean bile contains an increased content of Con A-binding nucleation promoting activity.
Interferon–α Delays S–Phase Progression in Human Hepatocellular Carcinoma Cells Via Inhibition of Specific Cyclin–Dependent Kinases
Authors: Murphy Derek , Detjen Katharina M. , Welzel Martina , Wiedenmann Bertram , Rosewicz Stefan
Published: August 2002

The potential antiproliferative effects of interferon–α (IFN–α) in the treatment of hepatocellular carcinoma (HCC) are controversial, and the growth inhibitory mechanisms remain poorly understood. Therefore, the current study was designed to delineate the molecular mechanisms responsible for direct antiproliferative actions of IFN–α in HCC cells. IFN–α receptor expression and signal transduction were examined by RT–PCR, immunoprecipitation, Western analysis, and transient transactivation assays. Effects of IFN–α on cell growth and cell–cycle distribution were evaluated based on cell numbers and flow cytometry. Composition and activity of cyclin–dependent kinase complexes were determined by immunoblotting and histone–H1–kinase assays. Expression of IFN–α receptors was found in all 3 HCC cell lines. IFN–α binding initiated phosphorylation of Jak1 and Tyk2 kinases leading to Stat1/Stat2 activation, nuclear translocation, and transactivation of an ISRE–luciferase reporter gene construct. IFN–α treatment resulted in a time– and dose–dependent reduction of proliferation. Cell cycle analysis of G1–synchronized, IFN–α–treated HCC cells revealed a substantial delay in S–phase progression but no alteration of G1/S–phase transition or evidence of apoptotic cell death. Reflecting the time course of S–phase accumulation, cell cycle–dependent induction of Cyclin A and Cyclin B was impaired, resulting in reduced activity of Cdk2 and Cdc2 kinases. Furthermore, Cdc25C was selectively down–regulated. IFN–α treatment inhibits growth of HCC cells by specifically delaying S–phase progression, most likely because of inhibition of Cyclin A induction, resulting in decreased activity of the associated Cdk2 and Cdc2 kinases.
Mechanism of Cell Death During Warm Hepatic Ischemia–Reperfusion in Rats: Apoptosis or Necrosis?
Authors: Gujral Jaspreet S. , Bucci Thomas J. , Farhood Anwar , Jaeschke Hartmut
Published: August 2002

Reperfusion injury can cause liver dysfunction after cold storage and warm ischemia. Recently it has been suggested that more than 50% of hepatocytes and sinusoidal endothelial cells (SEC) are undergoing apoptosis during the first 24 hours of reperfusion. The aim of our study was to quantify apoptotic and necrotic hepatocytes and apoptotic SEC after 60 or 120 minutes of warm, partial no–flow ischemia and 0 to 24 hours reperfusion in male SD rats. Apoptotic cells were identified by TUNEL assay in combination with morphological criteria. After 60 minutes of ischemia and 1 hour of reperfusion there was a significant increase of apoptotic hepatocytes (0.7 ± 0.1% vs. 0.3 ± 0.1% in controls) and SEC (1.5 ± 0.6% vs. 0.3 ± 0.1% in controls). The number of apoptotic SEC and hepatocytes was not different from controls at 6 hours or 24 hours of reperfusion. In contrast, the number of necrotic hepatocytes was quantified as 12 ± 2% at 1 hour, 34 ± 6% at 6 hours, and 57 ± 11% at 24 hours. These results correlated with the increase in plasma ALT levels at these time points. Longer (120 min) ischemia times did not affect the number of apoptotic cells but increased hepatocellular necrosis to 58 ± 4% at 6 hours reperfusion. No significant increase in caspase–3 activity and processing was detectable in any of these livers. Moreover, the caspase inhibitor Z–Asp–cmk (2 mg/kg IV) had no significant effect on reperfusion injury. Our results suggest that only a small minority of SEC and hepatocytes undergo apoptosis after 60 to 120 minutes of warm ischemia followed by 0 to 24 hours of reperfusion. Oncotic necrosis appears to be the principal mechanism of cell death for both cell types.
Cholestatic Expression Pattern of Sinusoidal and Canalicular Organic Anion Transport Systems in Primary Cultured Rat Hepatocytes
Authors: Rippin Jane S. , Hagenbuch Bruno , Meier Peter J. , Stieger Bruno
Published: August 2002

To maintain ongoing vectorial bile secretion, hepatocytes localize distinct transport systems at their basolateral and canalicular membrane domains. Here we compare the expression of the basolateral Na+–taurocholate cotransporter (Ntcp) and organic anion transporting polypetides 1 and 2 (Oatp1, Oatp2) and the canalicular bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2) in primary cultured rat hepatocytes. During 72 hours of culturing time the messenger RNA (mRNA) and protein levels of Ntcp and Oatp1 decreased in parallel to 2% to 7% of initial values at 3 hours. Although Oatp2 mRNA exhibited a similar down–regulation to 4%, Oatp2 protein and function were maintained at 25% to 47% of initial values. Furthermore, Bsep and Mrp2 protein levels were maintained at about 50%, while the Mrp2 mRNA showed a transient up–regulation to 154% at 24 and 48 hours. Also, induction of Mrp1 mRNA and protein was observed starting after 24 hours. These results indicate transcriptional down–regulation or decreased mRNA stability of Ntcp and Oatp1, transcriptional and posttranslational regulation of Oatp2, Bsep, and Mrp2 and transcriptional up–regulation of Mrp1 in primary cultured hepatocytes. Furthermore, and most importantly, the observed changes in transporter expression closely resemble the altered transporter phenotypes of cholestatic and proliferating hepatocytes in vivo, thus indicating that primary cultured hepatocytes acquire a cholestatic phenotype, and that the transporter expression might be a suitable differentiation marker for maintenance of hepatocytes in vitro.
Sustained Expression of Naked Plasmid Dna Encoding Hepatocyte Growth Factor in Mice Promotes Liver and Overall Body Growth
Authors: Yang Junwei , Chen Shiping , Huang Leaf , Michalopoulos George K. , Liu Youhua
Published: August 2002

To understand the physiological functions of exogenous hepatocyte growth factor (HGF) on normal adult animals, we delivered human HGF gene into mice by a hydrodynamics–based in vivo gene transfection approach using a naked plasmid vector. Systemic administration of naked plasmid containing HGF cDNA driven under cytomegalovirus promoter (pCMV–HGF) by rapid injection via the tail vein produced a remarkable level of human HGF protein in the circulation, beginning to appear at 4 hours and peaking at 12 hours following injection. Tissue distribution studies identified the liver as the organ with the highest level of transgene expression. Through weekly repeated injections of plasmid vector, we achieved sustained, long–term, high levels of exogenous HGF expression in mice for 8 weeks. Increases of more than 31% and 16% in liver and body weights were found, respectively, in the mice that received pCMV–HGF plasmid compared with that given the control vector for 8 weeks. Expression of exogenous HGF in vivo activated mitogen–activated protein kinases and induced proliferating cell nuclear antigen expression in normal adult liver and kidneys. These data suggest that systemic administration of naked plasmid vector is a convenient, safe, and highly efficient approach to introduce and maintain exogenous HGF gene expression in vivo in a controllable fashion. Our results also indicate that long–term expression of human HGF in mice markedly activates growth–related signal transduction events, promotes cell proliferation, and leads to liver and overall body growth in whole adult animals.
Cd45rc- γδ+ T–Cell Infiltration Is Associated With Immunologic Unresponsiveness Induced by Prior Donor–Specific Blood Transfusion in Rat Hepatic Allografts
Authors: Okabe Kazutoshi , Yamaguchi Yasuo , Takai Eiji , Ohshiro Hajime , Zhang Jia-Lin , Hidaka Hideki , Ishihara Kohjiroh , Uchino Shinichiro , Furuhashi Takashi , Yamada Shinwa , Mori Katsutaka , Ogawa Michio
Published: August 2002

Little is known regarding the role of γδ+ T cells in organ transplantation. We previously reported that immunologic unresponsiveness is induced by prior donor–specific blood transfusion (DST) in rat hepatic allografts. We investigated the phenotype and distribution of γδ+ T cells in the hepatic allograft, spleen, and peripheral blood of recipient rats with immunologic unresponsiveness induced by DST. γδ+ T cells were enumerated in allograft livers and spleens by immunostaining and in blood by flow cytometric analysis. The phenotype of γδ+ T cells was determined using CD45RC isoforms derived from alternative mRNA splicing. The cytokine profile of CD45RC+ and CD45RC- γδ+ T cells was analyzed by reverse transcription polymerase chain reaction. The number of γδ+ T cells in hepatic infiltrates in recipient rats pretreated with DST was significantly greater than in untreated animals. This correlated with significantly higher levels of γδ T cell receptor (TCR) mRNA in hepatic allografts of DST-treated rats as compared with untreated animals. The γδ+ T cell/αβ+ T–cell ratio increased in hepatic infiltrates in DST-treated recipient rats but not in untreated animals. CD45RC-γδ+ T cells were predominantly increased in DST-treated hepatic allografts compared with untreated allografts. Most of the intestinal intraepithelial T cells were CD45RC-γδ+. Interleukin (IL)–10 and IL–4 mRNA were detected more in CD45RC-γδ+ T cells than CD45RC+γδ+ T cells. CD45RC-γδ+ T cells infiltrating liver allografts produce Th2–type cytokines and are associated with immunologic unresponsiveness induced by DST.
Changes in Wnt/β–Catenin Pathway During Regulated Growth in Rat Liver Regeneration
Authors: Monga Satdarshan P.S. , Pediaditakis Peter , Mule Karen , Stolz Donna Beer , Michalopoulos George K.
Published: August 2002

The wnt/β–catenin pathway is important during embryogenesis and carcinogenesis. β–Catenin interaction with E–cadherin has been shown to be crucial in cell–cell adhesion. We report novel findings in the wnt pathway during rat liver regeneration after 70% partial hepatectomy using Western blot analyses, immunoprecipitation studies, and immunofluorescence. We found wnt–1 and β–catenin proteins to be predominantly localized in hepatocytes. Immediately following partial hepatectomy, we observed an initial increase in β–catenin protein during the first 5 minutes with its translocation to the nucleus. We show this increase to be the result of decreased degradation of β–catenin (decrease in serine phosphorylated β–catenin) as seen by immunoprecipitation studies. We observed activation of β–catenin degradation complex comprising of adenomatous polyposis coli gene product (APC) and serine–phosphorylated axin protein, beginning at 5 minutes after hepatectomy, leading to its decreased levels after this time. Quantitative changes observed in E–cadherin protein during liver regeneration are, in general, reverse to those seen in β–catenin. In addition, using immunoprecipitation, we observe elevated levels of tyrosine–phosphorylated β–catenin at 6 hours onward. Thus, changes in the wnt pathway during regulated growth seem to tightly regulate cytosolic β–catenin levels and may be contributing to induce cell proliferation and target gene expression. Furthermore, these changes might also be intended to negatively regulate cell–cell adhesion for structural reorganization during the process of liver regeneration.
The Phenobarbital Response Enhancer Module in the Human Bilirubin Udp–Glucuronosyltransferase Ugt1a1 Gene and Regulation by the Nuclear Receptor Car
Authors: Sugatani Junko , Kojima Hiroyuki , Ueda Akiko , Kakizaki Satoru , Yoshinari Kouichi , Gong Qi-Hui , Owens Ida S. , Negishi Masahiko , Sueyoshi Tatsuya
Published: August 2002

The UDP–glucuronosyltransferase, UGT1A1, is the critical enzyme responsible for detoxification of the potentially neurotoxic bilirubin by conjugating it with glucuronic acid. For decades, phenobarbital (PB) treatment for hyperbilirubinemia has been known to increase expression of the UGT1A1 gene in liver. We have now delineated the PB response activity to a 290–bp distal enhancer sequence (-3483/-3194) of the UGT1A1 gene. The enhancer contains 3 putative nuclear receptor motifs, and it was activated by the nuclear orphan receptor, human constitutive active receptor (hCAR), in cotransfected HepG2 cells. Bacterially expressed hCAR, acting as a heterodimer with in vitro-translated retinoid X receptor (RXRα), only bound to 1 of the 3 NR motifs, named gtNR1 in a gel–shift assay. Consistently, mutations of the gtNR1 site significantly decreased the activation by hCAR of the 290–bp DNA in transfection assays. Moreover, the 290–bp DNA was effectively activated in mouse primary hepatocytes in response to PB, offering an excellent clinical test for the examination of the responsiveness of the UGT1A1 to PB in the human population, particularly individuals with hyperbilirubinemia. (Hepatology 2001;33:1232–1238.)
Characterization of Liver T–Cell Receptor γδ+ T Cells Obtained From Individuals Chronically Infected With Hepatitis C Virus (Hcv): Evidence for These T Cells Playing A Role in the Liver Pathology Associated With Hcv Infections
Authors: Tseng Chien-Te K. , Miskovsky Emil , Houghton Michael , Klimpel Gary R.
Published: August 2002

The pathogenic mechanisms involved in viral hepatitis are not completely understood. Evidence suggests that the pathology associated with hepatitis C virus (HCV) and hepatitis B virus (HBV) infections are a result of the immune response in the liver to these viruses. The livers of patients with viral hepatitis have been shown to contain elevated numbers of T cells expressing the γ/δ form of the T–cell receptor for antigen (TCRγδ). In this study, we investigated whether liver biopsy specimens obtained from individuals with viral (HCV and/or HBV) or nonviral hepatitis contained TCRγδ+ T cells that could be expanded in vitro by cytokines. A high percentage of liver biopsy specimens obtained from HCV– and/or HBV–infected individuals contained high numbers of TCRγδ+ T cells. In contrast, T–cell lines generated from liver biopsy tissues obtained from individuals with nonviral hepatitis or from normal controls had no preferential expansion of TCRγδ+ T cells. Liver TCRγδ+ T–cell lines from HCV–infected individuals had high levels of non-major histocompatibility complex (MHC)-restricted cytotoxic activity against different targets including primary hepatocytes and produced interferon gamma (IFN–γ), tumor necrosis factor α (TNF–α), and interleukin 8 (IL–8) following activation by anti–CD3. Surprisingly, none of these liver TCRγδ+ T–cell lines could recognize any of the structural or nonstructural proteins of HCV and had no cytotoxic activity against cells infected with recombinant vaccinia viruses expressing different HCV proteins. However, the crosslinking of CD81, which has been shown to bind HCV particles and E2, resulted in significant levels of IFN–γ and TNF–α production by liver TCRγδ+ T cells. These results suggest that TCRγδ+ T cells may play a role in the liver pathology of HCV infections. (Hepatology 2001;33:1312–1320.)
Expression of Anti–Ov6 Antibody and Anti–N–Cam Antibody Along the Biliary Line of Normal and Diseased Human Livers
Authors: Van Den Heuvel Marius C. , Slooff Maarten J.H. , Visser Lydia , Muller Michael , De Jong Koert P. , Poppema Sibrand , Gouw Annette S.H.
Published: August 2002

Following hepatic injury, proliferation of anastomosing ductules can be observed. The origin of this ductular reaction is not completely clear, although there is considerable evidence for proliferation of a putative hepatic progenitor cell, reported to be located in the canals of Hering (CoH) and showing morphologic similarities with rat oval cells. In this study, we analyzed the immunophenotype of solitary oval cell-like cells (SOC), intralobular groups of cuboidal cells that might represent lining cells of CoH, bile ductular cells (BDC), bile duct epithelial cells (BEC), and hepatocytes. We used the antibodies OV6, CK19, and CD56 (NCAM) in a double–staining method in a series of 111 liver specimens. The series consisted of a variety of liver diseases, primary liver tumors, and normal livers. In normal livers, SOC, CoH, BDC, and BEC were uniformly and predominantly CK19+, OV6+, and CD56-. In diseased livers SOC and BDC were CK19+, OV6+, and also CD56+. Occasionally, BEC was CD56+ in damaged bile ducts in diseased liver, e.g., PSC. CoH lining cells were not present in cirrhotic nodules and were indistinguishable from BDC in the fibrous septa. The consistent and uniform staining patterns of SOC, CoH, and BDC support the concept that these cells share the same biliary lineage and might represent one biliary structure. The expression of CD56 on these cells in diseased livers indicates that CD56 is a useful marker for a reparative or regenerative state of the biliary liver–cell constituents but not to discriminate a putative hepatic stem cell.
Toll–Like Receptor 4 Is Involved in the Mechanism of Early Alcohol–Induced Liver Injury in Mice
Authors: Uesugi Takehiko , Froh Matthias , Arteel Gavin E. , Bradford Blair U. , Thurman Ronald G.
Published: August 2002

Chronic alcohol administration increases gut–derived endotoxin in the portal blood, which activates Kupffer cells and causes liver injury. Mice (C3H/HeJ) with mutations in toll–like receptor 4 (TLR4) are hyporesponsive to endotoxin. To test the hypothesis that TLR4 is involved in early alcohol–induced liver injury, the long–term intragastric ethanol feeding protocol developed by Tsukamoto and French for rats was adapted to mice. Animals with nonfunctional TLR4 and wild–type mice (C3H/HeOuJ) were compared. Two–month–old female mice were fed a high–fat liquid diet with either ethanol or isocaloric maltose–dextrin as control continuously for 4 weeks. There was no difference in mean urine alcohol concentrations between the groups. Dietary alcohol significantly increased liver–to–body weight ratios and serum alanine transaminase (ALT) levels in wild–type mice (109 ± 18 U/L) over high–fat controls (40 ± 3 U/L), effects that were blunted significantly in mice with a mutation of TLR4 (55 ± 9 U/L). While no significant pathologic changes were observed in high–fat controls, dietary ethanol caused steatosis, mild inflammation, and focal necrosis in wild–type animals (pathology score = 5.2 ± 1.2). These pathologic changes were significantly lower in TLR4–deficient mice fed ethanol (score = 2.0 ± 1.3). Endotoxin levels in the portal vein were increased significantly after 4 weeks in both groups fed ethanol. Moreover, ethanol increased tumor necrosis factor α (TNF–α) mRNA expression in wild–type, but not in TLR4–deficient, mice. These data are consistent with the hypothesis that Kupffer cell activation by endotoxin via TLR4 is involved in early alcohol–induced liver injury.
Expression and Role of Bcl–Xl in Human Hepatocellular Carcinomas
Authors: Takehara Tetsuo , Liu Xiaolong , Fujimoto Jiro , Friedman Scott L. , Takahashi Hiroshi
Published: August 2002

Transformed hepatocytes survive various apoptotic insults during their growth in vivo. However, molecular mechanisms that inhibit apoptosis and support their survival are not well understood. In this study, we investigated the expression and role of Bcl–xL, an antiapoptotic member of the Bcl–2 family, in human hepatocellular carcinoma (HCC). The Bcl–xL protein was expressed in HepG2, Hep3B, and Huh7 human hepatoma cell lines at high levels, but none of these cells expressed Bcl–2. Down–modulation of Bcl–xL by antisense oligonucleotide activated apoptosis in HepG2 cells in response to cellular stresses induced by staurosporine treatment or by serum starvation. Ectopic expression of transcriptionally active p53 alone was not sufficient for the activation of apoptosis in p53–null Hep3B cells, but apoptosis was induced when endogenous Bcl–xL was simultaneously inhibited by antisense oligonucleotide in these cells. Bcl–xL was expressed in all 20 surgically resected human HCC tissues when examined by Western blot analysis and immunohistochemistry, and levels of its expression were higher in a subset of HCC tissues than those of adjacent nontumor liver tissues or normal livers. We conclude that Bcl–xL expressed in human HCC cells inhibits apoptosis produced by various cellular stresses, such as staurosporine treatment, serum starvation, and p53 activation, and may play an important role in their survival.