bluuebunny/crossref_metadata_2025 huggingface dataset:
Competition among fishermen and fish causes the collapse of Barents Sea capelin
Authors: Hjermann Dag Ø , Ottersen Geir , Stenseth Nils Chr.
Published: July 2004

The vast majority of the world's fisheries are typically managed within a single-species perspective, ignoring the dynamic feedback mechanisms generated by the ecological web of which they are a part. Here we show that the dynamics of the Barents Sea capelin (
Mallotus villosus
), the world's largest stock of this species, is strongly influenced by both within-system ecological feedback mechanisms and the impact of harvesting. Both overexploitation and predation by herring (
Clupea harengus
) can cause the population to collapse, whereas predation by cod (
Gadus morhua
) is demonstrated a delay in the stock's recovery after a collapse. Such collapses, which have occurred twice in 20 years, affect the entire Barents Sea ecosystem, a region that for ages has provided food for all of Europe.
A eukaryotic BLUF domain mediates light-dependent gene expression in the purple bacterium
Rhodobacter sphaeroides
2.4.1
Authors: Han Yuchen , Braatsch Stephan , Osterloh Lisa , Klug Gabriele
Published: August 2004

The flavin-binding BLUF domain functions as a blue-light receptor in eukaryotes and bacteria. In the photoreceptor protein photo-activated adenylyl cyclase (PAC) from the flagellate
Euglena gracilis
, the BLUF domain is linked to an adenylyl cyclase domain. The PAC protein mediates a photophobic response. In the AppA protein of
Rhodobacter sphaeroides
, the BLUF domain is linked to a downstream domain without similarity to known proteins. AppA functions as a transcriptional antirepressor, controlling photosynthesis gene expression in the purple bacterium
R. sphaeroides
in response to light and oxygen. We fused the PACα1-BLUF domain from
Euglena
to the C terminus of AppA. Our results show that the hybrid protein is fully functional in light-dependent gene repression in
R. sphaeroides
, despite only ≈30% identity between the eukaryotic and the bacterial BLUF domains. Furthermore, the bacterial BLUF domain and the C terminus of AppA can transmit the light signal even when expressed as separated domains. This finding implies that the BLUF domain is fully modular and can relay signals to completely different output domains.
Modified amino acid copolymers suppress myelin basic protein 85–99-induced encephalomyelitis in humanized mice through different effects on T cells
Authors: Illés Zsolt , Stern Joel N. H. , Reddy Jayagopala , Waldner Hanspeter , Mycko Marcin P. , Brosnan Celia F. , Ellmerich Stephan , Altmann Daniel M. , Santambrogio Laura , Strominger Jack L. , Kuchroo Vijay K.
Published: August 2004

A humanized mouse bearing the HLA-DR2 (DRA/DRB1*1501) protein associated with multiple sclerosis (MS) and the myelin basic protein (MBP) 85–99-specific HLA-DR2-restricted T cell receptor from an MS patient has been used to examine the effectiveness of modified amino acid copolymers poly(F,Y,A,K)n and poly-(V,W,A,K)n in therapy of MBP 85–99-induced experimental autoimmune encephalomyelitis (EAE) in comparison to Copolymer 1 [Copaxone, poly(Y,E,A,K)n]. The copolymers were designed to optimize binding to HLA-DR2. Vaccination, prevention, and treatment of MBP-induced EAE in the humanized mice with copolymers FYAK and VWAK ameliorated EAE more effectively than Copolymer 1, reduced the number of pathological lesions, and prevented the up-regulation of human HLA-DR on CNS microglia. Moreover, VWAK inhibited MBP 85–99-specific T cell proliferation more efficiently than either FYAK or Copolymer 1 and induced anergy of HLA-DR2-restricted transgenic T cells as its principle mechanism. In contrast, FYAK induced proliferation and a pronounced production of the antiinflammatory T helper 2 cytokines IL-4 and IL-10 from nontransgenic T cells as its principle mechanism of immunosuppression. Thus, copolymers generated by using different amino acids inhibited disease using different mechanisms to regulate T cell responses.
Direct observation of RuvAB-catalyzed branch migration of single Holliday junctions
Authors: Amit Roee , Gileadi Opher , Stavans Joel
Published: August 2004

Holliday junctions form during DNA repair and homologous recombination processes. These processes entail branch migration, whereby the length of two arms of a cruciform increases at the expense of the two others. Branch migration is carried out in prokaryotic cells by the RuvAB motor complex. We study RuvAB-catalyzed branch migration by following the motion of a small paramagnetic bead tethered to a surface by two opposing arms of a single cruciform. The bead, pulled under the action of magnetic tweezers, exerts tension on the cruciform, which in turn transmits the force to a single RuvAB complex bound at the crossover point. This setup provides a unique means of measuring several kinetic parameters of interest such as the translocation rate, the processivity, and the force on the substrate against which the RuvAB complex cannot effect translocation. RuvAB-catalyzed branch migration proceeds with a small, discrete number of rates, supporting the view that the monomers comprising the RuvB hexameric rings are not functionally homogeneous and that dimers or trimers constitute the active subunits. The most frequently encountered rate, 98 ± 3 bp/sec, is approximately five times faster than previously estimated. The apparent processivity of branch migration between pauses of inactivity is ≈7,000 bp. Branch migration persists against opposing forces up to 23 pN.
Use of sequence duplication to engineer a ligand-triggered, long-distance molecular switch in T4 lysozyme
Authors: Yousef Mohammad S. , Baase Walter A. , Matthews Brian W.
Published: July 2004

We have designed a molecular switch in a T4 lysozyme construct that controls a large-scale translation of a duplicated helix. As shown by crystal structures of the construct with the switch on and off, the conformational change is triggered by the binding of a ligand (guanidinium ion) to a site that in the wild-type protein was occupied by the guanidino head group of an Arg. In the design template, a duplicated helix is flanked by two loop regions of different stabilities. In the “on” state, the N-terminal loop is weakly structured, whereas the C-terminal loop has a well defined conformation that is stabilized by means of nonbonded interactions with the Arg head group. The truncation of the Arg to Ala destabilizes this loop and switches the protein to the “off” state, in which the duplicated helix is translocated ≈20 Å. Guanidinium binding restores the key interactions, restabilizes the C-terminal loop, and restores the “on” state. Thus, the presence of an external ligand, which is unrelated to the catalytic activity of the enzyme, triggers the inserted helix to translate 20 Å away from the binding site. The results illustrate a proposed mechanism for protein evolution in which sequence duplication followed by point mutation can lead to the establishment of new function.
Resolution of organelle docking and fusion kinetics in a cell-free assay
Authors: Merz Alexey J. , Wickner William T.
Published: July 2004

In vitro
assays of compartment mixing have been key tools in the biochemical dissection of organelle docking and fusion. Many such assays measure compartment mixing through the enzymatic modification of reporter proteins. Homotypic fusion of yeast vacuoles is measured with a coupled assay of proteolytic maturation of pro-alkaline phosphatase (pro-ALP). A kinetic lag is observed between the end of docking, marked by the acquisition of resistance to anti-SNARE reagents, and ALP maturation. We therefore asked whether the time taken for pro-ALP maturation adds a kinetic lag to the measured fusion signal. Prb1p promotes ALP maturation; overproduction of Prb1p accelerates ALP activation in detergent lysates but does not alter the measured kinetics of docking or fusion. Thus, the lag between docking and ALP activation reflects a lag between docking and fusion. Many vacuoles in the population undergo multiple rounds of fusion; methods are presented for distinguishing the first round of fusion from ongoing rounds of fusion. A simple kinetic model distinguishes between two rates, the rate of fusion and the rate at which fusion competence is lost, and allows estimation of the number of rounds of fusion completed.
The Changing Nature of Adult Entertainment Districts: Between a Rock and a Hard Place or Going from Strength to Strength?
Authors: Ryder Andrew
Published: August 2004

Civic leaders and urban governments have tried to control adult entertainment activities with a variety of 'command-and-control' techniques, including vice laws, licensing, zoning and land use planning powers. However, despite persistent efforts, they have failed to eliminate such activities. Recently, civic authorities appear to have had some success in closing down or down-sizing adult entertainment districts in many cities, but this success is more apparent than real. Rather than attempting to legislate such activities out of existence, planners and law-makers might consider what factors draw 'vice' to certain areas and try to change those underlying conditions. Adult activities are suburbanising, and face growing competition from the Internet and other on-line services. This trend has been hastened by recent population growth in many cities, leading to increased rents and the expansion of residential and office districts into areas in which adult uses were concentrated. Adult entertainment districts are a retail service cluster, akin to antiques districts or other specialist districts. In addition, in recent years, adult entertainment has been redefined as a range of activities have become legitimate and the number of adult outlets has actually increased.
Cleansing the Metropolis: Sex Work and the Politics of Zero Tolerance
Authors: Hubbard Phil
Published: August 2004

Although red-light districts have long been characteristic features of Western cities, these spaces are periodically subject to forms of moral cleansing and purification enacted by the state and law. Focusing on central Paris and the West End of London, this paper describes recent 'Zero Tolerance' policies designed to displace sex work from such spaces. Exploring the motives for such actions, the paper argues that policy-makers are seeking to demonstrate their ability to assert moral order by reclaiming red-light districts from sex workers. Simultaneously, it is suggested that this process of 'purification' is intended to maximise the potential for capital accumulation in city centres via the promotion of family-oriented gentrification. The paper concludes by thinking through the implications of this, posing the question: is it right that sex workers are being excluded from our city centres?
The Risks of Street Prostitution: Punters, Police and Protesters
Authors: Sanders Teela
Published: August 2004

For female street sex workers in Britain, selling sex means managing risks. Violence from male clients, harassment from community protesters and criminalisation through overpolicing are daily hazards on the street. Using qualitative data and extensive field observations of the street market in Birmingham, UK, it is argued in this paper that street sex workers do not passively accept these risks but, instead, manage occupational hazards by manipulating, separating, controlling and resisting urban spaces. Women actively use space to inform their collective and individual working practices to minimise harm and maximise profits. However, the findings conclude that sites of street prostitution are made increasingly dangerous for women through punitive policing policies, conservative heterosexual discourses and a lack of realistic prostitution policy that addresses the central issues relating to commercial sex.
Homosexuality and the City: An Historical Overview
Authors: Aldrich Robert
Published: August 2004

Ever since the time of ancient Athens and the Biblical Sodom and Gomorrah, homosexuality has been associated with the city. Historians over the past decade have chronicled urban gay and lesbian groups in Renaissance Italy, Enlightenment France and Britain, and modern America and Australia, and social scientists have also identified emerging gay communities in Asia. Their researches show how homosexuals formed urban networks of sociability and solidarity, and how the presence of such minority communities impacted on urban development from Castro Street to Soho. Homosexuals often moved to cities to escape the sexual and social constraints of traditional life, and they played a major role in transforming the city and in creating a particular urban ethos. The city, in turn, is the site for the construction of much contemporary gay and lesbian culture.
Sexual Dissidence, Enterprise and Assimilation: Bedfellows in Urban Regeneration
Authors: Collins Alan
Published: August 2004

This paper offers a pragmatic, principally economic perspective on the body of work analysing the genesis and development of urban 'gay villages'. The Soho Gay Village in central London is presented as a case study. Its evolution and principal features are considered in the light of the existing corpus of research into gay agglomerations and the documented experiences of some other urban gay villages in England. It is suggested that, even with differing historical roots and widely differing levels and forms of municipal support, a recurrent developmental pattern seems to be discernible. This is characterised by an urban area in decline progressing through several broad stages of economic enterprise denoted by: sexual and legal liminality; gay male social and recreational opportunities; a widening service-sector business base; and, ultimately, the assimilation of the area into the fashionable mainstream.
Sex and Not the City? The Aspirations of the Thirty-something Working Woman
Authors: Brewis Joanna
Published: August 2004

In addressing the 'sex and the city' theme, this paper begins by suggesting that British women's lives have altered substantially since the late 1960s, given their rapidly increasing uptake of paid employment ('the city') and several important changes in their personal lives ('sex'). Moreover, 'the city' in its more literal sense of urban expanse is growing in world-wide importance. Other factors such as changing cultural representations of female urbanite professionals also point to it being an interesting juncture at which to explore this group's experiences. The paper therefore analyses female respondents' accounts of their relationships, their careers and their lives in London. It seeks to contribute to debates concerning urban (dis)content, liberal feminism and life-stages by connecting these stories to the relevant literatures.
Queer as Folk: Producing the Real of Urban Space
Authors: Skeggs Beverley , Moran Leslie , Tyrer Paul , Binnie Jon
Published: August 2004

This paper is about the construction of space and place in the urban imaginary. It draws on multidisciplinary empirical research—on Violence, Sexuality and Space—conducted in a place that was simultaneously represented in one of the most controversial programmes ever to be screened on British television— Queer as Folk. As the television programme intervened in the responses from the interviews and focus groups, we begin, through an exploration of spatial referents and characterisation, to explore the `authentic' underpinning for the queer imaginary. We examine the sexual politics of place and space as a politics of verisimilitude, affectivity and political claims-making.
Active Rho is localized to podosomes induced by oncogenic Src and is required for their assembly and function
Authors: Berdeaux Rebecca L. , Díaz Begoña , Kim Lomi , Martin G. Steven
Published: August 2004

Transformation of fibroblasts by oncogenic Src causes disruption of actin stress fibers and formation of invasive adhesions called podosomes. Because the small GTPase Rho stimulates stress fiber formation, Rho inactivation by Src has been thought to be necessary for stress fiber disruption. However, we show here that Rho[GTP] levels do not decrease after transformation by activated Src. Inactivation of Rho in Src-transformed fibroblasts by dominant negative RhoA or the Rho-specific inhibitor C3 exoenzyme disrupted podosome structure as judged by localization of podosome components F-actin, cortactin, and Fish. Inhibition of Rho strongly inhibited Src-induced proteolytic degradation of the extracellular matrix. Furthermore, development of an in situ Rho[GTP] affinity assay allowed us to detect endogenous Rho[GTP] at podosomes, where it colocalized with F-actin, cortactin, and Fish. Therefore, Rho is not globally inactivated in Src-transformed fibroblasts, but is necessary for the assembly and function of structures implicated in tumor cell invasion.
Differential transactivation of sphingosine-1-phosphate receptors modulates NGF-induced neurite extension
Authors: Toman Rachelle E. , Payne Shawn G. , Watterson Kenneth R. , Maceyka Michael , Lee Norman H. , Milstien Sheldon , Bigbee John W. , Spiegel Sarah
Published: August 2004

The process of neurite extension after activation of the TrkA tyrosine kinase receptor by nerve growth factor (NGF) involves complex signaling pathways. Stimulation of sphingosine kinase 1 (SphK1), the enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P), is part of the functional TrkA signaling repertoire. In this paper, we report that in PC12 cells and dorsal root ganglion neurons, NGF translocates SphK1 to the plasma membrane and differentially activates the S1P receptors S1P1 and S1P2 in a SphK1-dependent manner, as determined with specific inhibitors and small interfering RNA targeted to SphK1. NGF-induced neurite extension was suppressed by down-regulation of S1P1 expression with antisense RNA. Conversely, when overexpressed in PC12 cells, transactivation of S1P1 by NGF markedly enhanced neurite extension and stimulation of the small GTPase Rac, important for the cytoskeletal changes required for neurite extension. Concomitantly, differentiation down-regulated expression of S1P2 whose activation would stimulate Rho and inhibit neurite extension. Thus, differential transactivation of S1P receptors by NGF regulates antagonistic signaling pathways that modulate neurite extension.
Selective modulation of type 1 insulin-like growth factor receptor signaling and functions by β1 integrins
Authors: Goel Hira Lal , Fornaro Mara , Moro Loredana , Teider Natalia , Rhim Johng S. , King Michael , Languino Lucia R.
Published: August 2004

We show here that β1 integrins selectively modulate insulin-like growth factor type I receptor (IGF-IR) signaling in response to IGF stimulation. The β1A integrin forms a complex with the IGF-IR and insulin receptor substrate-1 (IRS-1); this complex does not promote IGF-I mediated cell adhesion to laminin (LN), although it does support IGF-mediated cell proliferation. In contrast, β1C, an integrin cytoplasmic variant, increases cell adhesion to LN in response to IGF-I and its down-regulation by a ribozyme prevents IGF-mediated adhesion to LN. Moreover, β1C completely prevents IGF-mediated cell proliferation and tumor growth by inhibiting IGF-IR auto-phosphorylation in response to IGF-I stimulation. Evidence is provided that the β1 cytodomain plays an important role in mediating β1 integrin association with either IRS-1 or Grb2-associated binder1 (Gab1)/SH2-containing protein-tyrosine phosphate 2 (Shp2), downstream effectors of IGF-IR: specifically, β1A associates with IRS-1 and β1C with Gab1/Shp2. This study unravels a novel mechanism mediated by the integrin cytoplasmic domain that differentially regulates cell adhesion to LN and cell proliferation in response to IGF.
Yeast cell death during DNA damage arrest is independent of caspase or reactive oxygen species
Authors: Wysocki Robert , Kron Stephen J.
Published: August 2004

CDC13 encodes a telomere-binding protein that prevents degradation of telomeres. cdc13-1 yeast grown at the nonpermissive temperature undergo G2/M arrest, progressive chromosome instability, and subsequent cell death. Recently, it has been suggested that cell death in the cdc13-1 mutant is an active process characterized by phenotypic hallmarks of apoptosis and caspase activation. In this work, we show that cell death triggered by cdc13-1 is independent of the yeast metacaspase Yca1p and reactive oxygen species but related to cell cycle arrest per se. Inactivating YCA1 or depleting reactive oxygen species does not increase viability of cdc13-1 cells. In turn, caspase activation does not precede cell death in the cdc13-1 mutant. Yca1p activity assayed by cell binding of mammalian caspase inhibitors is confounded by artifactual labeling of dead yeast cells, which nonspecifically bind fluorochromes. We speculate that during a prolonged cell cycle arrest, cdc13-1 cells reach a critical size and die by cell lysis.
Early Growth Response Gene 1–mediated Apoptosis Is Essential for Transforming Growth Factor β1–induced Pulmonary Fibrosis
Authors: Lee Chun Geun , Cho Soo Jung , Kang Min Jong , Chapoval Svetlana P. , Lee Patty J. , Noble Paul W. , Yehualaeshet Teshome , Lu Binfeng , Flavell Richard A. , Milbrandt Jeffrey , Homer Robert J. , Elias Jack A.
Published: August 2004

Fibrosis and apoptosis are juxtaposed in pulmonary disorders such as asthma and the interstitial diseases, and transforming growth factor (TGF)-β1 has been implicated in the pathogenesis of these responses. However, the in vivo effector functions of TGF-β1 in the lung and its roles in the pathogenesis of these responses are not completely understood. In addition, the relationships between apoptosis and other TGF-β1–induced responses have not been defined. To address these issues, we targeted bioactive TGF-β1 to the murine lung using a novel externally regulatable, triple transgenic system. TGF-β1 produced a transient wave of epithelial apoptosis that was followed by mononuclear-rich inflammation, tissue fibrosis, myofibroblast and myocyte hyperplasia, and septal rupture with honeycombing. Studies of these mice highlighted the reversibility of this fibrotic response. They also demonstrated that a null mutation of early growth response gene (Egr)-1 or caspase inhibition blocked TGF-β1–induced apoptosis. Interestingly, both interventions markedly ameliorated TGF-β1–induced fibrosis and alveolar remodeling. These studies illustrate the complex effects of TGF-β1 in vivo and define the critical role of Egr-1 in the TGF-β1 phenotype. They also demonstrate that Egr-1–mediated apoptosis is a prerequisite for TGF-β1–induced fibrosis and remodeling.
A Contribution of Mouse Dendritic Cell–Derived IL-2 for NK Cell Activation
Authors: Granucci Francesca , Zanoni Ivan , Pavelka Norman , van Dommelen Serani L.H. , Andoniou Christopher E. , Belardelli Filippo , Degli Esposti Mariapia A. , Ricciardi-Castagnoli Paola
Published: August 2004

Dendritic cells (DCs) play a predominant role in activation of natural killer (NK) cells that exert their functions against pathogen-infected and tumor cells. Here, we used a murine model to investigate the molecular mechanisms responsible for this process. Two soluble molecules produced by bacterially activated myeloid DCs are required for optimal priming of NK cells. Type I interferons (IFNs) promote the cytotoxic functions of NK cells. IL-2 is necessary both in vitro and in vivo for the efficient production of IFNγ, which has an important antimetastatic and antibacterial function. These findings provide new information about the mechanisms that mediate DC–NK cell interactions and define a novel and fundamental role for IL-2 in innate immunity.
Limited T Cell Receptor Diversity of HCV-specific T Cell Responses Is Associated with CTL Escape
Authors: Meyer-Olson Dirk , Shoukry Naglaa H. , Brady Kristen W. , Kim Helen , Olson Douglas P. , Hartman Kelly , Shintani Ayumi K. , Walker Christopher M. , Kalams Spyros A.
Published: August 2004

Escape mutations are believed to be important contributors to immune evasion by rapidly evolving viruses such as hepatitis C virus (HCV). We show that the majority of HCV-specific cytotoxic T lymphocyte (CTL) responses directed against viral epitopes that escaped immune recognition in HCV-infected chimpanzees displayed a reduced CDR3 amino acid diversity when compared with responses in which no CTL epitope variation was detected during chronic infection or with those associated with protective immunity. Decreased T cell receptor (TCR) CDR3 amino acid diversity in chronic infection could be detected long before the appearance of viral escape mutations in the plasma. In both chronic and resolved infection, identical T cell receptor clonotypes were present in liver and peripheral blood. These findings provide a deeper understanding of the evolution of CTL epitope variations in chronic viral infections and highlight the importance of the generation and maintenance of a diverse TCR repertoire directed against individual epitopes.
A new inequality for superdiffusions and its applications to nonlinear differential equations
Authors: Dynkin E.
Published: August 2004

Our motivation is the following problem: to describe all positive solutions of a semilinear elliptic equation

L
u
=

u
α

L u=u^\alpha

with

α
>
1

\alpha >1

in a bounded smooth domain

E
⊂

R

d

E\subset \mathbb {R}^d

. In 1998 Dynkin and Kuznetsov solved this problem for a class of solutions which they called

σ
\sigma

-moderate. The question if all solutions belong to this class remained open. In 2002 Mselati proved that this is true for the equation

Δ
u
=

u
2

\Delta u=u^2

in a domain of class

C
4

C^4

. His principal tool—the Brownian snake—is not applicable to the case

α
≠
2

\alpha \neq 2

. In 2003 Dynkin and Kuznetsov modified most of Mselati’s arguments by using superdiffusions instead of the snake. However a critical gap remained. A new inequality established in the present paper allows us to close this gap.
Bone Marrow Stromal Cells, Preadipocytes, and Dermal Fibroblasts Promote Epidermal Regeneration in Their Distinctive Fashions
Authors: Aoki Shigehisa , Toda Shuji , Ando Takashi , Sugihara Hajime
Published: August 2004

Mesenchymal cell types, under mesenchymal-epithelial interaction, are involved in tissue regeneration. Here we show that bone marrow stromal cells (BMSCs), subcutaneous preadipocytes, and dermal fibroblasts distinctively caused keratinocytes to promote epidermal regeneration, using a skin reconstruction model by their coculture with keratinocytes. Three mesenchymal cell types promoted the survival, growth, and differentiation of keratinocytes, whereas BMSCs and preadipocytes inhibited their apoptosis. BMSCs and preadipocytes induced keratinocytes to reorganize rete ridge- and epidermal ridge-like structures, respectively. Keratinocytes with fibroblasts or BMSCs expressed the greatest amount of interleukin (IL)-1α protein, which is critical for mesenchymal-epithelial cross-talk in skin. Keratinocytes with or without three mesenchymal supports displayed another cross-talk molecule, c-Jun protein. Without direct mesenchymal-epithelial contact, the rete ridge- and epidermal ridge-like structures were not replicated, whereas the other phenomena noted above were. DNA microarray analysis showed that the mesenchymal-epithelial interaction affected various gene expressions of keratinocytes and mesenchymal cell types. Our results suggest that not only skin-localized fibroblasts and preadipocytes but also BMSCs accelerate epidermal regeneration in complexes and that direct contact between keratinocytes and BMSCs or preadipocytes is required for the skin-specific morphogenesis above, through mechanisms that differ from the IL-1α/c-Jun pathway.
Elevated Endosomal Cholesterol Levels in Niemann-Pick Cells Inhibit Rab4 and Perturb Membrane Recycling
Authors: Choudhury Amit , Sharma Deepak K. , Marks David L. , Pagano Richard E.
Published: August 2004

In normal human skin fibroblasts (HSFs), fluorescent glycosphingolipid analogues are endocytosed and sorted into two pools, one that is recycled to the plasma membrane and one that is transported to the Golgi complex. Here, we investigated glycosphingolipid recycling in Niemann-Pick type A and C lipid storage disease fibroblasts (NPFs). Cells were incubated with a fluorescent analogue of lactosylceramide (LacCer) at 16°C to label early endosomes (EEs), shifted to 37°C, and lipid recycling was quantified. Using dominant negative rabs, we showed that, in normal HSFs, LacCer recycling was rapid (t1/2∼8 min) and mainly rab4-dependent. In NPFs, LacCer recycling was delayed (t1/2∼30–40 min), and rab4-dependent recycling was absent, whereas rab11-dependent recycling predominated. Transferrin recycling via the rab4 pathway was similarly perturbed in NPFs. Compared with normal HSFs, EEs in NPFs showed high cholesterol levels and an altered organization of rab4. In vitro extraction of rab4 (but not rab11) with GDP dissociation inhibitor was severely attenuated in NPF endosomal fractions. This impairment was reversed with cholesterol depletion of isolated endosomes or with high-salt treatment of endosomes. These data suggest that abnormal membrane recycling in NPFs results from specific inhibition of rab4 function by excess cholesterol in EEs.
The Fusome Mediates Intercellular Endoplasmic Reticulum Connectivity inDrosophilaOvarian Cysts
Authors: Snapp Erik L. , Iida Takako , Frescas David , Lippincott-Schwartz Jennifer , Lilly Mary A.
Published: August 2004

Drosophila ovarian cysts arise through a series of four synchronous incomplete mitotic divisions. After each round of mitosis, a membranous organelle, the fusome, grows along the cleavage furrow and the remnants of the mitotic spindle to connect all cystocytes in a cyst. The fusome is essential for the pattern and synchrony of the mitotic cyst divisions as well as oocyte differentiation. Using live cell imaging, greenfluorescent protein–tagged proteins, and photobleaching techniques, we demonstrate that fusomal endomembranes are part of a single continuous endoplasmic reticulum (ER) that is shared by all cystocytes in dividing ovarian cysts. Membrane and lumenal proteins of the common ER freely and rapidly diffuse between cystocytes. The fusomal ER mediates intercellular ER connectivity by linking the cytoplasmic ER membranes of all cystocytes within a cyst. Before entry into meiosis and onset of oocyte differentiation (between region 1 and region 2A), ER continuity between cystocytes is lost. Furthermore, analyses of hts and Dhc64c mutants indicate that intercellular ER continuity within dividing ovarian cysts requires the fusome cytoskeletal component and suggest a possible role for the common ER in synchronizing mitotic cyst divisions.
Characterization of vaginal microbial communities in adult healthy women using cultivation-independent methods
Authors: Zhou Xia , Bent Stephen J. , Schneider Maria G. , Davis Catherine C. , Islam Mohammed R. , Forney Larry J.
Published: August 2004

The normal microbial flora of the vagina plays an important role in preventing genital and urinary tract infections in women. Thus an accurate understanding of the composition and ecology of the ecosystem is important to understanding the aetiology of these diseases. Common wisdom is that lactobacilli dominate the normal vaginal microflora of post-pubertal women. However, this conclusion is based on methods that require cultivation of microbial populations; an approach that is known to yield a biased and incomplete assessment of microbial community structure. In this study cultivation-independent methods were used to analyse samples collected from the mid-vagina of five normal healthy Caucasian women between the ages of 28 and 44. Total microbial community DNA was isolated following resuspension of microbial cells from vaginal swabs. To identify the constituent numerically dominant populations in each community 16S rRNA gene libraries were prepared following PCR amplification using the 8f and 926r primers. From each library, the DNA sequences of approximately 200 16S rRNA clones were determined and subjected to phylogenetic analyses. The diversity and kinds of organisms that comprise the vaginal microbial community varied among women. Species ofLactobacillusappeared to dominate the communities in four of the five women. However, the community of one woman was dominated byAtopobiumsp., whereas a second woman had appreciable numbers ofMegasphaerasp.,Atopobiumsp. andLeptotrichiasp., none of which have previously been shown to be common members of the vaginal ecosystem. Of the women whose communities were dominated by lactobacilli, there were two distinct clusters, each of which consisted of a single species. One class consisted of two women with genetically divergent clones that were related toLactobacillus crispatus, whereas the second group of two women had clones ofLactobacillus inersthat were highly related to a single phylotype. These surprising results suggest that culture-independent methods can provide new insights into the diversity of bacterial species found in the human vagina, and this information could prove to be pivotal in understanding risk factors for various infectious diseases.
Genetic and functional analysis of the cytK family of genes in Bacillus cereus
Authors: Fagerlund Annette , Ween Ola , Lund Terje , Hardy Simon P. , Granum Per E.
Published: August 2004

CytK is a pore-forming toxin ofBacillus cereusthat has been linked to a case of necrotic enteritis. PCR products of the expected size were generated withcytKprimers in 13 of 29 strains. Six strains were PCR-positive for the related genehly-II, which encodes haemolysin II, a protein that is 37 % identical to the original CytK. Five of the strains were positive for both genes. The DNA sequences of putativecytKgenes from three positive strains were determined, and the deduced amino acid sequences were 89 % identical to that of the original CytK. The authors have designated this newcytKvariantcytK-2, and refer to the originalcytKascytK-1. The CytK-2 proteins from these three strains were isolated, and their identity was verified by N-terminal sequencing.blastanalysis using thecytK-2gene sequences revealed very high homology with twocytK-2sequences in the genomes ofB. cereusstrains ATCC 14579 and ATCC 10987. The differences between CytK-1 and the CytK-2 proteins were clustered to certain regions of the proteins. The isolated CytK-2 proteins were haemolytic and toxic towards human intestinal Caco-2 cells and Vero cells, although their toxicity was about 20 % of that of CytK-1. Both native and recombinant CytK-2 proteins fromB. cereus1230-88 were able to form pores in planar lipid bilayers, but the majority of the channels observed were of lower conductance than those created by CytK-1. It is likely that CytK-2 toxins contribute to the enterotoxicity of several strains ofB. cereus, although not all of the CytK-2 toxins may be as harmful as the CytK-1 originally isolated.
Effects of mutations in the rpoS gene on cell viability and global gene expression under nitrogen starvation in Escherichia coli
Authors: Kabir Md. Shahinur , Sagara Takehiro , Oshima Taku , Kawagoe Yuya , Mori Hirotada , Tsunedomi Ryouichi , Yamada Mamoru
Published: August 2004

Escherichia colibearing anrpoSamber or disrupted mutation exhibited a significant decrease in the number of colony-forming units (c.f.u.) when exposed to nitrogen starvation, which was not observed in cells bearing a functionalrpoSallele. The decrease in the number of c.f.u. that was observed about 25 h after initiation of nitrogen starvation was prevented by the addition of nitrogen within 3 h but not by the addition of nitrogen at more than 7 h after the initiation of nitrogen starvation, suggesting that a process leading to a decline in c.f.u. starts within this period. DNA microarray analysis of therpoSmutant showed that a large number of genes including many functionally undefined genes were affected by nitrogen starvation. The expression levels ofσSandσHregulon genes encoding acid-resistant proteins (hdeA,hdeB,gadAandgadB), DNA-binding protein (dps), chaperones (dnaK,ibpA,ibpB,dnaJandhtpG), chaperonins (mopBandmopA) and energy-metabolism-related proteins (hyaABCDFandgapA), and those of other genes encoding nucleotide-metabolism-related proteins (deoCanddeoB), cell-division protein (ftsL), outer-membrane lipoprotein (slp) and DNA-binding protein (stpA) were significantly decreased by 10 h nitrogen starvation. The genes encoding transport/binding proteins (nac,amtB,argT,artJ,potFandhisJ) and amino acid-metabolism-related proteins (glnA,trpB,argG,asnB,argC,gdhA,cstC,ntrB,asdandlysC) were significantly up-regulated under the same condition, some of which are known Ntr genes expressed under nitrogen limitation. On the basis of these results, possible causes of the decrease in the number of c.f.u. under nitrogen starvation are discussed.
The impact of different intensities of green light on the bacteriochlorophyll homologue composition of the chlorobiaceae Prosthecochloris aestuarii and Chlorobium phaeobacteroides
Authors: Massé Astrid , Airs Ruth L. , Keely Brendan J. , de Wit Rutger
Published: August 2004

Members of theChlorobiaceaeandChloroflexaceaeare unique among the phototrophic micro-organisms in having a remarkably rich chlorophyll pigment diversity. The physiological regulation of this diversity and its ecological implications are still enigmatic. The bacteriochlorophyll composition of the chlorobiaceaeProsthecochloris aestuariistrain CE 2404 andChlorobium phaeobacteroidesstrain UdG 6030 was therefore studied by both HPLC with photodiode array (PDA) detection and liquid chromatography-mass spectrometry (LC-MS). These strains were grown in liquid cultures under green light (480–615 nm) at different light intensities (0·2–55·7 μmol photons m−2 s−1), simulating the irradiance regime at different depths of the water column of deep lakes. The specific growth rates ofPtc. aestuariiunder green light achieved a maximum of 0·06 h−1at light intensities exceeding 6 μmol photons m−2 s−1, lower than the maximum observed under white light (approx. 0·1 h−1). The maximal growth rates ofChl. phaeobacteroidesunder green light were slightly higher (0·07 h−1) than observed forPtc. aestuariiand were achieved at 3·5 and 4·3 μmol photons m−2 s−1. LC-MS/MS analysis of pigment extracts revealed most (>90 %) BChlchomologues ofPtc. aestuariito be esterified with farnesol. The homologues differed in mass by multiples of 14 Da, reflecting different alkyl subsituents at positions C-8 and C-12 on the tetrapyrrole macrocycle. The relative proportions of the individual homologues varied only slightly among different light intensities. The specific content of BChlcwas maximal at 3–5 μmol photons m−2 s−1[400±150 nmol BChlc(mg protein)−1]. In the case ofChl. phaeobacteroides, the specific content of BChlewas maximal at 4·3 μmol photons m−2 s−1[115 nmol BChle(mg protein)−1], and this species was characterized by high carotenoid (isorenieratene) contents. The major BChleforms were esterified with a range of isoprenoid and straight-chain alcohols. The major isoprenoid alcohols comprised mainly farnesol and to a lesser extent geranylgeraniol. The straight-chain alcohols included C15, C15 : 1, C16, C16 : 1and C17. Interestingly, the proportion of straight alkyl chains over isoprenoid esterified side chains shifted markedly with increasing light intensity: the isoprenoid side chains dominated at low light intensities, while the straight-chain alkyl substituents dominated at higher light intensities. The authors propose that this phenomenon may be explained as a result of changing availability of reducing power, i.e. the highly reduced straight-chain alcohols have a higher biosynthetic demand for NADPH2than the polyunsaturated isoprenoid with the same number of carbon atoms.
Comparison of cytotoxin cytK promoters from Bacillus cereus strain ATCC 14579 and from a B. cereus food-poisoning strain
Authors: Brillard Julien , Lereclus Didier
Published: August 2004

The cytotoxin CytK produced by Bacillus cereus is believed to be involved in food-borne diseases. The transcriptional activity of the cytK promoter region in a food-poisoning strain was studied using a reporter gene and compared with that in the reference B. cereus strain ATCC 14579. In the food-poisoning strain, cytK is more strongly transcribed, possibly explaining the pathogenicity. The global regulator PlcR in B. cereus controls several putative virulence factors. It was found that PlcR regulates cytK in this clinical strain despite a mismatch in the PlcR recognition site, as currently defined. This suggests that the PlcR box consensus should be reconsidered and that the PlcR regulon might be larger than suspected. It is also shown that the high level of cytK transcription is not caused by a modification in the PlcR recognition site.
Killer toxin of Pichia membranifaciens and its possible use as a biocontrol agent against grey mould disease of grapevine
Authors: Santos A. , Marquina D.
Published: August 2004

The use ofPichia membranifaciensCYC 1106 killer toxin againstBotrytis cinereawas investigated. This strain exerted a broad-specificity killing action against other yeasts and fungi. At pH 4, optimal killer activity was observed at temperatures up to 20 °C. At 25 °C the toxic effect was reduced to 70 %. The killer activity was higher in acidic medium. Above about pH 4·5 activity decreased sharply and was barely noticeable at pH 6. The killer toxin protein fromP. membranifaciensCYC 1106 was purified to electrophoretic homogeneity. SDS-PAGE of the purified killer protein indicated an apparent molecular mass of 18 kDa. Killer toxin production was stimulated in the presence of non-ionic detergents. The toxin concentrations present in the supernatant during optimal production conditions exerted a fungicidal effect on a strain ofB. cinerea. The symptoms of infection and grey mould observed inVitis viniferaplants treated withB. cinereawere prevented in the presence of purifiedP. membranifacienskiller toxin. The results obtained suggest thatP. membranifaciensCYC 1106 killer toxin is of potential use in the biocontrol ofB. cinerea.
Characterization of a multicopper oxidase gene cluster in Phanerochaete chrysosporium and evidence of altered splicing of the mco transcripts
Authors: Larrondo Luis F. , González Bernardo , Cullen Dan , Vicuña Rafael
Published: August 2004

A cluster of multicopper oxidase genes (mco1,mco2,mco3,mco4) from the lignin-degrading basidiomycetePhanerochaete chrysosporiumis described. The four genes share the same transcriptional orientation within a 25 kb region.mco1,mco2andmco3 are tightly grouped, with intergenic regions of 2·3 and 0·8 kb, respectively, whereasmco4is located 11 kb upstream ofmco1. All are transcriptionally active, as shown by RT-PCR. Comparison of cDNAs and the corresponding genomic sequences identified 14–19 introns within each gene. Based on homology and intron composition, two subfamilies ofmcosequences could be identified. The sequences have copper-binding motifs similar to ferroxidase proteins, but different from fungal laccases. Thus, these sequences constitute a novel branch of the multicopper oxidase family. Analysis of several cDNA clones obtained from poly(A) RNA revealed the presence of transcripts of various lengths. Splice variants frommco2,mco3andmco4were characterized. They generally exhibited the presence of one to five introns, whereas other transcripts lacked some exons. In all cases, the presence of introns leads to frame shifts that give rise to premature stop codons. In aggregate, these investigations show thatP. chrysosporiumpossesses a novel family of multicopper oxidases which also feature clustering and incomplete processing of some of their transcripts, a phenomenon referred to in this paper as ‘altered splicing’.
Involvement of a Rab8-like protein of Dictyostelium discoideum, Sas1, in the formation of membrane extensions, secretion and adhesion during development
Authors: Powell Rhonda R. , Temesvari Lesly A.
Published: August 2004

Establishment of cell–cell adhesions, regulation of actin, and secretion are critical during development. Rab8-like GTPases have been shown to modulate these cellular events, suggesting an involvement in developmental processes. To further elucidate the function of Rab8-like GTPases in a developmental context, a Rab8-related protein (Sas1) ofDictyostelium discoideumwas examined, the expression of which increases at the onset of development.Dictyosteliumcell lines expressing inactive (N128I mutant) and constitutively active (Q74L mutant) Sas1 as green fluorescent protein (GFP)-Sas1 chimeras were generated. Cells expressing Sas1Q74L displayed numerous actin-rich membrane protrusions, increased secretion, and were unable to complete development. In particular, these cells demonstrated a reduction in adhesion as well as in the levels of a cell adhesion molecule, gp24 (DdCAD-1). In contrast, cells expressing Sas1N128I exhibited increased cell–cell adhesion and increased levels of gp24. Counting factor is a multisubunit signalling complex that is secreted in early development and controls aggregate size by negatively regulating the levels of cell adhesion molecules, including gp24. Interestingly, the Sas1Q74L mutant demonstrated increased levels of extracellular countin, a subunit of counting factor, suggesting that Sas1 may regulate trafficking of counting factor components. Together, the data suggest that Sas1 may be a key regulator of actin, adhesion and secretion during development.
Genomic organization and in vivo characterization of proteolytic activity of FtsH of Mycobacterium smegmatis SN2
Authors: Anilkumar Gopalakrishnapillai , Srinivasan Ramanujam , Ajitkumar Parthasarathi
Published: August 2004

TheftsHgene ofMycobacterium smegmatisSN2 (MsftsH) was cloned from two independent partial genomic DNA libraries and characterized, along with the identification ofephAandfolEas the neighbouring upstream and downstream genes respectively. The genomic organization of the MsftsHlocus was found to be identical to that of theMycobacterium tuberculosis ftsHgene (MtftsH) and similar to that of other bacterial genera, but with divergence in the upstream region. The MsftsHgene is 2·3 kb in size and encodes the AAA (ATPasesAssociated with diverse cellularActivities) family Zn2+-metalloprotease FtsH (MsFtsH) of 85 kDa molecular mass. This was demonstrated from the expression of the full-length recombinant gene inEscherichia coliJM109 cells and from the identification of native MsFtsH inM. smegmatisSN2 cell lysates by Western blotting with anti-MtFtsH and anti-EcFtsH antibodies respectively. The recombinant and the native MsFtsH proteins were found localized to the membrane ofE. coliandM. smegmatiscells respectively. Expression of MsFtsH protein inE. coliwas toxic and resulted in growth arrest and filamentation of cells. The MsftsHgene did not complement lethality of a ΔftsH3 : : kan mutation inE. coli, but when expressed inE. colicells, it efficiently degraded conventional FtsH substrates, namelyσ32protein and the protein translocase subunit SecY, ofE. colicells.
Cyclic di-GMP as a bacterial second messenger
Authors: D'Argenio David A. , Miller Samuel I.
Published: August 2004

Environmental signals trigger changes in the bacterial cell surface, including changes in exopolysaccharides and proteinaceous appendages that ultimately favour bacterial persistence and proliferation. Such adaptations are regulated in diverse bacteria by proteins with GGDEF and EAL domains. These proteins are predicted to regulate cell surface adhesiveness by controlling the level of a second messenger, the cyclic dinucleotide c-di-GMP. Genetic evidence suggests that the GGDEF domain acts as a nucleotide cyclase for c-di-GMP synthesis while the EAL domain is a good candidate for the opposing activity, a phosphodiesterase for c-di-GMP degradation.