bluuebunny/crossref_metadata_2025 huggingface dataset:
Epitope-Tagged P0Glycoprotein Causes Charcot-Marie-Tooth–Like Neuropathy in Transgenic Mice
Authors: Previtali Stefano C. , Quattrini Angelo , Fasolini Marina , Panzeri Maria Carla , Villa Antonello , Filbin Marie T. , Li Wenhui , Chiu Shing-Yan , Messing Albee , Wrabetz Lawrence , Feltri M. Laura
Published: July 2002

In peripheral nerve myelin, the intraperiod line results from compaction of the extracellular space due to homophilic adhesion between extracellular domains (ECD) of the protein zero (P0) glycoprotein. Point mutations in this region of P0 cause human hereditary demyelinating neuropathies such as Charcot-Marie-Tooth. We describe transgenic mice expressing a full-length P0 modified in the ECD with a myc epitope tag. The presence of the myc sequence caused a dysmyelinating peripheral neuropathy similar to two distinct subtypes of Charcot-Marie-Tooth, with hypomyelination, altered intraperiod lines, and tomacula (thickened myelin). The tagged protein was incorporated into myelin and was associated with the morphological abnormalities. In vivo and in vitro experiments showed that P0myc retained partial adhesive function, and suggested that the transgene inhibits P0-mediated adhesion in a dominant-negative fashion. These mice suggest new mechanisms underlying both the pathogenesis of P0 ECD mutants and the normal interactions of P0 in the myelin sheath.
Downregulation of an Aim-1 Kinase Couples with Megakaryocytic Polyploidization of Human Hematopoietic Cells
Authors: Kawasaki Akira , Matsumura Itaru , Miyagawa Jun-ichiro , Ezoe Sachiko , Tanaka Hirokazu , Terada Yasuhiko , Tatsuka Masaaki , Machii Takashi , Miyazaki Hiroshi , Furukawa Yusuke , Kanakura Yuzuru
Published: July 2002

During the late phase of megakaryopoiesis, megakaryocytes undergo polyploidization, which is characterized by DNA duplication without concomitant cell division. However, it remains unknown by which mechanisms this process occurs. AIM-1 and STK15 belong to the Aurora/increase-in-ploidy (Ipl)1 serine/threonine kinase family and play key roles in mitosis. In a human interleukin-3–dependent cell line, F-36P, the expressions of AIM-1 and STK15 mRNA were specifically observed at G2/M phase of the cell cycle during proliferation. In contrast, the expressions of AIM-1 and STK15 were continuously repressed during megakaryocytic polyploidization of human erythro/megakaryocytic cell lines (F-36P, K562, and CMK) treated with thrombopoietin, activated ras (H-rasG12V), or phorbol ester. Furthermore, their expressions were suppressed during thrombopoietin-induced polyploidization of normal human megakaryocytes. Activation of AIM-1 by the induced expression of AIM-1(wild-type) canceled TPA-induced polyploidization of K562 cells significantly, whereas that of STK15 did not. Moreover, suppression of AIM-1 by the induced expression of AIM-1 (K/R, dominant-negative type) led to polyploidization in 25% of K562 cells, whereas STK15(K/R) showed no effect. Also, the induced expression of AIM-1(K/R) in CMK cells provoked polyploidization up to 32N. These results suggested that downregulation of AIM-1 at M phase may be involved in abortive mitosis and polyploid formation of megakaryocytes.
Tethering and tickling
Authors: Somersan Selin , Bhardwaj Nina
Published: July 2002

Several receptors are implicated in apoptotic cell (AC) uptake by phagocytic cells; however, their relative dominance in mammalian systems remains to be established. New studies shed light on the role of the phosphatidyl serine (PS) receptor (PSR). Ligation of PSR by PS on AC surfaces is considered essential for signaling uptake of ACs that are tethered to phagocytes via other receptors.
The channel of death
Authors: Degterev Alexei , Boyce Michael , Yuan Junying
Published: July 2002

The proapoptotic members of the Bcl-2 family have been proposed to participate in the formation of a channel that releases these apoptogenic factors when mitochondria receive apoptotic signals. A recent study provides the first direct, biophysical measurement of a potentially apoptosis-specific mitochondrial channel, which is regulated by Bcl-2 family members and may play a primary role in the release of the proapoptotic factors.
Antibodies that Inhibit Malaria Merozoite Surface Protein–1 Processing and Erythrocyte Invasion Are Blocked by Naturally Acquired Human Antibodies
Authors: Guevara Patiño José A. , Holder Anthony A. , McBride Jana S. , Blackman Michael J.
Published: July 2002

Merozoite surface protein–1 (MSP-1) of the human malaria parasite Plasmodium falciparum undergoes at least two endoproteolytic cleavage events during merozoite maturation and release, and erythrocyte invasion. We have previously demonstrated that mAbs which inhibit erythrocyte invasion and are specific for epitopes within a membrane-proximal, COOH-terminal domain of MSP-1 (MSP-119) prevent the critical secondary processing step which occurs on the surface of the extracellular merozoite at around the time of erythrocyte invasion. Certain other anti–MSP-119 mAbs, which themselves inhibit neither erythrocyte invasion nor MSP-1 secondary processing, block the processing-inhibitory activity of the first group of antibodies and are termed blocking antibodies. We have now directly quantitated antibody-mediated inhibition of MSP-1 secondary processing and invasion, and the effects on this of blocking antibodies. We show that blocking antibodies function by competing with the binding of processing-inhibitory antibodies to their epitopes on the merozoite. Polyclonal rabbit antibodies specific for certain MSP-1 sequences outside of MSP-119 also act as blocking antibodies. Most significantly, affinity-purified, naturally acquired human antibodies specific for epitopes within the NH2-terminal 83-kD domain of MSP-1 very effectively block the processing-inhibitory activity of the anti-MSP-119 mAb 12.8. The presence of these blocking antibodies also completely abrogates the inhibitory effect of mAb 12.8 on erythrocyte invasion by the parasite in vitro. Blocking antibodies therefore (a) are part of the human response to malarial infection; (b) can be induced by MSP-1 structures unrelated to the MSP-119 target of processing-inhibitory antibodies; and (c) have the potential to abolish protection mediated by anti–MSP-119 antibodies. Our results suggest that an effective MSP-119–based falciparum malaria vaccine should aim to induce an antibody response that prevents MSP-1 processing on the merozoite surface.
Lifespan of γ/δ T Cells
Authors: Tough David F. , Sprent Jonathan
Published: July 2002

Information on the turnover and lifespan of murine γ/δ cells was obtained by administering the DNA precursor, bromodeoxyuridine (BrdU), in the drinking water and staining lymphoid cells for BrdU incorporation. For TCR-γ/δ (Vγ2) transgenic mice, nearly all γ/δ thymocytes became BrdU+ within 2 d and were released rapidly into the peripheral lymphoid tissues. These recent thymic emigrants (RTEs) underwent phenotypic maturation in the periphery for several days, but most of these cells died within 4 wk. In adult thymectomized (ATx) transgenic mice, only a small proportion of γ/δ cells survived as long-lived cells; most of these cells had a slow turnover and retained a naive phenotype. As in transgenic mice, the majority of RTEs generated in normal mice (C57BL/6) appeared to have a restricted lifespan as naive cells. However, in marked contrast to TCR transgenic mice, most of the γ/δ cells surviving in ATx normal mice had a rapid turnover and displayed an activated/memory phenotype, implying a chronic response to environmental antigens. Hence, in normal mice many γ/δ RTEs did not die but switched to memory cells.
Identification of C-C Chemokine Receptor 1 (CCR1) as the Monocyte Hemofiltrate C-C Chemokine (HCC)-1 Receptor
Authors: Tsou Chia-Lin , Gladue Ron P. , Carroll Laurie A. , Paradis Tim , Boyd James G. , Nelson Robin T. , Neote Kuldeep , Charo Israel F.
Published: July 2002

Hemofiltrate C-C chemokine (HCC)-1 is a recently cloned C-C chemokine that is structurally similar to macrophage inflammatory protein (MIP)-1α. Unlike most chemokines, it is constitutively secreted by tissues and is present at high concentrations in normal human plasma. Also atypical for chemokines, HCC-1 is reported not to be chemotactic for leukocytes. In this paper, we have investigated the chemokine receptor usage and downstream signaling pathways of HCC-1. Cross-desensitization experiments using THP-1 cells suggested that HCC-1 and MIP-1α activated the same receptor. Experiments using a panel of cloned chemokine receptors revealed that HCC-1 specifically activated C-C chemokine receptor (CCR)1, but not closely related receptors, including CCR5. HCC-1 competed with MIP-1α for binding to CCR1-transfected cells, but with a markedly reduced affinity (IC50 = 93 nM versus 1.3 nM for MIP-1α). Similarly, HCC-1 was less potent than MIP-1α in inducing inhibition of adenylyl cyclase in CCR1-transfected cells. HCC-1 induced chemotaxis of freshly isolated human monocytes, THP-1 cells, and CCR1-transfected cells, and the optimal concentration for cell migration (100 nM) was ∼100-fold lower than that of MIP-1α (1 nM). These data demonstrate that HCC-1 is a chemoattractant and identify CCR1 as a functional HCC-1 receptor on human monocytes.
Characterization of a Hemoglobin Protease Secreted by the Pathogenic Escherichia coli Strain EB1
Authors: Otto Ben R. , van Dooren Silvy J.M. , Nuijens Jan H. , Luirink Joen , Oudega Bauke
Published: July 2002

Many pathogenic bacteria can use heme compounds as a source of iron. Pathogenic Escherichia coli strains are capable of using hemoglobin as an iron source. However, the mechanism of heme acquisition from hemoglobin is not understood for this microorganism. We present the first molecular characterization of a hemoglobin protease (Hbp) from a human pathogenic E. coli strain. The enzyme also appeared to be a heme-binding protein. Affinity purification of this bifunctional protein enabled us to identify the extracellular gene product, and to clone and analyze its gene. A purification procedure developed for Hbp allowed us to perform functional studies. The protein interacted with hemoglobin, degraded it and subsequently bound the released heme. These results suggest that the protein is involved in heme acquisition by this human pathogen. Hbp belongs to the so-called IgA1 protease-like proteins, as indicated by the kinetics of its membrane transfer and DNA sequence similarity. The gene of this protein appears to be located on the large pColV-K30 episome, that only has been isolated from human and animal pathogens. All these characteristics indicate that Hbp may be an important virulence factor that may play a significant role in the pathogenesis of E. coli infections.
Melanoma Cells Present a MAGE-3 Epitope to CD4+ Cytotoxic T Cells in Association with Histocompatibility Leukocyte Antigen DR11
Authors: Manici Simona , Sturniolo Tiziana , Imro Maria Adele , Hammer Juergen , Sinigaglia Francesco , Noppen Christoph , Spagnoli Giulio , Mazzi Benedetta , Bellone Matteo , Dellabona Paolo , Protti Maria Pia
Published: July 2002

In this study we used TEPITOPE, a new epitope prediction software, to identify sequence segments on the MAGE-3 protein with promiscuous binding to histocompatibility leukocyte antigen (HLA)-DR molecules. Synthetic peptides corresponding to the identified sequences were synthesized and used to propagate CD4+ T cells from the blood of a healthy donor. CD4+ T cells strongly recognized MAGE-3281–295 and, to a lesser extent, MAGE-3141–155 and MAGE-3146–160. Moreover, CD4+ T cells proliferated in the presence of recombinant MAGE-3 after processing and presentation by autologous antigen presenting cells, demonstrating that the MAGE-3 epitopes recognized are naturally processed. CD4+ T cells, mostly of the T helper 1 type, showed specific lytic activity against HLA-DR11/MAGE-3–positive melanoma cells. Cold target inhibition experiments demonstrated indeed that the CD4+ T cells recognized MAGE-3281–295 in association with HLA-DR11 on melanoma cells. This is the first evidence that a tumor-specific shared antigen forms CD4+ T cell epitopes. Furthermore, we validated the use of algorithms for the prediction of promiscuous CD4+ T cell epitopes, thus opening the possibility of wide application to other tumor-associated antigens. These results have direct implications for cancer immunotherapy in the design of peptide-based vaccines with tumor-specific CD4+ T cell epitopes.
The Absence of Interleukin 1 Receptor–Related T1/St2 Does Not Affect T Helper Cell Type 2 Development and Its Effector Function
Authors: Hoshino Katsuaki , Kashiwamura Shin-ichiro , Kuribayashi Kozo , Kodama Taku , Tsujimura Tohru , Nakanishi Kenji , Matsuyama Tomohiro , Takeda Kiyoshi , Akira Shizuo
Published: July 2002

T1/ST2, an orphan receptor with homology with the interleukin (IL)-1 receptor family, is expressed constitutively and stably on the surface of T helper type 2 (Th2) cells, but not on Th1 cells. T1/ST2 is also expressed on mast cells, which are critical for Th2-mediated effector responses. To evaluate whether T1/ST2 is required for Th2 responses and mast cell function, we have generated T1/ST2-deficient (T1/ST2−/−) mice and examined the roles of T1/ST2. Naive CD4+ T cells isolated from T1/ST2−/− mice developed to Th2 cells in response to IL-4 in vitro. T1/ST2−/− mice showed normal Th2 responses after infection with the helminthic parasite Nippostrongylus brasiliensis as well as in the mouse model of allergen-induced airway inflammation. In addition, differentiation and function of bone marrow–derived cultured mast cells were unaffected. These findings demonstrate that T1/ST2 does not play an essential role in development and function of Th2 cells and mast cells.
Leukocyte Recruitment in the Cerebrospinal Fluid of Mice with Experimental Meningitis Is Inhibited by an Antibody to Junctional Adhesion Molecule (Jam)
Authors: Del Maschio Aldo , De Luigi Ada , Martin-Padura Ines , Brockhaus Manfred , Bartfai Tamas , Fruscella Paolo , Adorini Luciano , Martino GianVito , Furlan Roberto , De Simoni Maria Grazia , Dejana Elisabetta
Published: July 2002

The mechanisms that govern leukocyte transmigration through the endothelium are not yet fully defined. Junctional adhesion molecule (JAM) is a newly cloned member of the immunoglobulin superfamily which is selectively concentrated at tight junctions of endothelial and epithelial cells. A blocking monoclonal antibody (BV11 mAb) directed to JAM was able to inhibit monocyte transmigration through endothelial cells in in vitro and in vivo chemotaxis assays. In this study, we report that BV11 administration was able to attenuate cytokine-induced meningitis in mice. The intravenous injection of BV11 mAb significantly inhibited leukocyte accumulation in the cerebrospinal fluid and infiltration in the brain parenchyma. Blood–brain barrier permeability was also reduced by the mAb. We conclude that JAM may be a new target in limiting the inflammatory response that accompanies meningitis.
Inducible Costimulator Protein (Icos) Controls T Helper Cell Subset Polarization after Virus and Parasite Infection
Authors: Kopf Manfred , Coyle Anthony J. , Schmitz Nicole , Barner Marijke , Oxenius Annette , Gallimore Awen , Gutierrez-Ramos Jose-Carlos , Bachmann Martin F.
Published: July 2002

It has been shown that certain pathogens can trigger efficient T cell responses in the absence of CD28, a key costimulatory receptor expressed on resting T cells. Inducible costimulator protein (ICOS) is an inducible costimulator structurally and functionally related to CD28. Here, we show that in the absence of CD28 both T helper cell type 1 (Th1) and Th2 responses were impaired but not abrogated after infection with lymphocytic choriomeningitis virus (LCMV), vesicular stomatitis virus (VSV), and the nematode Nippostrongylus brasiliensis. Inhibition of ICOS in CD28-deficient mice further reduced Th1/Th2 polarization. Blocking of ICOS alone had a limited but significant capacity to downregulate Th subset development. In contrast, cytotoxic T lymphocyte (CTL) responses, which are regulated to a minor and major extent by CD28 after LCMV and VSV infection, respectively, remained unaffected by blocking ICOS. Together, our results demonstrate that ICOS regulates both CD28-dependent and CD28-independent CD4+ subset (Th1 and Th2) responses but not CTL responses in vivo.
Cc Chemokine Receptor (Ccr)2 Is Required for Langerhans Cell Migration and Localization of T Helper Cell Type 1 (Th1)-Inducing Dendritic Cells
Authors: Sato Naoko , Ahuja Sunil K. , Quinones Marlon , Kostecki Vannessa , Reddick Robert L. , Melby Peter C. , Kuziel William A. , Ahuja Seema S.
Published: July 2002

There is growing evidence that chemokines and their receptors regulate the movement and interaction of antigen-presenting cells such as dendritic cells (DCs) and T cells. We tested the hypothesis that the CC chemokine receptor (CCR)2 and CCR5 and the chemokine macrophage inflammatory protein (MIP)-1α, a ligand for CCR5, influence DC migration and localization. We found that deficiency of CCR2 but not CCR5 or MIP-1α led to distinct defects in DC biology. Langerhans cell (skin DC) density in CCR2-null mice was normal, and their ability to migrate into the dermis was intact; however, their migration to the draining lymph nodes was markedly impaired. CCR2-null mice had lower numbers of DCs in the spleen, and this was primarily due to a reduction in the CD8α1 T helper cell type 1 (Th1)-inducing subset of DCs. Additionally, there was a block in the Leishmania major infection–induced relocalization of splenic DCs from the marginal zone to the T cell areas. We propose that these DC defects, in conjunction with increased expression of B lymphocyte chemoattractant, a B cell–specific chemokine, may collectively contribute to the striking B cell outgrowth and Th2 cytokine–biased nonhealing phenotype that we observed in CCR2-deficient mice infected with L. major. This disease phenotype in mice with an L. major–resistant genetic background but lacking CCR2 is strikingly reminiscent of that observed typically in mice with an L. major–susceptible genetic background. Thus, CCR2 is an important determinant of not only DC migration and localization but also the development of protective cell-mediated immune responses to L. major.
Antibodies against Merozoite Surface Protein (Msp)-119 Are a Major Component of the Invasion-Inhibitory Response in Individuals Immune to Malaria
Authors: O'Donnell Rebecca A. , de Koning-Ward Tania F. , Burt Rachel A. , Bockarie Moses , Reeder John C. , Cowman Alan F. , Crabb Brendan S.
Published: July 2002

Antibodies that bind to antigens expressed on the merozoite form of the malaria parasite can inhibit parasite growth by preventing merozoite invasion of red blood cells. Inhibitory antibodies are found in the sera of malaria-immune individuals, however, the specificity of those that are important to this process is not known. In this paper, we have used allelic replacement to construct a Plasmodium falciparum parasite line that expresses the complete COOH-terminal fragment of merozoite surface protein (MSP)-119 from the divergent rodent malaria P. chabaudi. By comparing this transfected line with parental parasites that differ only in MSP-119, we show that antibodies specific for this domain are a major component of the inhibitory response in P. falciparum–immune humans and P. chabaudi–immune mice. In some individual human sera, MSP-119 antibodies dominated the inhibitory activity. The finding that antibodies to a small region of a single protein play a major role in this process has important implications for malaria immunity and is strongly supportive of further understanding and development of MSP-119–based vaccines.
Swift Development of Protective Effector Functions in Naive Cd8+ T Cells against Malaria Liver Stages
Authors: Sano Gen-ichiro , Hafalla Julius C.R. , Morrot Alexandre , Abe Ryo , Lafaille Juan J. , Zavala Fidel
Published: July 2002

We generated T cell receptor transgenic mice specific for the liver stages of the rodent malaria parasite Plasmodium yoelii and studied the early events in the development of in vivo effector functions in antigen-specific CD8+ T cells. Differently to activated/memory cells, naive CD8+ T cells are not capable of exerting antiparasitic activity unless previously primed by parasite immunization. While naive cells need to differentiate before achieving effector status, the time required for this process is very short. Indeed, interferon (IFN)-γ and perforin mRNA are detectable 24 h after immunization and IFN-γ secretion and cytotoxic activity are detected ex vivo 24 and 48 h after immunization, respectively. In contrast, the proliferation of CD8+ T cells begins after 24 h and an increase in the total number of antigen-specific cells is detected only after 48 h. Remarkably, a strong CD8+ T cell–mediated inhibition of parasite development is observed in mice challenged with viable parasites only 24 h after immunization with attenuated parasites. These results indicate that differentiation of naive CD8+ T cells does not begin only after extensive cell division, rather this process precedes or occurs simultaneously with proliferation.
The Role of Recombination Activating Gene (RAG) Reinduction in Thymocyte Development in Vivo
Authors: Yannoutsos Nikos , Wilson Patrick , Yu Wong , Chen Hua Tang , Nussenzweig Andre , Petrie Howard , Nussenzweig Michel C.
Published: July 2002

Assembly of T cell receptor (TCR)α/β genes by variable/diversity/joining (V[D]J) rearrangement is an ordered process beginning with recombination activating gene (RAG) expression and TCRβ recombination in CD4−CD8−CD25+ thymocytes. In these cells, TCRβ expression leads to clonal expansion, RAG downregulation, and TCRβ allelic exclusion. At the subsequent CD4+CD8+ stage, RAG expression is reinduced and V(D)J recombination is initiated at the TCRα locus. This second wave of RAG expression is terminated upon expression of a positively selected α/β TCR. To examine the physiologic role of the second wave of RAG expression, we analyzed mice that cannot reinduce RAG expression in CD4+CD8+ T cells because the transgenic locus that directs RAG1 and RAG2 expression in these mice is missing a distal regulatory element essential for reinduction. In the absence of RAG reinduction we find normal numbers of CD4+CD8+ cells but a 50–70% reduction in the number of mature CD4+CD8− and CD4−CD8+ thymocytes. TCRα rearrangement is restricted to the 5′ end of the Jα cluster and there is little apparent secondary TCRα recombination. Comparison of the TCRα genes expressed in wild-type or mutant mice shows that 65% of all α/β T cells carry receptors that are normally assembled by secondary TCRα rearrangement. We conclude that RAG reinduction in CD4+CD8+ thymocytes is not required for initial TCRα recombination but is essential for secondary TCRα recombination and that the majority of TCRα chains expressed in mature T cells are products of secondary recombination.
Reciprocal Activating Interaction between Natural Killer Cells and Dendritic Cells
Authors: Gerosa Franca , Baldani-Guerra Barbara , Nisii Carla , Marchesini Viviana , Carra Giuseppe , Trinchieri Giorgio
Published: July 2002

We analyzed the interaction between human peripheral blood natural killer (NK) cells and monocyte-derived immature dendritic cells (DC). Fresh NK cells were activated, as indicated by the induced expression of the CD69 antigen, and their cytolytic activity was strongly augmented by contact with lipopolysaccharide (LPS)-treated mature DC, or with immature DC in the presence of the maturation stimuli LPS, Mycobacterium tuberculosis or interferon (IFN)-α. Reciprocally, fresh NK cells cultured with immature DC in the presence of the maturation stimuli strongly enhanced DC maturation and interleukin (IL)-12 production. IL-2–activated NK cells directly induced maturation of DC and enhanced their ability to stimulate allogeneic naive CD4+ T cells. The effects of NK cells were cell contact dependent, although the secretion of IFN-γ and TNF also contributed to DC maturation. Within peripheral blood lymphocytes the reciprocal activating interaction with DC was restricted to NK cells, because the other lymphocyte subsets were neither induced to express CD69, nor induced to mature in contact with DC. These data demonstrated for the first time a bidirectional cross talk between NK cells and DC, in which NK cells activated by IL-2 or by mature DC induce DC maturation.
Human Dendritic Cells Activate Resting Natural Killer (NK) Cells and Are Recognized via the NKp30 Receptor by Activated NK Cells
Authors: Ferlazzo Guido , Tsang Ming L. , Moretta Lorenzo , Melioli Giovanni , Steinman Ralph M. , Münz Christian
Published: July 2002

During the innate response to many inflammatory and infectious stimuli, dendritic cells (DCs) undergo a differentiation process termed maturation. Mature DCs activate antigen-specific naive T cells. Here we show that both immature and mature DCs activate resting human natural killer (NK) cells. Within 1 wk the NK cells increase two– to fourfold in numbers, start secreting interferon (IFN)-γ, and acquire cytolytic activity against the classical NK target LCL721.221. The DC-activated NK cells then kill immature DCs efficiently, even though the latter express substantial levels of major histocompatibility complex (MHC) class I. Similar results are seen with interleukin (IL)-2–activated NK cell lines and clones, i.e., these NK cells kill and secrete IFN-γ in response to immature DCs. Mature DCs are protected from activated NK lysis, but lysis takes place if the NK inhibitory signal is blocked by a human histocompatibility leukocyte antigen (HLA)-A,B,C–specific antibody. The NK activating signal mainly involves the NKp30 natural cytotoxicity receptor, and not the NKp46 or NKp44 receptor. However, both immature and mature DCs seem to use a NKp30 independent mechanism to act as potent stimulators for resting NK cells. We suggest that DCs are able to control directly the expansion of NK cells and that the lysis of immature DCs can regulate the afferent limb of innate and adaptive immunity.
Vav1 Is a Component of Transcriptionally Active Complexes
Authors: Houlard Martin , Arudchandran Ramachandran , Regnier-Ricard Fabienne , Germani Antonia , Gisselbrecht Sylvie , Blank Ulrich , Rivera Juan , Varin-Blank Nadine
Published: July 2002

The importance of the hematopoietic protooncogene Vav1 in immune cell function is widely recognized, although its regulatory mechanisms are not completely understood. Here, we examined whether Vav1 has a nuclear function, as past studies have reported its nuclear localization. Our findings provide a definitive demonstration of Vav1 nuclear localization in a receptor stimulation–dependent manner and reveal a critical role for the COOH-terminal Src homology 3 (SH3) domain and a nuclear localization sequence within the pleckstrin homology domain. Analysis of DNA-bound transcription factor complexes revealed nuclear Vav1 as an integral component of transcriptionally active nuclear factor of activated T cells (NFAT)- and nuclear factor (NF)κB-like complexes, and the COOH-terminal SH3 domain as being critical in their formation. Thus, we describe a novel nuclear role for Vav1 as a component and facilitator of NFAT and NFκB-like transcriptional activity.
α-Helical Structural Elements within the Voltage-Sensing Domains of a K+ Channel
Authors: Li-Smerin Yingying , Hackos David H. , Swartz Kenton J.
Published: July 2002

Voltage-gated K+ channels are tetramers with each subunit containing six (S1–S6) putative membrane spanning segments. The fifth through sixth transmembrane segments (S5–S6) from each of four subunits assemble to form a central pore domain. A growing body of evidence suggests that the first four segments (S1–S4) comprise a domain-like voltage-sensing structure. While the topology of this region is reasonably well defined, the secondary and tertiary structures of these transmembrane segments are not. To explore the secondary structure of the voltage-sensing domains, we used alanine-scanning mutagenesis through the region encompassing the first four transmembrane segments in the drk1 voltage-gated K+ channel. We examined the mutation-induced perturbation in gating free energy for periodicity characteristic of α-helices. Our results are consistent with at least portions of S1, S2, S3, and S4 adopting α-helical secondary structure. In addition, both the S1–S2 and S3–S4 linkers exhibited substantial helical character. The distribution of gating perturbations for S1 and S2 suggest that these two helices interact primarily with two environments. In contrast, the distribution of perturbations for S3 and S4 were more complex, suggesting that the latter two helices make more extensive protein contacts, possibly interfacing directly with the shell of the pore domain.
Properties of Hyperpolarization-Activated Pacemaker Current Defined by Coassembly of Hcn1 and Hcn2 Subunits and Basal Modulation by Cyclic Nucleotide
Authors: Chen Shan , Wang Jing , Siegelbaum Steven A.
Published: July 2002

Members of the HCN channel family generate hyperpolarization-activated cation currents (Ih) that are directly regulated by cAMP and contribute to pacemaker activity in heart and brain. The four HCN isoforms show distinct but overlapping patterns of expression in different tissues. Here, we report that HCN1 and HCN2, isoforms coexpressed in neocortex and hippocampus that differ markedly in their biophysical properties, coassemble to generate heteromultimeric channels with novel properties. When expressed in Xenopus oocytes, HCN1 channels activate 5–10-fold more rapidly than HCN2 channels. HCN1 channels also activate at voltages that are 10–20 mV more positive than those required to activate HCN2. In cell-free patches, the steady-state activation curve of HCN1 channels shows a minimal shift in response to cAMP (+4 mV), whereas that of HCN2 channels shows a pronounced shift (+17 mV). Coexpression of HCN1 and HCN2 yields Ih currents that activate with kinetics and a voltage dependence that tend to be intermediate between those of HCN1 and HCN2 homomers, although the coexpressed channels do show a relatively large shift by cAMP (+14 mV). Neither the kinetics, steady-state voltage dependence, nor cAMP dose–response curve for the coexpressed Ih can be reproduced by the linear sum of independent populations of HCN1 and HCN2 homomers. These results are most simply explained by the formation of heteromeric channels with novel properties. The properties of these heteromeric channels closely resemble the properties of Ih in hippocampal CA1 pyramidal neurons, cells that coexpress HCN1 and HCN2. Finally, differences in Ih channel properties recorded in cell-free patches versus intact oocytes are shown to be due, in part, to modulation of Ih by basal levels of cAMP in intact cells.
S4 Charges Move Close to Residues in the Pore Domain during Activation in a K Channel
Authors: Elinder Fredrik , Männikkö Roope , Larsson H. Peter
Published: July 2002

Voltage-gated ion channels respond to changes in the transmembrane voltage by opening or closing their ion conducting pore. The positively charged fourth transmembrane segment (S4) has been identified as the main voltage sensor, but the mechanisms of coupling between the voltage sensor and the gates are still unknown. Obtaining information about the location and the exact motion of S4 is an important step toward an understanding of these coupling mechanisms. In previous studies we have shown that the extracellular end of S4 is located close to segment 5 (S5). The purpose of the present study is to estimate the location of S4 charges in both resting and activated states. We measured the modification rates by differently charged methanethiosulfonate regents of two residues in the extracellular end of S5 in the Shaker K channel (418C and 419C). When S4 moves to its activated state, the modification rate by the negatively charged sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES−) increases significantly more than the modification rate by the positively charged [2-(trimethylammonium)ethyl] methanethiosulfonate, bromide (MTSET+). This indicates that the positive S4 charges are moving close to 418C and 419C in S5 during activation. Neutralization of the most external charge of S4 (R362), shows that R362 in its activated state electrostatically affects the environment at 418C by 19 mV. In contrast, R362 in its resting state has no effect on 418C. This suggests that, during activation of the channel, R362 moves from a position far away (>20 Å) to a position close (8 Å) to 418C. Despite its close approach to E418, a residue shown to be important in slow inactivation, R362 has no effect on slow inactivation or the recovery from slow inactivation. This refutes previous models for slow inactivation with an electrostatic S4-to-gate coupling. Instead, we propose a model with an allosteric mechanism for the S4-to-gate coupling.
Bivariate version of the Hahn-Sonine theorem
Authors: Lee Jeongkeun
Published: July 2002

We consider orthogonal polynomials in two variables whose derivatives with respect to

x
x

are orthogonal. We show that they satisfy a system of partial differential equations of the form
<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" alttext="alpha left-parenthesis x comma y right-parenthesis partial-differential Subscript x Superscript 2 Baseline ModifyingAbove upper U With right-arrow Subscript n Baseline plus beta left-parenthesis x comma y right-parenthesis partial-differential Subscript x Baseline ModifyingAbove upper U With right-arrow Subscript n Baseline equals normal upper Lamda Subscript n Baseline ModifyingAbove upper U With right-arrow Subscript n Baseline comma">

α
(
x
,
y
)

∂

x

2

U
→

n

+
β
(
x
,
y
)

∂

x

U
→

n

=

Λ

n

U
→

n

,

\begin{equation*} \alpha (x,y)\partial _{x}^{2}\overrightarrow {U}\!_{n}+\beta (x,y)\partial _{x} \overrightarrow {U}\!_{n}=\Lambda _{n}\overrightarrow {U}\!_{n}, \end{equation*}

where

deg
⁡
α
≤
2

\deg \alpha \leq 2

,

deg
⁡
β
≤
1

\deg \beta \leq 1

,
<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" alttext="ModifyingAbove upper U With right-arrow Subscript n Baseline equals left-parenthesis upper U Subscript n Baseline 0 Baseline comma upper U Subscript n minus 1 comma 1 Baseline comma midline-horizontal-ellipsis comma upper U Subscript 0 n Baseline right-parenthesis">

U
→

n

=
(

U

n
0

,

U

n
−
1
,
1

,
⋯
,

U

0
n

)

\overrightarrow {U} _{n}=(U_{n0},U_{n-1,1},\cdots ,U_{0n})

is a vector of polynomials in

x
x

and

y
y

for

n
≥
0

n\geq 0

, and

Λ

n

\Lambda _{n}

is an eigenvalue matrix of order

(
n
+
1
)
×
(
n
+
1
)

(n+1)\times (n+1)

for

n
≥
0

n\geq 0

. Also we obtain several characterizations for these polynomials. Finally, we point out that our results are able to cover more examples than Bertran’s.
Continuity of the complex Monge-Ampère operator
Authors: Xing Yang
Published: July 2002

The complex Monge-Ampère operator

(
d

d
c

)
n

(dd^c)^n

is an important tool in complex analysis. It would be interesting to find the right notion of convergence

u
j

→
u

u_j\to u

such that

(
d

d
c

u
j

)
n

→
(
d

d
c

u

)
n

(dd^cu_j)^n\to (dd^cu)^n

in the weak topology. In this paper, using the

C

n
−
1

C_{n-1}

-capacity, we give a sufficient condition of the weak convergence

(
d

d
c

u
j

)
n

→
(
d

d
c

u

)
n

(dd^cu_j)^n\to (dd^cu)^n

. We also show that our condition is quite sharp in some case.
Harmonic analysis with respect to heat kernel measure
Authors: Hall Brian
Published: July 2002

This paper surveys developments over the last decade in harmonic analysis on Lie groups relative to a heat kernel measure. These include analogs of the Hermite expansion, the Segal-Bargmann transform, and the Taylor expansion. Some of the results can be understood from the standpoint of geometric quantization. Others are intimately related to stochastic analysis.
Trace alignment and some of its applications.
Authors: Mott R
Published: July 2002

MOTIVATION: Extra useful information can be extracted from a DNA chromatogram trace, over that contained in the base-called DNA sequence. Many sequencing applications can benefit from examination of these traces. RESULTS: An algorithm, based on dynamic programming, for aligning a DNA chromatogram to a DNA sequence is described and implemented. Its applications to vector clipping, EST alignment and mutation detection are discussed.
DeFries–Fulker multiple regression analysis of sibship
QTL data: a SAS®macro
Authors: Lessem J. L. , Cherny S. S.
Published: July 2002

Summary: A SAS\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(^{\mathrm{btc3811x.gif\ r{\circ}}}\) \end{document}macro package for performing multipoint QTL mapping using the DeFries–Fulker multiple regression method is presented.

Availability: The package is made available at http://qms2.sourceforge.net/.

Contact: Jeff.Lessem@Colorado.EDU

* To whom correspondence should be addressed.
DIVERGE: phylogeny-based analysis for
functional–structural divergence of a protein family
Authors: Gu Xun , Vander Velden Kent
Published: July 2002

Summary: DetectIng Variability in Evolutionary Rates among GEnes (DIVERGE) is a software system to study functional divergence of a protein family by detecting site-specific change in evolutionary rate using a multiple alignment of amino acid sequences for a given phylogenetic tree. The program first conducts a statistical test for site-specific rate shifts along the tree, and predicting candidate amino acid residues responsible for functional divergence based on posterior analysis. These results can then be mapped on the 3D protein structure if available.

Availability: DIVERGE is available free of charge from http://xgu1.zool.iastate.edu/. Distribution packages for both Linux and Microsoft Windows operating systems are available, including manual and example files.

Contact: xgu@iastate.edu; kent@iastate.edu

* To whom correspondence should be addressed.
HIV‐1 Tat protein down‐regulates CREB transcription factor expression in PC12 neuronal cells through a phosphatidylinositol 3‐kinase/AKT/cyclic nucleoside phosphodiesterase pathway
Authors: ZAULI GIORGIO , MILANI DANIELA , MIRANDOLA PRISCO , MAZZONI MERI , SECCHIERO PAOLA , MISCIA SEBASTIANO , CAPITANI SILVANO
Published: July 2002

The addition of low concentrations (0.1–1 nM) of extracellular HIV‐1 Tat protein to PC12 neuronal cells stimulated a rapid (peak at 5 min) elevation of the cAMP intracellular levels, which in turn induced the phosphorylation of CREB transcription factor (peak at 15 min) on serine‐133 (Ser‐133). On the contrary, at later time points (60–120 min) Tat induced a significant decline of intracellular cAMP with respect to the basal levels observed in control cells treated with bovine serum albumin. In blocking experiments performed with pharmacological inhibitors, Tat decreased the intracellular levels of cAMP and CREB Ser‐133 phosphorylation through a signal transduction pathway involving the sequential activation of phosphatidylinositol 3‐kinase, AKT, and cyclic nucleoside phosphodiesterases. Moreover, in transient transfection experiments, Tat inhibited transcription of CREB promoter in a manner strictly dependent on the presence of the cAMP‐responsive elements (CRE) in the CREB promoter. Consistently, the expression of endogenous CREB protein was significantly reduced in PC12 cells by prolonged (24–48 h) treatment with Tat. This decline in the expression of CREB, which plays an essential role in the survival and function of neuronal cells, anticipated a progressive increase of apoptosis in Tat‐treated cells. Although obtained in a neuronal cell line, our findings might help to explain some aspects of the pathogenesis of HIV‐1‐associated dementia.—Zauli, G., Milani, D., Mirandola, P., Mazzoni, M., Secchiero, P., Miscia, S., Capitani, S. HIV‐1 Tat protein downregulates CREB transcription factor expression in PC12 neuronal cells through a phosphatidylinositol 3‐kinase/AKT/cyclic nucleoside phosphodiesterase pathway. FASEB J. 15, 483‐491 (2001)
Bacterial population genetics, evolution and epidemiology
Authors: Spratt Brian G. , Maiden Martin C. J.
Published: July 2002

Asexual bacterial populations inevitably consist of an assemblage of distinct clonal lineages. However, bacterial populations are not entirely asexual since recombinational exchanges occur, mobilizing small genome segments among lineages and species. The relative contribution of recombination, as opposed tode novomutation, in the generation of new bacterial genotypes varies among bacterial populations and, as this contribution increases, the clonality of a given population decreases. In consequence, a spectrum of possible population structures exists, with few bacterial species occupying the extremes of highly clonal and completely non–clonal, most containing both clonal and non–clonal elements. The analysis of collections of bacterial isolates, which accurately represent the natural population, by nucleotide sequence determination of multiple housekeeping loci provides data that can be used both to investigate the population structure of bacterial pathogens and for the molecular characterization of bacterial isolates. Understanding the population structure of a given pathogen is important since it impacts on the questions that can be addressed by, and the methods and samples required for, effective molecular epidemiological studies.
Computational analysis of functional connectivity between areas of primate cerebral cortex
Authors: Stephan Klaas E. , Hilgetag Claus–C. , Burns Gully A. P. C. , O'Neill Marc A. , Young Malcolm P. , Kotter Rolf
Published: July 2002

Recent analyses of association fibre networks in the primate cerebral cortex have revealed a small number of densely intra–connected and hierarchically organized structural systems. Corresponding analyses of data on functional connectivity are required to establish the significance of these structural systems. W e therefore built up a relational database by systematically collating published data on the spread of activity after strychnine–induced disinhibition in the macaque cerebral cortex in vivo . After mapping these data to two different parcellation schemes, we used three independent methods of analysis which demonstrate that the cortical network of functional interactions is not homogeneous, but shows a clear segregation into functional assemblies of mutually interacting areas. The assemblies suggest a principal division of the cortex into visual, somatomotor and orbito–temporo–insular systems, while motor and somatosensory areas are inseparably interrelated. These results are largely compatible with corresponding analyses of structural data of mammalian cerebral cortex, and deliver the first functional evidence for ‘small–world’ architecture of primate cerebral cortex.
In vivoandin vitrostudies of immunoglobulin gene somatic hypermutation
Authors: Sale Julian E. , Bemark Mats , Williams Gareth T. , Jolly Christopher J. , Ehrenstein Michael R. , Rada Cristina , Milstein César , Neuberger Michael S.
Published: July 2002

Following antigen encounter, two distinct processes modify immunoglobulin genes. The variable region is diversified by somatic hypermutation while the constant region may be changed by class–switch recombination. Although both genetic events can occur concurrently within germinal centre B cells, there are examples of each occurring independently of the other. Here we compare the contributions of class–switch recombination and somatic hypermutation to the diversification of the serum immunoglobulin repertoire and review evidence that suggests that, despite clear differences, the two processes may share some aspects of their mechanism in common.
Why we see things the way we do: evidence for a wholly empirical strategy of vision
Authors: Purves Dale , Beau Lotto R. , Mark Williams S. , Nundy Surajit , Yang Zhiyong
Published: July 2002

Many otherwise puzzling aspects of the way we see brightness, colour, orientation and motion can be understood in wholly empirical terms. The evidence reviewed here leads to the conclusion that visual percepts are based on patterns of reflex neural activity shaped entirely by the past success (or failure) of visually guided behaviour in response to the same or a similar retinal stimulus. As a result, the images we see accord with what the sources of the stimuli have typically turned out to be, rather than with the physical properties of the relevant objects. If vision does indeed depend upon this operational strategy to generate optimally useful perceptions of inevitably ambiguous stimuli, then the underlying neurobiological processes will eventually need to be understood within this conceptual framework.
Evaluation of the energetic contribution of an ionic network to beta‐sheet stability
Authors: Lassila Kirsten S. , Datta Deepshikha , Mayo Stephen L.
Published: July 2002

We have evaluated the interaction energy of a three‐residue ionic network constructed on the β‐sheet surface of protein G using double mutant cycles. Although the two individual ion pairs were each stabilizing by ∼0.6 kcal/mol, the excess gain in stability for the triad was small (0.06 kcal/mol).
Decision theoretic steering and genetic algorithm optimization: Application to stereotactic radiosurgery treatment planning
Authors: Yu Yan , Schell M. C. , Zhang JB. Y.
Published: July 2002

Treatment planning for stereotactic radiosurgery and fractionated radiotherapy is currently a labor intensive, operator‐dependent process. Many degrees of freedom exist to make rigorous optimization intractable except by computationally intelligent techniques. The quality of a given plan is determined by an aggregate of clinical objectives, most of which are subject to competing tradeoffs. In this work, we present an autonomous scheme that couples decision theoretic guidance with a genetic algorithm for optimization. Ordinal ranking among a population of viable treatment plans is based on a generalized distance metric, which promotes a decreasing hyperfrontier of the efficient solution set. The solution set is driven toward efficiency by the genetic algorithm, which uses the tournament selection mechanism based on the ordinal ranking. Goals and satisficing conditions can be defined to signal the ultimate and the minimum achievement levels in a given objective. A conventionally challenging case in radiosurgery was used to demonstrate the practical utility and the problem‐solving power of the decision theoretic genetic algorithm. Treatment plans with one isocenter and four isocenters were derived under the autonomous scheme and compared to the actual treatment plan manually optimized by the expert planner. Quality assessment based on dose–volume histograms and normal tissue complication probabilities suggested that computational optimization could be driven to offer varying degrees of dosimetric improvement over a human‐guided optimization effort. Furthermore, it was possible to achieve a high degree of isodose conformity to the target volume in computational optimization by increasing the degree of freedom in the treatment parameters. The time taken to derive an efficient planning solution was comparable and usually shorter than in the manual planning process, and can be scaled down almost linearly with the number of processors. Overall, the autonomous genetic algorithm scheme was found to be powerful and versatile as a computationally intelligent counterpart to human‐guided strategies in treatment optimization for stereotactic radiosurgery and radiotherapy.