bluuebunny/crossref_metadata_2025 huggingface dataset:
Cbl-b Negatively Regulates B Cell Antigen Receptor Signaling in Mature B Cells through Ubiquitination of the Tyrosine Kinase Syk
Authors: Sohn Hae Won , Gu Hua , Pierce Susan K.
Published: June 2003

Members of the Cbl family of molecular adaptors play key roles in regulating tyrosine kinase-dependent signaling in a variety of cellular systems. Here we provide evidence that in B cells Cbl-b functions as a negative regulator of B cell antigen receptor (BCR) signaling during the normal course of a response. In B cells from Cbl-b–deficient mice cross-linking the BCRs resulted in sustained phosphorylation of Igα, Syk, and phospholipase C (PLC)-γ2, leading to prolonged Ca2+ mobilization, and increases in extracellular signal–regulated kinase (ERK) and c-Jun NH2-terminal protein kinase (JNK) phosphorylation and surface expression of the activation marker, CD69. Image analysis following BCR cross-linking showed sustained polarization of the BCRs into large signaling-active caps associated with phosphorylated Syk in Cbl-b–deficient B cells in contrast to the BCRs in Cbl-b–expressing B cells that rapidly proceeded to form small, condensed, signaling inactive caps. Significantly, prolonged phosphorylation of Syk correlated with reduced ubiquitination of Syk indicating that Cbl-b negatively regulates BCR signaling by targeting Syk for ubiquitination.
Classification and Prediction of Survival in Patients with the Leukemic Phase of Cutaneous T Cell Lymphoma
Authors: Kari Laszlo , Loboda Andrey , Nebozhyn Michael , Rook Alain H. , Vonderheid Eric C. , Nichols Calen , Virok Dezso , Chang Celia , Horng Wen-Hwai , Johnston James , Wysocka Maria , Showe Michael K. , Showe Louise C.
Published: June 2003

We have used cDNA arrays to investigate gene expression patterns in peripheral blood mononuclear cells from patients with leukemic forms of cutaneous T cell lymphoma, primarily Sezary syndrome (SS). When expression data for patients with high blood tumor burden (Sezary cells >60% of the lymphocytes) and healthy controls are compared by Student's t test, at P < 0.01, we find 385 genes to be differentially expressed. Highly overexpressed genes include Th2 cells–specific transcription factors Gata-3 and Jun B, as well as integrin β1, proteoglycan 2, the RhoB oncogene, and dual specificity phosphatase 1. Highly underexpressed genes include CD26, Stat-4, and the IL-1 receptors. Message for plastin-T, not normally expressed in lymphoid tissue, is detected only in patient samples and may provide a new marker for diagnosis. Using penalized discriminant analysis, we have identified a panel of eight genes that can distinguish SS in patients with as few as 5% circulating tumor cells. This suggests that, even in early disease, Sezary cells produce chemokines and cytokines that induce an expression profile in the peripheral blood distinctive to SS. Finally, we show that using 10 genes, we can identify a class of patients who will succumb within six months of sampling regardless of their tumor burden.
Preferential Signaling and Induction of Allergy-promoting Lymphokines Upon Weak Stimulation of the High Affinity IgE Receptor on Mast Cells
Authors: Gonzalez-Espinosa Claudia , Odom Sandra , Olivera Ana , Hobson J. Peyton , Martinez Maria Eugenia Cid , Oliveira-dos-Santos Antonio , Barra Lillian , Spiegel Sarah , Penninger Josef M. , Rivera Juan
Published: June 2003

Mast cell degranulation and de novo cytokine production is a consequence of antigen-aggregation of the immunoglobulin E (IgE)-occupied high affinity receptor for IgE (FcεRI). Herein, we report that lymphokines that promote allergic inflammation, like MCP-1, were potently induced at low antigen (Ag) concentrations or at low receptor occupancy with IgE whereas some that down-regulate this response, like interleukin (IL)-10, required high receptor occupancy. Weak stimulation of mast cells caused minimal degranulation whereas a half-maximal secretory response was observed for chemokines and, with the exception of TNF-α, a weaker cytokine secretory response was observed. The medium from weakly stimulated mast cells elicited a monocyte/macrophage chemotactic response similar to that observed at high receptor occupancy. Weak stimulation also favored the phosphorylation of Gab2 and p38MAPK, while LAT and ERK2 phosphorylation was induced by a stronger stimulus. Gab2-deficient mast cells were severely impaired in chemokine mRNA induction whereas LAT-deficient mast cells showed a more pronounced defect in cytokines. These findings demonstrate that perturbation of small numbers of IgE receptors on mast cells favors certain signals that contribute to a lymphokine response that can mediate allergic inflammation.
Inhibition of Interleukin 10 Signaling after Fc Receptor Ligation and during Rheumatoid Arthritis
Authors: Ji Jong-Dae , Tassiulas Ioannis , Park-Min Kyung-Hyun , Aydin Ani , Mecklenbräuker Ingrid , Tarakhovsky Alexander , Pricop Luminita , Salmon Jane E. , Ivashkiv Lionel B.
Published: June 2003

Interleukin-10 (IL-10) is a potent deactivator of myeloid cells that limits the intensity and duration of immune and inflammatory responses. The activity of IL-10 can be suppressed during inflammation, infection, or after allogeneic tissue transplantation. We investigated whether inflammatory factors suppress IL-10 activity at the level of signal transduction. Out of many factors tested, only ligation of Fc receptors by immune complexes inhibited IL-10 activation of the Jak-Stat signaling pathway. IL-10 signaling was suppressed in rheumatoid arthritis joint macrophages that are exposed to immune complexes in vivo. Activation of macrophages with interferon-γ was required for Fc receptor–mediated suppression of IL-10 signaling, which resulted in diminished activation of IL-10–inducible genes and reversal of IL-10–dependent suppression of cytokine production. The mechanism of inhibition involved decreased cell surface IL-10 receptor expression and Jak1 activation and was dependent on protein kinase C delta. These results establish that IL-10 signaling is regulated during inflammation and identify Fc receptors and interferon-γ as important regulators of IL-10 activity. Generation of macrophages refractory to IL-10 can contribute to pathogenesis of inflammatory and infectious diseases characterized by production of interferon-γ and immune complexes.
Identification of a Polymorphic Gene, BCL2A1, Encoding Two Novel Hematopoietic Lineage-specific Minor Histocompatibility Antigens
Authors: Akatsuka Yoshiki , Nishida Tetsuya , Kondo Eisei , Miyazaki Mikinori , Taji Hirohumi , Iida Hiroatsu , Tsujimura Kunio , Yazaki Makoto , Naoe Tomoki , Morishima Yasuo , Kodera Yoshihisa , Kuzushima Kiyotaka , Takahashi Toshitada
Published: June 2003

We report the identification of two novel minor histocompatibility antigens (mHAgs), encoded by two separate single nucleotide polymorphisms on a single gene, BCL2A1, and restricted by human histocompatibility leukocyte antigen (HLA)-A*2402 (the most common HLA-A allele in Japanese) and B*4403, respectively. Two cytotoxic T lymphocyte (CTL) clones specific for these mHAgs were first isolated from two distinct recipients after hematopoietic cell transplantation. Both clones lyse only normal and malignant cells within the hematopoietic lineage. To localize the gene encoding the mHAgs, two-point linkage analysis was performed on the CTL lytic patterns of restricting HLA-transfected B lymphoblastoid cell lines obtained from Centre d'Etude du Polymorphisme Humain. Both CTL clones showed a completely identical lytic pattern for 4 pedigrees and the gene was localized within a 3.6-cM interval of 15q24.3–25.1 region that encodes at least 46 genes. Of those, only BCL2A1 has been reported to be expressed in hematopoietic cells and possess three nonsynonymous nucleotide changes. Minigene transfection and epitope reconstitution assays with synthetic peptides identified both HLA-A*2402– and B*4403-restricted mHAg epitopes to be encoded by distinct polymorphisms within BCL2A1.
The Role of Intramolecular Epitope Spreading in the Pathogenesis of Endemic Pemphigus Foliaceus (Fogo Selvagem)
Authors: Li Ning , Aoki Valeria , Hans-Filho Gunter , Rivitti Evandro A. , Diaz Luis A.
Published: June 2003

We report here a relationship between intramolecular epitope spreading and the clinical onset of the endemic form of pemphigus foliaceus in a Brazilian community with a high prevalence and incidence of the disease. Also known as Fogo Selvagem (FS), this disease is characterized by severe skin blistering and pathogenic anti–desmoglein-1 (Dsg1) autoantibodies. These autoantibodies bind the Dsg1 ectodomain and trigger keratinocyte cell detachment, the hallmark of FS. We show that (a) sera from FS patients in the preclinical stage recognized epitopes on the COOH-terminal EC5 domain of Dsg1, (b) disease onset was associated with the emergence of antibodies specific for epitopes on the NH2-terminal EC1 and EC2 domains, (c) all sera from FS patients with active disease recognized the EC1 and/or EC2 domains, and (d) sera from FS patients in remission showed reactivity restricted to EC5. These results suggest that anti-Dsg1 autoantibodies in FS are initially raised against the COOH-terminal EC5 domain of Dsg1 in individuals without skin disease; in genetically predisposed subjects the autoimmune response may then undergo intramolecular epitope spreading toward epitopes on the NH2-terminal EC1 and EC2 domains of Dsg1 leading to disease onset. Moreover, intramolecular epitope spreading may also modulate remissions and relapses of FS.
Effect of Anatomical Distribution of Mast Cells on Their Defense Function against Bacterial Infections
Authors: Jippo Tomoko , Morii Eiichi , Ito Akihiko , Kitamura Yukihiko
Published: June 2003

Mast cells were depleted in the peritoneal cavity of WBB6F1-tg/tg mice that did not express a transcription factor, MITF. When acute bacterial peritonitis was induced in WBB6F1-+/+, WBB6F1-W/Wv, and WBB6F1-tg/tg mice, the proportion of surviving WBB6F1-+/+ mice was significantly higher than that of surviving WBB6F1-W/Wv or WBB6F1-tg/tg mice. The poor survival of WBB6F1-W/Wv and WBB6F1-tg/tg mice was attributed to the deficient influx of neutrophils into the peritoneal cavity. The injection of cultured mast cells (CMCs) derived from WBB6F1-+/+ mice normalized the neutrophil influx and reduced survival rate in WBB6F1-W/Wv mice, but not in WBB6F1-tg/tg mice. This was not attributable to a defect of neutrophils because injection of TNF-α increased the neutrophil influx and survival rate in both WBB6F1-W/Wv and WBB6F1-tg/tg mice. Although WBB6F1-+/+ CMCs injection normalized the number of mast cells in both the peritoneal cavity and mesentery of WBB6F1-W/Wv mice, it normalized the number of mast cells only in the peritoneal cavity of WBB6F1-tg/tg mice. Mast cells within the mesentery or mast cells in the vicinity of blood vessels appeared to play an important role against the acute bacterial peritonitis. WBB6F1-tg/tg mice may be useful for studying the effect of anatomical distribution of mast cells on their antiseptic function.
Gef1p and Scd1p, the Two GDP-GTP Exchange Factors for Cdc42p, Form a Ring Structure that Shrinks during Cytokinesis inSchizosaccharomyces pombe
Authors: Hirota Kouji , Tanaka Kayoko , Ohta Kunihiro , Yamamoto Masayuki
Published: June 2003

Fission yeast Cdc42p, a small GTPase of the Rho family, is essential for cell proliferation and maintenance of the rod-like cell morphology. Scd1/Ral1p is a GDP-GTP exchange factor (GEF) for Cdc42p. This study and a parallel study by others establish that Gef1p is another GEF for Cdc42p. Deletions of gef1 and scd1 are synthetically lethal, generating round dead cells, and hence mimic the phenotype of cdc42 deletion. Gef1p is localized mainly to the cell division site. Scd1p is also there, but it is also detectable in other parts of the cell, including the nucleus, growing ends, and the tips of conjugation tubes. Gef1p and Scd1p form a ring structure at the cell division site, which shrinks during cytokinesis following the contraction of the actomyosin ring. Formation of the Gef1p/Scd1p ring apparently depends on the integrity of the actomyosin ring. In turn, recruitment of Cdc42p to the cell division site follows the shrinking Gef1p/Scd1p ring; the Cdc42p accumulates like a closing iris. These observations suggest that Gef1p and Scd1p may have a role in mediating between contraction of the actomyosin ring and formation of the septum, by recruiting active Cdc42p to the septation site.
The Ubiquitin-associated Domain of hPLIC-2 Interacts with the Proteasome
Authors: Kleijnen Maurits F. , Alarcón Rodolfo M. , Howley Peter M.
Published: June 2003

The ubiquitin-like hPLIC proteins can associate with proteasomes, and hPLIC overexpression can specifically interfere with ubiquitin-mediated proteolysis ( Kleijnen et al., 2000 ). Because the hPLIC proteins can also interact with certain E3 ubiquitin protein ligases, they may provide a link between the ubiquitination and proteasomal degradation machineries. The amino-terminal ubiquitin-like (ubl) domain is a proteasome-binding domain. Herein, we report that there is a second proteasome-binding domain in hPLIC-2: the carboxyl-terminal ubiquitin-associated (uba) domain. Coimmunoprecipitation experiments of wild-type and mutant hPLIC proteins revealed that the ubl and uba domains each contribute independently to hPLIC-2–proteasome binding. There is specificity for the interaction of the hPLIC-2 uba domain with proteasomes, because uba domains from several other proteins failed to bind proteasomes. Furthermore, the binding of uba domains to polyubiquitinated proteins does not seem to be sufficient for the proteasome binding. Finally, the uba domain is necessary for the ability of full-length hPLIC-2 to interfere with the ubiquitin-mediated proteolysis of p53. The PLIC uba domain has been reported to bind and affect the functions of proteins such as GABAAreceptor and presenilins. It is possible that the function of these proteins may be regulated or mediated through proteasomal degradation pathways.
Exploring the Functional Interactions between Aurora B, INCENP, and Survivin in Mitosis
Authors: Honda Reiko , Körner Roman , Nigg Erich A.
Published: June 2003

The function of the Aurora B kinase at centromeres and the central spindle is crucial for chromosome segregation and cytokinesis, respectively. Herein, we have investigated the regulation of human Aurora B by its complex partners inner centromere protein (INCENP) and survivin. We found that overexpression of a catalytically inactive, dominant-negative mutant of Aurora B impaired the localization of the entire Aurora B/INCENP/survivin complex to centromeres and the central spindle and severely disturbed mitotic progression. Similar results were also observed after depletion, by RNA interference, of either Aurora B, INCENP, or survivin. These data suggest that Aurora B kinase activity and the formation of the Aurora B/INCENP/survivin complex both contribute to its proper localization. Using recombinant proteins, we found that Aurora B kinase activity was stimulated by INCENP and that the C-terminal region of INCENP was sufficient for activation. Under identical assay conditions, survivin did not detectably influence kinase activity. Human INCENP was a substrate of Aurora B and mass spectrometry identified three consecutive residues (threonine 893, serine 894, and serine 895) containing at least two phosphorylation sites. A nonphosphorylatable mutant (TSS893–895AAA) was a poor activator of Aurora B, demonstrating that INCENP phosphorylation is important for kinase activation.
Synchronization of Interphase Events Depends neither on Mitosis nor on cdk1
Authors: Laronne Ayelet , Rotkopf Shay , Hellman Asaf , Gruenbaum Yosef , Porter Andrew C.G. , Brandeis Michael
Published: June 2003

Human HT2-19 cells with a conditional cdk1 mutation stop dividing upon cdk1 inactivation and undergo multiple rounds of endoreplication. We show herein that major cell cycle events remain synchronized in these endoreplicating cells. DNA replication alternates with gap phases and cell cycle-specific cyclin E expression is maintained. Centrosomes duplicate in synchrony with chromosome replication, giving rise to polyploid cells with multiple centrosomes. Centrosome migration, a typical prophase event, also takes place in endoreplicating cells. The timing of these events is unaffected by cdk1 inactivation compared with normally dividing cells. Nuclear lamina breakdown, in contrast, previously shown to be dependent on cdk1, does not take place in endoreplicating HT2-19 cells. Moreover, breakdown of all other major components of the nuclear lamina, like the inner nuclear membrane proteins and nuclear pore complexes, seems also to depend on cdk1. Interestingly, the APC/C ubiquitin ligase is activated in these endoreplicating cells by fzr but not by fzy. The oscillations of interphase events are thus independent of cdk1 and of mitosis but may depend on APC/Cfzractivity.
RAM: A Conserved Signaling Network That Regulates Ace2p Transcriptional Activity and Polarized Morphogenesis
Authors: Nelson Bryce , Kurischko Cornelia , Horecka Joe , Mody Manali , Nair Pradeep , Pratt Lana , Zougman Alexandre , McBroom Linda D.B. , Hughes Timothy R. , Boone Charlie , Luca Francis C.
Published: June 2003

In Saccharomyces cerevisiae, polarized morphogenesis is critical for bud site selection, bud development, and cell separation. The latter is mediated by Ace2p transcription factor, which controls the daughter cell-specific expression of cell separation genes. Recently, a set of proteins that include Cbk1p kinase, its binding partner Mob2p, Tao3p (Pag1p), and Hym1p were shown to regulate both Ace2p activity and cellular morphogenesis. These proteins seem to form a signaling network, which we designate RAM for regulation of Ace2p activity and cellular morphogenesis. To find additional RAM components, we conducted genetic screens for bilateral mating and cell separation mutants and identified alleles of the PAK-related kinase Kic1p in addition to Cbk1p, Mob2p, Tao3p, and Hym1p. Deletion of each RAM gene resulted in a loss of Ace2p function and caused cell polarity defects that were distinct from formin or polarisome mutants. Two-hybrid and coimmunoprecipitation experiments reveal a complex network of interactions among the RAM proteins, including Cbk1p–Cbk1p, Cbk1p–Kic1p, Kic1p–Tao3p, and Kic1p–Hym1p interactions, in addition to the previously documented Cbk1p–Mob2p and Cbk1p–Tao3p interactions. We also identified a novel leucine-rich repeat-containing protein Sog2p that interacts with Hym1p and Kic1p. Cells lacking Sog2p exhibited the characteristic cell separation and cell morphology defects associated with perturbation in RAM signaling. Each RAM protein localized to cortical sites of growth during both budding and mating pheromone response. Hym1p was Kic1p- and Sog2p-dependent and Sog2p and Kic1p were interdependent for localization, indicating a close functional relationship between these proteins. Only Mob2p and Cbk1p were detectable in the daughter cell nucleus at the end of mitosis. The nuclear localization and kinase activity of the Mob2p–Cbk1p complex were dependent on all other RAM proteins, suggesting that Mob2p–Cbk1p functions late in the RAM network. Our data suggest that the functional architecture of RAM signaling is similar to the S. cerevisiae mitotic exit network and Schizosaccharomyces pombe septation initiation network and is likely conserved among eukaryotes.
The Yeast hnRNP-like Protein Hrp1/Nab4 Accumulates in the Cytoplasm after Hyperosmotic Stress: A Novel Fps1-dependent Response
Authors: Henry Michael F. , Mandel Daniel , Routson Valerie , Henry Pamela A.
Published: June 2003

The Hrp1/Nab4 shuttling protein belongs to a family of RNA binding proteins that bind to nascent RNA polymerase II transcripts and form hnRNP complexes. Members of this family function in a staggering array of cellular activities, ranging from transcription and pre-mRNA processing in the nucleus to cytoplasmic mRNA translation and turnover. It has recently been recognized that the yeast stress response can include alterations in hnRNP-mediated mRNA export. We now report that the steady-state localization of Hrp1p rapidly shifts from the nucleus to the cytoplasm in response to osmotic stress. In contrast to a general stress response resulting in a transient relocation, Hrp1p redistribution is specific to hyperosmotic stress and is only reversed after stress removal. Hrp1p relocalization requires both the CRM1/XPO1 exportin and the FPS1 glycerol transporter genes but is independent of ongoing RNA transcription and protein arginine methylation. However, mutations in the high osmolarity glycerol and protein kinase C osmosensing pathways do not impact the Hrp1p hyperosmotic response. We present a working model for the cytoplasmic accumulation of Hrp1 and discuss the implications of this relocalization on Hrp1p function.
Truth Half Told: Finding the Perfect Pitch for Advertising and Marketing in Socialist Yugoslavia, 1950–1991
Authors: Patterson Patrick Hyder
Published: June 2003

Beginning in the 1950s, advertising and marketing specialists in Yugoslavia campaigned aggressively to change public and official perceptions of their work. By casting commercial promotion in terms of categories already established as legitimate in the ideology of Yugoslav socialism, the industry gradually naturalized advertising and marketing, transforming them from suspect capitalist practices into apparent necessities of progressive, rational production and distribution. Although the rhetoric used in this campaign consistently appealed to socialist values, in practice Yugoslav advertising and marketing were largely based on Western models. Yugoslav commercial promotion was only superficially “socialist advertising”; practitioners' arguments tended to obscure the true qualities of the industry.
Symbolic Culture and Technological Change: The Cultural History of Aluminum as an Industrial Material
Authors: Schatzberg Eric
Published: June 2003

The history of aluminum illustrates how the concept of symbolic meanings can help connect culture with business history. Aluminum's symbolic meanings played a crucial role in its industrial history, largely through the enthusiasm that greeted the introduction and diffusion of the metal. Symbolic meanings influence technological innovation through their role in shaping expectations, a role understood by the historical actors who engage in struggles over the meanings of competing innovations. For aluminum, this struggle centered on the conflict between the material's two major meanings: aluminum as modern and aluminum as ersatz. This debate over meanings has played out differently in aviation, electric wiring, and automobiles.
Golden Opportunities: Jewelry Making in Birmingham between Mass Production and Specialty
Authors: Carnevali Francesca
Published: June 2003

Narratives of flexible specialization as an alternative to mass production are largely absent from the industrial history of twentieth-century Britain. In this article, I challenge the notion that we should relegate small firms and industrial districts to a marginal place in the historiography of this period. Drawing from a range of sources, I explore the history of Birmingham's jewelry makers to show how they adapted the traditional productive system of the district to respond in a dynamic way to the challenges of rapid product market differentiation. As jewelry increasingly became a commodity for mass consumption, the firms in the Birmingham district used a combination of specialty and mass production as a strategy to both satisfy and create demand.
The Culture of Credit in Eighteenth-Century Commerce: The English Textile Industry
Authors: Smail John
Published: June 2003

In this article, I seek to expand the relatively narrow focus of most work on commercial credit in eighteenth-century England by incorporating culture into an economic analysis. I argue that the various credit regimes that operated in the regional branches of the English wool textile industry are best understood as having a cultural dimension. A comparative analysis of business strategies in these regions suggests that the different cultures of credit had important implications for the development of the textile industry during the eighteenth century, shaping the character of the entrepreneurship of each region's merchants and producers.
The Diversity of Industrial Experience: Cabinet and Furniture Manufacture in Late Nineteenth-Century Ontario
Authors: Forster Ben , Inwood Kris
Published: June 2003

The diversity of paths to industrialization is illustrated by the example of the cabinet- and furniture-manufacturing industry in Ontario, Canada. Complex and unpredictable demand combined with smaller markets and lower incomes than those in the United States and the relative abundance of wood to limit mechanization and the size of enterprise in the Canadian industry. Small and unpowered workshops remained competitive throughout the nineteenth and into the twentieth century, creating a distinctive industrial experience that reflects the unique interaction of local demand and supply.
Transposition in Lactobacillus delbrueckii subsp. bulgaricus: identification of two thermosensitive replicons and two functional insertion sequences
Authors: Serror Pascale , Ilami Golnar , Chouayekh Hichem , Ehrlich S. Dusko , Maguin Emmanuelle
Published: June 2003

In this report, it is shown that the rolling circle replicon pG+host and the theta replicon pIP501 are thermosensitive in Lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus bulgaricus). Using a pIP501 derivative as a delivery vector for six insertion sequences originating from lactic acid bacteria, it is shown that IS1223 and IS1201 transpose in L. bulgaricus.
Conditional expression of Mycobacterium smegmatis ftsZ, an essential cell division gene
Authors: Dziadek Jaroslaw , Rutherford Stacey A. , Madiraju Murty V. , Atkinson Mark A. L. , Rajagopalan Malini
Published: June 2003

To understand the role of Mycobacterium smegmatis
ftsZ (ftsZsmeg
) in the cell division process, the ftsZ gene was characterized at the genetic level. This study shows that ftsZsmeg
is an essential gene in that it can only be disrupted in a merodiploid background carrying another functional copy. Expression of ftsZsmeg
in M. smegmatis from a constitutively active mycobacterial promoter resulted in lethality whereas that from a chemically inducible acetamidase (ami) promoter led to FtsZ accumulation, filamentation and cell lysis. To further understand the roles of ftsZ in cell division a conditionally complementing ftsZsmeg
mutant strain was constructed in which ftsZ expression is controlled by acetamide. Growth in the presence of 0·2 % acetamide increased FtsZ levels approximately 1·4-fold, but did not decrease viability or change cell length. Withdrawal of acetamide reduced FtsZ levels, decreased viability, increased cell length and eventually lysed the cells. Finally, it is shown that ftsZsmeg
function in M. smegmatis can be replaced with the Mycobacterium tuberculosis counterpart, indicating that heterologous FtsZ
tb
can independently initiate the formation of Z-rings and catalyse the septation process. It is concluded that optimal levels of M. smegmatis FtsZ are required to sustain cell division and that the cell division initiation mechanisms are similar in mycobacteria.
A single V317A or V317M substitution in Enzyme II of a newly identified β-glucoside phosphotransferase and utilization system of Corynebacterium glutamicum R extends its specificity towards cellobiose
Authors: Kotrba Pavel , Inui Masayuki , Yukawa Hideaki
Published: June 2003

A catabolic system involved in the utilization ofβ-glucosides inCorynebacterium glutamicumR and its spontaneous mutant variants allowing uptake of cellobiose were investigated. The system comprises aβ-glucoside-specific Enzyme IIBCA component (genebglF) of the phosphotransferase system (PTS), a phospho-β-glucosidase (bglA) and an antiterminator protein (bglG) from the BglG/SacY family of transcription regulators. The results suggest that transcription antitermination is involved in control of induction and carbon catabolite repression ofbglgenes, which presumably form an operon. Functional analysis of thebglFandbglAproducts revealed that they are simultaneously required for uptake, phosphorylation and breakdown of methylβ-glucoside, salicin and arbutin. Although cellobiose is not normally a substrate for BglF permease and is not utilized byC. glutamicumR, cellobiose-utilizing mutants can be obtained. The mutation responsible was mapped to thebgllocus and sequenced, and point mutations were found in codon 317 ofbglF. These led to substitutions V317A and/or V317M near the putative PTS active-site H313 in the membrane-spanning IIC domain of BglF and allowed BglF to act on cellobiose. Such results strengthen the evidence that the IIC domains can be regarded as selectivity filters of the PTS.
Intra-chromosomal heterogeneity between the four 16S rRNA gene copies in the genus Veillonella: implications for phylogeny and taxonomy
Authors: Marchandin Hélène , Teyssier Corinne , Siméon de Buochberg Michèle , Jean-Pierre Hélène , Carriere Christian , Jumas-Bilak Estelle
Published: June 2003

Among the seven species characterized within the genus Veillonella, three (Veillonella dispar, Veillonella parvula and Veillonella atypica) have so far been isolated from human flora and during infectious processes. Sequencing and analysis of 16S rDNA (rrs) has been described as the best method for identification of Veillonella strains at the species level since phenotypic characteristics are unable to differentiate between species. rrs sequencing for the three species isolated from humans showed more than 98 % identity between them. Four rrs copies were found in the reference strains and in all the clinical isolates studied. The sequences of each rrs were determined for the clinical strain ADV 360.1, and they showed a relatively high level of heterogeneity (1·43 %). In the majority of cases, polymorphic positions corresponded to nucleotides allowing differentiation between the three species isolated from humans. Moreover, variability observed between rrs copies was higher than that between 16S rDNA sequences of V. parvula and V. dispar. Phylogenetic analysis showed that polymorphism between rrs copies affected the position of strain ADV 360.1 in the tree. Variable positions occurred in stems and loops belonging to variable and hypervariable regions of the 16S rRNA secondary structure but did not change the overall structure of the 16S rRNA. PCR-RFLP experiments performed on 27 clinical isolates of Veillonella sp. suggested that inter-rrs heterogeneity occurs widely among the members of the genus Veillonella. These results, together with the lack of phenotypic criteria for species differentiation, give preliminary arguments for unification of V. dispar and V. parvula.
Structural and functional features of Rhodococcus ruber lipoarabinomannan
Authors: Gibson Kevin J. C. , Gilleron Martine , Constant Patricia , Puzo Germain , Nigou Jérôme , Besra Gurdyal S.
Published: June 2003

The genusRhodococcusis part of the phylogenetic group nocardioform actinomycetes, which also includes the genusMycobacterium. Members of this phylogenetic group have a characteristic cell envelope structure, which is dominated by various complex lipids. Among these, lipoglycans are of particular interest since mycobacterial lipoarabinomannans are important immunomodulatory molecules that are likely to be involved in the subsequent fate of mycobacterial bacilli once inside phagocytic cells.Rhodococcus ruberis a species closely related to an established opportunistic human pathogen,Rhodococcus equi. This paper reports the isolation and characterization ofR. ruberlipoarabinomannan, designated as RruLAM. SDS-PAGE and gas chromatography analyses revealed that RruLAM was of an intermediate size betweenMycobacterium tuberculosislipoarabinomannan and lipomannan. Using a combination of chemical degradation and1H,13C-NMR experiments, the carbohydrate structure of RruLAM was unambiguously shown to be composed of a linear (α1→6)-Manpbackbone substituted at someO-2 positions by a single t-α-Arafsugar unit. Integration of the anomeric proton signals provided an indication of the degree of branching as approximately 45 %. The RruLAM structure is much simpler than that established forM. tuberculosislipoarabinomannan but is also different from that determined for the closely related species and opportunistic human pathogen,R. equi. RruLAM was unable to induce the production of TNF-αby either human or murine macrophage cell lines, suggesting that more sophisticated structures, such as phosphoinositol capping motifs, are required for such activity.
The THI5 gene family of Saccharomyces cerevisiae: distribution of homologues among the hemiascomycetes and functional redundancy in the aerobic biosynthesis of thiamin from pyridoxine
Authors: Wightman Raymond , Meacock Peter A.
Published: June 2003

TheTHI5gene family ofSaccharomyces cerevisiaecomprises four highly conserved members namedTHI5(YFL058w),THI11(YJR156c),THI12(YNL332w) andTHI13(YDL244w). Each gene copy is located within the subtelomeric region of a different chromosome and all are homologues of theSchizosaccharomyces pombenmt1gene which is thought to function in the biosynthesis of hydroxymethylpyrimidine (HMP), a precursor of vitamin B1, thiamin. A comprehensive phylogenetic study has shown that the existence ofTHI5as a gene family is exclusive to those yeasts of theSaccharomyces sensustrictosubgroup. To determine the function and redundancy of each of theS. cerevisiaehomologues, all combinations of the single, double, triple and quadruple deletion mutants were constructed using a PCR-mediated gene-disruption strategy. Phenotypic analyses of these mutant strains have shown the four genes to be functionally redundant in terms of HMP formation for thiamin biosynthesis; each promotes synthesis of HMP from the pyridoxine (vitamin B6) biosynthetic pathway. Furthermore, growth studies with the quadruple mutant strain support a previous proposal of an alternative HMP biosynthetic pathway that operates in yeast under anaerobic growth conditions. Comparative analysis of mRNA levels has revealed subtle differences in the regulation of the four genes, suggesting that they respond differently to nutrient limitation.
The senX3–regX3 two-component regulatory system of Mycobacterium tuberculosis is required for virulence
Authors: Parish Tanya , Smith Debbie A. , Roberts Gretta , Betts Joanna , Stoker Neil G.
Published: June 2003

Two-component regulatory systems have been widely implicated in bacterial virulence. To investigate the role of one such system in Mycobacterium tuberculosis, a strain was constructed in which the senX3–regX3 system was deleted by homologous recombination. The mutant strain (Tame15) showed a growth defect after infection of macrophages and was attenuated in both immunodeficient and immunocompetent mice. Competitive hybridization of total RNA from the wild-type and mutant strains to a whole-genome microarray was used to identify changes in gene expression resulting from the deletion. One operon was highly up-regulated in the mutant, indicating that regX3 probably has a role as a repressor of this operon. Other genes which were up- or down-regulated were also identified. Many of the genes showing down-regulation are involved in normal growth of the bacterium, indicating that the mutant strain is subject to some type of growth slow-down or stress. Genes showing differential expression were further grouped according to their pattern of gene expression under other stress conditions. From this analysis 50 genes were identified which are the most likely to be controlled by RegX3. Most of these genes are of unknown function and no obvious motifs were found upstream of the genes identified. Thus, it has been demonstrated that the senX3–regX3 two-component system is involved in the virulence of M. tuberculosis and a number of genes controlled by this system have been identified.
Arming the enemy: the evolution of resistance to self-proteins
Authors: Bell Graham , Gouyon Pierre-Henri
Published: June 2003

A remarkable range of novel antibiotics is attracting increasing interest as a major new weapon in the campaign against bacterial infection. They are based on the toxic peptides that provide the innate immune system of animals, and it is claimed that bacteria will be unable to evolve resistance to them because they attack the ‘Achilles' heel’ of bacterial membrane structure. Both experimental evidence and theoretical arguments suggest that this claim is doubtful. If so, the introduction of these substances into general use may provoke the evolution of resistance to our own defence proteins and thus compromise our natural defences against infection.
Multiple effects on Clostridium perfringens binding, uptake and trafficking to lysosomes by inhibitors of macrophage phagocytosis receptors
Authors: O'Brien David K. , Melville Stephen B.
Published: June 2003

Clostridium perfringensis a Gram-positive, anaerobic bacterium that is the most common cause of gas gangrene (clostridial myonecrosis) in humans.C. perfringensproduces a variety of extracellular toxins that are thought to be the major virulence factors of the organism. However,C. perfringenshas recently been shown to have the ability to survive in a murine macrophage-like cell line, J774-33, even under aerobic conditions. In J774-33 cells,C. perfringenscan escape the phagosome and gain access to the cytoplasm. Since the receptor that is used for phagocytosis can determine the fate of an intracellular bacterium, we used a variety of inhibitors of specific receptors to identify those used by J774-33 cells to phagocytoseC. perfringens. It was found that the scavenger receptor and mannose receptor(s) were involved in the phagocytosis ofC. perfringens. In the presence of complement, the complement receptor (CR3) was also involved in the binding and/or uptake ofC. perfringens. Since the receptor inhibition studies indicated that the scavenger receptor played a major role in phagocytosis,C. perfringensbinding studies were performed with a Chinese hamster ovary (CHO) cell line expressing the mouse SR-A receptor. The cell line expressing the SR-A receptor showed a significant increase inC. perfringensbinding in comparison to the non-transfected CHO cells. In the absence of opsonizing antibodies, the Fc receptor was not used to phagocytoseC. perfringens. Forcing the macrophages to use a specific receptor by using combinations of different receptor inhibitors led to only a slight increase in co-localization of intracellularC. perfringenswith the late endosome-lysosome marker LAMP-1. Carbohydrate analysis ofC. perfringensstrain 13 extracellular polysaccharide confirmed the presence of mannose and negatively charged residues of glucuronic acid, which may provide the moieties that promote binding to the mannose and scavenger receptors, respectively.
AhlD, an N-acylhomoserine lactonase in Arthrobacter sp., and predicted homologues in other bacteria
Authors: Park Sun-Yang , Lee Sang Jun , Oh Tae-Kwang , Oh Jong-Won , Koo Bon-Tag , Yum Do-Young , Lee Jung-Kee
Published: June 2003

Quorum sensing is a signalling mechanism that controls diverse biological functions, including virulence, via N-acylhomoserine lactone (AHL) signal molecules in Gram-negative bacteria. With the aim of isolating strains or enzymes capable of blocking quorum sensing by inactivating AHL, bacteria were screened for AHL degradation by their ability to utilize N-3-oxohexanoyl-l-homoserine lactone (OHHL) as the sole carbon source. Among four isolates, strain IBN110, identified as Arthrobacter sp., was found to grow rapidly on OHHL, and to degrade various AHLs with different lengths and acyl side-chain substitutions. Co-culture of Arthrobacter sp. IBN110 and the plant pathogen Erwinia carotovora significantly reduced both the AHL amount and pectate lyase activity in co-culture medium, suggesting the possibility of applying Arthrobacter sp. IBN110 in the control of AHL-producing pathogenic bacteria. The ahlD gene from Arthrobacter sp. IBN110 encoding the enzyme catalysing AHL degradation was cloned, and found to encode a protein of 273 amino acids. A mass spectrometry analysis showed that AhlD probably hydrolyses the lactone ring of N-3-hexanoyl-l-homoserine lactone, indicating that AhlD is an N-acylhomoserine lactonase (AHLase). A comparison of AhlD with other known AHL-degrading enzymes, Bacillus sp. 240B1 AiiA, a Bacillus thuringiensis subsp. kyushuensis AiiA homologue and Agrobacterium tumefaciens AttM, revealed 25, 26 and 21 % overall identities, respectively, in the deduced amino acid sequences. Although these identities were relatively low, the HXDH≈H≈D motif was conserved in all the AHLases, suggesting that this motif is essential for AHLase activity. From a genome database search based on the conserved motif, putative AhlD-like lactonase genes were found in several other bacteria, and AHL-degrading activities were observed in Klebsiella pneumoniae and Bacillus stearothermophilus. Furthermore, it was verified that ahlK, an ahlD homologue, encodes an AHL-degrading enzyme in K. pneumoniae. Accordingly, the current results suggest the possibility that AhlD-like AHLases could exist in many other micro-organisms.
Lipoprotein signal peptidase of Streptococcus suis serotype 2
Authors: de Greeff Astrid , Hamilton Andrea , Sutcliffe Iain C. , Buys Herma , van Alphen Loek , Smith Hilde E.
Published: June 2003

This paper reports the complete coding sequence for a proliprotein signal peptidase (SP-ase) ofStreptococcus suis, Lsp. This is believed to be the first SP-ase described forS. suis. SP-ase II is involved in the removal of the signal peptide from glyceride-modified prolipoproteins. By usingin vitrotranscription/translation systems, it was shown that thelspgene was transcribedin vitro. Functionality of Lsp inEscherichia coliwas demonstrated by using anin vitroglobomycin resistance assay, to show that expression of Lsp inE. coliincreased the globomycin resistance. An isogenic mutant ofS. suisserotype 2 unable to produce Lsp was constructed and shown to process lipoproteins incorrectly, including anS. suishomologue of the pneumococcal PsaA lipoprotein. Five piglets were inoculated with a mixture of both strains in an experimental infection, to determine the virulence of the mutant strain relative to that of the wild-type strain in a competitive challenge experiment. The data showed that both strains were equally virulent, indicating that the knockout mutant oflspis not attenuatedin vivo.
Yeast Enhancer of Polycomb defines global Esa1-dependent acetylation of chromatin
Authors: Boudreault Alexandre A. , Cronier Dominique , Selleck William , Lacoste Nicolas , Utley Rhea T. , Allard Stéphane , Savard Julie , Lane William S. , Tan Song , Côté Jacques
Published: June 2003

Drosophila Enhancer of Polycomb, E(Pc), is a suppressor of position-effect variegation and an enhancer of both Polycomb and trithorax mutations. A homologous yeast protein, Epl1, is a subunit of the NuA4 histone acetyltransferase complex. Epl1 depletion causes cells to accumulate in G2/M and global loss of acetylated histones H4 and H2A. In relation to theDrosophilaprotein, mutation of Epl1 suppresses gene silencing by telomere position effect. Epl1 protein is found in the NuA4 complex and a novel highly active smaller complex named Piccolo NuA4 (picNuA4). The picNuA4 complex contains Esa1, Epl1, and Yng2 as subunits and strongly prefers chromatin over free histones as substrate. Epl1 conserved N-terminal domain bridges Esa1 and Yng2 together, stimulating Esa1 catalytic activity and enabling acetylation of chromatin substrates. A recombinant picNuA4 complex shows characteristics similar to the native complex, including strong chromatin preference. Cells expressing only the N-terminal half of Epl1 lack NuA4 HAT activity, but possess picNuA4 complex and activity. These results indicate that the essential aspect of Esa1 and Epl1 resides in picNuA4 function. We propose that picNuA4 represents a nontargeted histone H4/H2A acetyltransferase activity responsible for global acetylation, whereas the NuA4 complex is recruited to specific genomic loci to perturb locally the dynamic acetylation/deacetylation equilibrium.
Localized recruitment of a chromatin-remodeling activity by an activator in vivo drives transcriptional elongation
Authors: Corey Laura L. , Weirich Christine S. , Benjamin Ivor J. , Kingston Robert E.
Published: June 2003

To understand the role of chromatin-remodeling activities in transcription, it is necessary to understand how they interact with transcriptional activators in vivo to regulate the different steps of transcription. Human heat shock factor 1 (HSF1) stimulates both transcriptional initiation and elongation. We replaced mouse HSF1 in fibroblasts with wild-type and mutant human HSF1 constructs and characterized regulation of an endogenous mousehsp70gene. A mutation that diminished transcriptional initiation led to twofold reductions inhsp70mRNA induction and recruitment of a SWI/SNF remodeling complex. In contrast, a mutation that diminished transcriptional elongation abolished induction of full-length mRNA, SWI/SNF recruitment, and chromatin remodeling, but minimally impaired initiation from thehsp70promoter. Another remodeling factor, SNF2h, is constitutively present at the promoter irrespective of the genotype of HSF1. These data suggest that localized recruitment of SWI/SNF drives a specialized remodeling reaction necessary for the production of full-lengthhsp70mRNA.
Generation ofSki-knockdown mice by expressing a long double-strand RNA from an RNA polymerase II promoter
Authors: Shinagawa Toshie , Ishii Shunsuke
Published: June 2003

We have developed a new vector, named pDECAP, to express long double-strand RNA (ds-RNA) from an RNA polymerase II (Pol II) promoter. Because the transcripts from pDECAP lack both the 5′-cap structure and the 3′-poly(A) tail that facilitate ds-RNA export to the cytoplasm, long ds-RNA from pDECAP does not induce the interferon response. Transgenic mice embryos expressing long ds-RNA for the transcriptional corepressor Ski from this vector exhibited phenotypes that were remarkably similar to those ofSki-deficient embryos, including defects of neural tube closure and eye formation. Thus, this vector provides a new tool to efficiently generate tissue-specific knockdown mice for studying gene function in whole animal systems.
TRF1 is degraded by ubiquitin-mediated proteolysis after release from telomeres
Authors: Chang William , Dynek Jasmin N. , Smith Susan
Published: June 2003

Mammalian telomeres are coated by the sequence-specific, DNA-binding protein, TRF1, a negative regulator of telomere length. Previous results showed that ADP-ribosylation of TRF1 by tankyrase 1 released TRF1 from telomeres and promoted telomere elongation. We now show that loss of TRF1 from telomeres results in ubiquitination and degradation of TRF1 by the proteasome and that degradation is required to keep TRF1 off telomeres. Ubiquitination of TRF1 is regulated by its telomere-binding status; only the telomere-unbound form of TRF1 is ubiquitinated. Our findings suggest a novel mechanism of sequential post translational modification of TRF1 (ADP-ribosylation and ubiquitination) for regulating access of telomerase to telomeres.
Local spreading of MSL complexes fromroXgenes on theDrosophilaX chromosome
Authors: Oh Hyangyee , Park Yongkyu , Kuroda Mitzi I.
Published: June 2003

MSL proteins and noncodingroXRNAs form complexes to up-regulate hundreds of genes on theDrosophilamale X chromosome, and make X-linked gene expression equal in males and females. Altering the ratio of MSL proteins toroXRNA dramatically changes X-chromosome morphology. In protein excess, the MSL complex concentrates near sites ofroXtranscription and is depleted elsewhere. These results support a model for distribution of MSL complexes, in which local spreading incisfromroXgenes is balanced with diffusion of soluble complexes intrans. When overexpressed, MSL proteins can recognize the X chromosome, modify histones, and partially restore male viability even in the absence ofroXRNAs. Thus, the protein components can carry out all essential functions of dosage compensation, butroXRNAs facilitate the correct targeting of MSL complexes, in part by nucleation of spreading from their sites of synthesis.