bluuebunny/crossref_metadata_2025 huggingface dataset:
A Proteasome Cap Subunit Required for Spindle Pole Body Duplication in Yeast
Authors: McDonald Heather B. , Byers Breck
Published: July 2002

Proteasome-mediated protein degradation is a key regulatory mechanism in a diversity of complex processes, including the control of cell cycle progression. The selection of substrates for degradation clearly depends on the specificity of ubiquitination mechanisms, but further regulation may occur within the proteasomal 19S cap complexes, which attach to the ends of the 20S proteolytic core and are thought to control entry of substrates into the core. We have characterized a gene from Saccharomyces cerevisiae that displays extensive sequence similarity to members of a family of ATPases that are components of the 19S complex, including human subunit p42 and S. cerevisiae SUG1/ CIM3 and CIM5 products. This gene, termed PCS1 (for proteasomal cap subunit), is identical to the recently described SUG2 gene (Russell, S.J., U.G. Sathyanarayana, and S.A. Johnston. 1996. J. Biol. Chem. 271:32810– 32817). We have shown that PCS1 function is essential for viability. A temperature-sensitive pcs1 strain arrests principally in the second cycle after transfer to the restrictive temperature, blocking as large-budded cells with a G2 content of unsegregated DNA. EM reveals that each arrested pcs1 cell has failed to duplicate its spindle pole body (SPB), which becomes enlarged as in other monopolar mutants. Additionally, we have shown localization of a functional Pcs1–green fluorescent protein fusion to the nucleus throughout the cell cycle. We hypothesize that Pcs1p plays a role in the degradation of certain potentially nuclear component(s) in a manner that specifically is required for SPB duplication.
A Dominant Mutant of Inner Centromere Protein (INCENP), a Chromosomal Protein, Disrupts Prometaphase Congression and Cytokinesis
Authors: Mackay Alastair M. , Ainsztein Alexandra M. , Eckley D. Mark , Earnshaw William C.
Published: July 2002

INCENP is a tightly bound chromosomal protein that transfers to the spindle midzone at the metaphase/anaphase transition. Here, we show that an INCENP truncation mutant (INCENP382–839) associates with microtubules but does not bind to chromosomes, and coats the entire spindle throughout mitosis. Furthermore, an INCENP truncation mutant (INCENP43–839) previously shown not to transfer to the spindle at anaphase (Mackay, A.M., D.M. Eckley, C. Chue, and W.C. Earnshaw. 1993. J. Cell Biol. 123:373–385), is shown here to bind chromosomes, but is unable to target to the centromere. Thus, association with the chromosomes, and specifically with centromeres, appears to be essential for INCENP targeting to the correct spindle subdomain at anaphase. An INCENP truncation mutant (INCENP1–405) that targets to centromeres but lacks the microtubule association region acquires strong dominant-negative characteristics. INCENP1–405 interferes with both prometaphase chromosome alignment and the completion of cytokinesis. INCENP1–405 apparently exerts its effect by displacing the endogenous protein from centromeres. These experiments provide evidence of an unexpected link between this chromosomal protein and cytokinesis, and suggest that one function of INCENP may be to integrate the chromosomal and cytoskeletal events of mitosis.
Presence of Pre-rRNAs before Activation of Polymerase I Transcription in the Building Process of Nucleoli during Early Development of Xenopus laevis
Authors: Verheggen Céline , Le Panse Sophie , Almouzni Geneviève , Hernandez-Verdun Danièle
Published: July 2002

During the early development of Xenopus laevis, we followed in individual nuclei the formation of a nucleolus by examining simultaneously its structural organization and its transcriptional competence. Three distinct situations were encountered with different frequencies during development. During the first period of general transcriptional quiescence, the transcription factor UBF of maternal origin, was present in most nuclei at the ribosomal gene loci. In contrast, fibrillarin, a major protein of the processing machinery, was found in multiple prenucleolar bodies (PNBs) whereas nucleolin was dispersed largely in the nucleoplasm. During the second period, for most nuclei these PNBs had fused into two domains where nucleolin concentrated, generating a structure with most features expected from a transcriptionally competent nucleolus. However, RNA polymerase I–dependent transcription was not detected using run-on in situ assays whereas unprocessed ribosomal RNAs were observed. These RNAs were found to derive from a maternal pool. Later, during a third period, an increasing fraction of the nuclei presented RNA polymerase I–dependent transcription. Thus, the structural organization of the nucleolus preceded its transcriptional competence. We conclude that during the early development of X. laevis, the organization of a defined nucleolar structure, is not associated with the transcription process per se but rather with the presence of unprocessed ribosomal RNAs.
Hensin Remodels the Apical Cytoskeleton and Induces Columnarization of Intercalated Epithelial Cells: Processes that Resemble Terminal Differentiation
Authors: Vijayakumar S. , Takito Jiro , Hikita Chinami , Al-Awqati Qais
Published: July 2002

Intercalated epithelial cells exist in a spectrum of phenotypes; at one extreme, β cells secrete HCO3 by an apical Cl/HCO3 exchanger and a basolateral H+ ATPase. When an immortalized β cell line is seeded at high density it deposits in its extracellular matrix (ECM) a new protein, hensin, which can reverse the polarity of several proteins including the Cl/HCO3 exchanger (an alternately spliced form of band 3) and the proton translocating ATPase. When seeded at low density and allowed to form monolayers these polarized epithelial cells maintain the original distribution of these two proteins. Although these cells synthesize and secrete hensin, it is not retained in the ECM, but rather, hensin is present in a large number of intracellular vesicles. The apical cytoplasm of low density cells is devoid of actin, villin, and cytokeratin19. Scanning electron microscopy shows that these cells have sparse microvilli, whereas high density cells have exuberant apical surface infolding and microvilli. The apical cytoplasm of high density cells contains high levels of actin, cytokeratin19, and villin. The cell shape of these two phenotypes is different with high density cells being tall with a small cross-sectional area, whereas low density cells are low and flat. This columnarization and the remodeling of the apical cytoplasm is hensin-dependent; it can be induced by seeding low density cells on filters conditioned by high density cells and prevented by an antibody to hensin. The changes in cell shape and apical cytoskeleton are reminiscent of the processes that occur in terminal differentiation of the intestine and other epithelia. Hensin is highly expressed in the intestine and prostate (two organs where there is a continuous process of differentiation). The expression of hensin in the less differentiated crypt cells of the intestine and the basal cells of the prostate is similar to that of low density cells; i.e., abundant intracellular vesicles but no localization in the ECM. On the other hand, as in high density cells hensin is located exclusively in the ECM of the terminally differentiated absorptive villus cells and the prostatic luminal cell. These studies suggest that hensin is a critical new molecule in the terminal differentiation of intercalated cell and perhaps other epithelial cells.
The Unstable F-box Protein p58-Ctf13 Forms the Structural Core of the CBF3 Kinetochore Complex
Authors: Russell Iain D. , Grancell Adam S. , Sorger Peter K.
Published: July 2002

Kinetochores are smaller and more accessible experimentally in budding yeast than in any other eukaryote. Believing that simple and complex kinetochores have important structural and functional properties in common, we characterized the structure of CBF3, the essential centromere-binding complex that initiates kinetochore formation in Saccharomyces cerevisiae. We find that the four subunits of CBF3 are multimeric in solution: p23Skp1 and p58Ctf13 form a heterodimer, and p64Cep3 and p110Ndc10 form homodimers. Subcomplexes involving p58 and each of the other CBF3 subunits can assemble in the absence of centromeric DNA. In these subcomplexes, p58 appears to function as a structural core mediating stable interactions among other CBF3 proteins. p58 has a short half-life in yeast, being subject to ubiquitin-dependent proteolysis, but we find that it is much more stable following association with p64. We propose that p23Skp1-p58-p64 complexes constitute the primary pool of active p58 in yeast cells. These complexes can either dissociate, reexposing p58 to the degradation pathway, or can bind to p110 and centromeric DNA, forming a functional CBF3 complex in which p58 is fully protected from degradation. This pathway may constitute an editing mechanism preventing the formation of ectopic kinetochores and ensuring the fidelity of chromosome segregation.
Ubinuclein, a Novel Nuclear Protein Interacting with Cellular and Viral Transcription Factors
Authors: Aho Sirpa , Buisson Monique , Pajunen Tiina , Ryoo Young W. , Giot Jean-Francois , Gruffat Henry , Sergeant Alain , Uitto Jouni
Published: July 2002

The major target tissues for Epstein-Barr virus (EBV) infection are B lymphocytes and epithelial cells of the oropharyngeal zone. The product of the EBV BZLF1 early gene, EB1, a member of the basic leucine-zipper family of transcription factors, interacts with both viral and cellular promoters and transcription factors, modulating the reactivation of latent EBV infection. Here, we characterize a novel cellular protein interacting with the basic domains of EB1 and c-Jun, and competing of their binding to the AP1 consensus site. The transcript is present in a wide variety of human adult, fetal, and tumor tissues, and the protein is detected in the nuclei throughout the human epidermis and as either grainy or punctuate nuclear staining in the cultured keratinocytes. The overexpression of tagged cDNA constructs in keratinocytes revealed that the NH2 terminus is essential for the nuclear localization, while the central domain is responsible for the interaction with EB1 and for the phenotype of transfected keratinocytes similar to terminal differentiation. The gene was identified in tail-to-tail orientation with the periplakin gene (PPL) in human chromosome 16p13.3 and in a syntenic region in mouse chromosome 16. We designated this novel ubiquitously expressed nuclear protein as ubinuclein and the corresponding gene as UBN1.
Functional Redundancy in the Maize Meiotic Kinetochore
Authors: Yu Hong-Guo , Dawe R. Kelly
Published: July 2002

Kinetochores can be thought of as having three major functions in chromosome segregation: (a) moving plateward at prometaphase; (b) participating in spindle checkpoint control; and (c) moving poleward at anaphase. Normally, kinetochores cooperate with opposed sister kinetochores (mitosis, meiosis II) or paired homologous kinetochores (meiosis I) to carry out these functions. Here we exploit three- and four-dimensional light microscopy and the maize meiotic mutant absence of first division 1 (afd1) to investigate the properties of single kinetochores. As an outcome of premature sister kinetochore separation in afd1 meiocytes, all of the chromosomes at meiosis II carry single kinetochores. Approximately 60% of the single kinetochore chromosomes align at the spindle equator during prometaphase/metaphase II, whereas acentric fragments, also generated by afd1, fail to align at the equator. Immunocytochemistry suggests that the plateward movement occurs in part because the single kinetochores separate into half kinetochore units. Single kinetochores stain positive for spindle checkpoint proteins during prometaphase, but lose their staining as tension is applied to the half kinetochores. At anaphase, ∼6% of the kinetochores develop stable interactions with microtubules (kinetochore fibers) from both spindle poles. Our data indicate that maize meiotic kinetochores are plastic, redundant structures that can carry out each of their major functions in duplicate.
Mice lacking desmocollin 1 show epidermal fragility accompanied by barrier defects and abnormal differentiation
Authors: Chidgey Martyn , Brakebusch Cord , Gustafsson Erika , Cruchley Alan , Hail Chris , Kirk Sarah , Merritt Anita , North Alison , Tselepis Chris , Hewitt Jane , Byrne Carolyn , Fassler Reinhard , Garrod David
Published: July 2002

The desmosomal cadherin desmocollin (Dsc)1 is expressed in upper epidermis where strong adhesion is required. To investigate its role in vivo, we have genetically engineered mice with a targeted disruption in the Dsc1 gene. Soon after birth, null mice exhibit flaky skin and a striking punctate epidermal barrier defect. The epidermis is fragile, and acantholysis in the granular layer generates localized lesions, compromising skin barrier function. Neutrophils accumulate in the lesions and further degrade the tissue, causing sloughing (flaking) of lesional epidermis, but rapid wound healing prevents the formation of overt lesions. Null epidermis is hyperproliferative and overexpresses keratins 6 and 16, indicating abnormal differentiation. From 6 wk, null mice develop ulcerating lesions resembling chronic dermatitis. We speculate that ulceration occurs after acantholysis in the fragile epidermis because environmental insults are more stringent and wound healing is less rapid than in neonatal mice. This dermatitis is accompanied by localized hair loss associated with formation of utriculi and dermal cysts, denoting hair follicle degeneration. Possible resemblance of the lesions to human blistering diseases is discussed. These results show that Dsc1 is required for strong adhesion and barrier maintenance in epidermis and contributes to epidermal differentiation.
Vps34p differentially regulates endocytosis from the apical and basolateral domains in polarized hepatic cells
Authors: Tuma Pamela L. , Nyasae Lydia K. , Backer Jonathan M. , Hubbard Ann L.
Published: July 2002

Using a microinjection approach to study apical plasma membrane protein trafficking in hepatic cells, we found that specific inhibition of Vps34p, a class III phosphoinositide 3 (PI-3) kinase, nearly perfectly recapitulated the defects we reported for wortmannin-treated cells (Tuma, P.L., C.M. Finnegan, J.-H Yi, and A.L. Hubbard. 1999. J. Cell Biol. 145:1089–1102). Both wortmannin and injection of inhibitory Vps34p antibodies led to the accumulation of resident apical proteins in enlarged prelysosomes, whereas transcytosing apical proteins and recycling basolateral receptors transiently accumulated in basolateral early endosomes. To understand how the Vps34p catalytic product, PI(3)P, was differentially regulating endocytosis from the two domains, we examined the PI(3)P binding protein early endosomal antigen 1 (EEA1). We determined that EEA1 distributed to two biochemically distinct endosomal populations: basolateral early endosomes and subapical endosomes. Both contained rab5, although the latter also contained late endosomal markers but was distinct from the transcytotic intermediate, the subapical compartment. When PI(3)P was depleted, EEA1 dissociated from basolateral endosomes, whereas it remained on subapical endosomes. From these results, we conclude that PI(3)P, via EEA1, regulates early steps in endocytosis from the basolateral surface in polarized WIF-B cells. However, PI(3)P must use different machinery in its regulation of the apical endocytic pathway, since later steps are affected by Vps34p inhibition.
The integrin cytoplasmic domain-associated protein ICAP-1 binds and regulates Rho family GTPases during cell spreading
Authors: Degani Simona , Balzac Fiorella , Brancaccio Mara , Guazzone Simona , Retta Saverio Francesco , Silengo Lorenzo , Eva Alessandra , Tarone Guido
Published: July 2002

Using two-hybrid screening, we isolated the integrin cytoplasmic domain-associated protein (ICAP-1), an interactor for the COOH terminal region of the β1A integrin cytoplasmic domain. To investigate the role of ICAP-1 in integrin-mediated adhesive function, we expressed the full-length molecule in NIH3T3 cells. ICAP-1 expression strongly prevents NIH3T3 cell spreading on extracellular matrix. This inhibition is transient and can be counteracted by coexpression of a constitutively activated mutant of Cdc42, suggesting that ICAP-1 acts upstream of this GTPase. In addition, we found that ICAP-1 binds both to Cdc42 and Rac1 in vitro, and its expression markedly inhibits activation of these GTPases during integrin-mediated cell adhesion to fibronectin as detected by PAK binding assay. In the attempt to define the molecular mechanism of this inhibition, we show that ICAP-1 reduces both the intrinsic and the exchange factor–induced dissociation of GDP from Cdc42; moreover, purified ICAP-1 displaces this GTPase from cellular membranes. Together, these data show for the first time that ICAP-1 regulates Rho family GTPases during integrin-mediated cell matrix adhesion, acting as guanine dissociation inhibitor.
Have the bloody cells gone to our heads?
Authors: Meletis Konstantinos , Frisén Jonas
Published: July 2002

Recent studies have shown that cells expressing neuronal antigens can be derived from a bone marrow transplant. A new report lends support to and extends these previous results by presenting compelling morphological evidence for the generation and integration of highly differentiated bone marrow–derived neurons.
Allelic Exclusion in pTα-deficient Mice: No Evidence for Cell Surface Expression of Two T Cell Receptor (TCR)-β Chains, but Less Efficient Inhibition of Endogeneous Vβ→ (D)Jβ Rearrangements in the Presence of a Functional TCR-β Transgene
Authors: Krotkova Anna , Boehmer Harald von , Fehling Hans Jörg
Published: July 2002

Although individual T lymphocytes have the potential to generate two distinct T cell receptor (TCR)-β chains, they usually express only one allele, a phenomenon termed allelic exclusion. Expression of a functional TCR-β chain during early T cell development leads to the formation of a pre-T cell receptor (pre-TCR) complex and, at the same developmental stage, arrest of further TCR-β rearrangements, suggesting a role of the pre-TCR in mediating allelic exclusion. To investigate the potential link between pre-TCR formation and inhibition of further TCR-β rearrangements, we have studied the efficiency of allelic exclusion in mice lacking the pre-TCR-α (pTα) chain, a core component of the pre-TCR. Staining of CD3+ thymocytes and lymph node cells with antibodies specific for Vβ6 or Vβ8 and a pool of antibodies specific for most other Vβ elements, did not reveal any violation of allelic exclusion at the level of cell surface expression. This was also true for pTα-deficient mice expressing a functionally rearranged TCR-β transgene. Interestingly, although the transgenic TCR-β chain significantly influenced thymocyte development even in the absence of pTα, it was not able to inhibit fully endogeneous TCR-β rearrangements either in total thymocytes or in sorted CD25+ pre-T cells of pTα−/− mice, clearly indicating an involvement of the pre-TCR in allelic exclusion.
Interaction of Chemokine Receptor CCR5 with its Ligands: Multiple Domains for HIV-1 gp120 Binding and a Single Domain for Chemokine Binding
Authors: Wu Lijun , LaRosa Greg , Kassam Nasim , Gordon Cynthia J. , Heath Heidi , Ruffing Nancy , Chen Howard , Humblias Jason , Samson Michel , Parmentier Marc , Moore John P. , Mackay Charles R.
Published: July 2002

CCR5 is a chemokine receptor expressed by T cells and macrophages, which also functions as the principal coreceptor for macrophage (M)-tropic strains of HIV-1. To understand the molecular basis of the binding of chemokines and HIV-1 to CCR5, we developed a number of mAbs that inhibit the various interactions of CCR5, and mapped the binding sites of these mAbs using a panel of CCR5/CCR2b chimeras. One mAb termed 2D7 completely blocked the binding and chemotaxis of the three natural chemokine ligands of CCR5, RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1α, and MIP-1β, to CCR5 transfectants. This mAb was a genuine antagonist of CCR5, since it failed to stimulate an increase in intracellular calcium concentration in the CCR5 transfectants, but blocked calcium responses elicited by RANTES, MIP-1α, or MIP-1β. This mAb inhibited most of the RANTES and MIP-1α chemotactic responses of activated T cells, but not of monocytes, suggesting differential usage of chemokine receptors by these two cell types. The 2D7 binding site mapped to the second extracellular loop of CCR5, whereas a group of mAbs that failed to block chemokine binding all mapped to the NH2-terminal region of CCR5. Efficient inhibition of an M-tropic HIV-1–derived envelope glycoprotein gp120 binding to CCR5 could be achieved with mAbs recognizing either the second extracellular loop or the NH2-terminal region, although the former showed superior inhibition. Additionally, 2D7 efficiently blocked the infectivity of several M-tropic and dual-tropic HIV-1 strains in vitro. These results suggest a complicated pattern of HIV-1 gp120 binding to different regions of CCR5, but a relatively simple pattern for chemokine binding. We conclude that the second extracellular loop of CCR5 is an ideal target site for the development of inhibitors of either chemokine or HIV-1 binding to CCR5.
A Small-molecule Inhibitor Directed against the Chemokine Receptor CXCR4 Prevents its Use as an HIV-1 Coreceptor
Authors: Doranz Benjamin J. , Grovit-Ferbas Kathie , Sharron Matthew P. , Mao Si-Hua , Goetz Matthew Bidwell , Daar Eric S. , Doms Robert W. , O'Brien William A.
Published: July 2002

The chemokine receptor CXCR4 is the major coreceptor used for cellular entry by T cell– tropic human immunodeficiency virus (HIV)-1 strains, whereas CCR5 is used by macrophage (M)-tropic strains. Here we show that a small-molecule inhibitor, ALX40-4C, inhibits HIV-1 envelope (Env)-mediated membrane fusion and viral entry directly at the level of coreceptor use. ALX40-4C inhibited HIV-1 use of the coreceptor CXCR4 by T- and dual-tropic HIV-1 strains, whereas use of CCR5 by M- and dual-tropic strains was not inhibited. Dual-tropic viruses capable of using both CXCR4 and CCR5 were inhibited by ALX40-4C only when cells expressed CXCR4 alone. ALX40-4C blocked stromal-derived factor (SDF)-1α–mediated activation of CXCR4 and binding of the monoclonal antibody 12G5 to cells expressing CXCR4. Overlap of the ALX40-4C binding site with that of 12G5 and SDF implicates direct blocking of Env interactions, rather than downregulation of receptor, as the mechanism of inhibition. Thus, ALX40-4C represents a small-molecule inhibitor of HIV-1 infection that acts directly against a chemokine receptor at the level of Env-mediated membrane fusion.
T Cell Receptor (TCR) Engagement in Apoptosis-defective, but Interleukin 2 (IL-2)–producing, T Cells Results in Impaired ZAP70/CD3-ζ Association
Authors: Sahuquillo Almudena G. , Roumier Anne , Teixeiro Emma , Bragado Rafael , Alarcón Balbino
Published: July 2002

We have previously shown that a tyrosine to leucine replacement in the transmembrane region of T cell receptor (TCR)-β results in a deficient induction of CD95-L and apoptosis upon TCR triggering in a transfected T cell line. By contrast, interleukin (IL)-2 production and the expression of CD25 and CD69 were normally induced. Since the mutation in TCR-β also resulted in impaired association of CD3-ζ, it was proposed that this chain is specifically required for the induction of apoptosis. We now show that the deficient induction of CD95-L and apoptosis does not derive from a general lower production of second messengers, since intracellular Ca2+ fluxes and tyrosine phosphorylation of total proteins were elicited at wild-type levels. Unlike in T cell clones stimulated with partial agonists, both p21 and p18 forms of tyrosine-phosphorylated CD3-ζ were detected, although the overall level of tyrosine-phosphorylated CD3-ζ was low. More strikingly, inducible association of ZAP70 to CD3-ζ was strongly inhibited, despite a normal induction of ZAP70 tyrosine phosphorylation. Finally, ZAP70 was not concentrated near the plasma membrane in the apoptosis-deficient cells. These results suggest that CD3-ζ is necessary for engagement of a specific signaling pathway leading to CD95-L expression that also needs the recruitment of ZAP70.
Targeted Disruption of the Plasmodium berghei Ctrp Gene Reveals Its Essential Role in Malaria Infection of the Vector Mosquito
Authors: Yuda Masao , Sakaida Hiroshi , Chinzei Yasuo
Published: July 2002

CTRP (circumsporozoite protein and thrombospondin-related adhesive protein [TRAP]-related protein) of the rodent malaria parasite Plasmodium berghei (PbCTRP) makes up a protein family together with other apicomplexan proteins that are specifically expressed in the host-invasive stage 1. PbCTRP is produced in the mosquito-invasive, or ookinete, stage and is a protein candidate for a role in ookinete adhesion and invasion of the mosquito midgut epithelium. To demonstrate involvement of PbCTRP in the infection of the vector, we performed targeting disruption experiments with this gene. PbCTRP disruptants showed normal exflagellation rates and development into ookinetes. However, no oocyst formation was observed in the midgut after ingestion of these parasites, suggesting complete loss of their invasion ability. On the other hand, when ingested together with wild-type parasites, disruptants were able to infect mosquitoes, indicating that the PbCTRP gene of the wild-type parasite rescued infectivity of disruptants when they heterologously mated in the mosquito midgut lumen. Our results show that PbCTRP plays a crucial role in malaria infection of the mosquito midgut and suggest that similar molecular mechanisms are used by malaria parasites to invade cells in the insect vector and the mammalian host.
Expression Levels of B Cell Surface Immunoglobulin Regulate Efficiency of Allelic Exclusion and Size of Autoreactive B-1 Cell Compartment
Authors: Watanabe Norihiko , Nisitani Sazuku , Ikuta Koichi , Suzuki Misao , Chiba Tsutomu , Honjo Tasuku
Published: July 2002

Surface-expressed immunoglobulin (Ig) has been shown to have a critical role in allelic exclusion of Ig heavy (H) and light (L) chains. Although various degrees of suppression of endogenous Ig expression are observed in Ig transgenic (Tg) mice, it was not clear whether this difference is due to different onsets of Tg expression or to different levels of Tg expression, which are obviously affected by integration sites of the transgene. In this study we generated antierythrocyte antibody Tg mice that carry tandem joined H and L chain transgenes (H+L) and confirmed that homozygosity of the transgene loci enhances the level of transgene expression as compared with heterozygosity. Suppression of endogenous H and L chain gene expression was stronger in homozygous than in heterozygous Tg mice. Similar results were obtained in control Tg mice carrying the H chain only. These results suggest that there is a threshold of the B cell receptor expression level that induces allelic exclusion. In addition, despite the same B cell receptor specificity, the size of Tg autoreactive B-1 cell compartment in the peritoneal cavity is larger in homozygous than in heterozygous mice, although the number of the Tg B-2 cell subset decreased in the spleen and bone marrow of homozygous Tg mice as compared with heterozygous Tg mice. By contrast, homozygosity of the H chain alone Tg line, which does not recognize self-antigens, did not increase the size of the peritoneal B-1 subset. These results suggest that the size of the B-1 cell subset in the Tg mice may depend on strength of signals through B cell receptors triggered by self-antigens.
Unexpected Rearrangement and Expression of the Immunoglobulin λ1 Locus in Scid Mice
Authors: Ruetsch Norman R. , Bosma Gayle C. , Bosma Melvin J.
Published: July 2002

In severe combined immunodeficient (scid) mice, V(D)J recombination is severely impaired due to a recessive mutation (scid). Thus, we were surprised to find in this study that Vλ1–Jλ1 rearrangement is routinely detectable in scid fetal liver, adult bone marrow, and spleen in the apparent absence of completed VH–DJH and Vκ–Jκ rearrangements. Particularly surprising, we found the level of Vλ1–Jλ1 rearrangement in scid fetal liver to be comparable to that in fetal liver of wild-type mice. The majority of scid Vλ1–Jλ1 rearrangements contained abnormal deletions at the VJ junction, consistent with the known effect of scid. However, ∼15% of Vλ1–Jλ1 rearrangements lacked abnormal deletions. Productive λ1 transcripts resulting from in-frame rearrangements were readily detectable in scid adult bone marrow and spleen, consistent with our ability to detect λ1-expressing cells by flow cytometry in the spleens of bcl-2–transgenic scid mice. Strikingly, λ1 transcripts from individual scid mice often showed VJ junctional sequences with the same recurring palindromic (P) additions of three, four, or five nucleotides. To account for these findings, we suggest that (a) nonhomologous end joining of Vλ1 and Jλ1 coding ends in fetal B lineage cells may not be (severely) impaired by scid; (b) recurring P additions in scid λ1 transcripts may reflect certain molecular constraints imposed by scid on the resolution of Vλ1 and Jλ1 hairpin coding ends; and (c), scid lymphocytes with productively rearranged Vλ1 and Jλ1 elements may differentiate into recombinase-inactive cells and emigrate from bone marrow to spleen.
Blockade of Transforming Growth Factor β/Smad Signaling in T Cells by Overexpression of Smad7 Enhances Antigen-Induced Airway Inflammation and Airway Reactivity
Authors: Nakao Atsuhito , Miike Satoshi , Hatano Masahiko , Okumura Ko , Tokuhisa Takeshi , Ra Chisei , Iwamoto Itsuo
Published: July 2002

Transforming growth factor (TGF)-β has been implicated in immunosuppression. However, it remains obscure whether regulation of T cells by TGF-β contributes to the immunosuppression in vivo. To address this issue, we developed transgenic mice expressing Smad7, an intracellular antagonist of TGF-β/Smad signaling, selectively in mature T cells using a plasmid construct coding a promoter element (the distal lck promoter) that directs high expression in peripheral T cells. Peripheral T cells were not growth inhibited by TGF-β in Smad7 transgenic mice. Although Smad7 transgenic mice did not spontaneously show a specific phenotype, antigen-induced airway inflammation and airway reactivity were enhanced in Smad7 transgenic mice associated with high production of both T helper cell type 1 (Th1) and Th2 cytokines. Thus, blockade of TGF-β/Smad signaling in mature T cells by expression of Smad7 enhanced airway inflammation and airway reactivity, suggesting that regulation of T cells by TGF-β was crucial for negative regulation of the inflammatory (immune) response. Our findings also implicated TGF-β/Smad signaling in mature T cells as a regulatory component of allergic asthma.
Predominant Role of T Cell Receptor (TCR)-α Chain in Forming Preimmune TCR Repertoire Revealed by Clonal TCR Reconstitution System
Authors: Yokosuka Tadashi , Takase Kan , Suzuki Misao , Nakagawa Yohko , Taki Shinsuke , Takahashi Hidemi , Fujisawa Takehiko , Arase Hisashi , Saito Takashi
Published: July 2002

The CDR3 regions of T cell receptor (TCR)-α and -β chains play central roles in the recognition of antigen (Ag)-MHC complex. TCR repertoire is created on the basis of Ag recognition specificity by CDR3s. To analyze the potential spectrum of TCR-α and -β to exhibit Ag specificity and generate TCR repertoire, we established hundreds of TCR transfectants bearing a single TCR-α or -β chain derived from a cytotoxic T cell (CTL) clone, RT-1, specific for HIVgp160 peptide, and randomly picked up TCR-β or -α chains. Surprisingly, one-third of such TCR-β containing random CDR3β from naive T cells of normal mice could reconstitute the antigen-reactive TCR coupling with RT-1 TCR-α. A similar dominant function of TCR-α in forming Ag-specific TCR, though low-frequency, was obtained for lymphocytic choriomeningitis virus–specific TCR. Subsequently, we generated TCR-α and/or -β transgenic (Tg) mice specific for HIVgp160 peptide, and analyzed the TCR repertoire of Ag-specific CTLs. Similar to the results from TCR reconstitution, TCR-α Tg generated CTLs with heterogeneous TCR-β, whereas TCR-β Tg-induced CTLs bearing a single TCR-α. These findings of Ag recognition with minimum involvement of CDR3β expand our understanding regarding the flexibility of the spectrum of TCR and suggest a predominant role of TCR-α chain in determining the preimmune repertoire of Ag-specific TCR.
Disruption of Transforming Growth Factor β Signaling by a Novel Ligand-dependent Mechanism
Authors: Fernandez Tania , Amoroso Stephanie , Sharpe Shellyann , Jones Gary M. , Bliskovski Valery , Kovalchuk Alexander , Wakefield Lalage M. , Kim Seong-Jin , Potter Michael , Letterio John J.
Published: July 2002

Transforming growth factor (TGF)-β is the prototype in a family of secreted proteins that act in autocrine and paracrine pathways to regulate cell development and function. Normal cells typically coexpress TGF-β receptors and one or more isoforms of TGF-β, thus the synthesis and secretion of TGF-β as an inactive latent complex is considered an essential step in regula-ting the activity of this pathway. To determine whether intracellular activation of TGF-β results in TGF-β ligand–receptor interactions within the cell, we studied pristane-induced plasma cell tumors (PCTs). We now demonstrate that active TGF-β1 in the PCT binds to intracellular TGF-β type II receptor (TβRII). Disruption of the expression of TGF-β1 by antisense TGF-β1 mRNA restores localization of TβRII at the PCT cell surface, indicating a ligand-induced impediment in receptor trafficking. We also show that retroviral expression of a truncated, dominant-negative TβRII (dnTβRII) effectively competes for intracellular binding of active ligand in the PCT and restores cell surface expression of the endogenous TβRII. Analysis of TGF-β receptor–activated Smad2 suggests the intracellular ligand–receptor complex is not capable of signaling. These data are the first to demonstrate the formation of an intracellular TGF-β–receptor complex, and define a novel mechanism for modulating the TGF-β signaling pathway.
Differential Occurrence of Reluctant Openings in G-Protein–Inhibited N- and P/Q-Type Calcium Channels
Authors: Colecraft Henry M. , Patil Parag G. , Yue David T.
Published: July 2002

Voltage-dependent inhibition of N- and P/Q-type calcium channels by G proteins is crucial for presynaptic inhibition of neurotransmitter release, and may contribute importantly to short-term synaptic plasticity. Such calcium-channel modulation could thereby impact significantly the neuro-computational repertoire of neural networks. The differential modulation of N and P/Q channels could even further enrich their impact upon synaptic tuning. Here, we performed in-depth comparison of the G-protein inhibition of recombinant N and P/Q channels, expressed in HEK 293 cells with the m2 muscarinic receptor. While both channel types display classic features of G-protein modulation (kinetic slowing of activation, prepulse facilitation, and voltage dependence of inhibition), we confirmed previously reported quantitative differences, with N channels displaying stronger inhibition and greater relief of inhibition by prepulses. A more fundamental, qualitative difference in the modulation of these two channels was revealed by a modified tail-activation paradigm, as well as by a novel “slope” analysis method comparing time courses of slow activation and prepulse facilitation. The stark contrast in modulatory behavior can be understood within the context of the “willing–reluctant” model, in which binding of G-protein βγ subunits to channels induces a reluctant mode of gating, where stronger depolarization is required for opening. Our experiments suggest that only N channels could be opened in the reluctant mode, at voltages normally spanned by neuronal action potentials. By contrast, P/Q channels appear to remain closed, especially over these physiological voltages. Further, the differential occurrence of reluctant openings is not explained by differences in the rate of G-protein unbinding from the two channels. These two scenarios predict very different effects of G-protein inhibition on the waveform of Ca2+ entry during action potentials, with potentially important consequences for the timing and efficacy of synaptic transmission.
Polar and coisotropic actions on Kähler manifolds
Authors: Podestà Fabio , Thorbergsson Gudlaugur
Published: July 2002

The main result of the paper is that a polar action on a compact irreducible homogeneous Kähler manifold is coisotropic. This is then used to give new examples of polar actions and to classify coisotropic and polar actions on quadrics.
Rounding errors in solving block Hessenberg systems
Authors: von Matt Urs , Stewart G.
Published: July 2002

A rounding error analysis is presented for a divide-and-conquer algorithm to solve linear systems with block Hessenberg matrices. Conditions are derived under which the algorithm computes a stable solution. The algorithm is shown to be stable for block diagonally dominant matrices and for M-matrices.
Constructing optimal maps for Monge’s transport problem as a limit of strictly convex costs
Authors: Caffarelli Luis , Feldman Mikhail , McCann Robert
Published: July 2002

Given two densities on

R

n

\mathbf {R}^n

with the same total mass, the Monge transport problem is to find a Borel map

s
:

R

n

→

R

n

s:\mathbf {R}^n \to \mathbf {R}^n

rearranging the first distribution of mass onto the second, while minimizing the average distance transported. Here distance is measured by a norm with a uniformly smooth and convex unit ball. This paper gives a complete proof of the existence of optimal maps under the technical hypothesis that the distributions of mass be compactly supported. The maps are not generally unique. The approach developed here is new, and based on a geometrical change-of-variables technique offering considerably more flexibility than existing approaches.
SAWTED: Structure Assignment With Text
Description—Enhanced detection of remote homologues with
automated SWISS-PROT annotation comparisons
Authors: MacCallum Robert M. , Kelley Lawrence A. , Sternberg Michael J. E.
Published: July 2002

Motivation: Sequence database search methods often identify putative sub-threshold hits of known function or structure for a given query sequence. It is widespread practice to filter these hits by hand using knowledge of function and other factors; to the expert, some hits may appear more sensible than others. SAWTED (Structure Assignment With Text Description) is an automated solution to this post-filtering problem which will be applicable to large scale genome assignments.

Results: A standard document comparison algorithm is applied to text descriptions extracted from SWISS-PROT annotations. The added value of SAWTED in combination with PSI-BLAST has been shown with a benchmark of difficult remote homologues taken from the SCOP structure database.

Availability: A SAWTED PSI-BLAST Web server is available to perform sensitive searches against the protein structure database (http://www.bmm.icnet.uk/servers/sawted).

Contact: R.MacCallum@icrf.icnet.uk
PIR: a new resource for bioinformatics
Authors: McGarvey Peter B. , Huang Hongzhan , Barker Winona C. , Orcutt Bruce C. , Garavelli John S. , Srinivasarao Geetha Y. , Yeh Lai-Su L. , Xiao Chunlin , Wu Cathy H.
Published: July 2002

Summary: The Protein Information Resource (PIR) has greatly expanded its Web site and developed a set of interactive search and analysis tools to facilitate the analysis, annotation, and functional identification of proteins. New search engines have been implemented to combine sequence similarity search results with database annotation information. The new PIR search systems have proved very useful in providing enriched functional annotation of protein sequences, determining protein superfamily-domain relationships, and detecting annotation errors in genomic database archives.

Availability: http://pir.georgetown.edu/.

Contact: mcgarvey@nbrf.georgetown.edu
Hens, cocks and avian sex determination
Authors: Ellegren Hans
Published: July 2002

The sex of an individual is generally determined genetically by genes on one of the two sex chromosomes. In mammals, for instance, the presence of the male‐specific Y chromosome confers maleness, whereas in Drosophila melanogaster and Caenorhabditis elegans it is the number of X chromosomes that matters. For birds (males ZZ, females ZW), however, the situation remains unclear. The recent discovery that the Z‐linked DMRT1 gene, which is conserved across phyla as a gene involved in sexual differentiation, is expressed early in male development suggests that it might be the number of Z chromosomes that regulate sex in birds. On the other hand, the recent identification of the first protein unique to female birds, encoded by the W‐linked PKCIW gene, and the observation that it is expressed early in female gonads, suggests that the W chromosome plays a role in avian sexual differentiation. Clearly defining the roles of the DMRT1 and PKC1W genes in gonadal development, and ultimately determining whether avian sex is dependent on Z or W, will require transgenic experiments.
Drugs for a new millennium
Authors: Snyder Solomon H.
Published: July 2002

A millennium, a century, even a decade is a long time–frame for speculation about anything. Advances in biomedical research in the last few decades have been so extraordinary and escalating at an ever–accelerating pace that any prophecy is a risky proposition. However, it is possible to divine the big, unanswered questions and envisage ways in which they might reasonably be approached in the next few decades, a task which I will try to essay. So many drugs treat so many different medical conditions that a detailed and comprehensive coverage would probably be tiresome. Instead, I will address certain broad themes and diseases that offer both immense challenges and great potential for advances. Rather than review detailed experimental issues, I will confine myself to the ‘big picture’ issues, providing examples of specific research only in a few instances drawing largely from areas I know best.
Picking out parallels: plant circadian clocks in context
Authors: McWatters Harriet G. , Roden Laura C. , Staiger Dorothee
Published: July 2002

Molecular models have been described for the circadian clocks of representatives of several different taxa. Much of the work on the plant circadian system has been carried out using the thale cress,Arabidopsis thaliana, as a model. We discuss the roles of genes implicated in the plant circadian system, with special emphasis onArabidopsis. Plants have an endogenous clock that regulates many aspects of circadian and photoperiodic behaviour. Despite the discovery of components that resemble those involved in the clocks of animals or fungi, no coherent model of the plant clock has yet been proposed. In this review, we aim to provide an overview of studies of theArabidopsiscircadian system. We shall compare these with results from different taxa and discuss them in the context of what is known about clocks in other organisms.
Characterization of bothrojaracin interaction with human prothrombin
Authors: Monteiro Robson Q. , Bock Paul E. , Bianconi M. Lucia , Zingali Russolina B.
Published: July 2002

Bothrojaracin (BJC) is a 27‐kD snake venom protein from Bothrops jararaca that has been characterized as a potent thrombin inhibitor. BJC binds to exosites I and II, with a dissociation constant of 0.7 nM, and influences but does not block the proteinase catalytic site. BJC also binds prothrombin through an interaction that has not been characterized. In the present work we characterize the interaction of BJC with prothrombin quantitatively for the first time, and identify the BJC binding site on human prothrombin. Gel filtration chromatography demonstrated calcium‐independent, 1:1 complex formation between fluorescein‐labeled BJC ([5F]BJC) and prothrombin, whereas no interactions were observed with activation fragments 1 or 2 of prothrombin. Isothermal titration calorimetry showed that binding of BJC to prothrombin is endothermic, with a dissociation constant of 76 ± 32 nM. The exosite I‐specific ligand, hirudin54–65 (Hir54–65 (SO3−), displaced competitively [5F]BJC from prothrombin. Titration of the fluorescent hirudin54–65 derivative, [5F]Hir54–65(SO3−), with human prothrombin showed a dissociation constant of 7.0 ± 0.2 μM, indicating a ∼100‐fold lower binding affinity than that exhibited by BJC. Both ligands, however, displayed a similar, ∼100‐fold increase in affinity for exosite I when prothrombin was activated to thrombin. BJC efficiently displaced [5F]Hir54–65(SO3−) from complexes formed with thrombin or prothrombin with dissociation constants of 0.7 ± 0.9 nM and 11 ± 80 nM, respectively, indicating that BJC and Hir54–65(SO3−) compete for the same exosite on these molecules. The results indicate that BJC is a potent and specific probe of the partially exposed anion‐binding exosite (proexosite I) of human prothrombin.
The optimum intensities for multiple static multileaf collimator field compensation
Authors: Evans Philip M. , Hansen Vibeke N. , Swindell William
Published: July 2002

A method of determining the optimum beam intensities for compensation using multiple static multileaf collimator fields is presented. In this method a histogram of the number of beam pixels against beam intensity is generated for the intensity‐modulated beam (IMB). The intensity of each beam to be used is chosen to minimize the mean square deviation between each bin in the histogram and the closest beam intensity. This method has been applied to sample IMBs possessing one maximum and two maxima. For both cases, the use of uniform beam intensity increments is shown to be close to optimal. In the case with two maxima, the efficacy of irradiating both peaks simultaneously, rather than separately, has been studied and shown to be of potential benefit. The optimum intensities for an IMB for breast radiotherapy are also presented.
Fast iterative algorithms for three‐dimensional inverse treatment planning
Authors: Xing L. , Hamilton R. J. , Spelbring D. , Pelizzari C. A. , Chen G. T. Y. , Boyer A. L.
Published: July 2002

Three types of iterative algorithms, algebraic inverse treatment planning (AITP), simultaneous iterative inverse treatment planning (SIITP), and iterative least‐square inverse treatment planning (ILSITP), differentiated according to their updating sequences, were generalized to three dimension with true beam geometry and dose model. A rapid ray‐tracing approach was developed to optimize the primary beam components. Instead of recalculating the dose matrix at each iteration, the dose distribution was generated by scaling up or down the dose matrix elements of the previous iteration. This significantly increased the calculation speed. The iterative algorithms started with an initial intensity profile for each beam, specified by a two‐dimensional pixel beam map of M elements. The calculation volume was divided into N voxels, and the calculation was done by repeatedly comparing the calculated and desired doses and adjusting the values of the beam map elements to minimize an objective function. In AITP, the iteration is performed voxel by voxel. For each voxel, the dose discrepancy was evaluated and the contributing pencil beams were updated. In ILSITP and SIITP, the iteration proceeded pencil beam by pencil beam instead of voxel by voxel. In all cases, the iteration procedure was repeated until the best possible dose distribution was achieved. The algorithms were applied to two examples and the results showed that the iterative techniques were able to produce superior isodose distributions.