bluuebunny/crossref_metadata_2025 huggingface dataset:
Thiamin deficiency and brain disorders
Authors: Butterworth Roger F.
Published: January 2004

Thiamin plays a key role in the maintenance of brain function. Thiamin diphosphate is cofactor for several enzymes involved in glucose metabolism whereas thiamin triphosphate has distinct properties at the neuronal membrane. Thiamin metabolism in the brain is compartmented between neurons and neighbouring glial cells. Thiamin deficiency is commonly encountered in severe malnutrition associated with chronic alcoholism, HIV–AIDS and gastrointestinal disease where it frequently results in Wernicke's encephalopathy (the Wernicke–Korsakoff syndrome). Wernicke's encephalopathy is severely underdiagnosed according to clinical criteria in both alcoholic and HIV–AIDS patients. Magnetic resonance imaging reveals bilateral ventricular enlargement, mammillary body atrophy and cerebellar degeneration indicative of selective neuronal loss that is characteristic of Wernicke's encephalopathy. Several mechanisms have been proposed to explain this selective loss of neurons including a cerebral energy deficit resulting from reductions in activity of thiamin diphosphate-dependent enzymes, oxidative stress and N-methyl-D-aspartate receptor-mediated excitotoxicity. Both microglia and perivascular endothelial cells are sources of NO and oxidative stress in thiamin deficiency. Decreased activities of thiamin diphosphate-dependent enzymes (in particular α-ketoglutarate dehydrogenase) have also been reported in neurodegenerative diseases such as Alzheimer's and Parkinson's diseases independent of patient malnutrition. In these cases, decreased activities result from direct toxic actions of oxidative stress and β-amyloid produced as part of the neuronal cell death cascade in these disorders.
The impact of low concentrations of aflatoxin, deoxynivalenol or fumonisin in diets on growing pigs and poultry
Authors: Dersjant-Li Yueming , Verstegen Martin W.A. , Gerrits Walter J.J.
Published: January 2004

In the present review, the quantitative impact of dietary aflatoxin, deoxynivalenol (DON) and fumonisin concentrations on performance of pigs and broilers is evaluated, with special emphasis on low concentrations of these toxins. Also, responses in performance of pigs and broilers to these three toxins are related to their absorption and elimination kinetics. By applying simple linear regression, information from many literature sources is integrated and condensed into, for example, estimates of depression in rates of weight gain, relative to non-contaminated diets, with increasing toxin concentrations. It was estimated that with each mg/kg increase of aflatoxin in the diet, the growth rate would be depressed by 16 % for pigs and 5 % for broilers. For DON, with each mg/kg increase in the diet, the growth depression was estimated at about 8 % for pigs, while broilers showed no response to DON concentrations below 16 mg/kg. Fumonisin showed the lowest impact on growth performance; with each mg/kg increase, the depression in growth rate was estimated at 0·4 and 0·0 % for pigs and broilers, respectively. Dietary concentrations that cause a 5 % reduction in growth rate were estimated at 0·3 and 1·0 mg/kg for aflatoxin for pigs and broilers, respectively; 1·8 and 0·6 mg/kg for pure and naturally contaminated DON for pigs, respectively; 21 and 251 mg/kg for fumonisin for pigs and broilers, respectively.
The combined use of triacylglycerols containing medium-chain fatty acids and exogenous lipolytic enzymes as an alternative to in-feed antibiotics in piglets: concept, possibilities and limitations. An overview
Authors: Decuypere J. A. , Dierick N. A.
Published: January 2004

In the search for alternatives to banned in-feed antibiotics, a concept was developed based on studies with medium-chain fatty acid-containing triacylglycerols (MCTAG) and selected lipases forin situgeneration of diacylglycerols, monoacylglycerols and medium-chain fatty acids (MCFA) in the stomach and proximal gut of piglets. MCFA are known to have strong antibacterial properties but can hardly be used as such because of their repellent odour and taste. Those problems could be overcome by the generation of MCFAin situ. The concept was testedin vitroand validatedin vivowith gastric-cannulated piglets and under field conditions, including effects on zootechnical performance, with classical antibacterial growth promoters or organic acids acting as positive controls. Furthermore, the metabolic and dietary constraints on the nutritional and nutritive use of MCTAG and/or MCFA (for example, the effects on digestive physiology, gut flora, feed intake, performance, carcass composition) are reviewed. The role of natural preduodenal lipase activity, the presence of endogenous plant lipase activity in raw materials and the feasibility for exogenous lipase addition to the feed are discussed, in order to optimize the concept. The present review illustrates the similarity of the action of MCFA and commonly used antimicrobials on the flora (total flora, Gram-positive flora, Gram-negative flora, potential pathogens) and epithelial morphology and histology in the foregut. These observations are believed to be the basis for obtaining optimal growth performances. In addition, these naturally occurring antimicrobial agents have little or no human or animal toxicity and induce no problems of residues and cross-resistance induction. They are proposed as a valuable alternative to in-feed antibiotics, used for growth promotion, and even for the preventive and curative treatment of gastrointestinal diseases.
The uniformity of phagosome maturation in macrophages
Authors: Henry Rebecca M. , Hoppe Adam D. , Joshi Nikhil , Swanson Joel A.
Published: January 2004

Many studies of endocytosis and phagocytosis presume that organelles containing a single kind of internalized particle exhibit invariant patterns of protein and phospholipid association as they mature inside cells. To test this presumption, fluorescent protein chimeras were expressed in RAW 264.7 macrophages, and time-lapse ratiometric fluorescence microscopy was used to measure the maturation dynamics of individual phagosomes containing IgG-opsonized erythrocytes. Quantitative analysis revealed consistent patterns of association for YFP chimeras of β-actin, Rab5a, Rab7, and LAMP-1, and no association of YFP chimeras marking endoplasmic reticulum or Golgi. YFP-2xFYVE, recognizing phosphatidylinositol 3-phosphate (PI(3)P), showed two patterns of phagosome labeling. Some phagosomes increased labeling quickly after phagosome closure and then lost the label within 20 min, whereas others labeled more slowly and retained the label for several hours. The two patterns of PI(3)P on otherwise identical phagosomes indicated that organelle maturation does not necessarily follow a single path and that some features of phagosome maturation are integrated over the entire organelle.
Intraflagellar transport (IFT) cargo
Authors: Qin Hongmin , Diener Dennis R. , Geimer Stefan , Cole Douglas G. , Rosenbaum Joel L.
Published: January 2004

Intraflagellar transport (IFT) is the bidirectional movement of multisubunit protein particles along axonemal microtubules and is required for assembly and maintenance of eukaryotic flagella and cilia. One posited role of IFT is to transport flagellar precursors to the flagellar tip for assembly. Here, we examine radial spokes, axonemal subunits consisting of 22 polypeptides, as potential cargo for IFT. Radial spokes were found to be partially assembled in the cell body, before being transported to the flagellar tip by anterograde IFT. Fully assembled radial spokes, detached from axonemal microtubules during flagellar breakdown or turnover, are removed from flagella by retrograde IFT. Interactions between IFT particles, motors, radial spokes, and other axonemal proteins were verified by coimmunoprecipitation of these proteins from the soluble fraction of Chlamydomonas flagella. These studies indicate that one of the main roles of IFT in flagellar assembly and maintenance is to transport axonemal proteins in and out of the flagellum.
Breaking Tolerance to Double Stranded DNA, Nucleosome, and Other Nuclear Antigens Is Not Required for the Pathogenesis of Lupus Glomerulonephritis
Authors: Waters Samuel T. , McDuffie Marcia , Bagavant Harini , Deshmukh Umesh S. , Gaskin Felicia , Jiang Chao , Tung Kenneth S.K. , Fu Shu Man
Published: January 2004

In lupus-prone NZM2328 mice, a locus Cgnz1 on chromosome 1 was linked to chronic glomerulonephritis, severe proteinuria, and early mortality in females. A locus Adnz1 on chromosome 4 was linked to antinuclear antibody (ANA) and anti–double stranded DNA (dsDNA) antibody (Ab) production. In this investigation, two congenic strains, NZM2328.C57L/Jc1 (NZM.C57Lc1) and NZM2328.C57L/Jc4 (NZM.C57Lc4), were generated by replacing the respective genetic intervals containing either Cgnz1 or Adnz1 with those from C57L/J, a nonlupus-prone strain. The NZM.C57Lc1 females had markedly reduced incidence of chronic glomerulonephritis and severe proteinuria. NZM.C57Lc4 females had chronic glomerulonephritis and severe proteinuria without circulating ANA, anti-dsDNA, and antinucleosome Ab. These data confirm the linkage analysis. Unexpectedly, NZM.C57Lc1 females had little anti-dsDNA and related Ab, suggesting the presence of a second locus Adnz2 on chromosome 1. The diseased NZM.C57Lc4 kidneys had immune complexes by immunofluorescence and electron microscopy. The eluates from these kidneys did not contain ANA, anti-dsDNA, and antinucleosome Ab, indicative of the presence of non–anti-dsDNA nephritogenic Ab. Thus, breaking tolerance to dsDNA and chromatin is not required for the pathogenesis of lupus nephritis. These results reaffirm that anti-dsDNA and related Ab production and chronic glomerulonephritis are under independent genetic control. These findings have significant implications in the pathogenesis of systemic lupus erythematosus.
Aiolos Is Required for the Generation of High Affinity Bone Marrow Plasma Cells Responsible for Long-Term Immunity
Authors: Cortés Marta , Georgopoulos Katia
Published: January 2004

Antigenic encounter generates long-term immunity sustained by long-lived high affinity plasma cells resident in the bone marrow (BM). Here we show that the Ikaros family member, Aiolos, is specifically required for the generation of these plasma cells. Failure to generate high affinity plasma cells in the BM and to sustain serum antibody titers is apparent after both primary and secondary immunization of Aiolos−/− mice with a range of hapten concentrations. Chimera reconstitutions demonstrate that the BM plasma cell defect is B cell intrinsic. Lack of Aiolos does not alter expression of any of the previously described factors required for general plasma cell differentiation. No defect in somatic hypermutation, the generation of memory B cells, or short-lived high affinity plasma cells in the spleen was observed upon rechallenge. These studies support a model by which the high affinity plasma cell population in the BM undergoes a unique differentiation program that is dependent on Aiolos.
Ionic Currents Mediated by a Prokaryotic Homologue of CLC Cl− Channels
Authors: Accardi Alessio , Kolmakova-Partensky Ludmila , Williams Carole , Miller Christopher
Published: January 2004

CLC-ec1 is an E. coli homologue of the CLC family of Cl− channels, which are widespread throughout eukaryotic organisms. The structure of this membrane protein is known, and its physiological role has been described, but our knowledge of its functional characteristics is severely limited by the absence of electrophysiological recordings. High-density reconstitution and incorporation of crystallization-quality CLC-ec1 in planar lipid bilayers failed to yield measurable CLC-ec1 currents due to porin contamination. A procedure developed to prepare the protein at a very high level of purity allowed us to measure macroscopic CLC-ec1 currents in lipid bilayers. The current is Cl− selective, and its pH dependence mimics that observed with a 36Cl− flux assay in reconstituted liposomes. The unitary conductance is estimated to be <0.2 pS. Surprisingly, the currents have a subnernstian reversal potential in a KCl gradient, indicating imperfect selectivity for anions over cations. Mutation of a conserved glutamate residue found in the selectivity filter eliminates the pH-dependence of both currents and 36Cl− flux and appears to trap CLC-ec1 in a constitutively active state. These effects correlate well with known characteristics of eukaryotic CLC channels. The E148A mutant displays nearly ideal Cl− selectivity.
A Cyclic GMP–dependent Calcium-activated Chloride Current in Smooth-muscle Cells from Rat Mesenteric Resistance Arteries
Authors: Matchkov Vladimir V. , Aalkjaer Christian , Nilsson Holger
Published: January 2004

We have previously demonstrated the presence of a cyclic GMP (cGMP)-dependent calcium-activated inward current in vascular smooth-muscle cells, and suggested this to be of importance in synchronizing smooth-muscle contraction. Here we demonstrate the characteristics of this current. Using conventional patch-clamp technique, whole-cell currents were evoked in freshly isolated smooth-muscle cells from rat mesenteric resistance arteries by elevation of intracellular calcium with either 10 mM caffeine, 1 μM BAY K8644, 0.4 μM ionomycin, or by high calcium concentration (900 nM) in the pipette solution. The current was found to be a calcium-activated chloride current with an absolute requirement for cyclic GMP (EC50 6.4 μM). The current could be activated by the constitutively active subunit of PKG. Current activation was blocked by the protein kinase G antagonist Rp-8-Br-PET-cGMP or with a peptide inhibitor of PKG, or with the nonhydrolysable ATP analogue AMP-PNP. Under biionic conditions, the anion permeability sequence of the channel was SCN− > Br− > I− > Cl− > acetate > F− >> aspartate, but the conductance sequence was I− > Br− > Cl− > acetate > F− > aspartate = SCN−. The current had no voltage or time dependence. It was inhibited by nickel and zinc ions in the micromolar range, but was unaffected by cobalt and had a low sensitivity to inhibition by the chloride channel blockers niflumic acid, DIDS, and IAA-94. The properties of this current in mesenteric artery smooth-muscle cells differed from those of the calcium-activated chloride current in pulmonary myocytes, which was cGMP-independent, exhibited a high sensitivity to inhibition by niflumic acid, was unaffected by zinc ions, and showed outward current rectification as has previously been reported for this current. Under conditions of high calcium in the patch-pipette solution, a current similar to the latter could be identified also in the mesenteric artery smooth-muscle cells. We conclude that smooth-muscle cells from rat mesenteric resistance arteries have a novel cGMP-dependent calcium-activated chloride current, which is activated by intracellular calcium release and which has characteristics distinct from other calcium-activated chloride currents.
Experimenting with musical intervals
Authors: Michael C LoPresto
Published: January 2004

When two tuning forks of different frequency are
sounded simultaneously the result is a complex wave with a
repetition frequency that is the fundamental of the harmonic
series to which both frequencies belong. The ear perceives this
'musical interval' as a single musical pitch with a sound
quality produced by the harmonic spectrum responsible for the
waveform. This waveform can be captured and displayed with data
collection hardware and software. The fundamental frequency can
then be calculated and compared with what would be expected from
the frequencies of the tuning forks. Also, graphing software can be
used to determine equations for the waveforms and predict their
shapes. This experiment could be used in an introductory physics
or musical acoustics course as a practical lesson in
superposition of waves, basic Fourier series and the
relationship between some of the ear's subjective perceptions of
sound and the physical properties of the waves that cause
them.
Polarized gas targets
Authors: Erhard Steffens  , Willy Haeberli
Published: January 2004

This review describes the development and present performance of nuclear polarized gas targets in nuclear or particle physics experiments. After a brief account of the various development steps, the design principles for the relevant light nucleon targets, hydrogen, deuterium and3He, for storage rings, are discussed. Most of these targets make use of so-called storage cells in order to enhance the target thickness. The performance of targets used in medium and high-energy ion and electron storage rings is reviewed. Finally, future trends and possible improvements are discussed.
Nano-biotechnology: carbon nanofibres as improved neural
and orthopaedic implants
Authors: Webster Thomas J , Waid Michael C , McKenzie Janice L , Price Rachel L , Ejiofor Jeremiah U
Published: January 2004

For the continuous monitoring, diagnosis, and treatment of neural tissue, implantable
probes are required. However, sometimes such neural probes (usually composed of
silicon) become encapsulated with non-conductive, undesirable glial scar tissue.
Similarly for orthopaedic implants, biomaterials (usually titanium and/or titanium
alloys) often become encapsulated with undesirable soft fibrous, not hard bony,
tissue. Although possessing intriguing electrical and mechanical properties for
neural and orthopaedic applications, carbon nanofibres/nanotubes have not been widely
considered for these applications to date. The present work developed a carbon
nanofibre reinforced polycarbonate urethane (PU) composite in an attempt to determine
the possibility of using carbon nanofibres (CNs) as either neural or orthopaedic
prosthetic devices. Electrical and mechanical characterization studies determined
that such composites have properties suitable for neural and orthopaedic
applications. More importantly, cell adhesion experiments revealed for the first time
the promise these materials have to increase neural (nerve cell) and osteoblast
(bone-forming cell) functions. In contrast, functions of cells that contribute to
glial scar-tissue formation for neural prostheses (astrocytes) and fibrous-tissue
encapsulation events for bone implants (fibroblasts) decreased on PU composites
containing increasing amounts of CNs. In this manner, this study provided the first
evidence of the future that CN formulations may have towards interacting with neural
and bone cells which is important for the design of successful neural probes and
orthopaedic implants, respectively.
Yeast Lacking theSRO7/SOP1-encoded Tumor Suppressor Homologue Show Increased Susceptibility to Apoptosis-like Cell Death on Exposure to NaCl Stress
Authors: Wadskog Ingrid , Maldener Corinna , Proksch Astrid , Madeo Frank , Adler Lennart
Published: January 2004

Yeast cells deleted for the SRO7/SOP1 encoded tumor suppressor homologue show increased sensitivity to NaCl stress. On exposure to growth-inhibiting NaCl concentrations, sro7Δ mutants display a rapid loss in viability that is associated with markers of apoptosis: accumulation of reactive oxygen species, DNA breakage, and nuclear fragmentation. Additional deletion of the yeast metacaspase gene YCA1 prevents the primary fast drop in viability and diminishes nuclear fragmentation and DNA breakage. We also observed that NaCl induced loss in viability of wild-type cells is Yca1p dependent. However, a yeast strain deleted for both SRO7 and its homologue SRO77 exhibits NaCl-induced cell death that is independent on YCA1. Likewise, sro77Δ single mutants do not survive better after additional deletion of the YCA1 gene, and both sro77Δ and sro77Δyca1Δ mutants display apoptotic characteristics when exposed to growth-inhibiting salinity, suggesting that yeast possesses Yca1p-independent pathway(s) for apoptosis-like cell death. The activity of Yca1p increases with increasing NaCl stress and sro7Δ mutants achieve levels that are higher than in wild-type cells. However, mutants lacking SRO77 do not enhance caspase activity when subject to NaCl stress, suggesting that Sro7p and Sro77p exert opposing effects on the cellular activity of Yca1p.
CRB3 Binds Directly to Par6 and Regulates the Morphogenesis of the Tight Junctions in Mammalian Epithelial Cells
Authors: Lemmers Céline , Michel Didier , Lane-Guermonprez Lydie , Delgrossi Marie-Hélène , Médina Emmanuelle , Arsanto Jean-Pierre , Le Bivic André
Published: January 2004

Crumbs is an apical transmembrane protein crucial for epithelial morphogenesis in Drosophila melanogaster embryos. A protein with all the characteristics for a Crumbs homologue has been identified from patients suffering from retinitis pigmentosa group 12, but this protein (CRB1) is only expressed in retina and some parts of the brain, both in human and mouse. Here, we describe CRB3, another Crumbs homologue that is preferentially expressed in epithelial tissues and skeletal muscles in human. CRB3 shares the conserved cytoplasmic domain with other Crumbs but exhibits a very short extracellular domain without the EGF- and laminin A-like G repeats present in the other Crumbs. CRB3 is localized to the apical and subapical area of epithelial cells from the mouse and human intestine, suggesting that it could play a role in epithelial morphogenesis. Indeed, expression of CRB3 or of a chimera containing the extracellular domain of the neurotrophin receptor p75NTR and the transmembrane and cytoplasmic domains of CRB3 led to a slower development of functional tight junctions in Madin-Darby canine kidney cells. This phenotype relied on the presence of CRB3 four last amino acids (ERLI) that are involved in a direct interaction with Par6, a regulator of epithelial polarity and tight junction formation. Thus, CRB3, through its cytoplasmic domain and its interactors, plays a role in apical membrane morphogenesis and tight junction regulation.
A Polycystin-1 Multiprotein Complex Is Disrupted in Polycystic Kidney Disease Cells
Authors: Roitbak Tamara , Ward Christopher J. , Harris Peter C. , Bacallao Robert , Ness Scott A. , Wandinger-Ness Angela
Published: January 2004

Autosomal dominant polycystic kidney disease (ADPKD) is typified by the accumulation of fluid-filled cysts and abnormalities in renal epithelial cell function. The disease is principally caused by mutations in the gene encoding polycystin-1, a large basolateral plasma membrane protein expressed in kidney epithelial cells. Our studies reveal that, in normal kidney cells, polycystin-1 forms a complex with the adherens junction protein E-cadherin and its associated catenins, suggesting a role in cell adhesion or polarity. In primary cells from ADPKD patients, the polycystin-1/polycystin-2/E-cadherin/β-catenin complex was disrupted and both polycystin-1 and E-cadherin were depleted from the plasma membrane as a result of the increased phosphorylation of polycystin-1. The loss of E-cadherin was compensated by the transcriptional upregulation of the normally mesenchymal N-cadherin. Increased cell surface N-cadherin in the disease cells in turn stabilized the continued plasma membrane localization of β-catenin in the absence of E-cadherin. The results suggest that enhanced phosphorylation of polycystin-1 in ADPKD cells precipitates changes in its localization and its ability to form protein complexes that are critical for the stabilization of adherens junctions and the maintenance of a fully differentiated polarized renal epithelium.
Cardiac Steroids Induce Changes in Recycling of the Plasma Membrane in Human NT2 Cells
Authors: Rosen Haim , Glukhman Vladimir , Feldmann Tomer , Fridman Eleonora , Lichtstein David
Published: January 2004

Cardiac steroids (CSs) are specific inhibitors of Na+, K+-ATPase activity. Although the presence of CS-like compounds in animal tissues has been established, their physiological role is not evident. In the present study, treatment of human NT2 cells with physiological concentrations (nanomolar) of CSs caused the accumulation of large vesicles adjacent to the nucleus. Experiments using N-(3-triethylammonium propyl)-4-(dibutilamino)styryl-pyrodinum dibromide, transferrin, low-density lipoprotein, and selected anti-transferrin receptor and Rab protein antibodies revealed that CSs induced changes in endocytosis-dependent membrane traffic. Our data indicate that the CS-induced accumulation of cytoplasmic membrane components is a result of inhibited recycling within the late endocytic pathway. Furthermore, our results support the notion that the CS-induced changes in membrane traffic is mediated by the Na+, K+-ATPase. These phenomena were apparent in NT2 cells at nanomolar concentrations of CSs and were observed also in other human cell lines, pointing to the generality of this phenomenon. Based on these observations, we propose that the endogenous CS-like compounds are physiological regulators of recycling of endocytosed membrane proteins and cargo.
TopBP1 and ATR Colocalization at Meiotic Chromosomes: Role of TopBP1/Cut5 in the Meiotic Recombination Checkpoint
Authors: Perera David , Perez-Hidalgo Livia , Moens Peter B. , Reini Kaarina , Lakin Nicholas , Syväoja Juhani E. , San-Segundo Pedro A. , Freire Raimundo
Published: January 2004

Mammalian TopBP1 is a BRCT domain–containing protein whose function in mitotic cells is linked to replication and DNA damage checkpoint. Here, we study its possible role during meiosis in mice. TopBP1 foci are abundant during early prophase I and localize mainly to histone γ-H2AX–positive domains, where DNA double–strand breaks (required to initiate recombination) occur. Strikingly, TopBP1 showed a pattern almost identical to that of ATR, a PI3K-like kinase involved in mitotic DNA damage checkpoint. In the synapsis-defective Fkbp6-/-mouse, TopBP1 heavily stains unsynapsed regions of chromosomes. We also tested whether Schizosaccharomyces pombe Cut5 (the TopBP1 homologue) plays a role in the meiotic recombination checkpoint, like spRad3, the ATR homologue. Indeed, we found that a cut5 mutation suppresses the checkpoint-dependent meiotic delay of a meiotic recombination defective mutant, indicating a direct role of the Cut5 protein in the meiotic checkpoint. Our findings suggest that ATR and TopBP1 monitor meiotic recombination and are required for activation of the meiotic recombination checkpoint.
Fibronectin Matrix Assembly Regulates α5β1-mediated Cell Cohesion
Authors: Robinson Elizabeth E. , Foty Ramsey A. , Corbett Siobhan A.
Published: January 2004

Integrin-extracellular matrix (ECM) interactions in two-dimensional (2D) culture systems are widely studied (Goldstein and DiMilla, 2002. J Biomed. Mater. Res. 59, 665–675; Koo et al., 2002. J. Cell Sci. 115, 1423–1433). Less understood is the role of the ECM in promoting intercellular cohesion in three-dimensional (3D) environments. We have demonstrated that the α5β1-integrin mediates strong intercellular cohesion of 3D cellular aggregates (Robinson et al., 2003. J. Cell Sci. 116, 377–386). To further investigate the mechanism of α5β1-mediated cohesivity, we used a series of chimeric α5β1-integrin–expressing cells cultured as multilayer cellular aggregates. In these cell lines, the α5 subunit cytoplasmic domain distal to the GFFKR sequence was truncated, replaced with that of the integrin α4, the integrin α2, or maintained intact. Using these cells, α5β1-integrin–mediated cell aggregation, compaction and cohesion were determined and correlated with FN matrix assembly. The data presented demonstrate that cells cultured in the absence of external mechanical support can assemble a FN matrix that promotes integrin-mediated aggregate compaction and cohesion. Further, inhibition of FN matrix assembly blocks the intercellular associations required for compaction, resulting in cell dispersal. These results demonstrate that FN matrix assembly contributes significantly to tissue cohesion and represents an alternative mechanism for regulating tissue architecture.
TOGp, the Human Homolog of XMAP215/Dis1, Is Required for Centrosome Integrity, Spindle Pole Organization, and Bipolar Spindle Assembly
Authors: Cassimeris Lynne , Morabito Justin
Published: January 2004

The XMAP215/Dis1 MAP family is thought to regulate microtubule plus-end assembly in part by antagonizing the catastrophe-promoting function of kin I kinesins, yet XMAP215/Dis1 proteins localize to centrosomes. We probed the mitotic function of TOGp (human homolog of XMAP215/Dis1) using siRNA. Cells lacking TOGp assembled multipolar spindles, confirming results of Gergely et al. (2003. Genes Dev. 17, 336–341). Eg5 motor activity was necessary to maintain the multipolar morphology. Depletion of TOGp decreased microtubule length and density in the spindle by ∼20%. Depletion of MCAK, a kin I kinesin, increased MT lengths and density by ∼20%, but did not disrupt spindle morphology. Mitotic cells lacking both TOGp and MCAK formed bipolar and monopolar spindles, indicating that TOGp and MCAK contribute to spindle bipolarity, without major effects on MT stability. TOGp localized to centrosomes in the absence of MTs and depletion of TOGp resulted in centrosome fragmentation. TOGp depletion also disrupted MT minus-end focus at the spindle poles, detected by localizations of NuMA and the p150 component of dynactin. The major functions of TOGp during mitosis are to focus MT minus ends at spindle poles, maintain centrosome integrity, and contribute to spindle bipolarity.
Adenylyl Cyclase G Is Activated by an Intramolecular Osmosensor
Authors: Saran Shweta , Schaap Pauline
Published: January 2004

Adenylyl cyclase G (ACG) is activated by high osmolality and mediates inhibition of spore germination by this stress factor. The catalytic domains of all eukaryote cyclases are active as dimers and dimerization often mediates activation. To investigate the role of dimerization in ACG activation, we coexpressed ACG with an ACG construct that lacked the catalytic domain (ACGΔcat) and was driven by a UV-inducible promoter. After UV induction of ACGΔcat, cAMP production by ACG was strongly inhibited, but osmostimulation was not reduced. Size fractionation of native ACG showed that dimers were formed between ACG molecules and between ACG and ACGΔcat. However, high osmolality did not alter the dimer/monomer ratio. This indicates that ACG activity requires dimerization via a region outside the catalytic domain but that dimer formation does not mediate activation by high osmolality. To establish whether ACG required auxiliary sensors for osmostimulation, we expressed ACG cDNA in a yeast adenylyl cyclase null mutant. In yeast, cAMP production by ACG was similarly activated by high osmolality as in Dictyostelium. This strongly suggests that the ACG osmosensor is intramolecular, which would define ACG as the first characterized primary osmosensor in eukaryotes.
Phospholipase D2 Localizes to the Plasma Membrane and Regulates Angiotensin II Receptor Endocytosis
Authors: Du Guangwei , Huang Ping , Liang Bruce T. , Frohman Michael A.
Published: January 2004

Phospholipase D (PLD) is a key facilitator of multiple types of membrane vesicle trafficking events. Two PLD isoforms, PLD1 and PLD2, exist in mammals. Initial studies based on overexpression studies suggested that in resting cells, human PLD1 localized primarily to the Golgi and perinuclear vesicles in multiple cell types. In contrast, overexpressed mouse PLD2 was observed to localize primarily to the plasma membrane, although internalization on membrane vesicles was observed subsequent to serum stimulation. A recent report has suggested that the assignment of PLD2 to the plasma membrane is in error, because the endogenous isoform in rat secretory cells was imaged and found to be present primarily in the Golgi apparatus. We have reexamined this issue by using a monoclonal antibody specific for mouse PLD2, and find, as reported initially using overexpression studies, that endogenous mouse PLD2 is detected most readily at the plasma membrane in multiple cell types. In addition, we report that mouse, rat, and human PLD2 when overexpressed all similarly localize to the plasma membrane in cell lines from all three species. Finally, studies conducted using overexpression of wild-type active or dominant-negative isoforms of PLD2 and RNA interference-mediated targeting of PLD2 suggest that PLD2 functions at the plasma membrane to facilitate endocytosis of the angiotensin II type 1 receptor.
The Differential Roles of Budding Yeast Tem1p, Cdc15p, and Bub2p Protein Dynamics in Mitotic Exit
Authors: Molk Jeffrey N. , Schuyler Scott C. , Liu Jenny Y. , Evans James G. , Salmon E. D. , Pellman David , Bloom Kerry
Published: January 2004

In the budding yeast Saccharomyces cerevisiae the mitotic spindle must be positioned along the mother-bud axis to activate the mitotic exit network (MEN) in anaphase. To examine MEN proteins during mitotic exit, we imaged the MEN activators Tem1p and Cdc15p and the MEN regulator Bub2p in vivo. Quantitative live cell fluorescence microscopy demonstrated the spindle pole body that segregated into the daughter cell (dSPB) signaled mitotic exit upon penetration into the bud. Activation of mitotic exit was associated with an increased abundance of Tem1p-GFP and the localization of Cdc15p-GFP on the dSPB. In contrast, Bub2p-GFP fluorescence intensity decreased in mid-to-late anaphase on the dSPB. Therefore, MEN protein localization fluctuates to switch from Bub2p inhibition of mitotic exit to Cdc15p activation of mitotic exit. The mechanism that elevates Tem1p-GFP abundance in anaphase is specific to dSPB penetration into the bud and Dhc1p and Lte1p promote Tem1p-GFP localization. Finally, fluorescence recovery after photobleaching (FRAP) measurements revealed Tem1p-GFP is dynamic at the dSPB in late anaphase. These data suggest spindle pole penetration into the bud activates mitotic exit, resulting in Tem1p and Cdc15p persistence at the dSPB to initiate the MEN signal cascade.
Two Functionally Identical Modular Enhancers inDrosophilaTroponin T Gene Establish the Correct Protein Levels in Different Muscle Types
Authors: Mas José-Antonio , García-Zaragoza Elena , Cervera Margarita
Published: January 2004

The control of muscle-specific expression is one of the principal mechanisms by which diversity is generated among muscle types. In an attempt to elucidate the regulatory mechanisms that control fiber diversity in any given muscle, we have focused our attention on the transcriptional regulation of the Drosophila Troponin T gene. Two, nonredundant, functionally identical, enhancer-like elements activate Troponin T transcription independently in all major muscles of the embryo and larvae as well as in adult somatic and visceral muscles. Here, we propose that the differential but concerted interaction of these two elements underlies the mechanism by which a particular muscle-type establish the correct levels of Troponin T expression, adapting these levels to their specific needs. This mechanism is not exclusive to the Troponin T gene, but is also relevant to the muscle-specific Troponin I gene. In conjunction with in vivo transgenic studies, an in silico analysis of the Troponin T enhancer-like sequences revealed that both these elements are organized in a modular manner. Extending this analysis to the Troponin I and Tropomyosin regulatory elements, the two other components of the muscle-regulatory complex, we have discovered a similar modular organization of phylogenetically conserved domains.
The Transcriptome of Prematurely Aging Yeast Cells Is Similar to That of Telomerase-deficient Cells
Authors: Lesur Isabelle , Campbell Judith L.
Published: January 2004

To help define the pathologies associated with yeast cells as they age, we analyzed the transcriptome of young and old cells isolated by elutriation, which allows isolation of biochemical quantities of old cells much further advanced in their life span than old cells prepared by the biotin-streptavidin method. Both 18-generation-old wild-type yeast and 8-generation-old cells from a prematurely aging mutant (dna2-1), with a defect in DNA replication, were evaluated. Genes involved in gluconeogenesis, the glyoxylate cycle, lipid metabolism, and glycogen production are induced in old cells, signifying a shift toward energy storage. We observed a much more extensive generalized stress response known as the environmental stress response (ESR), than observed previously in biotin-streptavidin-isolated cells, perhaps because the elutriated cells were further advanced in their life span. In addition, there was induction of DNA repair genes that fall in the so-called DNA damage “signature” set. In the dna2-1 mutant, energy production genes were also induced. The response in the dna2-1 strain is similar to the telomerase delete response, genes whose expression changes during cellular senescence in telomerase-deficient cells. We propose that these results suggest, albeit indirectly, that old cells are responding to genome instability.
Distinct Functional Domains within Nucleoporins Nup153 and Nup98 Mediate Transcription-dependent Mobility
Authors: Griffis Eric R. , Craige Branch , Dimaano Christian , Ullman Katharine S. , Powers Maureen A.
Published: January 2004

Despite the apparent overall structural stability of the nuclear pore complex during interphase, at least two nucleoporins have been shown to move dynamically on and off the pore. It is not yet certain what contribution nucleoporin mobility makes to the process of nuclear transport or how such mobility is regulated. Previously, we showed that Nup98 dynamically interacts with the NPC as well as bodies within the nucleus in a transcription-dependent manner. We have extended our studies of dynamics to include Nup153, another mobile nucleoporin implicated in RNA export. In both cases, we found that although only one domain is essential for NPC localization, other regions of the protein significantly affect the stability of association with the pore. Interestingly, like Nup98, the exchange of Nup153 on and off the pore is inhibited when transcription by Pol I and Pol II is blocked. We have mapped the regions required to link Nup98 and Nup153 mobility to transcription and found that the requirements differ depending on which polymerases are inhibited. Our data support a model whereby transcription of RNA is coupled to nucleoporin mobility, perhaps ultimately linking transport of RNAs to a cycle of remodeling at the nuclear pore basket.
The Yeast Elongator Histone Acetylase Requires Sit4-dependent Dephosphorylation for Toxin-Target Capacity
Authors: Jablonowski Daniel , Fichtner Lars , Stark Michael J.R. , Schaffrath Raffael
Published: January 2004

Kluyveromyces lactis zymocin, a heterotrimeric toxin complex, imposes a G1 cell cycle block on Saccharomyces cerevisiae that requires the toxin-target (TOT) function of holo-Elongator, a six-subunit histone acetylase. Here, we demonstrate that Elongator is a phospho-complex. Phosphorylation of its largest subunit Tot1 (Elp1) is supported by Kti11, an Elongator-interactor essential for zymocin action. Tot1 dephosphorylation depends on the Sit4 phosphatase and its associators Sap185 and Sap190. Zymocin-resistant cells lacking or overproducing Elongator-associator Tot4 (Kti12), respectively, abolish or intensify Tot1 phosphorylation. Excess Sit4·Sap190 antagonizes the latter scenario to reinstate zymocin sensitivity in multicopy TOT4 cells, suggesting physical competition between Sit4 and Tot4. Consistently, Sit4 and Tot4 mutually oppose Tot1 de-/phosphorylation, which is dispensable for integrity of holo-Elongator but crucial for the TOT-dependent G1 block by zymocin. Moreover, Sit4, Tot4, and Tot1 cofractionate, Sit4 is nucleocytoplasmically localized, and sit4Δ-nuclei retain Tot4. Together with the findings that sit4Δ and totΔ cells phenocopy protection against zymocin and the ceramide-induced G1 block, Sit4 is functionally linked to Elongator in cell cycle events targetable by antizymotics.
Expression Profiling of Mammalian Male Meiosis and Gametogenesis Identifies Novel Candidate Genes for Roles in the Regulation of Fertility
Authors: Schlecht Ulrich , Demougin Philippe , Koch Reinhold , Hermida Leandro , Wiederkehr Christa , Descombes Patrick , Pineau Charles , Jégou Bernard , Primig Michael
Published: January 2004

We report a comprehensive large-scale expression profiling analysis of mammalian male germ cells undergoing mitotic growth, meiosis, and gametogenesis by using high-density oligonucleotide microarrays and highly enriched cell populations. Among 11,955 rat loci investigated, 1268 were identified as differentially transcribed in germ cells at subsequent developmental stages compared with total testis, somatic Sertoli cells as well as brain and skeletal muscle controls. The loci were organized into four expression clusters that correspond to somatic, mitotic, meiotic, and postmeiotic cell types. This work provides information about expression patterns of ∼200 genes known to be important during male germ cell development. Approximately 40 of those are included in a group of 121 transcripts for which we report germ cell expression and lack of transcription in three somatic control cell types. Moreover, we demonstrate the testicular expression and transcriptional induction in mitotic, meiotic, and/or postmeiotic germ cells of 293 as yet uncharacterized transcripts, some of which are likely to encode factors involved in spermatogenesis and fertility. This group also contains potential germ cell-specific targets for innovative contraceptives. A graphical display of the data is conveniently accessible through the GermOnline database at http://www.germonline.org .
A BAC- and BIBAC-Based Physical Map of the Soybean Genome
Authors: Wu Chengcang , Sun Shuku , Nimmakayala Padmavathi , Santos Felipe A. , Meksem Khalid , Springman Rachael , Ding Kejiao , Lightfoot David A. , Zhang Hong-Bin
Published: January 2004

Genome-wide physical maps are crucial to many aspects of advanced genome research. We report a genome-wide, bacterial artificial chromosome (BAC) and plant-transformation-competent binary large-insert plasmid clone (hereafter BIBAC)-based physical map of the soybean genome. The map was constructed from 78,001 clones from five soybean BAC and BIBAC libraries representing 9.6 haploid genomes and three cultivars, and consisted of 2905 BAC/BIBAC contigs, estimated to span 1408 Mb in physical length. We evaluated the reliability of the map contigs using different contig assembly strategies, independent contig building methods, DNA marker hybridization, and different fingerprinting methods, and the results showed that the contigs were assembled properly. Furthermore, we tested the feasibility of integrating the physical map with the existing soybean composite genetic map using 388 DNA markers. The results further confirmed the nature of the ancient tetraploid origin of soybean and indicated that it is feasible to integrate the physical map with the linkage map even though greater efforts are needed. This map represents the first genome-wide, BAC/BIBAC-based physical map of the soybean genome and would provide a platform for advanced genome research of soybean and other legume species. The inclusion of BIBACs in the map would streamline the utility of the map for positional cloning of genes and QTLs, and functional analysis of soybean genomic sequences.
Expression Profiling and Comparative Genomics Identify a Conserved Regulatory Region Controlling Midline Expression in the Zebrafish Embryo
Authors: Dickmeis Thomas , Plessy Charles , Rastegar Sepand , Aanstad Pia , Herwig Ralf , Chalmel Frédéric , Fischer Nadine , Strähle Uwe
Published: January 2004

Differential gene transcription is a fundamental regulatory mechanism of biological systems during development, body homeostasis, and disease. Comparative genomics is believed to be a rapid means for the identification of regulatory sequences in genomes. We tested this approach to identify regulatory sequences that control expression in the midline of the zebrafish embryo. We first isolated a set of genes that are coexpressed in the midline of the zebrafish embryo during somitogenesis stages by gene array analysis and subsequent rescreens by in situ hybridization. We subjected 45 of these genes to Compare and DotPlot analysis to detect conserved sequences in noncoding regions of orthologous loci in the zebrafish and Takifugu genomes. The regions of homology that were scored in nonconserved regions were inserted into expression vectors and tested for their regulatory activity by transient transgenesis in the zebrafish embryo. We identified one conserved region from the connective tissue growth factor gene (ctgf), which was able to drive expression in the midline of the embryo. This region shares sequence similarity with other floor plate/notochord-specific regulatory regions. Our results demonstrate that an unbiased comparative approach is a relevant method for the identification of tissue-specific cis-regulatory sequences in the zebrafish embryo.
Flux Coupling Analysis of Genome-Scale Metabolic Network Reconstructions
Authors: Burgard Anthony P. , Nikolaev Evgeni V. , Schilling Christophe H. , Maranas Costas D.
Published: January 2004

In this paper, we introduce the Flux Coupling Finder (FCF) framework for elucidating the topological and flux connectivity features of genome-scale metabolic networks. The framework is demonstrated on genome-scale metabolic reconstructions of Helicobacter pylori, Escherichia coli, and Saccharomyces cerevisiae. The analysis allows one to determine whether any two metabolic fluxes, v1 and v2, are (1) directionally coupled, if a non-zero flux for v1 implies a non-zero flux for v2 but not necessarily the reverse; (2) partially coupled, if a non-zero flux for v1 implies a non-zero, though variable, flux for v2 and vice versa; or (3) fully coupled, if a non-zero flux for v1 implies not only a non-zero but also a fixed flux for v2 and vice versa. Flux coupling analysis also enables the global identification of blocked reactions, which are all reactions incapable of carrying flux under a certain condition; equivalent knockouts, defined as the set of all possible reactions whose deletion forces the flux through a particular reaction to zero; and sets of affected reactions denoting all reactions whose fluxes are forced to zero if a particular reaction is deleted. The FCF approach thus provides a novel and versatile tool for aiding metabolic reconstructions and guiding genetic manipulations.
Closing the Gaps on Human Chromosome 19 Revealed Genes With a High Density of Repetitive Tandemly Arrayed Elements
Authors: Leem Sun-Hee , Kouprina Natalay , Grimwood Jane , Kim Jung-Hyun , Mullokandov Michael , Yoon Young-Ho , Chae Ji-Youn , Morgan Jenna , Lucas Susan , Richardson Paul , Detter Chris , Glavina Tijana , Rubin Eddy , Barrett J. Carl , Larionov Vladimir
Published: January 2004

The reported human genome sequence includes about 400 gaps of unknown sequence that were not found in the bacterial artificial chromosome (BAC) and cosmid libraries used for sequencing of the genome. These missing sequences correspond to ∼1% of euchromatic regions of the human genome. Gap filling is a laborious process because it relies on analysis of random clones of numerous genomic BAC or cosmid libraries. In this work we demonstrate that closing the gaps can be accelerated by a selective recombinational capture of missing chromosomal segments in yeast. The use of both methodologies allowed us to close the four remaining gaps on the human chromosome 19. Analysis of the gap sequences revealed that they contain several abnormalities that could result in instability of the sequences in microbe hosts, including large blocks of micro- and minisatellites and a high density of Alu repeats. Sequencing of the gap regions, in both BAC and YAC forms, allowed us to generate a complete sequence of four genes, including the neuronal cell signaling gene SCK1/SLI. The SCK1/SLI gene contains a record number of minisatellites, most of which are polymorphic and transmitted through meiosis following a Mendelian inheritance. In conclusion, the use of the alternative recombinational cloning system in yeast may greatly accelerate work on closing the remaining gaps in the human genome (as well as in other complex genomes) to achieve the goal of annotation of all human genes.