bluuebunny/crossref_metadata_2025 huggingface dataset:
Intake of dietary iron is low in patients with Crohn's disease: a case–control study
Authors: Lomer Miranda C. E. , Kodjabashia Kamelia , Hutchinson Carol , Greenfield Simon M. , Thompson Richard P. H. , Powell Jonathan J.
Published: January 2004

Patients with Crohn's disease (CD) often experience Fe deficiency (ID) and frequently alter their diet to relieve abdominal symptoms. The present study set out to assess whether patients with CD have dietary habits that lead to low Fe intakes and/or reduced bioavailable Fe compared with control subjects. Patients with asymptomatic CD were matched to controls (n91/group). Dietary intakes of Fe and contributions from different food groups were compared using a 7 d food diary. Promoters and inhibitors of non-haem Fe absorption were investigated and a recently published algorithm was applied to assess bioavailable Fe. Fewer patients than controls met the reference nutrient intake for Fe (32 % CD patientsv. 42 % controls). Overall, patients had significantly lower mean Fe intakes (by 2·3 mg/d) and Fe density (by 0·26 mg/MJ (1·1 mg/1000 kcal)) compared with controls (bothP<0·001). Differences were mainly due to a preference among CD patients for low-fibre non-Fe fortified cereals, particularly breakfast cereals. In particular, control subjects had higher Fe intakes than matched CD subjects for men (P<0·001) and women less than 50 years (P=0·03). Intakes of both ascorbic acid (P<0·001) and phytic acid (P<0·01), but not animal tissue (P=1·0), were lower in patients with CD, but these had no overall effect on the predicted percentage of bioavailable Fe. Thus total bioavailable Fe was reduced in patients with CD due to lower intakes (P<0·01). Dietary Fe intakes are low in CD patients, which may contribute to an increased risk of ID and anaemia. Changing dietary advice may compromise perceived symptoms of the disease so the need for Fe supplementation should be carefully considered.
Market Town Regeneration; Challenges for Policy and Implementation
Authors: Caffyn Alison
Published: January 2004

Many challenges face local authorities and communities as they engage in the Government's Market Towns Initiative, launched in 2001. This article first considers vertical relationships and the conflicts which emerged when national and regional agencies attempted to roll out a top-down programme that is intended to be essentially bottom-up and community driven. Horizontal relationships and issues are then examined such as integrating across sectors, engaging rural hinterlands and working with neighbouring towns. The article uses examples from the West Midlands and identifies key issues that could improve policy guidance and assist future implementation of the Market Towns Initiative.
Visualising the Operating Behaviour of SMEs in Sector and Cluster: Evidence from the West Midlands
Authors: Tully Janet , Berkeley Nigel
Published: January 2004

Cluster policy in the UK, pursued by the Regional Development Agencies (RDAs), has readily adopted a simplistic definition based upon industrial sectors and location quotients. Evidence drawn from a study of the operating behaviour of SMEs belonging to two traditional manufacturing industries within the West Midlands—automotive components and clothing—provides a critique of this approach. Whilst the automotive components industry has been designated part of a key, high priority cluster, the clothing industry has not. Using case studies from both industries, this paper shows firms both within and outside RDA cluster definitions display a remarkably similar range of behavioural characteristics. Yet, based on weakly defined cluster policy, one industry enjoys considerably more policy support than the other. The paper begins to question the logic of RDA cluster policy and to ask whether a more sophisticated and locally sympathetic manner of visualising clusters and business behaviour rather than an emphasis on employment numbers would have a greater impact for policy.
Decentralisation of Competencies and Local Development Agencies in North-Eastern Italy
Authors: Solari Stefano
Published: January 2004

Despite the economic success of north-eastern Italy, some of its areas, such as those closest to the Adriatic coast, are still experiencing difficulties in industrialisation. Since the 1960s, some development policies have been implemented to stimulate economic restructuring. After the disappointing performance of centrally planned initiatives, most development policies are now the responsibility of local authorities. In fact, institutional and constitutional reforms have increased the competencies and the autonomy of lower levels of government, which have also been allowed greater responsibility in development initiatives. The establishment of development agencies is therefore increasingly left to local collective action.1 This paper examines the effectiveness of such bottom-up initiatives driven by local institutions, and attempts to identify the best forms and the best regulatory framework for such activities.
Responding to a Coalfield Closure: Old Issues for a New Regional Development Agency?
Authors: Henderson Roger , Shutt John
Published: January 2004

This paper examines the response of a new Regional Development Agency, Yorkshire Forward, to the sudden announcement that the Selby coalfield in North Yorkshire would close with the potential loss of 4000 regional jobs. Once the ‘jewel in the crown’ of the UK coal industry, its demise has repercussions beyond the Selby district. Following an examination of coal industry restructuring, previous regeneration efforts and issues, the paper considers the impact of closure. It is argued that by rapid analysis of the situation and by leading a Task Force to engage a range of interested parties and experts, the RDA has demonstrated a coordinated sectoral and spatial response to crisis management.
Inhibition of Hepatocyte Lipogenesis by Nitric Oxide Donor: Could Nitric Oxide Regulate Lipid Synthesis?
Authors: Roediger William E. , Hems Reginald , Wiggins David , Gibbons Geoffrey F.
Published: January 2004

Tissue lipogenesis is variably controlled by substrate supply and hormones. The possibility that nitric oxide (NO) might regulate lipogenesis derives from the action of NO on coenzyme A (CoA) to produce metabolically inactive S‐nitrosoCoA. The effect of the nitric oxide donor S‐nitrosoglutathione (GSNO) on long chain fatty acid and cholesterol synthesis was measured in isolated cultured rat hepatocytes. [1‐14C] Butyrate was used as substrate to measure 14C incorporation into lipids as butyrate is twice as effective as acetate in hepatic lipogenesis and is ketogenic via the Lynen cycle. NO very significantly (P < 0.01) impaired long chain fatty acid and cholesterol synthesis an observation dependent upon time of exposure (3 h pre‐incubation or 6 h continuous exposure) and concentration of GSNO (500 μM to 2.0 mM). Decrease in hepatic lipogenesis was paralleled by decrease in ketogenesis. ATP levels remained unchanged following short term exposure to GSNO. Exposure of hepatocytes to GSNO together with 2.0 mM glutathione significantly diminished the inhibition of lipogenesis induced by GSNO alone. Impairment of lipogenesis by GSNO appears not to be limited by energy supply and now adduced, but not proven, to be operative via the degree of inactivation of cytosolic CoA. NO control of lipogenesis could be clinically important where NO production is increased as in demyelinating diseases, chronic arthritis or colitis and in wasting diseases such as AIDS.
IUBMB Life, 56: 35‐40, 2004
Platelet Interactions in Thrombosis
Authors: Andrews Robert K. , Gardiner Elizabeth E. , Shen Yang , Berndt Michael C.
Published: January 2004

Patho/physiological platelet aggregate (thrombus) formation is initiated by engagement of platelet surface receptors, glycoprotein (GP)Ib‐IX‐V and GPVI that bind von Willebrand factor or collagen. Although beneficial in response to vascular injury by preventing blood loss (haemostasis), platelet aggregation in a sclerotic coronary artery or other diseased blood vessel (thrombosis) can cause thrombotic diseases like heart attack and stroke. At the molecular level, ligand interactions with GPIb‐IX‐V or GPVI trigger signalling responses, including elevation of cytosolic Ca2 +, dissociation of calmodulin from their cytoplasmic domains, cytoskeletal actin‐filament rearrangements, activation of src‐family kinases or PI 3‐kinase, and 'inside‐out' activation of the integrin, αIIbβ3 (GPIIb‐IIIa), that binds von Willebrand factor or fibrinogen and mediates platelet aggregation. Furthermore, emerging evidence supports a topographical co‐association of these receptors of the leucine‐rich repeat family (GPIb‐IX‐V) and immunoglobulin superfamily (GPVI) in an adhesive cluster or 'adhesosome'. This arrangement may underlie common mechanisms of initiating thrombus formation in haemostasis or thrombotic disease.
IUBMB Life, 56: 13‐18, 2004
ACYP1 Gene Possesses Two Alternative Splicing Forms That Induce Apoptosis
Authors: Degl'Innocenti Donatella , Marzocchini Riccardo , Malentacchi Francesca , Ramazzotti Matteo , Raugei Giovanni , Ramponi Giampietro
Published: January 2004

In human cell lines two products of the ACYP1 gene were detected by RT ‐ PCR. In addition to the expected amplicon corresponding to the CT form of acylphosphatase (320 bp) a second unexpected one (400 bp) was characterized as the result of an alternative splicing in which an extra 79 bp long exon is inserted between the two known exons. This new product, indicated as CTsv, was cloned and expressed. We performed the ectopic expression of the two alternative splicing forms. Both CT and CTsv products were able to induce a proapoptotic effect when expressed in HeLa cell line, despite the fact that the CTsv protein did not show any acylphosphatase activity.
IUBMB Life, 56: 29‐33, 2004
Sending RNAs into the Future: RNA Localization and Germ Cell Fate
Authors: Zhou Yi , King Mary Lou
Published: January 2004

RNA localization is a cellular mechanism used to localize proteins to subcellular domains and to control protein synthesis regionally. In oocytes, RNA localization has profound implications for development, setting up local concentrations of regulatory proteins that will establish regional fates in the future embryo. One such fate is that of the germ cell lineage. In a diverse number of organisms, including Drosophila and Xenopus, the germ cell lineage is determined by the inheritance of germ plasm assembled during oogenesis. This process requires the recruitment of specific RNAs, many now identified, to the germ plasm. Complex signals located in the 3' UTR direct RNAs to their destinations. These signals are sites for protein binding and assembly into particles competent to localize. Three different mechanisms have been described that operate during oogenesis or embryogenesis to localize RNAs in the germ plasm: motor driven transport, differential stability, and entrapment. Each of these localization mechanisms must be coordinated with translation and anchoring mechanisms to achieve functional germ plasms. Here we review what is known about these processes in germ cells, but the cellular mechanisms that select and transport RNAs are likely to be conserved among somatic cells as well.
IUBMB Life, 56: 19‐27, 2004
Neurogenesis: Is the Adult Stem Cell Young or Old?
Authors: Ricard Jerome , Liebl Daniel J.
Published: January 2004

Stem cell biology is one of the most exciting, controversial, and debated fields in science today. It has been suggested that neuronal replacement therapy using stem cell transplants may be one possible answer to a host of neuropathological disorders including spinal cord injury, stroke, and neurodegenerative diseases. Important sources for stem cells include the developing embryo and adult central nervous system, but will these populations of cells exhibit similar behavior and responses to stimuli? This review will discuss some important similarities and differences between the embryonic and adult stem cell, as well as the basis for developing therapeutic approaches for stem cell replacement.
IUBMB Life, 56: 1‐6, 2004
Platelet‐Activating Factor Mediates Helicobacter pylori Lipopolysaccharide Interference with Gastric Mucin Synthesis
Authors: Slomiany Bronislaw L. , Slomiany Amalia
Published: January 2004

Platelet‐activating factor (PAF) is now recognized as the most proximal mediator of cellular events triggered by bacterial infection. In this study, we report that a specific PAF antagonist, BN52020, impedes the reduction in mucin synthesis evoked in gastric mucosal cells by H. pylori LPS. The impedance by BN52020 of the LPS inhibitory effect on mucin synthesis was blocked by wortmannin, an inhibitor of phosphatidylinositol 3‐kinase (PI3K), which also obviated the inhibitory effect of BN52020 on the LPS‐induced upregulation in apoptosis, TNF‐α, and NO generation. A reduction in the impedance by BN52020 of the LPS detrimental effect on mucin synthesis was also attained with cNOS inhibitor, L‐NNA, whereas NOS‐2 inhibitor, 1400W caused a potentiation in the impedance effect of BN52020. However, while 1400W and BN52020 countered the potentiating effect of wortmannin on the LPS‐induced decrease in mucin synthesis, a further exacerbation of the potentiating effect of wortmannin was attained in the presence of cNOS inhibitor, L‐NNA. Our findings suggest that PAF, through the interference with PI3K‐dependent cNOS activation, plays a critical role in influencing the extent of pathological consequences of H. pylori infection on the synthesis of gastric mucin.
IUBMB Life, 56: 41‐46, 2004
Reactions of Nitric Oxide with Copper Containing Oxidases; Cytochrome c Oxidase and Laccase
Authors: Wilson Michael T. , Torres Jaume
Published: January 2004

The reactions of nitric oxide with copper containing oxidases such as cytochrome c oxidase and laccase are described and discussed in the present review.
IUBMB Life, 56: 7‐11, 2004
Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation
Authors: Palumbo Roberta , Sampaolesi Maurilio , De Marchis Francesco , Tonlorenzi Rossana , Colombetti Sara , Mondino Anna , Cossu Giulio , Bianchi Marco E.
Published: January 2004

High mobility group box 1 (HMGB1) is an abundant chromatin protein that acts as a cytokine when released in the extracellular milieu by necrotic and inflammatory cells. Here, we show that extracellular HMGB1 and its receptor for advanced glycation end products (RAGE) induce both migration and proliferation of vessel-associated stem cells (mesoangioblasts), and thus may play a role in muscle tissue regeneration. In vitro, HMGB1 induces migration and proliferation of both adult and embryonic mesoangioblasts, and disrupts the barrier function of endothelial monolayers. In living mice, mesoangioblasts injected into the femoral artery migrate close to HMGB1-loaded heparin-Sepharose beads implanted in healthy muscle, but are unresponsive to control beads. Interestingly, α-sarcoglycan null dystrophic muscle contains elevated levels of HMGB1; however, mesoangioblasts migrate into dystrophic muscle even if their RAGE receptor is disabled. This implies that the HMGB1–RAGE interaction is sufficient, but not necessary, for mesoangioblast homing; a different pathway might coexist. Although the role of endogenous HMGB1 in the reconstruction of dystrophic muscle remains to be clarified, injected HMGB1 may be used to promote tissue regeneration.
WASp is required for the correct temporal morphogenesis of rhabdomere microvilli
Authors: Zelhof Andrew C. , Hardy Robert W.
Published: January 2004

Microvilli are actin-based fingerlike membrane projections that form the basis of the brush border of enterocytes and the Drosophila melanogaster photoreceptor rhabdomere. Although many microvillar cytoskeletal components have been identified, the molecular basis of microvillus formation is largely undefined. Here, we report that the Wiskott-Aldrich syndrome protein (WASp) is necessary for rhabdomere microvillus morphogenesis. We show that WASp accumulates on the photoreceptor apical surface before microvillus formation, and at the time of microvillus initiation WASp colocalizes with amphiphysin and moesin. The loss of WASp delays the enrichment of F-actin on the apical photoreceptor surface, delays the appearance of the primordial microvillar projections, and subsequently leads to malformed rhabdomeres.
Signaling switches and bistability arising from multisite phosphorylation in protein kinase cascades
Authors: Markevich Nick I. , Hoek Jan B. , Kholodenko Boris N.
Published: January 2004

Mitogen-activated protein kinase (MAPK) cascades can operate as bistable switches residing in either of two different stable states. MAPK cascades are often embedded in positive feedback loops, which are considered to be a prerequisite for bistable behavior. Here we demonstrate that in the absence of any imposed feedback regulation, bistability and hysteresis can arise solely from a distributive kinetic mechanism of the two-site MAPK phosphorylation and dephosphorylation. Importantly, the reported kinetic properties of the kinase (MEK) and phosphatase (MKP3) of extracellular signal–regulated kinase (ERK) fulfill the essential requirements for generating a bistable switch at a single MAPK cascade level. Likewise, a cycle where multisite phosphorylations are performed by different kinases, but dephosphorylation reactions are catalyzed by the same phosphatase, can also exhibit bistability and hysteresis. Hence, bistability induced by multisite covalent modification may be a widespread mechanism of the control of protein activity.
Mating type–dependent constraints on the mobility of the left arm of yeast chromosome III
Authors: Bressan Debra A. , Vazquez Julio , Haber James E.
Published: January 2004

Mating-type gene (MAT) switching in budding yeast exhibits donor preference. MATa preferentially recombines with HML near the left telomere of chromosome III, whereas MATα prefers HMR near the right telomere. Donor preference is controlled by the recombination enhancer (RE) located proximal to HML. To test if HML is constrained in pairing with MATα, we examined live-cell mobility of LacI-GFP–bound lactose operator (lacO) arrays inserted at different chromosomal sites. Without induction of recombination, lacO sequences adjacent to HML are strongly constrained in both MATα and RE-deleted MATa strains, compared with MATa. In contrast, chromosome movement at HMR or near a telomere of chromosome V is mating-type independent. HML is more constrained in MATa Δre and less constrained in MATa RE+ compared with other sites. Although HML and MATa are not prealigned before inducing recombination, the three-dimensional configuration of MAT, HML, and HMR is mating-type dependent. These data suggest there is constitutive tethering of HML, which is relieved in MATa cells through the action of RE.
Noninvasive two-photon imaging reveals retinyl ester storage structures in the eye
Authors: Imanishi Yoshikazu , Batten Matthew L. , Piston David W. , Baehr Wolfgang , Palczewski Krzysztof
Published: January 2004

Visual sensation in vertebrates is triggered when light strikes retinal photoreceptor cells causing photoisomerization of the rhodopsin chromophore 11-cis-retinal to all-trans-retinal. The regeneration of preillumination conditions of the photoreceptor cells requires formation of 11-cis-retinal in the adjacent retinal pigment epithelium (RPE). Using the intrinsic fluorescence of all-trans-retinyl esters, noninvasive two-photon microscopy revealed previously uncharacterized structures (6.9 ± 1.1 μm in length and 0.8 ± 0.2 μm in diameter) distinct from other cellular organelles, termed the retinyl ester storage particles (RESTs), or retinosomes. These structures form autonomous all-trans-retinyl ester-rich intracellular compartments distinct from other organelles and colocalize with adipose differentiation-related protein. As demonstrated by in vivo experiments using wild-type mice, the RESTs participate in 11-cis-retinal formation. RESTs accumulate in Rpe65−/− mice incapable of carrying out the enzymatic isomerization, and correspondingly, are absent in the eyes of Lrat−/− mice deficient in retinyl ester synthesis. These results indicate that RESTs located close to the RPE plasma membrane are essential components in 11-cis-retinal production.
Defective angiogenesis and fatal embryonic hemorrhage in mice lacking core 1–derived O-glycans
Authors: Xia Lijun , Ju Tongzhong , Westmuckett Andrew , An Guangyu , Ivanciu Lacramioara , McDaniel J. Michael , Lupu Florea , Cummings Richard D. , McEver Rodger P.
Published: January 2004

The core 1 β1-3-galactosyltransferase (T-synthase) transfers Gal from UDP-Gal to GalNAcα1-Ser/Thr (Tn antigen) to form the core 1 O-glycan Galβ1-3GalNAcα1-Ser/Thr (T antigen). The T antigen is a precursor for extended and branched O-glycans of largely unknown function. We found that wild-type mice expressed the NeuAcα2-3Galβ1-3GalNAcα1-Ser/Thr primarily in endothelial, hematopoietic, and epithelial cells during development. Gene-targeted mice lacking T-synthase instead expressed the nonsialylated Tn antigen in these cells and developed brain hemorrhage that was uniformly fatal by embryonic day 14. T-synthase–deficient brains formed a chaotic microvascular network with distorted capillary lumens and defective association of endothelial cells with pericytes and extracellular matrix. These data reveal an unexpected requirement for core 1–derived O-glycans during angiogenesis.
Insulin Induces the Release of Vasodilator Compounds From Platelets by a Nitric Oxide–G Kinase–VAMP-3–dependent Pathway
Authors: Randriamboavonjy Voahanginirina , Schrader Jürgen , Busse Rudi , Fleming Ingrid
Published: January 2004

Insulin-induced vasodilatation is sensitive to nitric oxide (NO) synthase (NOS) inhibitors. However, insulin is unable to relax isolated arteries or to activate endothelial NOS in endothelial cells. Since insulin can enhance platelet endothelial NOS activity, we determined whether insulin-induced vasodilatation can be attributed to a NO-dependent, platelet-mediated process.

Insulin failed to relax endothelium-intact rings of porcine coronary artery. The supernatant from insulin-stimulated human platelets induced complete relaxation, which was prevented by preincubation of platelets with a NOS inhibitor, the soluble guanylyl cyclase inhibitor, NS 2028, or the G kinase inhibitor, KT 5823, and was abolished by an adenosine A2A receptor antagonist. Insulin induced the release of adenosine trisphosphate (ATP), adenosine, and serotonin from platelet-dense granules in a NO-dependent manner. This response was not detected using insulin-stimulated platelets from endothelial NOS−/− mice, although a NO donor elicited ATP release. Insulin-induced ATP release from human platelets correlated with the association of syntaxin 2 with the vesicle-associated membrane protein 3 but was not associated with the activation of αIIbβ3 integrin. Thus, insulin elicits the release of vasoactive concentrations of ATP and adenosine from human platelets via a NO–G kinase–dependent signaling cascade. The mechanism of dense granule secretion involves the G kinase–dependent association of syntaxin 2 with vesicle-associated membrane protein 3.
Asymmetric Division and Lineage Commitment at the Level of Hematopoietic Stem Cells
Authors: Takano Hina , Ema Hideo , Sudo Kazuhiro , Nakauchi Hiromitsu
Published: January 2004

How hematopoietic stem cells (HSCs) commit to a particular lineage is unclear. A high degree of HSC purification enabled us to address this issue at the clonal level. Single-cell transplantation studies revealed that 40% of the CD34−/low, c-Kit+, Sca-1+, and lineage marker− (CD34−KSL) cells in adult mouse bone marrow were able, as individual cells, to reconstitute myeloid and B- and T-lymphoid lineages over the long-term. Single-cell culture showed that >40% of CD34−KSL cells could form neutrophil (n)/macrophage (m)/erythroblast (E)/megakaryocyte (M) (nmEM) colonies. Assuming that a substantial portion of long-term repopulating cells can be detected as nmEM cells within this population, we compared differentiation potentials between individual pairs of daughter and granddaughter cells derived in vitro from single nmEM cells. One of the two daughter or granddaughter cells remained an nmEM cell. The other showed a variety of combinations of differentiation potential. In particular, an nmEM cell directly gave rise, after one cell division, to progenitor cells committed to nm, EM, or M lineages. The probability of asymmetric division of nmEM cells depended on the cytokines used. These data strongly suggest that lineage commitment takes place asymmetrically at the level of HSCs under the influence of external factors.
Chemokine Receptor CCR9 Contributes to the Localization of Plasma Cells to the Small Intestine
Authors: Pabst Oliver , Ohl Lars , Wendland Meike , Wurbel Marc-André , Kremmer Elisabeth , Malissen Bernard , Förster Reinhold
Published: January 2004

Humoral immunity in the gut-associated lymphoid tissue is characterized by the production of immunoglobulin A (IgA) by antibody-secreting plasma cells (PCs) in the lamina propria. The chemokine CCL25 is expressed by intestinal epithelial cells and is capable of inducing chemotaxis of IgA+ PCs in vitro. Using a newly generated monoclonal antibody against murine CCR9, we show that IgA+ PCs express high levels of CCR9 in the mesenteric lymph node (MLN) and Peyer's patches (PPs), but down-regulate CCR9 once they are located in the small intestine. In CCR9-deficient mice, IgA+ PCs are substantially reduced in number in the lamina propria of the small intestine. In adoptive transfer experiments, CCR9-deficient IgA+ PCs show reduced migration into the small intestine compared with wild-type controls. Furthermore, CCR9 mutants fail to mount a regular IgA response to an orally administered antigen, although the architecture and cell type composition of PPs and MLN are unaffected and are functional for the generation of IgA PCs. These findings provide profound in vivo evidence that CCL25/CCR9 guides PCs into the small intestine.
Massive Thymic Deletion Results in Systemic Autoimmunity through Elimination of CD4+ CD25+ T Regulatory Cells
Authors: Shih Fei F. , Mandik-Nayak Laura , Wipke Brian T. , Allen Paul M.
Published: January 2004

Incomplete deletion of KRN T cells that recognize the ubiquitously expressed self-antigen glucose-6-phosphate-isomerase (GPI) initiates an anti-GPI autoimmune cascade in K/BxN mice resulting in a humorally mediated arthritis. Transgenic (Tg) expression of a KRN T cell receptor (TCR) agonist under the major histocompatibility complex class II promoter resulted in thymic deletion with loss of anti-GPI T and B cell responses and attenuated arthritis course. However, double Tg mice succumbed to systemic autoimmunity with multiorgan inflammation and autoantibody production. Extensive thymic deletion resulted in lymphopenia and elimination of CD4+ CD25+ regulatory T cells (Tregs), but spared some CD4+ T cells expressing endogenous TCR, which oligoclonally expanded in the periphery. Disease was transferred by these T cells and prevented by cotransfer of CD4+ CD25+ Tregs. Moreover, we extended our findings to another TCR system (anti–hen egg lysozyme [HEL] TCR/HEL mice) where similarly extensive thymic deletion also resulted in disease. Thus, our studies demonstrated that central tolerance can paradoxically result in systemic autoimmunity through differential susceptibility of Tregs and autoreactive T cells to thymic deletion. Therefore, too little or too much negative selection to a self-antigen can result in systemic autoimmunity and disease.
The Metallo-β-Lactamase/β-CASP Domain of Artemis Constitutes the Catalytic Core for V(D)J Recombination
Authors: Poinsignon Catherine , Moshous Despina , Callebaut Isabelle , de Chasseval Régina , Villey Isabelle , de Villartay Jean-Pierre
Published: January 2004

The V(D)J recombination/DNA repair factor Artemis belongs to the metallo-β-lactamase (β-Lact) superfamily of enzymes. Three regions can be defined within the Artemis protein sequence: (a) the β-Lact homology domain, to which is appended (b) the β-CASP region, specific of members of the β-Lact superfamily acting on nucleic acids, and (c) the COOH-terminal domain. Using in vitro mutagenesis, here we show that the association of the β-Lact and the β-CASP regions suffices for in vivo V(D)J recombination of chromosome-integrated substrates. Single amino acid mutants point to critical catalytic residues for V(D)J recombination activity. The results presented here define the β-Lact/β-CASP domain of Artemis as the minimal core catalytic domain needed for V(D)J recombination and suggest that Artemis uses one or two Zn(II) ions to exert its catalytic activity, like bacterial class B β-Lact enzymes hydrolyzing β-lactam compounds.
Phylogenetic Analysis of Sec7-Domain–containing Arf Nucleotide Exchangers
Authors: Cox Randal , Mason-Gamer Roberta J , Jackson Catherine L. , Segev Nava
Published: January 2004

The eukaryotic family of ADP-ribosylation factor (Arf) GTPases plays a key role in the regulation of protein trafficking, and guanine-nucleotide exchange is crucial for Arf function. Exchange is stimulated by members of another family of proteins characterized by a 200-amino acid Sec7 domain, which alone is sufficient to catalyze exchange on Arf. Here, we analyzed the phylogeny of Sec7-domain–containing proteins in seven model organisms, representing fungi, plants, and animals. The phylogenetic tree has seven main groups, of which two include members from all seven model systems. Three groups are specific for animals, whereas two are specific for fungi. Based on this grouping, we propose a phylogenetically consistent set of names for members of the Sec7-domain family. Each group, except for one, contains proteins with known Arf exchange activity, implying that all members of this family have this activity. Contrary to the current convention, the sensitivity of Arf exchange activity to the inhibitor brefeldin A probably cannot be predicted by group membership. Multiple alignment reveals group-specific domains outside the Sec7 domain and a set of highly conserved amino acids within it. Determination of the importance of these conserved elements in Arf exchange activity and other cellular functions is now possible.
Sti1 and Cdc37 Can Stabilize Hsp90 in Chaperone Complexes with a Protein Kinase
Authors: Lee Paul , Shabbir Arsalan , Cardozo Christopher , Caplan Avrom J.
Published: January 2004

Hsp90 functions in association with several cochaperones for folding of protein kinases and transcription factors, although the relative contribution of each to the overall reaction is unknown. We assayed the role of nine different cochaperones in the activation of Ste11, a Saccharomyces cerevisiae mitogen-activated protein kinase kinase kinase. Studies on signaling via this protein kinase pathway was measured by α-factor-stimulated induction of FIG1 or lacZ, and repression of HHF1. Several cochaperone mutants tested had reduced FIG1 induction or HHF1 repression, although to differing extents. The greatest defects were in cpr7Δ, sse1Δ, and ydj1Δ mutants. Assays of Ste11 kinase activity revealed a pattern of defects in the cochaperone mutant strains that were similar to the gene expression studies. Overexpression of CDC37, a chaperone required for protein kinase folding, suppressed defects the sti1Δ mutant back to wild-type levels. CDC37 overexpression also restored stable Hsp90 binding to the Ste11 protein kinase domain in the sti1Δ mutant strain. These data suggest that Cdc37 and Sti1 have functional overlap in stabilizing Hsp90:client complexes. Finally, we show that Cns1 functions in MAP kinase signaling in association with Cpr7.
O-Glycosylation as a Sorting Determinant for Cell Surface Delivery in Yeast
Authors: Proszynski Tomasz J. , Simons Kai , Bagnat Michel
Published: January 2004

Little is known about the mechanisms that determine localization of proteins to the plasma membrane in Saccharomyces cerevisiae. The length of the transmembrane domains and association of proteins with lipid rafts have been proposed to play a role in sorting to the cell surface. Here, we report that Fus1p, an O-glycosylated integral membrane protein involved in cell fusion during yeast mating, requires O-glycosylation for cell surface delivery. In cells lacking PMT4, encoding a mannosyltransferase involved in the initial step of O-glycosylation, Fus1p was not glycosylated and accumulated in late Golgi structures. A chimeric protein lacking O-glycosylation motif was missorted to the vacuole and accumulated in late Golgi in wild-type cells. Exocytosis of this protein could be restored by addition of a 33-amino acid portion of an O-glycosylated sequence from Fus1p. Our data suggest that O-glycosylation functions as a sorting determinant for cell surface delivery of Fus1p.
Dinitroanilines Bind α-Tubulin to Disrupt Microtubules
Authors: Morrissette Naomi S. , Mitra Arpita , Sept David , Sibley L. David
Published: January 2004

Protozoan parasites are remarkably sensitive to dinitroanilines such as oryzalin, which disrupt plant but not animal microtubules. To explore the basis of dinitroaniline action, we isolated 49 independent resistant Toxoplasma gondii lines after chemical mutagenesis. All 23 of the lines that we examined harbored single point mutations in α-tubulin. These point mutations were sufficient to confer resistance when transfected into wild-type parasites. Several mutations were in the M or N loops, which coordinate protofilament interactions in the microtubule, but most of the mutations were in the core of α-tubulin. Docking studies predict that oryzalin binds with an average affinity of 23 nM to a site located beneath the N loop of Toxoplasma α-tubulin. This binding site included residues that were mutated in several resistant lines. Moreover, parallel analysis of Bos taurus α-tubulin indicated that oryzalin did not interact with this site and had a significantly decreased, nonspecific affinity for vertebrate α-tubulin. We propose that the dinitroanilines act through a novel mechanism, by disrupting M-N loop contacts. These compounds also represent the first class of drugs that act on α-tubulin function.
Syntaxin-6 SNARE Involvement in Secretory and Endocytic Pathways of Cultured Pancreatic β-Cells
Authors: Kuliawat Regina , Kalinina Elena , Bock Jason , Fricker Lloyd , McGraw Timothy E. , Kim Se Ryoung , Zhong Jiayu , Scheller Richard , Arvan Peter
Published: January 2004

In pancreatic β-cells, the syntaxin 6 (Syn6) soluble N-ethylmaleimide-sensitive factor attachment protein receptor is distributed in the trans-Golgi network (TGN) (with spillover into immature secretory granules) and endosomes. A possible Syn6 requirement has been suggested in secretory granule biogenesis, but the role of Syn6 in live regulated secretory cells remains unexplored. We have created an ecdysone-inducible gene expression system in the INS-1 β-cell line and find that induced expression of a membrane-anchorless, cytosolic Syn6 (called Syn6t), but not full-length Syn6, causes a prominent defect in endosomal delivery to lysosomes, and the TGN, in these cells. The defect occurs downstream of the endosomal branchpoint involved in transferrin recycling, and upstream of the steady-state distribution of mannose 6-phosphate receptors. By contrast, neither acquisition of stimulus competence nor the ultimate size of β-granules is affected. Biosynthetic effects of dominant-interfering Syn6 seem limited to slowed intragranular processing to insulin (achieving normal levels within 2 h) and minor perturbation of sorting of newly synthesized lysosomal proenzymes. We conclude that expression of the Syn6t mutant slows a rate-limiting step in endosomal maturation but provides only modest and potentially indirect interference with regulated and constitutive secretory pathways, and in TGN sorting of lysosomal enzymes.
Clustering and Redistribution of Late Endocytic Compartments in Response toHelicobacter pyloriVacuolating Toxin
Authors: Li Yi , Wandinger-Ness Angela , Goldenring James R. , Cover Timothy L.
Published: January 2004

Helicobacter pylori VacA is a secreted protein toxin that may contribute to the pathogenesis of peptic ulcer disease and gastric adenocarcinoma. When added to cultured mammalian cells in the presence of weak bases (e.g., ammonium chloride), VacA induces the formation of large cytoplasmic vacuoles. Here, we report a previously unrecognized capacity of VacA to induce clustering and perinuclear redistribution of late endocytic compartments. In contrast to VacA-induced cell vacuolation, VacA-induced clustering and redistribution of late endocytic compartments are not dependent on the presence of weak bases and are not inhibited by bafilomycin A1. VacA mutant toxins defective in the capacity to form anion-selective membrane channels fail to cause clustering and redistribution. VacA-induced clusters of late endocytic compartments undergo transformation into vacuoles after the addition of ammonium chloride. VacA-induced clustering and redistribution of late endocytic compartments occur in cells expressing wild-type or constitutively active Rab7, but not in cells expressing dominant-negative mutant Rab7. In VacA-treated cells containing clustered late endocytic compartments, overexpression of dominant-negative Rab7 causes reversion to a nonclustered distribution. Redistribution of late endocytic compartments to the perinuclear region requires a functional microtubule cytoskeleton, whereas clustering of these compartments and vacuole formation do not. These data provide evidence that clustering of late endocytic compartments is a critical mechanistic step in the process of VacA-induced cell vacuolation. We speculate that VacA-induced alterations in late endocytic membrane traffic contribute to the capacity of H. pylori to persistently colonize the human gastric mucosa.
Identification of Protein Complexes Required for Efficient Sister Chromatid Cohesion
Authors: Mayer Melanie L. , Pot Isabelle , Chang Michael , Xu Hong , Aneliunas Victoria , Kwok Teresa , Newitt Rick , Aebersold Ruedi , Boone Charles , Brown Grant W. , Hieter Philip
Published: January 2004

Ctf8p is a component of Ctf18-RFC, an alternative replication factor C-like complex required for efficient sister chromatid cohesion in Saccharomyces cerevisiae. We performed synthetic genetic array (SGA) analysis with a ctf8 deletion strain as a primary screen to identify other nonessential genes required for efficient sister chromatid cohesion. We then assessed proficiency of cohesion at three chromosomal loci in strains containing deletions of the genes identified in the ctf8 SGA screen. Deletion of seven genes (CHL1, CSM3, BIM1, KAR3, TOF1, CTF4, and VIK1) resulted in defective sister chromatid cohesion. Mass spectrometric analysis of immunoprecipitated complexes identified a physical association between Kar3p and Vik1p and an interaction between Csm3p and Tof1p that we confirmed by coimmunoprecipitation from cell extracts. These data indicate that synthetic genetic array analysis coupled with specific secondary screens can effectively identify protein complexes functionally related to a reference gene. Furthermore, we find that genes involved in mitotic spindle integrity and positioning have a previously unrecognized role in sister chromatid cohesion.
The FAM Deubiquitylating Enzyme Localizes to Multiple Points of Protein Trafficking in Epithelia, where It Associates with E-cadherin and β-catenin
Authors: Murray Rachael Z. , Jolly Lachlan A. , Wood Stephen A.
Published: January 2004

Ubiquitylation is a necessary step in the endocytosis and lysosomal trafficking of many plasma membrane proteins and can also influence protein trafficking in the biosynthetic pathway. Although a molecular understanding of ubiquitylation in these processes is beginning to emerge, very little is known about the role deubiquitylation may play. Fat Facets in mouse (FAM) is substrate-specific deubiquitylating enzyme highly expressed in epithelia where it interacts with its substrate, β-catenin. Here we show, in the polarized intestinal epithelial cell line T84, FAM localized to multiple points of protein trafficking. FAM interacted with β-catenin and E-cadherin in T84 cells but only in subconfluent cultures. FAM extensively colocalized with β-catenin in cytoplasmic puncta but not at sites of cell-cell contact as well as immunoprecipitating with β-catenin and E-cadherin from a higher molecular weight complex (∼500 kDa). At confluence FAM neither colocalized with, nor immunoprecipitated, β-catenin or E-cadherin, which were predominantly in a larger molecular weight complex (∼2 MDa) at the cell surface. Overexpression of FAM in MCF-7 epithelial cells resulted in increased β-catenin levels, which localized to the plasma membrane. Expression of E-cadherin in L-cell fibroblasts resulted in the relocalization of FAM from the Golgi to cytoplasmic puncta. These data strongly suggest that FAM associates with E-cadherin and β-catenin during trafficking to the plasma membrane.
Ectopic EphA4 Receptor Induces Posterior Protrusions via FGF Signaling inXenopusEmbryos
Authors: Park Eui Kyun , Warner Neil , Bong Yong-Sik , Stapleton David , Maeda Ryu , Pawson Tony , Daar Ira O.
Published: January 2004

The Eph family of receptor tyrosine kinases regulates numerous biological processes. To examine the biochemical and developmental contributions of specific structural motifs within Eph receptors, wild-type or mutant forms of the EphA4 receptor were ectopically expressed in developing Xenopus embryos. Wild-type EphA4 and a mutant lacking both the SAM domain and PDZ binding motif were constitutively tyrosine phosphorylated in vivo and catalytically active in vitro. EphA4 induced loss of cell adhesion, ventro-lateral protrusions, and severely expanded posterior structures in Xenopus embryos. Moreover, mutation of a conserved SAM domain tyrosine to phenylalanine (Y928F) enhanced the ability of EphA4 to induce these phenotypes, suggesting that the SAM domain may negatively regulate some aspects of EphA4 activity in Xenopus. Analysis of double mutants revealed that the Y928F EphA4 phenotypes were dependent on kinase activity; juxtamembrane sites of tyrosine phosphorylation and SH2 domain-binding were required for cell dissociation, but not for posterior protrusions. The induction of protrusions and expansion of posterior structures is similar to phenotypic effects observed in Xenopus embryos expressing activated FGFR1. Furthermore, the budding ectopic protrusions induced by EphA4 express FGF-8, FGFR1, and FGFR4a. In addition, antisense morpholino oligonucleotide-mediated loss of FGF-8 expression in vivo substantially reduced the phenotypic effects in EphA4Y928F expressing embryos, suggesting a connection between Eph and FGF signaling.
Calcium Signaling Is Involved in Dynein-dependent Microtubule Organization
Authors: Adamíková L'ubica , Straube Anne , Schulz Irene , Steinberg Gero
Published: January 2004

The microtubule cytoskeleton supports cellular morphogenesis and polar growth, but the underlying mechanisms are not understood. In a screen for morphology mutants defective in microtubule organization in the fungus Ustilago maydis, we identified eca1 that encodes a sarcoplasmic/endoplasmic calcium ATPase. Eca1 resides in the endoplasmic reticulum and restores growth of a yeast mutant defective in calcium homeostasis. Deletion of eca1 resulted in elevated cytosolic calcium levels and a severe growth and morphology defect. While F-actin and myosin V distribution is unaffected, Δeca1 mutants contain longer and disorganized microtubules that show increased rescue and reduced catastrophe frequencies. Morphology can be restored by inhibition of Ca2+/calmodulin-dependent kinases or destabilizing microtubules, indicating that calcium-dependent alterations in dynamic instability are a major cause of the growth defect. Interestingly, dynein mutants show virtually identical changes in microtubule dynamics and dynein-dependent ER motility was drastically decreased in Δeca1. This indicates a connection between calcium signaling, dynein, and microtubule organization in morphogenesis of U. maydis.
SNAP-23 Functions in Docking/Fusion of Granules at Low Ca2+
Authors: Chieregatti Evelina , Chicka Michael C. , Chapman Edwin R. , Baldini Giulia
Published: January 2004

Ca2+-triggered exocytosis of secretory granules mediates the release of hormones from endocrine cells and neurons. The plasma membrane protein synaptosome-associated protein of 25 kDa (SNAP-25) is thought to be a key component of the membrane fusion apparatus that mediates exocytosis in neurons. Recently, homologues of SNAP-25 have been identified, including SNAP-23, which is expressed in many tissues, albeit at different levels. At present, little is known concerning functional differences among members of this family of proteins. Using an in vitro assay, we show here that SNAP-25 and SNAP-23 mediate the docking of secretory granules with the plasma membrane at high (1 μM) and low (100 nM) Ca2+levels, respectively, by interacting with different members of the synaptotagmin family. In intact endocrine cells, expression of exogenous SNAP-23 leads to high levels of hormone secretion under basal conditions. Thus, the relative expression levels of SNAP-25 and SNAP-23 might control the mode (regulated vs. basal) of granule release by forming docking complexes at different Ca2+thresholds.
Mutant Telomere Sequences Lead to Impaired Chromosome Separation and a Unique Checkpoint Response
Authors: Lin Jue , Smith Dana L. , Blackburn Elizabeth H.
Published: January 2004

Mutation of the template region in the RNA component of telomerase can cause incorporation of mutant DNA sequences at telomeres. We made all 63 mutant sequence combinations at template positions 474–476 of the yeast telomerase RNA, TLC1. Mutants contained faithfully incorporated template mutations, as well as misincorporated sequences in telomeres, a phenotype not previously reported for Saccharomyces cerevisiae telomerase template mutants. Although growth rates and telomere profiles varied widely among the tlc1 mutants, chromosome separation and segregation were always aberrant. The mutants showed defects in sister chromatid separation at centromeres as well as telomeres, suggesting activation of a cell cycle checkpoint. Deletion of the DNA damage response genes DDC1, MEC3, or DDC2/SML1 failed to restore chromosome separation in the tlc1 template mutants. These results suggest that mutant telomere sequences elicit a checkpoint that is genetically distinct from those activated by deletion of telomerase or DNA damage.
Positive and Negative Regulation of a SNARE Protein by Control of Intracellular Localization
Authors: Nakanishi Hideki , de los Santos Pablo , Neiman Aaron M.
Published: January 2004

In Saccharomyces cerevisiae, the developmentally regulated Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein Spo20p mediates the fusion of vesicles with the prospore membrane, which is required for the formation of spores. Spo20p is subject to both positive and negative regulation by separate sequences in its aminoterminal domain. We report that the positive activity is conferred by a short, amphipathic helix that is sufficient to confer plasma membrane or prospore membrane localization to green fluorescent protein. In vitro, this helix binds to acidic phospholipids, and mutations that reduce or eliminate phospholipid binding in vitro inactivate Spo20p in vivo. Genetic manipulation of phospholipid pools indicates that the likely in vivo ligand of this domain is phosphatidic acid. The inhibitory activity is a nuclear targeting signal, which confers nuclear localization in vegetative cells and in cells entering meiosis. However, as cells initiate spore formation, fusions containing the inhibitory domain exit the nucleus and localize to the nascent prospore membrane. Thus, the SNARE Spo20p is both positively and negatively regulated by control of its intracellular localization.