bluuebunny/crossref_metadata_2025 huggingface dataset:
HES Presidential Address Hayek: Right for the Wrong Reasons?
Authors: Caldwell Bruce
Published: July 2002

The controversial question of whether the millennium began on January 1, 2000 or on January 1, 2001 weighed heavily on my mind as I composed my talk. The dilemma is, I should think, evident: Should a historian of thought want to give the last address of the old millennium, or the first address of the new one? I couldn't make up my mind about this, so I decided to consult my friends, particularly those whom I felt had given the question their considered attention. I asked David Colander, and he assured me that my preferences didn't matter, since he in fact had certainly given the final presidential address of the last millennium when he spoke in Greensboro the previous June. I then asked John Davis, and he assured me that my preferences didn't matter since he, in fact, was certainly going to give the first one of the new millennium, in 2001. Now being not nearly so stupid as I look, it occurred to me that if I listened to both of these individuals, there was a chance that I was going to give neither the last address of the old nor the first address of the new millennium. Feeling that this was, to put it mildly, an unacceptable outcome, I decided to disregard the ill-considered advice of my so-called friends and take firm control of the situation. Ladies and gentleman, I am very proud to be before you here today, delivering both the last presidential address of the old millennium and the first of the new one.
A reversibly immortalized human hepatocyte cell line as a source of hepatocyte‐based biological support
Authors: Kobayashi Naoya , Westerman Karen A. , Tanaka Noriaki , Fox Ira J. , Leboulch Philippe
Published: July 2002

The application of hepatocyte transplantation (HTX) is increasingly envisioned for temporary metabolic support during acute liver failure and provision of specific liver functions in inherited liver‐based metabolic diseases. Compared with whole liver transplantation, HTX is a technically simple procedure and hepatocytes can be cryopreserved for future use. A major limitation of this form of therapy in humans is the worldwide shortage of human livers for isolating an adequate number of transplantable human hepatocyes when needed. Furthermore, the numbers of donor livers available for hepatocyte isolation is limited by competition for their use in whole organ transplantation. Considering the cost of hepatocyte isolation and the need for immediate preparation of consistent and functional cells, it is unlikely that human hepatocytes can be obtained on such a scale to treat a large number of patients with falling liver functions. The utilization of xenogenic hepatocytes will result in additional concerns regarding transmission of infectious pathogens and immunological and physiological incompatibilities between animals and humans. An attractive alternative to primary human hepatocytes is the use of tightly regulated human hepatocyte cell lines. Such cell lines can provide the advantages of unlimited availability, sterility and uniformity. We describe here methods for creating transplantable human hepatocyte cell lines using currently available cell cultures and gene transfer technology.
Association analysis of polymorphisms in the mu opioid gene and heroin abuse in Chinese subjects
Authors: Li Tao , Liu Xiehe , Zhu Zuo Hong , Zhao Jinghua , Hu Xun , Sham Pak C. , Collier David A.
Published: July 2002

We examined four polymorphisms in the mu opioid receptor gene in 282 Chinese heroin addicts from Sichuan Province, Southwest China and compared the allele and genotype frequencies to those in 258 normal controls from the same geographic region. Two of these polymorphisms (Ala6Val and Ser147Cys) were not polymorphic in the Chinese, with only Ala6 and Ser147 observed. The frequencies of the two other polymorphisms were significantly different from those observed in Caucasians, African Americans and Native Americans. The Asn40Asp and IVS2 + 691G/C polymorphisms did not differ significantly for allele (p= 0.16; p = 0.21), genotype (p= 0.32; p = 0.09) or haplotype frequencies (p= 0.24) between the Chinese heroin‐addicted cases and normal controls. Similarly, we did not detect any association when the population was stratified by gender, route of administration (nasal inhalation and/or injection) and age‐at‐onset (above or below 25 years). This indicates that the mu opioid receptor is not likely to be a major genetic risk factor for heroin abuse in this population.
Alcohol and gastrointestinal cancer: pathogenic mechanisms
Authors: SEITZ HELMUT , POSCHL GUDRUN
Published: July 2002

Abstract Chronic heavy alcohol consumption leads to a significantly increased risk of cancer in the oropharynx, larynx and the oesophagus. In the liver, chronic alcohol abuse results in cirrhosis, a precursor of hepatocellular cancer. More recentepidemiologic studies also demonstrate that regular alcohol consumption, even in low amounts, has an enhanced risk for rectal cancer and cancer of the breast. Alcohol by itself is not a carcinogen. However, alcohol can increase the susceptibility of various organs to chemical carcinogens by a variety of mechanisms. Among these, increased activation of procarcinogens through microsomal enzyme induction, a change in the metabolism and/or distribution of carcinogens, interference with the system that repairs carcinogen‐induced DNA alkylations, direct mucosal tissue damage with consecutive stimulation of cellular regeneration and alcohol‐mediated malnutrition may be of importance. In the upper gastrointestinal tract the production of acetaldehyde and free radicals via cytochrome P450 2E1 and via alcohol dehydrogenase may lead to tissue damage and to secondary hyper‐regeneration. In addition, local mechanisms may also be involved in the co‐carcinogenic process. In the rectal mucosa acetaldehyde seems to be an important factor in carcinogenesis and may be predominantly produced by faecal bacteria.
Butyrylcholinesterase: an enzyme antidote for cocaine intoxication
Authors: MATTES CAROL E. , BELENDIUK GEORGE W. , LYNCH THOMAS J. , BRADY ROSCOE O. , DRETCHEN KENNETH L.
Published: July 2002

Cocaine‐associated toxicity is the result of effects on the cardiovascular and central nervous systems. Since the primary route of cocaine inactivation is enzymatic degradation by butyrylcholinesterase (BChE), we sought to determine if the administration of purified human enzyme would ameliorate the lethal effects of cocaine. While the cardiovascular, autonomic or central nervous systems were unaffected by BChE, the enzyme reduced the adverse effects of cocaine including hypertension, hyperactivity and convulsions. BChE decreased both the brain and blood levels of cocaine and shifted the metabolites towards the production of the inactive product ecgonine methyl ester and away from the physiologically active metabolites, norcocaine and benzoylecgonine. We conclude that BChE would appear to be an ideal antidote in the treatment of cocaine intoxication and has potential therapeutic application.
Patterns of phonological disability in Cantonese‐speaking children in Hong Kong
Authors: Cheung Pamela , Abberton Evelyn
Published: July 2002

Tone, vowel and consonant production are described for a large group of Cantonese‐speaking children assessed in speech and language therapy clinics in Hong Kong. The patterns of disability follow predictions made on the basis of work on normal phonological development in Cantonese, and on psychoacoustic factors in acquisition: consonants account for more disability than vowels, and tones are least problematic. Possible articulatory and auditory contributions to explanation of the observed patterns are discussed.
Vascular Bed–specific Expression of an Endothelial Cell Gene Is Programmed by the Tissue Microenvironment
Authors: Aird William C. , Edelberg Jay M. , Weiler-Guettler Hartmut , Simmons William W. , Smith Thomas W. , Rosenberg Robert D.
Published: July 2002

The endothelium is morphologically and functionally adapted to meet the unique demands of the underlying tissue. At the present time, little is known about the molecular basis of endothelial cell diversity. As one approach to this problem, we have chosen to study the mechanisms that govern differential expression of the endothelial cell–restricted von Willebrand factor (vWF) gene. Transgenic mice were generated with a fragment of the vWF gene containing 2,182 bp of 5′ flanking sequence, the first exon and first intron coupled to the LacZ reporter gene. In multiple independent lines of mice, β-galactosidase expression was detected within endothelial cells in the brain, heart, and skeletal muscle. In isogeneic transplantation models, LacZ expression in host-derived auricular blood vessels was specifically induced by the microenvironment of the heart. In in vitro coculture assays, expression of both the transgene and the endogenous vWF gene in cardiac microvascular endothelial cells (CMEC) was upregulated in the presence of cardiac myocytes. In contrast, endothelial cell levels of thrombomodulin protein and mRNA were unchanged by the addition of ventricular myocytes. Moreover, CMEC expression of vWF was not influenced by the addition of 3T3 fibroblasts or mouse hepatocytes. Taken together, the results suggest that the vWF gene is regulated by vascular bed–specific pathways in response to signals derived from the local microenvironment.
Cystic Fibrosis Transmembrane Conductance Regulator–associated ATP Release Is Controlled by a Chloride Sensor
Authors: Jiang Qinshi , Mak Daniel , Devidas Sreenivas , Schwiebert Erik M. , Bragin Alvina , Zhang Yulong , Skach William R. , Guggino William B. , Foskett J. Kevin , Engelhardt John F.
Published: July 2002

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis, and has also been closely associated with ATP permeability in cells. Using a Xenopus oocyte cRNA expression system, we have evaluated the molecular mechanisms that control CFTR-modulated ATP release. CFTR-modulated ATP release was dependent on both cAMP activation and a gradient change in the extracellular chloride concentration. Activation of ATP release occurred within a narrow concentration range of external Cl− that was similar to that reported in airway surface fluid. Mutagenesis of CFTR demonstrated that Cl− conductance and ATP release regulatory properties could be dissociated to different regions of the CFTR protein. Despite the lack of a need for Cl− conductance through CFTR to modulate ATP release, alterations in channel pore residues R347 and R334 caused changes in the relative ability of different halides to activate ATP efflux (wtCFTR, Cl >> Br; R347P, Cl >> Br; R347E, Br >> Cl; R334W, Cl = Br). We hypothesize that residues R347 and R334 may contribute a Cl− binding site within the CFTR channel pore that is necessary for activation of ATP efflux in response to increases of extracellular Cl−. In summary, these findings suggest a novel chloride sensor mechanism by which CFTR is capable of responding to changes in the extracellular chloride concentration by modulating the activity of an unidentified ATP efflux pathway. This pathway may play an important role in maintaining fluid and electrolyte balance in the airway through purinergic regulation of epithelial cells. Insight into these molecular mechanisms enhances our understanding of pathogenesis in the cystic fibrosis lung.
Integral Membrane Protein Sorting to Vacuoles in Plant Cells: Evidence for Two Pathways
Authors: Jiang Liwen , Rogers John C.
Published: July 2002

Plant cells may contain two functionally distinct vacuolar compartments. Membranes of protein storage vacuoles (PSV) are marked by the presence of α-tonoplast intrinsic protein (TIP), whereas lytic vacuoles (LV) are marked by the presence of γ-TIP. Mechanisms for sorting integral membrane proteins to the different vacuoles have not been elucidated. Here we study a chimeric integral membrane reporter protein expressed in tobacco suspension culture protoplasts whose traffic was assessed biochemically by following acquisition of complex Asn-linked glycan modifications and proteolytic processing, and whose intracellular localization was determined with confocal immunofluorescence. We show that the transmembrane domain of the plant vacuolar sorting receptor BP-80 directs the reporter protein via the Golgi to the LV prevacuolar compartment, and attaching the cytoplasmic tail (CT) of γ-TIP did not alter this traffic. In contrast, the α-TIP CT prevented traffic of the reporter protein through the Golgi and caused it to be localized in organelles separate from ER and from Golgi and LV prevacuolar compartment markers. These organelles had a buoyant density consistent with vacuoles, and α-TIP protein colocalized in them with the α-TIP CT reporter protein when the two were expressed together in protoplasts. These results are consistent with two separate pathways to vacuoles for membrane proteins: a direct ER to PSV pathway, and a separate pathway via the Golgi to the LV.
Phosphorylation of Adducin by Rho-Kinase Plays a Crucial Role in Cell Motility
Authors: Fukata Yuko , Oshiro Noriko , Kinoshita Nagatoki , Kawano Yoji , Matsuoka Yoichiro , Bennett Vann , Matsuura Yoshiharu , Kaibuchi Kozo
Published: July 2002

Adducin is a membrane skeletal protein that binds to actin filaments (F-actin) and thereby promotes the association of spectrin with F-actin to form a spectrin-actin meshwork beneath plasma membranes such as ruffling membranes. Rho-associated kinase (Rho- kinase), which is activated by the small guanosine triphosphatase Rho, phosphorylates α-adducin and thereby enhances the F-actin–binding activity of α-adducin in vitro. Here we identified the sites of phosphorylation of α-adducin by Rho-kinase as Thr445 and Thr480. We prepared antibody that specifically recognized α-adducin phosphorylated at Thr445, and found by use of this antibody that Rho-kinase phosphorylated α-adducin at Thr445 in COS7 cells in a Rho-dependent manner. Phosphorylated α-adducin accumulated in the membrane ruffling area of Madin-Darby canine kidney (MDCK) epithelial cells and the leading edge of scattering cells during the action of tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF). The microinjection of Botulinum C3 ADP-ribosyl-transferase, dominant negative Rho-kinase, or α-adducinT445A,T480A (substitution of Thr445 and Thr480 by Ala) inhibited the TPA-induced membrane ruffling in MDCK cells and wound-induced migra- tion in NRK49F cells. α-AdducinT445D,T480D (substi- tution of Thr445 and Thr480 by Asp), but not α-adducinT445A,T480A, counteracted the inhibitory effect of the dominant negative Rho-kinase on the TPA-induced membrane ruffling in MDCK cells. Taken together, these results indicate that Rho-kinase phosphorylates α-adducin downstream of Rho in vivo, and that the phosphorylation of adducin by Rho-kinase plays a crucial role in the regulation of membrane ruffling and cell motility.
Epsin 1 Undergoes Nucleocytosolic Shuttling and Its Eps15 Interactor Nh2-Terminal Homology (Enth) Domain, Structurally Similar to Armadillo and Heat Repeats, Interacts with the Transcription Factor Promyelocytic Leukemia Zn2+ Finger Protein (Plzf)
Authors: Hyman Joel , Chen Hong , Di Fiore Pier Paolo , De Camilli Pietro , Brunger Axel T.
Published: July 2002

Epsin (Eps15 interactor) is a cytosolic protein involved in clathrin-mediated endocytosis via its direct interactions with clathrin, the clathrin adaptor AP-2, and Eps15. The NH2-terminal portion of epsin contains a phylogenetically conserved module of unknown function, known as the ENTH domain (epsin NH2-terminal homology domain). We have now solved the crystal structure of rat epsin 1 ENTH domain to 1.8 Å resolution. This domain is structurally similar to armadillo and Heat repeats of β-catenin and karyopherin-β, respectively. We have also identified and characterized the interaction of epsin 1, via the ENTH domain, with the transcription factor promyelocytic leukemia Zn2+ finger protein (PLZF). Leptomycin B, an antifungal antibiotic, which inhibits the Crm1- dependent nuclear export pathway, induces an accumulation of epsin 1 in the nucleus. These findings suggest that epsin 1 may function in a signaling pathway connecting the endocytic machinery to the regulation of nuclear function.
Gap Junctional Communication in the Early Xenopus Embryo
Authors: Landesman Yosef , Goodenough Daniel A. , Paul David L.
Published: July 2002

In the Xenopus embryo, blastomeres are joined by gap junctions that allow the movement of small molecules between neighboring cells. Previous studies using Lucifer yellow (LY) have reported asymmetries in the patterns of junctional communication suggesting involvement in dorso-ventral patterning. To explore that relationship, we systematically compared the transfer of LY and neurobiotin in embryos containing 16–128 cells. In all cases, the junction-permeable tracer was coinjected with a fluorescent dextran that cannot pass through gap junctions. Surprisingly, while LY appeared to transfer in whole-mount embryos, in no case did we observe junctional transfer of LY in fixed and sectioned embryos. The lack of correspondence between data obtained from whole-mounts and from sections results from two synergistic effects. First, uninjected blastomeres in whole-mounts reflect and scatter light originating from the intensely fluorescent injected cell, creating a diffuse background interpretable as dye transfer. Second, the heavier pigmentation in ventral blastomeres masks this scattered signal, giving the impression of an asymmetry in communication. Thus, inspection of whole-mount embryos is an unreliable method for the assessment of dye transfer between embryonic blastomeres. A rigorous and unambiguous demonstration of gap junctional intercellular communication demands both the coinjection of permeant and impermeant tracers followed by the examination of sectioned specimens. Whereas LY transfer was never observed, neurobiotin was consistently transferred in both ventral and dorsal aspects of the embryo, with no apparent asymmetry. Ventralization of embryos by UV irradiation and dorsalization by Xwnt-8 did not alter the patterns of communication. Thus, our results are not compatible with current models for a role of gap junctional communication in dorso-ventral patterning.
Patterns of Immunodominance in HIV-1–specific Cytotoxic T Lymphocyte Responses in Two Human Histocompatibility Leukocyte Antigens (HLA)-identical Siblings with HLA-A*0201 Are Influenced by Epitope Mutation
Authors: Goulder P.J.R. , Sewell A.K. , Lalloo D.G. , Price D.A. , Whelan J.A. , Evans J. , Taylor G.P. , Luzzi G. , Giangrande P. , Phillips R.E. , McMichael A.J.
Published: July 2002

Primary human immunodeficiency virus (HIV) infection is controlled principally by HIV-specific cytotoxic T lymphocytes (CTL) to a steady-state level of virus load, which strongly influences the ultimate rate of progression to disease. Epitope selection by CTL may be an important determinant of the degree of immune control over the virus. This report describes the CTL responses of two HLA-identical hemophiliac brothers who were exposed to identical batches of Factor VIII and became seropositive within 10 wk of one another. Both have HLA-A*0201. The CTL responses of the two siblings were very dissimilar, one donor making strong responses to two epitopes within p17 Gag (HLA-A*0201–restricted SLYNTVATL and HLA-A3–restricted RLRPGGKKK). The sibling responded to neither epitope, but made strong responses to two epitopes presented by HLA-B7. This was not the result of differences in presentation of the epitopes. However, mutations in both immunodominant epitopes of the p17 Gag responder were seen in proviral sequences of the nonresponder. We then documented the CTL responses to two HLA-A*0201–restricted epitopes, in Gag (SLYNTVATL) and Pol (ILKEPVHGV) in 22 other HIV-infected donors with HLA-A*0201. The majority (71%) generated responses to the Gag epitope. In the 29% of donors failing to respond to the Gag epitope in standard assays, there was evidence of low frequency memory CTL responses using peptide stimulation of PBMC, and most of these donors also showed mutations in or around the Gag epitope. We concluded that HLA class I genotype determines epitope selection initially but that mutation in immunodominant epitopes can profoundly alter the pattern of CTL response.
Human Macrophage–derived Chemokine (MDC), a Novel Chemoattractant for Monocytes, Monocyte-derived Dendritic Cells, and Natural Killer Cells
Authors: Godiska Ronald , Chantry David , Raport Carol J. , Sozzani Silvano , Allavena Paola , Leviten Dina , Mantovani Alberto , Gray Patrick W.
Published: July 2002

A cDNA encoding a novel human chemokine was isolated by random sequencing of cDNA clones from human monocyte-derived macrophages. This protein has been termed macrophagederived chemokine (MDC) because it appears to be synthesized specifically by cells of the macrophage lineage. MDC has the four-cysteine motif and other highly conserved residues characteristic of CC chemokines, but it shares <35% identity with any of the known chemokines. Recombinant MDC was expressed in Chinese hamster ovary cells and purified by heparin– Sepharose chromatography. NH2-terminal sequencing and mass spectrophotometry were used to verify the NH2 terminus and molecular mass of recombinant MDC (8,081 dalton). In microchamber migration assays, monocyte-derived dendritic cells and IL-2–activated natural killer cells migrated to MDC in a dose-dependent manner, with a maximal chemotactic response at 1 ng/ml. Freshly isolated monocytes also migrated toward MDC, but with a peak response at 100 ng/ml MDC. Northern analyses indicated MDC is highly expressed in macrophages and in monocyte-derived dendritic cells, but not in monocytes, natural killer cells, or several cell lines of epithelial, endothelial, or fibroblast origin. High expression was also detected in normal thymus and less expression in lung and spleen. Unlike most other CC chemokines, MDC is encoded on human chromosome 16. MDC is thus a unique member of the CC chemokine family that may play a fundamental role in the function of dendritic cells, natural killer cells, and monocytes.
Itk and Fyn Make Independent Contributions to T Cell Activation
Authors: Liao X. Charlene , Littman Dan R. , Weiss Arthur
Published: July 2002

Itk is a member of the Btk/Tec/Itk family of nonreceptor protein tyrosine kinases (PTKs), and has been implicated in T cell antigen receptor (TCR) signal transduction. Lck and Fyn are the Src-family nonreceptor PTKs that are involved in TCR signaling. To address the question of how these members of different families of PTKs functionally contribute to T cell development and to T cell activation, mice deficient for both Itk and either Lck or Fyn were generated. The Itk/Lck doubly deficient mice exhibited a phenotype similar to that of Lck-deficient mice. The phenotype of the Itk/Fyn doubly deficient mice was similar to that of Itk deficient mice. However the Itk/Fyn doubly deficient mice exhibited a more severe defect in TCR-induced proliferation of thymocytes and peripheral T cells than did mice deficient in either kinase alone. These data support the notion that Itk and Fyn both make independent contributions to TCR-induced T cell activation.
Extrathymic T Cell Deletion and Allogeneic Stem Cell Engraftment Induced with Costimulatory Blockade Is Followed by Central T Cell Tolerance
Authors: Wekerle Thomas , Sayegh Mohamed H. , Hill Joshua , Zhao Yong , Chandraker Anil , Swenson Kirsten G. , Zhao Guiling , Sykes Megan
Published: July 2002

A reliable, nontoxic method of inducing transplantation tolerance is needed to overcome the problems of chronic organ graft rejection and immunosuppression-related toxicity. Treatment of mice with single injections of an anti-CD40 ligand antibody and CTLA4Ig, a low dose (3 Gy) of whole body irradiation, plus fully major histocompatibility complex–mismatched allogeneic bone marrow transplantation (BMT) reliably induced high levels (>40%) of stable (>8 mo) multilineage donor hematopoiesis. Chimeric mice permanently accepted donor skin grafts (>100 d), and rapidly rejected third party grafts. Progressive deletion of donor-reactive host T cells occurred among peripheral CD4+ lymphocytes, beginning as early as 1 wk after bone marrow transplantation. Early deletion of peripheral donor-reactive host CD4 cells also occurred in thymectomized, similarly treated marrow recipients, demonstrating a role for peripheral clonal deletion of donor-reactive T cells after allogeneic BMT in the presence of costimulatory blockade. Central intrathymic deletion of newly developing T cells ensued after donor stem cell engraftment had occurred. Thus, we have shown that high levels of chimerism and systemic T cell tolerance can be reliably achieved without myeloablation or T cell depletion of the host. Chronic immunosuppression and rejection are avoided with this powerful, nontoxic approach to inducing tolerance.
CD4 T Cell Tolerance to Human C-reactive Protein, an Inducible Serum Protein, Is Mediated by Medullary Thymic Epithelium
Authors: Klein Ludger , Klein Thomas , Rüther Ulrich , Kyewski Bruno
Published: July 2002

Inducible serum proteins whose concentrations oscillate between nontolerogenic and tolerogenic levels pose a particular challenge to the maintenance of self-tolerance. Temporal restrictions of intrathymic antigen supply should prevent continuous central tolerization of T cells, in analogy to the spatial limitation imposed by tissue-restricted antigen expression. Major acute-phase proteins such as human C-reactive protein (hCRP) are typical examples for such inducible self-antigens. The circulating concentration of hCRP, which is secreted by hepatocytes, is induced up to 1,000-fold during an acute-phase reaction. We have analyzed tolerance to hCRP expressed in transgenic mice under its autologous regulatory regions. Physiological regulation of basal levels (<10−9 M) and inducibility (>500-fold) are preserved in female transgenics, whereas male transgenics constitutively display induced levels. Surprisingly, crossing of hCRP transgenic mice to two lines of T cell receptor transgenic mice (specific for either a dominant or a subdominant epitope) showed that tolerance is mediated by intrathymic deletion of immature thymocytes, irrespective of widely differing serum levels. In the absence of induction, hCRP expressed by thymic medullary epithelial cells rather than liver-derived hCRP is necessary and sufficient to induce tolerance. Importantly, medullary epithelial cells also express two homologous mouse acute-phase proteins. These results support a physiological role of “ectopic” thymic expression in tolerance induction to acute-phase proteins and possibly other inducible self-antigens and have implications for delineating the relative contributions of central versus peripheral tolerance.
Inhibition of Angiogenesis by Interleukin 4
Authors: Volpert Olga V. , Fong Tim , Koch Alisa E. , Peterson Jeffrey D. , Waltenbaugh Carl , Tepper Robert I. , Bouck Noël P.
Published: July 2002

Interleukin (IL)-4, a crucial modulator of the immune system and an active antitumor agent, is also a potent inhibitor of angiogenesis. When incorporated at concentrations of 10 ng/ml or more into pellets implanted into the rat cornea or when delivered systemically to the mouse by intraperitoneal injection, IL-4 blocked the induction of corneal neovascularization by basic fibroblast growth factor. IL-4 as well as IL-13 inhibited the migration of cultured bovine or human microvascular cells, showing unusual dose–response curves that were sharply stimulatory at a concentration of 0.01 ng/ml but inhibitory over a wide range of higher concentrations. Recombinant cytokine from mouse and from human worked equally well in vitro on bovine and human endothelial cells and in vivo in the rat, showing no species specificity. IL-4 was secreted at inhibitory levels by activated murine T helper (TH0) cells and by a line of carcinoma cells whose tumorigenicity is known to be inhibited by IL-4. Its ability to cause media conditioned by these cells to be antiangiogenic suggested that the antiangiogenic activity of IL-4 may play a role in normal physiology and contribute significantly to its demonstrated antitumor activity.
A Human Minor Histocompatibility Antigen Specific for B Cell Acute Lymphoblastic Leukemia
Authors: Dolstra Harry , Fredrix Hanny , Maas Frans , Coulie Pierre G. , Brasseur Francis , Mensink Ewald , Adema Gosse J. , de Witte Theo M. , Figdor Carl G. , van de Wiel-van Kemenade Elly
Published: July 2002

Human minor histocompatibility antigens (mHags) play an important role in the induction of cytotoxic T lymphocyte (CTL) reactivity against leukemia after human histocompatibility leukocyte antigen (HLA)-identical allogeneic bone marrow transplantation (BMT). As most mHags are not leukemia specific but are also expressed by normal tissues, antileukemia reactivity is often associated with life-threatening graft-versus-host disease (GVHD). Here, we describe a novel mHag, HB-1, that elicits donor-derived CTL reactivity in a B cell acute lymphoblastic leukemia (B-ALL) patient treated by HLA-matched BMT. We identified the gene encoding the antigenic peptide recognized by HB-1–specific CTLs. Interestingly, expression of the HB-1 gene was only observed in B-ALL cells and Epstein-Barr virus–transformed B cells. The HB-1 gene–encoded peptide EEKRGSLHVW is recognized by the CTL in association with HLA-B44. Further analysis reveals that a polymorphism in the HB-1 gene generates a single amino acid exchange from His to Tyr at position 8 within this peptide. This amino acid substitution is critical for recognition by HB-1–specific CTLs. The restricted expression of the polymorphic HB-1 Ag by B-ALL cells and the ability to generate HB-1–specific CTLs in vitro using peptide-loaded dendritic cells offer novel opportunities to specifically target the immune system against B-ALL without the risk of evoking GVHD.
Homeostatic Competition Among T Cells Revealed by Conditional Inactivation of the Mouse Cd4 Gene
Authors: Wang Qi , Strong Julie , Killeen Nigel
Published: July 2002

Absence of CD4 impairs the efficiency of T cell receptor (TCR) signaling in response to major histocompatibility complex (MHC) class II–presented peptides. Here we use mice carrying a conditional Cd4 allele to study the consequences of impaired TCR signaling after the completion of thymocyte development. We show that loss of CD4 decreases the steady-state proliferation of T cells as monitored by in vivo labeling with bromo-deoxyuridine. Moreover, T cells lacking CD4 compete poorly with CD4-expressing T cells during proliferative expansion after transfer into lymphopenic recipients. The data suggest that T cells compete with one another during homeostatic proliferation, and indicate that the basis of this competition is TCR signaling.
Progress Toward a Human CD4/CCR5 Transgenic Rat Model for De Novo Infection by Human Immunodeficiency Virus Type 1
Authors: Keppler Oliver T. , Welte Frank J. , Ngo Tuan A. , Chin Peggy S. , Patton Kathryn S. , Tsou Chia-Lin , Abbey Nancy W. , Sharkey Mark E. , Grant Robert M. , You Yun , Scarborough John D. , Ellmeier Wilfried , Littman Dan R. , Stevenson Mario , Charo Israel F. , Herndier Brian G. , Speck Roberto F. , Goldsmith Mark A.
Published: July 2002

The development of a permissive small animal model for the study of human immunodeficiency virus type (HIV)-1 pathogenesis and the testing of antiviral strategies has been hampered by the inability of HIV-1 to infect primary rodent cells productively. In this study, we explored transgenic rats expressing the HIV-1 receptor complex as a susceptible host. Rats transgenic for human CD4 (hCD4) and the human chemokine receptor CCR5 (hCCR5) were generated that express the transgenes in CD4+ T lymphocytes, macrophages, and microglia. In ex vivo cultures, CD4+ T lymphocytes, macrophages, and microglia from hCD4/hCCR5 transgenic rats were highly susceptible to infection by HIV-1 R5 viruses leading to expression of abundant levels of early HIV-1 gene products comparable to those found in human reference cultures. Primary rat macrophages and microglia, but not lymphocytes, from double-transgenic rats could be productively infected by various recombinant and primary R5 strains of HIV-1. Moreover, after systemic challenge with HIV-1, lymphatic organs from hCD4/hCCR5 transgenic rats contained episomal 2–long terminal repeat (LTR) circles, integrated provirus, and early viral gene products, demonstrating susceptibility to HIV-1 in vivo. Transgenic rats also displayed a low-level plasma viremia early in infection. Thus, transgenic rats expressing the appropriate human receptor complex are promising candidates for a small animal model of HIV-1 infection.
Murine CD9 Is the Receptor for Pregnancy-specific Glycoprotein 17
Authors: Waterhouse Roseann , Ha Cam , Dveksler Gabriela S.
Published: July 2002

Pregnancy-specific glycoproteins (PSGs) are a family of highly similar secreted proteins produced by the placenta. PSG homologs have been identified in primates and rodents. Members of the human and murine PSG family induce secretion of antiinflammatory cytokines in mononuclear phagocytes. For the purpose of cloning the receptor, we screened a RAW 264.7 cell cDNA expression library. The PSG17 receptor was identified as the tetraspanin, CD9. We confirmed binding of PSG17 to CD9 by ELISA, flow cytometry, alkaline phosphatase binding assays, and in situ rosetting. Anti-CD9 monoclonal antibody inhibited binding of PSG17 to CD9-transfected cells and RAW 264.7 cells. Moreover, PSG17 binding to macrophages from CD9-deficient mice was significantly reduced. We then tested whether PSG17 binds to other members of the murine tetraspanin family. PSG17 did not bind to cells transfected with CD53, CD63, CD81, CD82, or CD151, suggesting that PSG17–CD9 binding is a specific interaction. We have identified the first receptor for a murine PSG as well as the first natural ligand for a member of the tetraspanin superfamily.
Steady State Relation between Cytoplasmic Free Ca2+ Concentration and Force in Intact Frog Skeletal Muscle Fibers
Authors: Konishi Masato , Watanabe Masaru
Published: July 2002

The steady state relation between cytoplasmic Ca2+ concentration ([Ca2+]i) and force was studied in intact skeletal muscle fibers of frogs. Intact twitch fibers were injected with the dextran-conjugated Ca2+ indicator, fura dextran, and the fluorescence signals of fura dextran were converted to [Ca2+]i using calibration parameters previously estimated in permeabilized muscle fibers (Konishi and Watanabe. 1995. J. Gen. Physiol. 106:1123–1150). In the first series of experiments, [Ca2+]i and isometric force were simultaneously measured during high K+ depolarization. Slow changes in [Ca2+]i and force induced by 15–30 mM K+ appeared to be in equilibrium, as instantaneous [Ca2+]i versus force plot tracked the common path in the rising and relaxation phases of K+ contractures. In the second series of experiments, 2,5-di-tert-butylhydroquinone (TBQ), an inhibitor of the sarcoplasmic reticulum Ca2+ pump, was used to decrease the rate of decline of [Ca2+]i after tetanic stimulation. The decay time courses of both [Ca2+]i and force were dose-dependently slowed by TBQ up to 5 μM; the instantaneous [Ca2+]i– force relations were nearly identical at ≥1 μM TBQ, suggesting that the change in [Ca2+]i was slow enough to reach equilibrium with force. The [Ca2+]i–force data obtained from the two types of experiments were consistent with the Hill curve using a Hill coefficient of 3.2–3.9 and [Ca2+]i for half activation (Ca50) of 1.5–1.7 μM. However, if fura dextran reacts with Ca2+ with a 2.5-fold greater Kd as previously estimated from the kinetic fitting (Konishi and Watanabe. 1995. J. Gen. Physiol. 106:1123–1150), Ca50 would be 3.7–4.2 μM. We also studied the [Ca2+]–force relation in skinned fibers under similar experimental conditions. The average Hill coefficient and Ca50 were estimated to be 3.3 and 1.8 μM, respectively. Although uncertainties remain about the precise levels of [Ca2+]i, we conclude that the steady state force is a 3rd to 4th power function of [Ca2+]i, and Ca50 is in the low micromolar range in intact frog muscle fibers, which is in reasonable agreement with results obtained from skinned fibers.
Single-Channel Properties of IKs Potassium Channels
Authors: Yang Youshan , Sigworth Fred J.
Published: July 2002

Expressed in Xenopus oocytes, KvLQT1 channel subunits yield a small, rapidly activating, voltage- dependent potassium conductance. When coexpressed with the minK gene product, a slowly activating and much larger potassium current results. Using fluctuation analysis and single-channel recordings, we have studied the currents formed by human KvLQT1 subunits alone and in conjunction with human or rat minK subunits. With low external K+, the single-channel conductances of these three channel types are estimated to be 0.7, 4.5, and 6.5 pS, respectively, based on noise analysis at 20 kHz bandwidth of currents at +50 mV. Power spectra computed over the range 0.1 Hz–20 kHz show a weak frequency dependence, consistent with current interruptions occurring on a broad range of time scales. The broad spectrum causes the apparent single-channel current value to depend on the bandwidth of the recording, and is mirrored in very “flickery” single-channel events of the channels from coexpressed KvLQT1 and human minK subunits. The increase in macroscopic current due to the presence of the minK subunit is accounted for by the increased apparent single-channel conductance it confers on the expressed channels. The rat minK subunit also confers the property that the outward single-channel current is increased by external potassium ions.
Analysis of third-order methods for secular equations
Authors: Melman A.
Published: July 2002

Third-order numerical methods are analyzed for secular equations. These equations arise in several matrix problems and numerical linear algebra applications. A closer look at an existing method shows that it can be considered as a classical method for an equivalent problem. This not only leads to other third-order methods, it also provides the means for a unifying convergence analysis of these methods and for their comparisons. Finally, we consider approximated versions of the aforementioned methods.
Almost optimal convergence of the point vortex method for vortex sheets using numerical filtering
Authors: Caflisch Russel , Hou Thomas , Lowengrub John
Published: July 2002

Standard numerical methods for the Birkhoff-Rott equation for a vortex sheet are unstable due to the amplification of roundoff error by the Kelvin-Helmholtz instability. A nonlinear filtering method was used by Krasny to eliminate this spurious growth of round-off error and accurately compute the Birkhoff-Rott solution essentially up to the time it becomes singular. In this paper convergence is proved for the discretized Birkhoff-Rott equation with Krasny filtering and simulated roundoff error. The convergence is proved for a time almost up to the singularity time of the continuous solution. The proof is in an analytic function class and uses a discrete form of the abstract Cauchy-Kowalewski theorem. In order for the proof to work almost up to the singularity time, the linear and nonlinear parts of the equation, as well as the effects of Krasny filtering, are precisely estimated. The technique of proof applies directly to other ill-posed problems such as Rayleigh-Taylor unstable interfaces in incompressible, inviscid, and irrotational fluids, as well as to Saffman-Taylor unstable interfaces in Hele-Shaw cells.
Which are the simplest algebraic varieties?
Authors: Kollár János
Published: July 2002

This paper is a slightly revised version of the notes prepared in connection with the AMS Colloquium Lectures delivered in New Orleans, January 2001.
Automated protein sequence database classification. II. Delineation Of domain boundaries from sequence similarities.
Authors: Gracy J , Argos P
Published: July 2002

MOTIVATION: Decomposing each protein into modular domains is a basic prerequisite to classify accurately structural units in biological molecules. Boundaries between domains are indicated by two similar amino acid sequence segments located within the same protein (repeats) or within homologous proteins at notably different distances from their respective N- or C-termini. RESULTS: We have developed an automated method that combines such positional constraints derived from various detected pairwise sequence similarities to delineate the modular organization of proteins. The procedure has been applied to a non-redundant data set of 26 990 proteins whose sequences were taken from the PIR and SWISS-PROT databanks and shared <60% sequence identity amongst pairs. The resultant clustering, delineation and multiple alignment of 24 380 sequence fragments yielded a new database of 4364 domain families. Comparison of the domain collection with that of PRODOM indicates a clear improvement in the number and size of domain families, domain boundaries and multiple sequence alignments. The accuracy and sensitivity of the method are illustrated by results obtained for ankyrin-like repeats and EGF-like modules. AVAILABILITY: The resulting database, called DOMO, is available through the database search routine SRS at Infobiogen (http://www.infobiogen.fr/srs5/), EBI (http://srs.ebi.ac.uk:5000/) and EMBL (http://www.embl-heidelberg.de/srs5/) World Wide Web sites. CONTACT: gracy@infobiogen.fr
Control of gene expression in intestinal epithelial cells
Authors: Traber Peter G.
Published: July 2002

Coordination of gene transcription is a critical regulatory step in orchestrating developmental, differentiation and adaptation processes in the mammalian intestinal epithelium. Insight into these mechanisms has been gained by the study of transcriptional regulation of the sucrase–isomaltase gene. An understanding of the regulatory network of nuclear proteins that direct transcriptional initiation of intestinal genes such as sucrase–isomaltase will provide insight into the mechanisms of normal development and differentiation as well as disease processes such as neoplasia.
Appropriate targets for monoclonal antibodies in the induction of transplantation tolerance
Authors: Lechler Robert , Chai Jian-Guo , Marelli-Berg Federica , Lombard Giovanna
Published: July 2002

There are many routes to exploiting tolerance processes to ensure long–term graft survival. Complete tolerance although attractive as a goal, may not be the most practical in the clinic. Instead simple and low–impact procedures that harness tolerance processes used in conjunction with low doses of immunosuppressive drugs may prove the most reliable and user–friendly of approaches.
Tiptoeing to chromosome tips: facts, promises and perils of today's human telomere biology
Authors: Fajkus J. , Šimíčková M. , Maláska J.
Published: July 2002

The past decade has witnessed an explosion of knowledge concerning the structure and function of chromosome terminal structures—telomeres. Today's telomere research has advanced from a pure descriptive approach of DNA and protein components to an elementary understanding of telomere metabolism, and now to promising applications in medicine. These applications include ‘passive’ ones, among which the use of analysis of telomeres and telomerase (a cellular reverse transcriptase that synthesizes telomeres) for cancer diagnostics is the best known. The ‘active’ applications involve targeted downregulation or upregulation of telomere synthesis, either to mortalize immortal cancer cells, or to rejuvenate mortal somatic cells and tissues for cellular transplantations, respectively. This article reviews the basic data on structure and function of human telomeres and telomerase, as well as both passive and active applications of human telomere biology.
Domain-Level Differences in Microsatellite Distribution and Content Result from Different Relative Rates of Insertion and Deletion Mutations
Authors: Metzgar David , Liu Li , Hansen Christian , Dybvig Kevin , Wills Christopher
Published: July 2002

Microsatellites (short tandem polynucleotide repeats) are found throughout eukaryotic genomes at frequencies many orders of magnitude higher than the frequencies predicted to occur by chance. Most of these microsatellites appear to have evolved in a generally neutral manner. In contrast, microsatellites are generally absent from bacterial genomes except in locations where they provide adaptive functional variability, and these appear to have evolved under selection. We demonstrate a mutational bias towards deletion (repeat contraction) in a native chromosomal microsatellite of the bacterium Mycoplasma gallisepticum, through the collection and analysis of independent mutations in the absence of natural selection. Using this and similar existing data from two other bacterial species and four eukaryotic species, we find strong evidence that deletion biases resulting in repeat contraction are common in bacteria, while eukaryotic microsatellites generally experience unbiased mutation or a bias towards insertion (repeat expansion). This difference in mutational bias suggests that eukaryotic microsatellites should generally expand wherever selection does not exclude them, whereas bacterial microsatellites should be driven to extinction by mutational pressure wherever they are not maintained by selection. This is consistent with observed bacterial and eukaryotic microsatellite distributions. Hence, mutational biases that differ between eukaryotes and bacteria can account for many of the observed differences in microsatellite DNA content and distribution found in these two groups of organisms.
Side-Specificity of Olfactory Learning in the Honeybee: Generalization between Odors and Sides
Authors: Sandoz Jean-Christophe , Menzel Randolf
Published: July 2002

Honeybees (Apis mellifera) can be trained to associate an odor stimulus with a sucrose reward. The neural structures involved in the detection and integration of olfactory stimuli are represented bilaterally in the brain. Little is known about the respective roles of the two sides of the brain in olfactory learning. Does each side learn independently of the other, or do they communicate, and if so, to what extent and at what level of neural integration? We addressed these questions using the proboscis extension response (PER) conditioning paradigm applied in a preparation that allows the separation of the two input sides during olfactory stimulations. Bees conditioned to two odorants A and B, one being learned on each side (A+/B+ training), showed in extinction tests rather unspecific responses: They responded to both odorants on both sides. This could be attributable to either a transfer of the learned information between sides, or to a generalization between odorants on each side. By subjecting bees to conditioning on one side only (A+/0 training), we found that the learned information is indeed transferred between sides. However, when bees were trained explicitly to give opposite values to the two odorants on the two sides (A+B−/B+A− training), they showed clear side-specific response patterns to these odorants. These results are used in the elaboration of a functional model of laterality of olfactory learning and memory processing in the honeybee brain.