bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of Saccharomyces cerevisiae in the identification of novel transcription factor DNA binding specificities
Authors: Henry Kenneth W. , Carey Brenna , Howard William R. , Hoefner Daniel , Noonan Daniel J.
Published: August 2002

Members of the steroid/hormone nuclear receptor superfamily regulate target gene transcription via recognition and association with specific cis‐acting sequences of DNA, called hormone response elements (HREs). The identification of novel HREs is fundamental to understanding the physiological function of nuclear receptor‐mediated signalling pathways. A number of these receptors are transcriptionally active, or can be induced to an active state, when expressed in the yeast strain Saccharomyces cerevisiae. This aspect of nuclear receptor activity was used to screen random rat genomic DNA fragments for their ability to function as a HRE for the farnesoid X‐activated receptor (FXR). An isolated genomic fragment mediated FXR transcriptional activation without the co‐expression of the retinoid‐X receptor (RXR), a receptor previously thought to be an obligate heterodimer partner for FXR function. This genomic sequence of DNA contained a pair of highly conserved HRE half‐sites arranged in an everted orientation and separated by 3 bp (ER3). Furthermore, it was located 240 bp from a highly conserved TATA box motif. A minimal ER3 sequence of DNA was further demonstrated to function as a FXR HRE and was bound in vitro by FXR‐expressing yeast extracts. Using RT–PCR, an expressed mRNA fragment was identified within an 8 kb region downstream of the putative TATA box motif. This sequence of DNA was observed to bear homology to a cDNA found in mouse blastocyst. These findings define a novel FXR DNA binding specificity but, more importantly, these data suggest that this strategy might be universally applied to any transcription system that can be reconstituted in yeast. Copyright © 2001 John Wiley & Sons, Ltd.
A Saccharomyces servazzii clone homologous to Saccharomyces cerevisiae chromosome III spanning KAR4, ARS 304 and SPB1 lacks the recombination enhancer but contains an unknown ORF
Authors: Zhou Zhiqi , Sun Kaiming , Lipstein Ellen A. , Haber James E.
Published: August 2002

In order to learn about the evolutionary conservation of the recombination enhancer (RE) that controls donor preference during mating type switching in Saccharomyces cerevisiae, we have cloned a 13 kb region from S. servazzii. We find that the order of four genes surrounding the RE in S. cerevisiae (PRD1, KAR4, SPB1 and PBN1) is preserved in S.servazzii. However, there is an additional ORF in S. servazzii between PRD1 and KAR4 that is not homologous to any gene in S. cerevisiae or to genes in other organisms. Despite a 75–79% amino acid identity for KAR4 and SPB1, respectively, the S. servazzii sequence did not carry a well‐conserved RE sequence and these sequences lacked RE function when introduced into S. cerevisiae. The S. servazzii region contains a sequence that supports autonomous DNA replication in S. cerevisiae and may represent a homologue of ARS304. The S. servazziii sequence has Genbank Accession No. BankIt359091 AF307954. Copyright © 2001 John Wiley & Sons, Ltd.
Functional characterization of Gms1p/UDP‐galactose transporter in Schizosaccharomyces pombe
Authors: Tanaka Naotaka , Takegawa Kaoru
Published: August 2002

Galactosylation of glycoproteins in the fission yeast Schizosaccharomyces pombe requires the transport of UDP‐galactose as substrate for the galactosyltransferase into the lumen of the Golgi apparatus, which is achieved by the UDP‐galactose transporter. We isolated a mutant (gms1) that is deficient in galactosylation of cell surface glycoproteins in Sz.pombe, and found that the gms1+ gene encodes a UDP‐galactose transporter. In the prediction of secondary structure of the Gms1 protein, an eight‐membrane‐spanning structure was obtained. Fluorescent microscopy revealed the functional Gms1–GFP fusion protein to be stably localized at the Golgi membrane. Sequencing analysis of the coding region of Gms1p derived from galactosylation‐defective mutants identified a single amino acid mutation (A102T or A258E) located within the putative transmembrane region, helix 2 or helix 7, respectively. The mutagenized Gms1(A102T or A258E)p exhibited loss of UDP‐galactose transport activity but no change in the localization to the Golgi membrane. The C‐terminal truncated Gms1p mutants demonstrated that the C‐terminal hydrophilic region was dispensable for targeting and function as UDP‐galactose transporter at the Golgi membrane.We suggest that the putative eighth (the most C‐terminus‐proximal) transmembrane helix of Gms1p is critical to targeting from ER to the Golgi membrane. Copyright © 2001 John Wiley & Sons, Ltd.
Functional analysis of the Saccharomyces cerevisiae YFR021w/YGR223c/YPL100w ORF family suggests relations to mitochondrial/peroxisomal functions and amino acid signalling pathways
Authors: Georgakopoulos Tassos , Koutroubas George , Vakonakis Ioannis , Tzermia Maria , Prokova Vassiliki , Voutsina Alexandra , Alexandraki Despina
Published: August 2002

Saccharomyces cerevisiae YFR021w, YGR223c and YPL100w are paralogous ORFs of unknown function. Phenotypic analysis of overexpression, single‐, double‐ and triple‐ORF deletion strains under various growth conditions indicated mitochondria‐related functions for all three ORFs. Two‐hybrid screens of a yeast genomic library identified potentially interacting proteins for the three ORFs. Among these, the transcriptional activator Rtg3p interacted with both Yfr021wp and Ypl100wp and both ORF single deletions reduced the constitutive expression of the RTG‐regulated CIT2 and DLD3 genes and caused typical retrograde response of CIT2 and DLD3 under growth conditions requiring functional mitochondria, indicating that YFR021w and YPL100w are also involved in unidentified mitochondrial functions. Ptr3p, a component of the amino acid sensor Ssy1p/Ptr3p, was also found as a two‐hybrid interactant of Yfr021wp. Of the three single‐ORF deletions, ypl100wΔ exhibited ptr3Δ‐similar phenotypes. These findings, combined with the fact that RTG‐dependent expression is modulated by specific amino acids, suggested possible relations of Yfr021wp and Ypl100wp to amino acid signalling pathways. Under most conditions examined, the effects of the single‐ and double‐ORF deletions indicated that YFR021w, YPL100w and YGR223c are not parts of the same pathway. We found no unique phenotype attributed to the deletion of YGR223c. However, its function interferes with the function of the other two ORFs, as revealed by the effects of double‐ and triple‐ORF deletions. Copyright © 2001 John Wiley & Sons, Ltd.
The mei3 region of the Schizosaccharomyces pombe genome
Authors: Aves Stephen J. , Hunt Cherryl , Xiang Zheng , Lyne Michael H. , Wood Valerie , Rajandream Marie‐Adèle , Skelton Jason , Churcher Carol M. , Warren Timothy , Harris David , Gwilliam Rhian , Barrell Barclay G.
Published: August 2002

Expression of the mei3 gene is sufficient to induce meiosis in the fission yeast Schizosaccharomyces pombe. The mei3 gene is located 0.64 Mb from the telomere of the left arm of Sz. pombe chromosome II. We have sequenced and analysed 107 kb of DNA from the mei3 genomic region. The sequence includes 14 known genes (bag1‐B, csh3, dps1, gpt1, mei3, mfm3, pac1, prp31, rpl38‐1, rpn3, rti1, spa1, spm1 and ubc4) and 26 other open reading frames (ORFs) longer than 100 codons: a density of one protein‐coding gene per 2.7 kb. Twenty‐one of the 40 ORFs (53%) have introns. In addition there is one lone Tf1 transposon long terminal repeat (LTR), tRNATrp and tRNASer genes and a 5S rRNA gene. 14 of the novel ORFs show sequence similarities which suggest functions of their products, including a coatomer α‐subunit, a catechol O‐methyltransferase, protein kinase, asparagine synthetase, zinc metalloprotease, acetyltransferase, phosphatidylinositol 4‐kinase, inositol polyphosphate phosphatase, GTPase‐activating protein, permease, pre‐mRNA splicing factor, 20S proteasome component and a thioredoxin‐like protein. One predicted protein has similarity to the human Cockayne syndrome protein CSA and one with human GTPase XPA binding protein XAB1. Three ORFs are likely to code for proteins because they have sequence similarity with hypothetical proteins, three encode predicted coiled‐coil proteins and four are sequence orphans. The sequence is derived from cosmid clones c4664, c119 and c557 and has been submitted to the EMBL database under Accession Nos AL591302, AL022117, AL110506 and AL023634 (part). Copyright © 2002 John Wiley & Sons, Ltd.
The textbook
Authors: DeAngelo Christine
Published: August 2002

A review of Cetacean Societies: Field Studies of Dolphins and Whales, edited by Janet Mann, Richard C. Connor, Peter L. Tyack, and Hal Whitehead. Chicago: The University of Chicago Press, 2000, 433 pp., $80.00, hard bound; $35.00, paperback.
Chimpocentrism revisited
Authors: Stoinski Tara S.
Published: August 2002

A review of The Mentalities of Gorillas and Orangutans: Comparative Perspectives, edited by S.T. Parker, R.W. Mitchell, and H.L. Miles. Cambridge: Cambridge University Press, 1999, 419 pp., $85 hardbound
The relative effects of mutation accumulation versus inbreeding depression on fitness in experimental populations of the housefly
Authors: Reed David H. , Bryant Edwin H.
Published: August 2002

Loss of fitness due to inbreeding depression in small captive populations of endangered species is widely appreciated. Populations of all sizes may also experience loss in fitness when environmental conditions are ameliorated because deleterious alleles may be rendered neutral and accumulate rapidly. Few data exist, however, to demonstrate loss in fitness due to relaxed selection. Loss of fitness in life‐history traits were compared between LARGE (Ne ≥ 500) and SMALL (Ne = 50) populations of the housefly Musca domestica L that were subjected to curtailed life span at 21 days to remove selection on late‐acting deleterious alleles. During the early part of the life history (≤21 days), the rate of decline in fecundity and progeny production over 24 generations was greater in the small (1.5%) than in the large populations
<0.2%), but rate of loss in late‐life fecundity and progeny production (>21 days) was equivalent across populations, consistent with neutral theory, and amounted to 1.7% per generation. This rate of loss due to relaxed selection was equivalent to the rate of loss due to inbreeding in populations with an effective size of 50 individuals. Even if captive populations are kept large to avoid inbreeding, breeding them in benign environments where the forces of natural selection are curtailed may jeopardize the capability of these populations to exist in natural environments within few generations. Zoo Biol 20:145–156, 2001. © 2001 Wiley‐Liss, Inc.
Influence of Exogenous Fat Emulsion on Pulmonary Gas Exchangeafter Major Surgery
Authors: Sane Souji , Baba Masamichi , Kusano Chikara , Shirao Kazusada , Yamada Haruhiko , Aikou Takashi
Published: August 2002

The use of fat emulsion in totalparenteral nutrition (TPN) is closely related to changes in respiratoryfunction. The aim of this study was to evaluate the influence ofexogenous fat emulsion on pulmonary gas exchange in the early periodafter major surgery. Total parenteral nutrition was administered to 18patients for 6 days after esophagectomy for carcinoma. Half of thepatients received glucose (glucose group), and the other half receivedglucose and fat (fat group). The fat emulsion was continuously infusedfor 24 hours over 6 days. Glucose utilization was significantly higherin the glucose group than in the fat group. Fat utilization wassignificantly higher in the fat group than in the glucose group. Carbondioxide (CO2) production and respiratory quotient weresignificantly decreased in the fat group compared to the glucose group.There were no differences in the pulmonary vascular resistance index oralveolar–arterial difference in oxygen tension between the two groups.Although exogenous fat emulsion utilized as energy substrate decreasesCO2 production after major surgery, it does not clinicallyinfluence the pulmonary hemodynamics or diffusion capacity.
Small Bowel Intussusception in Symptomatic Pediatric Patients: Experiences with 19 Surgically Proven Cases
Authors: Ko Sheung‐Fat , Lee Tze‐Yu , Ng Shu‐Hang , Wan Yung‐Liang , Chen Min‐Chi , Tiao Mao‐Meng , Liang Chi‐Di , Shieh Chie‐Song , Chuang Jiin‐Haur
Published: August 2002

Nineteen cases of surgically proven symptomatic pediatric small bowel intussusceptions (SBI) were retrospectively reviewed. Clinical presentations included vomiting
(89.5%), abdominal pain and/or irritable crying (89.5%), fever
(52.6%), bloody stools (26.3%), palpable abdominal masses (15.8%),
hematemesis (10.5%), jaundice (5.3%), and seizures (5.3%). The duration between symptom onset and hospitalization ranged between 20
and 336 hours (average 75.8 hours). Two patients with suspected appendicitis and small bowel obstruction were operated on promptly.
Sonograms revealed target lesions (average diameter 2.9 cm) suggestive of intussusception in 13 out of 17 patients, with 10 lesions located in the paraumbilical or left abdominal regions. Barium enemas in 12 of these 13 patients demonstrated no colonic lesions. Diagnosis and surgery were delayed in 16 patients (average delay = 32 hours).
The remaining 1 patient with positive sonographic findings underwent early surgery after computed tomographic (CT) confirmation of SBI.
Surgery revealed ileoileal intussusceptions in 11 patients,
jejunojejunal in 4, jejunoileal in 3, and duodenojejunal in 1. Eight patients had lead points. Bowel complications (ischemia, necrosis, or perforation) occurred in 8 patients. The duration between symptom onset and surgery in patients with bowel complications was significantly longer than for patients without complications (p =
0.0026). In conclusion, delayed diagnosis and surgical treatment in symptomatic pediatric patients with SBI were common, leading to a high rate (42%) of bowel complications. Sonographic demonstration of a 2–3
cm target lesion, especially if paraumbilical or left abdominal, is suggestive of SBI and may obviate the need for a barium enema; however,
CT is helpful for confirming SBI. In symptomatic SBI, once diagnosed,
early surgical referral is strongly recommended.
Blood Purification for Prevention and Treatment of Multiple Organ Failure
Authors: Hirasawa Hiroyuki , Sugai Takao , Ohtake Yoshio , Oda Shigetoshi , Matsuda Kenichi , Kitamura Nobuya
Published: August 2002

Blood purification has been applied conventionally as an artificial kidney or artificial liver in the management of patients with multiple organ failure (MOF), and most blood purifications have been performed intermittently. Recent advances in medical engineering made it possible to perform such blood purifications continuously (i.e., 24 hours a day, 7 days a week if necessary) even in critically ill patients. This modality is referred to as continuous renal replacement therapy (CRRT) or continuous blood purification (CBP). Among many kinds of CBP, continuous hemodiafiltration (CHDF) is most useful for management of MOF, as it can be performed without serious or hazardous side effects, and improvement can be expected with it. Recently, CHDF and polymyxin B immobilized endotoxin adsorption columns were used for the prevention or treatment of MOF, with the expectation that such therapy can be effective as a countermeasure against the pathophysiologic causes of MOF. Our data and that of others clearly indicate that continuous blood purification, such as with CHDF and endotoxin adsorption, can remove or decrease the blood levels of humoral mediators, including proinflammatory cytokines, and can improve tissue oxygenation, especially oxygen consumption (VO2) among critically ill patients including those with MOF. Blood purification is also useful in the careful management of fluid, electrolytes, and acid‐base balance and for the removal of metabolic wastes. Blood purification is now considered to be one of the basic therapeutic tools of critical care, equal to nutritional support with total parenteral nutrition and respiratory support without a ventilator.
Operative Experience of U.S. General Surgery Residents with Diseases of the Adrenal Glands, Endocrine Pancreas, and Other Less Common Endocrine Organs
Authors: Harness Jay K. , Organ Claude H. , Thompson Norman W.
Published: August 2002

The aim of this study was to determine if the experience of general surgery residents is adequate and effective. The Resident Statistic Summaries (Report C) of the Residence Review Committee (Surgery) for eight academic years from 1986 to 1994 were analyzed. The main outcome measurements were total number of residents and programs, average number of operations performed, maximum number of operations performed, standard deviation, and the most common number of operations performed. For adrenalectomy, the average per resident was 0.98. The maximum range was from 7 to 15. The standard deviations ranged from 1.12 to 2.00. For pancreatic endocrine operations the average per resident was 0.15 with maximums of 3 to 10. For other endocrine procedures (nonthyroid and nonparathyroid) the average per resident was 0.14, with the maximums ranging from 7 to 19. The most common number of any of these procedures performed by U.S. graduates was 0. The number of adrenal, endocrine pancreas, and other less common endocrine procedures available for graduates of U.S. residency training programs is limited. As a consequence, most U.S. resident graduates have little or no experience with any of these procedures. Our findings suggest a strong need for fellowship training for any surgeon hoping to develop expertise in the management of these unusual and infrequent endocrine surgical diseases.
Microwave Coagulonecrotic Therapy for Hepatocellular Carcinoma
Authors: Yamanaka Naoki , Tanaka Tsuneo , Oriyama Takeshi , Furukawa Kazutaka , Tanaka Wataru , Okamoto Eizo
Published: August 2002

The present study reports on the usefulness of microwave coagulonecrotic therapy (MCT) as a treatment option for hepatocellular carcinoma (HCC) with poor hepatic reserve. From June 1992 to March 1995, MCT using a microwave electrode was employed on 8 patients using laparoscopic control and 19 with the open method, and wedge resection (Hx) was applied to the 23 patients. All patients had HCC with poor hepatic reserve. Radiation output was 100 watts with a mean radiation duration of about 30 minutes. The severity of liver dysfunction and the regional characteristics of the tumor (tumor size, multiplicity, portal invasion, tumor depth) were comparable between the MCT and Hx groups. The operative time was significantly shorter for the MCT group than the Hx group. The mean blood loss was 1570 ml in the Hx group but negligible in the MCT group. There was no operative mortality in the MCT group in contrast to 4.3% (1 of 23) in the Hx group. Complications were observed in 11.1% (3 of 27) and 34.8% (8 of 23), respectively, for the MCT and Hx groups. The postoperative total bilirubin had lower values and the start of diet was earlier in the MCT group than the Hx group. The 3‐year crude and disease‐free survival rates were 86% and 44%, respectively, for patients who underwent MCT, which were comparable to 75% and 14% for those with Hx. MCT can achieve long‐term results equivalent to those obtained by wedge resections, but it is less invasive and technically easier. Therefore it can be an alternative option in place of limited resection for HCC with poor hepatic reserve.
Biliopancreatic Diversion with Duodenal Switch
Authors: Marceau P. , Hould F.‐S. , Simard S. , Lebel S. , Bourque R.‐A. , Potvin M. , Biron S.
Published: August 2002

In 1990 Scopinaro's technique of biliopancreatic diversion with distal gastrectomy (DG) and gastroileostomy was modified. A sleeve gastrectomy with duodenal switch (DS) was used instead of the distal gastrectomy; and the length of the common channel was made 100 cm instead of 50 cm. A questionnaire and a prescription for blood work were sent to 252 patients who underwent DG a mean 8.3 years ago (range 6–13 years) and 465 patients who underwent DS 4.1 years ago (range 1.7–6.0 years). The questionnaire response rate was 93%, and laboratory work was completed for 65% of both groups. The mean weight loss after DG was 37 ± 21 kg and after DS 46 ± 20 kg. There were fewer side effects after DS: The number of daily stools was lower (p < 0.0002), as was the prevalence of diarrhea (p < 0.01), vomiting (p < 0.001), and bone pain (p < 0.001). Greater benefits related to several aspects of life were reported after DS than DG (p < 0.0001). The mean serum levels of ferritin, calcium, and vitamin A were higher (p < 0.001), and parathyroid hormone was lower. The yearly revision rate for excessive malabsorption was 1.7% per year after DG and 0.1% per year after DS. The two procedures were equally efficient for treating co‐morbid conditions such as diabetes, hypertension, and hypercholesterolemia. Biliopancreatic diversion with sleeve gastrectomy/duodenal switch and a 100‐cm common limb was shown to produce greater weight loss with fewer side effects.
Hypomagnesemia in Trauma Patients
Authors: Frankel Heidi , Haskell Robin , Lee Sun Yong , Miller David , Rotondo Michael , Schwab C. William
Published: August 2002

The objective of this study was to determine the incidence of hypomagnesemia in injured patients (versus a general hospital cohort) and to compare total and ionized values with each other and with the incidence of injury severity and ethanol level. It was a descriptive study of consecutive injured patients at a level II trauma center. For 3 months subjects underwent admission paired analysis of blood total magnesium (TMg) and ionized magnesium (IMg). IMg was determined by microanalysis of whole blood (Nova Biomedical, NovaSP9). During the same time period, all hospital samples for TMg (m= 1308) underwent simultaneous IMg testing. Pearson correlation coefficients were determined for IMg versus TMg, Injury Severity Score (ISS), Trauma Score (TS), Glasgow Coma Scale (GCS), and blood units transfused. By convention, hypomagnesemia was defined as TMg ≤ 1.6 mg/dl and IMg ≤ 0.5 mg/dl. Altogether 43% of 113 trauma patients had low magnesium levels compared to 30% of noninjured cohorts (p < 0.05). The correlation coefficient (r2) for TMg and IMg was 0.74 for TMg values > 1.6, but for TMg ≤ 1.6 the r2 was 0.35. Coefficients for IMg and ISS, TS, GCS, units transfused, and ethanol level were 0.06, 0.08, 0.10, 0.04, and 0.01, respectively. Mean IMg was 0.57 ± 0.09 mg/dl with ethanol ingestion and 0.56 ± 0.06 mg/dl without ethanol ingestion (mean ± SD, p > 0.05). It was concluded that hypomagnesia is common in injured patients but does not correlate with indices of injury severity or ethanol level. TMg is not a good predictor of IMg at low levels. Trauma patients may benefit from determination of IMg for accurate diagnosis of a low Mg level to facilitate repletion.
Value of Routine Computed Tomography in the Preoperative Assessment of Abdominal Aneurysm Replacement
Authors: Lamah Marc , Darke Simon
Published: August 2002

This study compares the findings of conventional computed tomography (CT) and ultrasound (US) imaging in the preoperative evaluation of patients with abdominal aortic aneurysm (AAA). It also assesses the impact of preoperative CT‐derived information on operative strategy. A prospective study was conducted of 96 patients who were considered for aortic aneurysm surgery, and the operative notes and US and CT reports were analyzed to assess correlation of findings and influence of CT on operative tactics. Agreement between CT and US in sizing the aneurysm was generally good. CT was more accurate than US for defining the upper and lower extent of the aneurysm (75% and 83%, respectively, with CT, compared to 47% and 41% with US), although it had a high false positive rate (48%) for juxtarenal disease. Its advantage over US in regard to showing other intraabdominal pathology was only marginal, and it predicted an inflammatory reaction in only two of the five cases. Its influence on operative strategy was minimal: Of the 25 cases where a juxtarenal aneurysm was predicted by CT, only 2 patients did not have surgery as a result. CT is a relatively expensive and time‐consuming procedure, and its ionizing radiation, however small, and potential side effects from contrast material hypersensitivity cannot be ignored. In light of the above findings, we suggest that CT scanning need not be routinely employed in the preoperative workup of elective aortic aneurysm repair but should be used only in selected cases.
Slowdown estimates and central limit theorem for random walks in random environment
Authors: Sznitman Alain-Sol
Published: August 2002

Abstract. This work is concerned with asymptotic properties of multi-dimensional random walks in random environment. Under Kalikow's condition, we show a central limit theorem for random walks in random environment on Êd, when dS2. We also derive tail estimates on the probability of slowdowns. These latter estimates are of special interest due to the natural interplay between slowdowns and the presence of traps in the medium. The tail behavior of the renewal time constructed in [25] plays an important role in the investigation of both problems. This article also improves the previous work of the author [24], concerning estimates of probabilities of slowdowns for walks which are neutral or biased to the right.
A general host–pathogen model with free–living infective stages and differing rates of uptake of the infective stages by infected and susceptible hosts
Authors: Boots Michael
Published: August 2002

In addition to their lethal effects, pathogens can cause a number of other debilitating effects on infected hosts. A population dynamical model of the interaction between an invertebrate host and a pathogen is constructed to examine the importance of one such debilitating effect on the host population dynamics. Specifically the feeding rate and therefore the uptake of pathogen free‐living infective particles by infected individuals is reduced as a consequence of the pathogen infection. The pathogen is more likely to regulate the host and the equilibrium population density of the host is reduced. Less intuitively there is also an increased chance of the pathogen causing cyclic population dynamics in the host.
The Hepatitis B Virus X Protein (Hbx) Induces A Migratory Phenotype in A Cd44–Dependent Manner: Possible Role of Hbx in Invasion and Metastasis
Authors: Lara-Pezzi Enrique , Serrador Juan M. , Montoya María C. , Zamora David , Yáñez-Mó María , Carretero Marta , Furthmayr Heinz , Sánchez-Madrid Francisco , López-Cabrera Manuel
Published: August 2002

The hepatitis B virus X protein (HBx) of the hepatitis B virus (HBV) has been involved in the development of hepatocellular carcinoma (HCC). However, its possible contribution to the metastatic spreading of liver tumors has not been explored so far. We report here the ability of HBx to enhance cell motility, both alone and in synergy with growth factors, and to induce a migratory phenotype in transformed cells. HBx altered the cellular morphology by inducing the formation of pseudopodial protrusions and cytoskeletal rearrangements, which was accompanied by the polarization of cell–surface adhesion molecules, including the hyaluronan (HA) receptor, CD44. Furthermore, HBx induced the redistribution to the pseudopodial tips of F–actin-binding proteins of the ezrin/radixin/moesin (ERM) family in a Rho– and Rac–dependent manner and increased the association of CD44 with moesin. The migration of HBx–bearing cells in response to HA and growth factors was impaired by a blocking anti–CD44 monoclonal antibody (mAb), suggesting that the HBx–induced cell motility is partially mediated by CD44. Interestingly, HBx–bearing cells showed increased HA–interaction efficiency as assessed under laminar flow conditions, which was the result, at least in part, of an enhanced binding affinity of CD44. HBx may therefore contribute to the acquisition of metastatic properties by modifying the migratory behavior of transformed hepatocytes and by increasing their ability to bind HA in the outer margin of the tumors or in secondary target organs. (Hepatology 2001;33:1270–1281.)
Hepatitis B Virus Dane Particles Bind to Human Plasma Apolipoprotein H
Authors: Stefas Ilias , Rucheton Marcel , D'Angeac Arnaud Dupuy , Morel-Baccard Christine , Seigneurin Jean M. , Zarski Jean P. , Martin Marianne , Cerutti Martine , Bossy Jean Pierre , Missé Dorothée , Graafland Hubert , Veas Francisco
Published: August 2002

Human apolipoprotein H (apo H) was found to bind specifically to hepatitis B surface antigen (HBsAg) from hepatitis B virus (HBV)–infected individuals. We used recombinant HBsAg proteins to analyze HBV domains recognized by apo H. We showed that the myristylated pre–S1 domain of HBsAg strongly interacted with apo H. This binding involved phospholipid components of the HBV envelope because their removal by detergent prevented apo H–HBsAg interaction. The opposite effects of iron and zinc metal ions on binding suggest that the oxidation of phospholipids also affects apo H–HBsAg interaction. After fractionation of viral particles on a sucrose gradient, and their addition to microtiter plates coated with apo H or anti–HBsAg, we observed that the maximal anti–HBsAg capture activity corresponded to a sucrose concentration of 36%, whereas the maximal apo H capture activity corresponded to a concentration of 39%. Electron microscopy and polymerase chain reaction (PCR) Southern blot studies of these fractions showed that the fraction with maximal apo H binding predominantly contained full Dane particles. Finally, we studied apo H–HBsAg binding relative to the presence of hepatitis B virus markers and observed that apo H binding activity for HBsAg was higher in sera from patients in the active virus replication phase.
A Case–Control Study for Clinical and Molecular Biological Differences Between Hepatitis B Viruses of Genotypes B and C
Authors: Orito Etsuro , Mizokami Masashi , Sakugawa Hiroshi , Michitaka Kojiro , Ishikawa Kazuyoshi , Ichida Takafumi , Okanoue Takeshi , Yotsuyanagi Hiroshi , Iino Shiro
Published: August 2002

Clinical and molecular virological differences were evaluated in 50 Japanese patients chronically infected with HBV of genotype B and C who were matched for age and sex as well as the severity of liver disease in a case–control study. Hepatitis B e antigen (HBeAg) was significantly less frequent (16% vs. 42%, P < .01), whereas antibody to HBeAg (anti–HBe) was significantly more common (84% vs. 56%, P < .01) in genotype B than C patients. The predominance of mutants with G–to–A mutation at nucleotide (nt) 1896 in the precore region (A1896) over the wild–type was comparable between genotype B and C patients (60% and 62%, respectively), and it correlated with anti–HBe. The double mutation in the basic core promoter (A–to–T at nt 1762 and G–to–A at nt 1764), however, was significantly more frequent in genotype C than B patients (58% vs. 16%, P < .01), and it did not correlate with anti–HBe or HBeAg. By the multiple logistic regression analysis, the double mutation in the basic core promoter (T1762/A1764) was significantly associated with genotype C [odds ratio (OR), 9.3; 95% confidence interval (CI), 3.4–25.1]], age ≥ 35 years (OR, 5.5; CI, 1.5–20.5), and more advanced liver disease (OR, 4.1; CI, 1.6–10.2), but it was not associated with sex, HBeAg, HBV DNA, or the precore mutation (A1896). These results suggest a role of the double mutation in the basic core promoter in association with genotype C and a longer duration of infection in the aggravation of chronic hepatitis B.
Hla Class Ii Genes Determine the Natural Variance of Hepatitis C Viral Load
Authors: Fanning Liam J. , Levis John , Kenny-Walsh Elizabeth , Whelton Michael , O'Sullivan Kathleen , Shanahan Fergus
Published: August 2002

The aim of this study was to investigate the relationship between human leukocyte antigen (HLA) class II genes and the natural fluctuations in hepatitis C viral load in a homogeneous patient population. The study group consisted of 57 viremic (hepatitis C virus [HCV] 1b) women for whom HLA class II DRB1 and DQB1 haplotyping, virologic, histologic, and biochemical markers of disease activity were available. All patients were infected with HCV 1b from the same source of hepatitis C–contaminated anti–D immunoglobulin during the period from May 1977 to November 1978. The mean slope of change of viral load was 0.34 (SD ± 0.73) log10 viral copies/mL/year, which is significantly different from zero, P < 10-9. Analysis of the relationship between the slope of change of viral load and HLA class II haplotype indicated a significantly different slope of change of viral load between the alleles of (1) DRB1*15 and DRB1*0701, and (2) DQB1*0602 and DQB1*0201, P
c = .036 and P
c = .026 after Bonferroni correction for multiple comparisons, respectively. Significant differences for grade and stage of disease at liver biopsy were observed for DQB1*0501 and DQB1*0201 alleles; P = .019, r
s = .64, and P = .047, r
s = .57, respectively. In addition, significant differences in stage of disease were found to exist between DRB1*13 and DRB1*0701, P = .031, r
s = -.71. Our results define an association between the slope of change of viral load and HLA class II haplotype in patients infected with genotype 1b of HCV. This suggests a role for host immunogenetic factors in HCV infection in this homogeneous group.
Involvement of P38 Mitogen–Activated Protein Kinase Signaling in Transformed Growth of A Cholangiocarcinoma Cell Line
Authors: Tadlock Laura , Patel Tushar
Published: August 2002

Although mitogen–activated protein kinase (MAPK) pathways play a key role in cell growth, their role in mediating the altered growth phenotype of transformed cells remains unclear. The p44/p42 MAPK signaling cascades are activated by mitogenic stimulation of human cholangiocytes. In contrast, the p38 MAPK pathway is activated by mitogen stimulation of malignant, but not nonmalignant cholangiocytes. Thus, our aims were to determine the role of p38 MAPK signaling in mediating the growth phenotype of transformed cholangiocytes. KMCH–1 malignant human cholangiocytes required the presence of serum for proliferation, but were able to grow in reduced serum conditions. Inhibition of p38 MAPK decreased serum–dependent proliferation of KMCH–1 cells. Furthermore, inhibition of p38 MAPK, but not of p44/p42 MAPK, reduced anchorage–independent growth of KMCH–1 cells. Although both p38 and p44/p42 MAPK are activated in response to mitogens, they have divergent effects on anchorage–independent growth. Inhibition of p38 MAPK, but not of p44/p42 MAPK signaling, decreased cell cycle progression and increased expression of the cyclin–dependent kinase inhibitor p21WAF1/CIPl. However, expression of p27KIP1 or p16INK4A was not altered by either pathway. Thus, mitogen activation of p38 MAPK decreases expression of p21WAF1/CIP1 and mediates growth independent of anchorage signals, whereas mitogen activation of p44/p42 MAPK mediates an anchorage signal–dependent growth pathway. These data provide a link between aberrant stress–activated cell signaling and the altered growth phenotype of transformed cells that may be important for the development of therapies to limit transformed cell growth.
Wound–Induced Migration of Rat Hepatic Stellate Cells Is Modulated by Endothelin–1 Through Rho–Kinase–Mediated Alterations in the Acto–Myosin Cytoskeleton
Authors: Tangkijvanich Pisit , Tam Steven P. , Yee Hal F.
Published: August 2002

Although migration of stellate cells during hepatic injury is essential for wound–healing and fibrosis of the liver, the extracellular and intracellular signals that regulate stellate cell migration are incompletely understood. In this study we tested the hypothesis that wound–induced migration of stellate cells is modulated by endothelin–1 (ET–1) through rho–kinase–mediated alterations in the acto–myosin cytoskeleton. To address this hypothesis, a method was established for direct visualization of wound–induced migration of culture–activated stellate cells with subcellular resolution. Migration in response to wounding was characterized by (1) plasma membrane ruffling and protrusion into the wound, (2) lamellipodia formation at the leading edge, (3) focal adhesion and stress fiber assembly, and (4) myosin reorganization. Exogenous ET–1 accelerated wound–induced migration of stellate cells, but did not alter wound–induced proliferation. Experiments using ET–1 antagonists in the absence of exogenous ET–1 showed that wound–induced migration was also stimulated by endogenous ET–1. Selective inhibition of rho–associated kinase decelerated migration in response to wounding. Moreover, inhibition of rho–associated kinase was distinguished by abrogation of focal adhesion formation, stress fiber assembly, and myosin reorganization. This study shows that rho–kinase–dependent alterations in the acto–myosin cytoskeleton contribute to wound–induced stellate cell migration, which is accelerated by both exogenous and endogenous ET–1. Consequently, these results provide important new evidence suggesting that, migration of stellate cells is modulated by paracrine and autocrine ET–1 stimulation via the action of rho–kinase on the acto–myosin cytoskeleton.
Formation of Normal Desmin Intermediate Filaments in Mouse Hepatic Stellate Cells Requires Vimentin
Authors: Geerts Albert , Eliasson Camilla , Niki Toshiro , Wielant Annemie , Vaeyens Freja , Pekny Milos
Published: August 2002

Increased desmin synthesis and formation of desmin–containing intermediate filaments (IFs) is one of the hallmarks of transdifferentiation of hepatic stellate cells into myofibroblast–like cells. These desmin–enriched myofibroblast–like cells are the major sources of fibrotic extracellular matrix in chronically diseased liver. Myofibroblast–like cells are also involved in the contraction of sinusoids, which leads to increased intrahepatic pressure and portal hypertension. To address the requirements for the formation of desmin–containing IFs both in quiescent and in transdifferentiated stellate cells, we used mice deficient for glial fibrillary acidic protein (GFAP) and/or vimentin, which are additional IF proteins present in stellate cells. In this study, we show that desmin cannot form full–length bundles of IFs in the absence of both GFAP and vimentin. Quiescent and transdifferentiated GFAP-/-vim-/- stellate cells are devoid of normal bundles of IFs. Instead, they exhibit only residual IF bundles restricted to subcortical cytoplasm, although these cells contain equal desmin mRNA and protein levels as wild–type cells. The absence of vimentin alone restricts formation of desmin–containing IF bundles to the perinuclear region, while both the distal processes in quiescent stellate cells and the subcortical zone in myofibroblast–like cells remain free of desmin–containing IF bundles. The absence of GFAP alone does not interfere with the formation of desmin–containing IFs. Thus, to form normal IFs in stellate cells, desmin is required to partnerize with vimentin. In addition, these mouse models will prove to be instrumental in addressing the role of IFs in the process of stellate cell transdifferentiation.
Altered Expression of Heme Oxygenase–1 in the Livers of Patients With Portal Hypertensive Diseases
Authors: Makino Nobuya , Suematsu Makoto , Sugiura Yoshiaki , Morikawa Hiroyasu , Shiomi Susumu , Goda Nobuhito , Sano Tsuyoshi , Nimura Yuji , Sugimachi Keizo , Ishimura Yuzuru
Published: August 2002

This study was designed to determine changes in expression of heme oxygenase (HO)–1, the stress–inducible and carbon monoxide-producing enzyme, in normotensive and portal hypertensive human livers. GTS–1, a monoclonal antibody against rat HO–1 cross–reacted with the human HO–1 and blocked its enzyme activity, allowing us to examine the activity and localization of HO–1. In controls, approximately 50% of the total HO activity was from HO–1 as judged by the sensitivity to GTS–1, while the rest of activity was from other isozymes such as HO–2. HO–1 was expressed mainly in a subpopulation of Kupffer cells, and the expression in hepatic stellate cells, sinusoidal endothelial cells, and hepatocytes was little, if any. The HO–1 expression exhibited quite different pictures in the livers of portal hypertensive diseases. In cirrhotic livers, which undergo portal hypertension through increases in intrasinusoidal resistance and regenerative changes in the parenchyma, HO–1 occurred in a majority of Kupffer cells and was also observed in hepatocytes. Consequently, the total HO–1 activities became significantly greater in these tissues than those from normal individuals. By contrast, livers of idiopathic portal hypertension that are characterized by an increase in presinusoidal resistance displayed a significant decrease in the HO–1 expression in Kupffer cells, and its hepatocellular expression was not detectable. Although factors involved in altered HO–1 expression in these cells remain unknown, the results suggest that Kupffer cells could alter their expression of HO–1 in response to local hemodynamic changes associated with chronic portal hypertension in humans.
Which Patients With Cirrhosis Should Undergo Endoscopic Screening for Esophageal Varices Detection?
Authors: Schepis Filippo , Cammà Calogero , Niceforo Domenico , Magnano Antonio , Pallio Socrate , Cinquegrani Maurizio , D'Amico Gennaro , Pasta Linda , Craxì Antonio , Saitta Antonino , Raimondo Giovanni
Published: August 2002

Our aims were to develop a noninvasive predictive tool to identify cirrhotic patients with esophageal varices and to evaluate whether portal Doppler ultrasonographic parameters may improve the value of other predictors. One hundred forty–three consecutive compensated cirrhotic patients underwent upper gastrointestinal endoscopy. Fourteen clinical, biochemical, ultrasonographic, and Doppler ultrasonographic parameters of each patient were also recorded. Esophageal varices were detected in 63 of the 143 patients examined (44%; 95% confidence interval [CI] 36.2–52.6). Medium and large esophageal varices were observed in 28 subjects (44%; 95% CI 31.4–58.4). Using stepwise logistic regression, presence of esophageal varices was independently predicted by prothrombin activity less than 70% (odds ratio [OR]: 5.83; 95% CI: 2.6–12.8), ultrasonographic portal vein diameter greater than 13 mm (OR: 2.92; 95% CI: 1.3–6.4), and platelet count less than 100 × 109/L (OR: 2.83; 95% CI: 1.27–6.28). Variables included in the model were used to generate a simple incremental rule to evaluate each individual patient. The discriminating ability of the prediction rule was relevant (area under the curve: 0.80) and did not change by replacing ultrasonographic portal vein diameter with congestion index of portal vein. We concluded that compensated cirrhotic patients should be screened by upper gastrointestinal endoscopy when prothrombin activity less than 70%, platelet count less than 100 × 109/L, and ultrasonographic portal vein diameter greater than 13 mm are observed, whereas those without any of these predictors should not undergo endoscopy. The contribution provided by portal Doppler ultrasonographic parameters does not appear of practical utility. (Hepatology 2001;33:333–338.)
Long–Term Mortality and Morbidity of Transfusion–Associated Non–A, Non–B, and Type C Hepatitis: A National Heart, Lung, and Blood Institute Collaborative Study
Authors: Seeff Leonard B. , Hollinger Blaine F. , Alter Harvey J. , Wright Elizabeth C. , Cain Casey M.B. , Buskell Zelma J. , Ishak Kamal G. , Iber Frank L. , Toro Doris , Samanta Arun , Koretz Ronald L. , Perrillo Robert P.
Published: August 2002

Persons with non–A, non–B hepatitis (cases) identified in 5 transfusion studies in the early 1970s have been followed ever since and compared for outcome with matched, transfused, non–hepatitis controls from the same studies. Previously, we reported no difference in all–cause mortality but slightly increased liver–related mortality between these cohorts after 18 years follow–up. We now present mortality and morbidity data after approximately 25 years of follow–up, restricted to the 3 studies with archived original sera. All–cause mortality was 67% among 222 hepatitis C–related cases and 65% among 377 controls (P = NS). Liver–related mortality was 4.1% and 1.3%, respectively (P = .05). Of 129 living persons with previously diagnosed transfusion–associated hepatitis (TAH), 90 (70%) had proven TAH–C, and 39 (30%), non–A–G hepatitis. Follow–up of the 90 TAH–C cases revealed viremia with chronic hepatitis in 38%, viremia without chronic hepatitis in 39%, anti–HCV without viremia in 17%, and no residual HCV markers in 7%. Thirty–five percent of 20 TAH–C patients biopsied for biochemically defined chronic hepatitis displayed cirrhosis, representing 17% of all those originally HCV–infected. Clinically evident liver disease was observed in 86% with cirrhosis but in only 23% with chronic hepatitis alone. Thirty percent of non–A, non–B hepatitis cases were unrelated to hepatitis viruses A,B,C, and G, suggesting another unidentified agent. In conclusion, all–cause mortality approximately 25 years after acute TAH–C is high but is no different between cases and controls. Liver–related mortality attributable to chronic hepatitis C, though low (<3%), is significantly higher among the cases. Among living patients originally HCV–infected, 23% have spontaneously lost HCV RNA.
The Peroxisomal Proliferator Clofibrate Enhances the Hepatic Cytoplasmic Movement of Fatty Acids in Rats
Authors: Milliano Michael T. , Luxon Bruce A.
Published: August 2002

The role of cytosolic fatty acid binding protein (FABP) in cellular fatty acid metabolism remains poorly defined. The intracellular movement of fatty acids is thought to be facilitated through codiffusion with FABP. Peroxisomal proliferators like clofibrate induce FABP and may stimulate fatty acid use by increasing cytoplasmic diffusion rates. Our aim was to determine if induction of FABP by clofibrate increases the cytoplasmic transport of a fluorescent fatty acid NBD–stearate. Male rats were fed clofibrate for 9 days to increase the hepatic concentration of FABP. Hepatocytes were isolated using collagenase perfusion. Cytosolic FABP was measured by enzyme–linked immunosorbent assay (ELISA) using a specific antibody to hepatic FABP. Two–dimensional laser photobleaching (FRAP) was used to measure the cytoplasmic movement of NBD–stearate in hepatocytes. Cytoplasmic transport of NBD–stearate occurred by isotropic diffusion with no evidence for convective or directed transport. Treatment with clofibrate increased FABP levels, the fraction of NBD–stearate found in cytosol, and the observed cytoplasmic diffusion rate. The increase in cytoplasmic movement exceeded that predicted from FABP binding changes suggesting that clofibrate also enhances fatty acid diffusion within intracellular membranes. Nonspecific effects of clofibrate on cytoplasmic viscosity or pore size were not observed. These data suggest that clofibrate treatment induces cytosolic FABP and stimulates the intracellular movement of fatty acids by 2 distinct mechanisms. Soluble proteins like FABP promote the cytoplasmic transport of fatty acids by increasing diffusion. These data further support a role for intracellular binding proteins in facilitating the intracellular movements of fatty acids.