bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of the Southern Annular Mode on Southern Ocean circulation and biology
Authors: Lovenduski Nicole S. , Gruber Nicolas
Published: June 2005

We investigate the impact of the Southern Annular Mode (SAM) on surface wind, sea surface temperature (SST), and surface chlorophyll concentration on intraseasonal to interannual timescales in the Southern Ocean using 8‐day average satellite observations. Positive phases of the SAM are associated with enhanced westerly winds over the Antarctic Zone (AZ) and Polar Frontal Zone, driving increased equatorward Ekman transport and cold SST anomalies in these regions. Positive SAM is also associated with easterly wind and warm SST anomalies in the Subtropical Zone. South of the Antarctic Polar Front (APF), chlorophyll concentration anomalies are positively correlated with the SAM, however this correlation is negative north of the APF. We suggest that the positive correlation in the AZ is due to the increased supply of iron by upwelling, while the negative correlation north of the APF is caused by stronger light limitation as a consequence of deeper mixed layers.
Measuring surface currents in lakes with high spatial resolution thermal infrared imagery
Authors: Steissberg Todd E. , Hook Simon J. , Schladow S. Geoffrey
Published: June 2005

High spatial resolution thermal infrared satellite images acquired 38 minutes apart from Landsat ETM+ and ASTER were used to measure the surface currents and circulation in Lake Tahoe, California‐Nevada, USA. Mean currents of 5–10 cm/s were measured, with maximum currents approaching 35 cm/s. The eastward transport of an upwelling surface jet was clearly apparent, with 15–30 cm/s currents. Three gyres were evident in the vector fields. The satellite image‐derived surface currents and circulation patterns are consistent with surface drifter measurements acquired during a different period. These results modify the findings of an earlier study using 1.1 km AVHRR imagery, which concluded there were two counter‐rotating gyres in the opposite sense to the two dominant large gyres measured in this study. The high spatial resolution and small time separation allow the surface currents and general circulation in lakes and coastal environments to be accurately quantified using the maximum cross‐correlation method.
Bright and dark ocean whitecaps observed in the infrared
Authors: Marmorino G. O. , Smith G. B.
Published: June 2005

Airborne infrared imagery of breaking ocean waves shows that whitecaps on actively breaking wave crests appear relatively bright while residual whitecaps appear dark compared to the ambient water surface. These differences in relative brightness, which appear to arise from changes in both emissivity and physical temperature, provide a means to investigate the evolution of a breaking wave and may be of some relevance to infrared and microwave remote sensing.
Middle Pliocene sea surface temperature variability
Authors: Dowsett Harry J. , Chandler Mark A. , Cronin Thomas M. , Dwyer Gary S.
Published: June 2005

Estimates of sea surface temperature (SST) based upon foraminifer, diatom, and ostracod assemblages from ocean cores reveal a warm phase of the Pliocene between about 3.3 and 3.0 Ma. Pollen records and plant megafossils, although not as well dated, show evidence for a warmer climate at about the same time. Increased greenhouse forcing and altered ocean heat transport are the leading candidates for the underlying cause of Pliocene global warmth. Despite being a period of global warmth, this interval encompasses considerable variability. Two new SST reconstructions are presented that are designed to provide a climatological error bar for warm peak phases of the Pliocene and to document the spatial distribution and magnitude of SST variability within the mid‐Pliocene warm period. These data suggest long‐term stability of low‐latitude SST and document greater variability in regions of maximum warming.
Degenerate mitochondria
Authors: van der Giezen Mark , Tovar Jorge
Published: June 2005

Mitochondria are the main sites of biological energy generation in eukaryotes. These organelles are remnants of a bacterial endosymbiont that took up residence inside a host cell over 1,500 million years ago. Comparative genomics studies suggest that the mitochondrion is monophyletic in origin. Thus, the original mitochondrial endosymbiont has evolved independently in anaerobic and aerobic environments that are inhabited by diverse eukaryotic lineages. This process has resulted in a collection of morphologically, genetically and functionally heterogeneous organelle variants that include anaerobic and aerobic mitochondria, hydrogenosomes and mitosomes. Current studies aim to determine whether a central common function drives the retention of mitochondrial organelles in different eukaryotic organisms.
The effects of histone deacetylase inhibitors on heterochromatin: implications for anticancer therapy?
Authors: Taddei Angela , Roche Danièle , Bickmore Wendy A. , Almouzni Geneviève
Published: June 2005

Histone acetylation regulates many chromosome functions, such as gene expression and chromosome segregation. Histone deacetylase inhibitors (HDACIs) induce growth arrest, differentiation and apoptosis of cancer cells ex vivo, as well as in vivo in tumour‐bearing animal models, and are now undergoing clinical trials as anti‐tumour agents. However, little attention has been paid to how HDACIs function in these biological settings and why different cells respond in different ways. Here, we discuss the consequences of inhibiting histone deacetylases in cycling versus non‐cycling cells, in light of the dynamics of histone acetylation patterns with a specific emphasis on heterochromatic regions of the genome.
The membrane-anchored serine protease, TMPRSS2, activates PAR-2 in prostate cancer cells
Authors: WILSON Susan , GREER Brett , HOOPER John , ZIJLSTRA Andries , WALKER Brian , QUIGLEY James , HAWTHORNE Susan
Published: June 2005

TMPRSS2 is a type II transmembrane-bound serine protease that has gained interest owing to its highly localized expression in the prostate and its overexpression in neoplastic prostate epithelium. Once activated, the serine protease domain of TMPRSS2 is released from the cell surface into the extracellular space. PAR (protease-activated receptor)-2 belongs to a family of G-protein-coupled receptors (PAR-1–4) that are activated by specific serine proteases, which are expressed in many normal and malignant cell types. Previous in vitro studies on prostate cancer cells suggest a role for PAR-2 in prostate cancer metastasis. A polyclonal anti-human TMPRSS2 antibody was generated against the TMPRSS2 serine protease domain. The antibody showed specific reactivity with recombinant expressed TMPRSS2, and so was used to extract and purify the cleaved active TMPRSS2 protease from prostate cancer cells. Reverse transcriptase PCR and Western blot analysis were used to show the expression of both TMPRSS2 and PAR-2 in the androgen-dependent LNCaP prostate cancer cell line. Treatment of LNCaP cells with the cellular immunopurified TMPRSS2 protease induced a transient increase in intracellular calcium, which is indicative of G-protein-coupled-receptor activation. This calcium mobilization was inhibited by cellular pre-treatment with a specific PAR-2 antagonist, but not with a PAR-1 antagonist; inhibition of the protease activity also failed to mobilize calcium, suggesting that TMPRSS2 is capable of cleaving and thereby activating the PAR-2 receptor. The calcium mobilization was also inhibited by cellular pre-treatment with suramin or 2-APB (2-aminoethoxydiphenyl borate), indicating that a G-protein pathway is involved and that subsequent calcium release is mainly from intracellular stores. The present study describes how TMPRSS2 may contribute to prostate tumour metastasis via the activation of PAR-2.
High-throughput metabolic state analysis: the missing link in integrated functional genomics of yeasts
Authors: VILLAS-BÔAS Silas G. , MOXLEY Joel F. , ÅKESSON Mats , STEPHANOPOULOS Gregory , NIELSEN Jens
Published: June 2005

The lack of comparable metabolic state assays severely limits understanding the metabolic changes caused by genetic or environmental perturbations. The present study reports the application of a novel derivatization method for metabolome analysis of yeast, coupled to data-mining software that achieve comparable throughput, effort and cost compared with DNA arrays. Our sample workup method enables simultaneous metabolite measurements throughout central carbon metabolism and amino acid biosynthesis, using a standard GC-MS platform that was optimized for this purpose. As an implementation proof-of-concept, we assayed metabolite levels in two yeast strains and two different environmental conditions in the context of metabolic pathway reconstruction. We demonstrate that these differential metabolite level data distinguish among sample types, such as typical metabolic fingerprinting or footprinting. More importantly, we demonstrate that this differential metabolite level data provides insight into specific metabolic pathways and lays the groundwork for integrated transcription–metabolism studies of yeasts.
Biosynthesis of agmatine in isolated mitochondria and perfused rat liver: studies with 15N-labelled arginine
Authors: HORYN Oksana , LUHOVYY Bohdan , LAZAROW Adam , DAIKHIN Yevgeny , NISSIM Ilana , YUDKOFF Marc , NISSIM Itzhak
Published: June 2005

An important but unresolved question is whether mammalian mitochondria metabolize arginine to agmatine by the ADC (arginine decarboxylase) reaction. 15N-labelled arginine was used as a precursor to address this question and to determine the flux through the ADC reaction in isolated mitochondria obtained from rat liver. In addition, liver perfusion system was used to examine a possible action of insulin, glucagon or cAMP on a flux through the ADC reaction. In mitochondria and liver perfusion, 15N-labelled agmatine was generated from external 15N-labelled arginine. The production of 15N-labelled agmatine was time- and dose-dependent. The time-course of [U-15N4]agmatine formation from 2 mM [U-15N4]arginine was best fitted to a one-phase exponential curve with a production rate of approx. 29 pmol·min−1·(mg of protein)−1. Experiments with an increasing concentration (0– 40 mM) of [guanidino-15N2]arginine showed a Michaelis constant Km for arginine of 46 mM and a Vmax of 3.7 nmol·min−1·(mg of protein)−1 for flux through the ADC reaction. Experiments with broken mitochondria showed little changes in Vmax or Km values, suggesting that mitochondrial arginine uptake had little effect on the observed Vmax or Km values. Experiments with liver perfusion demonstrated that over 95% of the effluent agmatine was derived from perfusate [guanidino-15N2]arginine regardless of the experimental condition. However, the output of 15N-labelled agmatine (nmol·min−1·g−1) increased by approx. 2-fold (P<0.05) in perfusions with cAMP. The findings of the present study provide compelling evidence that mitochondrial ADC is present in the rat liver, and suggest that cAMP may stimulate flux through this pathway.
Distinct requirements for the Sprouty domain for functional activity of Spred proteins
Authors: KING James A. J. , STRAFFON Andrew F. L. , D'ABACO Giovanna M. , POON Carole L. C. , I Stacey T. T. , SMITH Craig M. , BUCHERT Michael , CORCORAN Niall M. , HALL Nathan E. , CALLUS Bernard A. , SARCEVIC Boris , MARTIN Daniel , LOCK Peter , HOVENS Christopher M.
Published: June 2005

Sprouty and Spred {Sprouty-related EVH1 [Ena/VASP (vasodilator-stimulated phosphoprotein) homology 1] domain} proteins have been identified as antagonists of growth factor signalling pathways. We show here that Spred-1 and Spred-2 appear to have distinct mechanisms whereby they induce their effects, as the Sprouty domain of Spred-1 is not required to block MAPK (mitogen-activated protein kinase) activation, while that of Spred-2 is required. Similarly, deletion of the C-terminal Sprouty domain of Spred-1 does not affect cell-cycle progression of G0-synchronized cells through to S-phase following growth factor stimulation, while the Sprouty domain is required for Spred-2 function. We also demonstrate that the inhibitory function of Spred proteins is restricted to the Ras/MAPK pathway, that tyrosine phosphorylation is not required for this function, and that the Sprouty domain mediates heterodimer formation of Spred proteins. Growth-factor-mediated activation of the small GTPases, Ras and Rap1, was able to be regulated by Spred-1 and Spred-2, without affecting receptor activation. Taken together, these results highlight the potential for different functional roles of the Sprouty domain within the Spred family of proteins, suggesting that Spred proteins may use different mechanisms to induce inhibition of the MAPK pathway.
Nop53p is a novel nucleolar 60S ribosomal subunit biogenesis protein
Authors: SYDORSKYY Yaroslav , DILWORTH David J. , HALLORAN Brendan , YI Eugene C. , MAKHNEVYCH Taras , WOZNIAK Richard W. , AITCHISON John D.
Published: June 2005

Ribosome biogenesis in Saccharomyces cerevisiae occurs primarily in a specialized nuclear compartment termed the nucleolus within which the rRNA genes are transcribed by RNA polymerase I into a large 35 S rRNA precursor. The ensuing association/dissociation and catalytic activity of numerous trans-acting protein factors, RNAs and ribosomal proteins ultimately leads to the maturation of the precursor rRNAs into 25, 5.8 and 18 S rRNAs and the formation of mature cytoplasmic 40 and 60 S ribosomal subunits. Although many components involved in ribosome biogenesis have been identified, our understanding of this essential cellular process remains limited. In the present study we demonstrate a crucial role for the previously uncharacterized nucleolar protein Nop53p (Ypl146p) in ribosome biogenesis. Specifically, Nop53p appears to be most important for biogenesis of the 60 S subunit. It physically interacts with rRNA processing factors, notably Cbf5p and Nop2p, and co-fractionates specifically with pre-60 S particles on sucrose gradients. Deletion or mutations within NOP53 cause significant growth defects and display significant 60 S subunit deficiencies, an imbalance in the 40 S:60 S ratio, as revealed by polysome profiling, and defects in progression beyond the 27 S stage of 25 S rRNA maturation during 60 S biogenesis.
H11 has dose-dependent and dual hypertrophic and proapoptotic functions in cardiac myocytes
Authors: HASE Makoto , DEPRE Christophe , VATNER Stephen F. , SADOSHIMA Junichi
Published: June 2005

We have shown previously that H11, a serine/threonine kinase, is up-regulated in a heart subjected to ischaemia/reperfusion. In the present study, we have characterized the cellular function of H11, using neonatal rat cardiac myocytes. Although transduction of adenovirus harbouring H11 at low doses increased the cell size, at higher doses it induced apoptosis in cardiac myocytes. Apoptosis was not observed when adenovirus harbouring H11-KI (kinase-inactive mutant of H11) was used, suggesting that the proapoptotic effect of H11 is kinase-dependent. The hypertrophic effect of H11 at high doses was unmasked when apoptosis was inhibited by the caspase inhibitor DEVD-CHO, suggesting that H11 stimulates both hypertrophy and apoptosis in parallel. H11-KI induced hypertrophy even at high doses, indicating that H11 stimulates hypertrophy through kinase-independent mechanisms. H11-KI activated Akt, and cardiac hypertrophy induced by H11-KI was blocked by LY294002, an inhibitor of phosphoinositide 3-kinase. Co-immunoprecipitation analyses indicated that H11 interacts with the α subunit of CK2 (casein kinase 2). Overexpression of H11 decreased the kinase activity of CK2. DRB (5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole), an inhibitor of CK2, mimicked the effect of H11, whereas DRB and H11 failed to exhibit additive effects on apoptosis, suggesting that H11 and DRB utilize a common mechanism to induce apoptosis, namely inhibition of CK2. In summary, H11 is a dual-function kinase in cardiac cells: it induces hypertrophy at low doses through kinase-independent activation of Akt, whereas it causes apoptosis at high doses through protein kinase-dependent mechanisms, in particular by physical interaction with and subsequent inhibition of CK2.
A new type of scorpion Na+-channel-toxin-like polypeptide active on K+ channels
Authors: SRAIRI-ABID Najet , GUIJARRO Joseba Iñaki , BENKHALIFA Rym , MANTEGAZZA Massimo , CHEIKH Amani , BEN AISSA Manel , HAUMONT Pierre-Yves , DELEPIERRE Muriel , EL AYEB Mohamed
Published: June 2005

We have purified and characterized two peptides, named KAaH1 and KAaH2 (AaH polypeptides 1 and 2 active on K+ channels, where AaH stands for Androctonus australis Hector), from the venom of A. australis Hector scorpions. Their sequences contain 58 amino acids including six half-cysteines and differ only at positions 26 (Phe/Ser) and 29 (Lys/Gln). Although KAaH1 and KAaH2 show important sequence similarity with anti-mammal β toxins specific for voltage-gated Na+ channels, only weak β-like effects were observed when KAaH1 or KAaH2 (1 μM) were tested on brain Nav1.2 channels. In contrast, KAaH1 blocks Kv1.1 and Kv1.3 channels expressed in Xenopus oocytes with IC50 values of 5 and 50 nM respectively, whereas KAaH2 blocks only 20% of the current on Kv1.1 and is not active on Kv1.3 channels at a 100 nM concentration. KAaH1 is thus the first member of a new subfamily of long-chain toxins mainly active on voltage-gated K+ channels. NMR spectra of KAaH1 and KAaH2 show good dispersion of signals but broad lines and poor quality. Self-diffusion NMR experiments indicate that lines are broadened due to a conformational exchange on the millisecond time scale. NMR and CD indicate that both polypeptides adopt a similar fold with α-helical and β-sheet structures. Homology-based molecular models generated for KAaH1 and KAaH2 are in accordance with CD and NMR data. In the model of KAaH1, the functionally important residues Phe26 and Lys29 are close to each other and are located in the α-helix. These residues may constitute the so-called functional dyad observed for short α-KTx scorpion toxins in the β-sheet.
C/EBPα activates the transcription of triacylglycerol hydrolase in 3T3-L1 adipocytes
Authors: WEI Enhui , LEHNER Richard , VANCE Dennis E.
Published: June 2005

TGH (triacylglycerol hydrolase) catalyses the lipolysis of intracellular stored triacylglycerol. To explore the mechanisms that regulate TGH expression in adipose tissue, we studied the expression of TGH during the differentiation of 3T3-L1 adipocytes. TGH mRNA and protein levels increased dramatically in 3T3-L1 adipocytes compared with pre-adipocytes. Electrophoretic mobility shift assays demonstrated enhanced binding of nuclear proteins of adipocytes to the distal murine TGH promoter region (−542/−371 bp), yielding one adipocyte-specific migrating complex. Competitive and supershift assays demonstrated that the distal TGH promoter fragment bound C/EBPα (CCAAT/enhancer-binding protein α). Transient transfections of different mutant TGH promoter–luciferase constructs into 3T3-L1 adipocytes and competitive electromobility shift assays showed that the C/EBP-binding elements at positions −470/−459 bp and −404/−390 bp are important for transcriptional activation. Co-transfection with C/EBPα cDNA and TGH promoter constructs in 3T3-L1 pre-adipocytes demonstrated that C/EBPα increased TGH promoter activity. Ectopic expression of C/EBPα in NIH 3T3 cells activated TGH mRNA expression without causing differentiation into adipocytes. These experiments directly link increased TGH expression in adipocytes to transcriptional regulation by C/EBPα. This is the first evidence that C/EBPα participates directly in the regulation of an enzyme associated with lipolysis.
YB-1 represses AP1-dependent gene transactivation and interacts with an AP-1 DNA sequence
Authors: SAMUEL Shaija , TWIZERE Jean-Claude , BERNSTEIN Lori R.
Published: June 2005

Involvement of the AP-1 (activator protein-1) transcription factor has been demonstrated previously in the regulation of cell proliferation and cell-cycle progression, in the control of cell migration, invasion and metastasis, and in signal transduction, stress responsiveness, DNA replication and DNA repair. YB-1 (Y-box-binding protein-1) has also been implicated in many of these processes. However, the mechanism by which YB-1 mediates these processes is poorly understood. In the present study, we report that overexpression of a transfected gene encoding YB-1 in human HeLa cervical carcinoma cells significantly represses the transactivation of a minimal AP-1 reporter construct in response to the tumour promoter PMA. YB-1 also represses mRNA expression and PMA-induced promoter transactivation of the endogenous AP-1 target gene encoding matrix metalloproteinase-12 (metalloelastase). YB-1 transrepression of both the minimal and matrix metalloproteinase-12 promoter reporter constructs is dependent on the AP-1 sequence. To identify new nuclear proteins that bind specifically to the AP-1 DNA-binding site, we devised a DNA-affinity-chromatography-based assay termed NAPSTER (nucleotide-affinity preincubation specificity test of recognition) and discovered a 49 kDa protein from human cancer cells that binds in a sequence-specific manner to the AP-1 DNA sequence. By tandem MS fragmentation sequencing analyses we determined that p49 is a YB-1. Immunoblotting of the NAPSTER-purified p49 protein using anti-YB-1 antibodies confirmed YB-1 binding to the AP-1 DNA sequence, as did gel mobility-supershift assays using YB-1 antibodies. This is the first report of YB-1 transrepression and interaction at the AP-1 DNA-binding site.
αII-Spectrin interacts with Tes and EVL, two actin-binding proteins located at cell contacts
Authors: ROTTER Björn , BOURNIER Odile , NICOLAS Gael , DHERMY Didier , LECOMTE Marie-Christine
Published: June 2005

The spectrin-based membrane skeleton, a multi-protein scaffold attached to diverse cellular membranes, is presumed to be involved in the stabilization of membranes, the establishment of membrane domains as well as in vesicle trafficking and nuclear functions. Spectrin tetramers made of α- and β-subunits are linked to actin microfilaments, forming a network that binds a multitude of proteins. The most prevalent α-spectrin subunit in non-erythroid cells, αII-spectrin, contains two particular spectrin repeats in its central region, α9 and α10, which host an Src homology 3 domain, a tissue-specific spliced sequence of 20 residues, a calmodulin-binding site and major cleavage sites for caspases and calpains. Using yeast two-hybrid screening of kidney libraries, we identified two partners of the α9-α10 repeats: the potential tumour suppressor Tes, an actin-binding protein mainly located at focal adhesions; and EVL (Ena/vasodilator-stimulated phosphoprotein-like protein), another actin-binding protein, equally recruited at focal adhesions. Interactions between spectrin and overexpressed Tes and EVL were confirmed by co-immunoprecipitation. In vitro studies showed that the interaction between Tes and spectrin is mediated by a LIM (Lin-11, Isl-1 and Mec3) domain of Tes and by the α10 repeat of αII-spectrin whereas EVL interacts with the Src homology 3 domain located within the α9 repeat. Moreover, we describe an in vitro interaction between Tes and EVL, and a co-localization of these two proteins at focal adhesions. These interactions between αII-spectrin, Tes and EVL indicate new functions for spectrin in actin dynamics and focal adhesions.
Glucosamine induces cell-cycle arrest and hypertrophy of mesangial cells: implication of gangliosides
Authors: MASSON Elodie , WIERNSPERGER Nicolas , LAGARDE Michel , BAWAB Samer El
Published: June 2005

Alterations in proliferation and hypertrophy of renal mesangial cells are typical features of diabetic nephropathy. The HP (hexosamine pathway) has been proposed as a biochemical hypothesis to explain microvascular alterations due to diabetic nephropathy; however, involvement of HP in the regulation of mesangial cell growth or hypertrophy has been poorly studied. Although gangliosides are known to regulate cell proliferation, their potential role in mesangial cell-growth perturbations has hardly been explored. In the present study, we investigated the effects of the HP activation, mimicked by GlcN (glucosamine) treatment, on mesangial cell growth and hypertrophy and the potential implication of gangliosides in these processes. Our results indicate that GlcN induced hypertrophy of mesangial cells, as measured by an increase in the protein/cell ratio, and it caused cell-cycle arrest by an increase in the expression of cyclin-dependent kinase inhibitor p21Waf1/Cip1. Furthermore, GlcN treatment resulted in a massive increase in the levels of gangliosides GM2 and GM1. Treatment of cells with exogenous GM2 and GM1 reproduced the effects of 0.5 mM GlcN on p21Waf1/Cip1 expression, cell-cycle arrest and hypertrophy, suggesting that gangliosides GM2 and GM1 are probably involved in mediating GlcN effects. These results document a new role of the HP in the regulation of mesangial cell growth and hypertrophy. They also suggest a potential new mechanism of action of the HP through modulation of ganglioside levels.
Interaction of U-box E3 ligase SNEV with PSMB4, the β7 subunit of the 20 S proteasome
Authors: LÖSCHER Marlies , FORTSCHEGGER Klaus , RITTER Gustav , WOSTRY Martina , VOGLAUER Regina , SCHMID Johannes A. , WATTERS Steven , RIVETT A. Jennifer , AJUH Paul , LAMOND Angus I. , KATINGER Hermann , GRILLARI Johannes
Published: June 2005

Recognition of specific substrates for degradation by the ubiquitin–proteasome pathway is ensured by a cascade of ubiquitin transferases E1, E2 and E3. The mechanism by which the target proteins are transported to the proteasome is not clear, but two yeast E3s and one mammalian E3 ligase seem to be involved in the delivery of targets to the proteasome, by escorting them and by binding to the 19 S regulatory particle of the proteasome. In the present study, we show that SNEV (senescence evasion factor), a protein with in vitro E3 ligase activity, which is also involved in DNA repair and splicing, associates with the proteasome by directly binding to the β7 subunit of the 20 S proteasome. Upon inhibition of proteasome activity, SNEV does not accumulate within the cells although its co-localization with the proteasome increases significantly. Since immunofluorescence microscopy also shows increased co-localization of SNEV with ubiquitin after proteasome inhibition, without SNEV being ubiquitinated by itself, we suggest that SNEV shows E3 ligase activity not only in vitro but also in vivo and escorts its substrate to the proteasome. Since the yeast homologue of SNEV, Prp19, also interacts with the yeast β7 subunit of the proteasome, this mechanism seems to be conserved during evolution. Therefore these results support the hypothesis that E3 ligases might generally be involved in substrate transport to the proteasome. Additionally, our results provide the first evidence for a physical link between components of the ubiquitin–proteasome system and the spliceosome.
Mechanism of feedback regulation of insulin receptor substrate-1 phosphorylation in primary adipocytes
Authors: HERS Ingeborg , TAVARÉ Jeremy M.
Published: June 2005

Serine and threonine phosphorylation of IRS-1 (insulin receptor substrate-1) has been reported to decrease its ability to be tyrosine-phosphorylated by the insulin receptor. Insulin itself may negatively regulate tyrosine phosphorylation of IRS-1 through a PI3K (phosphoinositide 3-kinase)-dependent feedback pathway. In the present study, we examined the regulation and role of IRS-1 serine phosphorylation in the modulation of IRS-1 tyrosine phosphorylation in physiologically relevant cells, namely freshly isolated primary adipocytes. We show that insulin-stimulated phosphorylation of Ser312 and Ser616 in IRS-1 was relatively slow, with maximal phosphorylation achieved after 20 and 5 min respectively. The effect of insulin on phosphorylation of both these sites required the activation of PI3K and the MAPKs (mitogen-activated protein kinases) ERK1/2 (extracellular-signal-regulated kinase 1 and 2), but not the activation of mTOR (mammalian target of rapamycin)/p70S6 kinase, JNK (c-Jun N-terminal kinase) or p38MAPK. Although inhibition of PI3K and ERK1/2 both substantially decreased insulin-stimulated phosphorylation of Ser312 and Ser616, only wortmannin enhanced insulin-stimulated tyrosine phosphorylation of IRS-1. Furthermore, inhibition of mTOR/p70S6 kinase, JNK or p38MAPK had no effect on insulin-stimulated IRS-1 tyrosine phosphorylation. The differential effect of inhibition of ERK1/2 on insulin-stimulated IRS-1 phosphorylation of Ser312/Ser616 and tyrosine indicates that these events are independent of each other and that phosphorylation of Ser312/Ser616 is not responsible for the negative regulation of IRS-1 tyrosine phosphorylation mediated by PI3K in primary adipocytes.
Roles of Rac and p38 kinase in the activation of cytosolic phospholipase A2 in response to PMA
Authors: YOU Hye Jin , WOO Chang-Hoon , CHOI Eun-Young , CHO Sung-Hoon , YOO Yung Joon , KIM Jae-Hong
Published: June 2005

The roles of Rac and p38 kinase in the activation of cPLA2 (cytosolic PLA2) in Rat-2 fibroblasts were investigated. In the present study, we found that PMA activates cPLA2 by a Rac-p38 kinase-dependent pathway. Consistent with this, Rac, if activated, was shown to stimulate cPLA2 in a p38 kinase-dependent manner. In another experiment to understand the signalling mechanism by which the Rac-p38 kinase cascade mediates cPLA2 activation in response to PMA, we observed that PMA-induced cPLA2 translocation to the perinuclear region is completely inhibited by the expression of Rac1N17 or treatment with SB203580 (inhibitor of p38 kinase), suggesting that Rac-p38 kinase cascade acts in this instance by mediating the translocation of cPLA2. The mediatory role of p38 kinase in cPLA2 activation was further demonstrated after a treatment with anisomycin, a very effective activator of p38 kinase. Consistent with the mediatory role of p38 kinase in stimulating cPLA2, anisomycin induced the translocation and activation of cPLA2 in a p38 kinase-dependent manner.
Evidence for the occurrence of membrane-type serine protease 1/matriptase on the basolateral sides of enterocytes
Authors: TSUZUKI Satoshi , MURAI Nobuhito , MIYAKE Yuka , INOUYE Kuniyo , HIRAYASU Hirofumi , IWANAGA Toshihiko , FUSHIKI Tohru
Published: June 2005

MT-SP1 (membrane-type serine protease 1)/matriptase is an epithelial-derived integral membrane enzyme. The purpose of the present study was to examine whether the enzyme exists on the basolateral side of simple columnar epithelial cells, such as enterocytes, of normal adult animals. Using COS-1 monkey kidney cells transiently transfected with rat MT-SP1/matriptase expression plasmids, we found that the enzyme is post-translationally processed by the cleavage between Gly149 and Ser150, that a portion of the C-terminal part (Ser150–Val855) remains in the cells by association with the NTF (N-terminal fragment) (Met1–Gly149), while the other portions are released into the medium and that the release is increased on activation by co-expression with hepatocyte growth factor activator inhibitor type-1. Western-blot analysis of crude membranes prepared from rat jejunum demonstrated the presence of the NTF but negligible or no occurrence of the C-terminal part of the protein. Fractionation of the crude membranes by ultracentrifugation with Percoll followed by Western-blot analysis showed that the fractionation profile of the NTF correlated significantly with that of E-cadherin, an adhesion molecule on the lateral membrane. Immunostaining of the jejunum demonstrated the occurrence of the NTF on the lateral membranes but not on the apical membranes. These results suggest that considerable MT-SP1/matriptase molecules occur on the basolateral sides of normal epithelial cells and support our hypothesis that a possible physiological function of this enzyme is the control of epithelial-cell turnover by regulating cell–cell and/or cell–substratum adhesions.
Anti-inflammatory potential of CB1-mediated cAMP elevation in mast cells
Authors: SMALL-HOWARD Andrea L. , SHIMODA Lori M. N. , ADRA Chaker N. , TURNER Helen
Published: June 2005

Cannabinoids are broadly immunosuppressive, and anti-inflammatory properties have been reported for certain marijuana constituents and endogenously produced cannabinoids. The CB2 cannabinoid receptor is an established constituent of immune system cells, and we have recently established that the CB1 cannabinoid receptor is expressed in mast cells. In the present study, we sought to define a role for CB1 in mast cells and to identify the signalling pathways that may mediate the suppressive effects of CB1 ligation on mast cell activation. Our results show that CB1 and CB2 mediate diametrically opposed effects on cAMP levels in mast cells. The observed long-term stimulation of cAMP levels by the Gαi/o-coupled CB1 is paradoxical, and our results indicate that it may be attributed to CB1-mediated transcriptional regulation of specific adenylate cyclase isoenzymes that exhibit superactivatable kinetics. Taken together, these results reveal the complexity in signalling of natively co-expressed cannabinoid receptors and suggest that some anti-inflammatory effects of CB1 ligands may be attributable to sustained cAMP elevation that, in turn, causes suppression of mast cell degranulation.
Multiple biological responses are induced by glycosylation-deficient hepatocyte growth factor
Authors: FUKUTA Kazuhiro , MATSUMOTO Kunio , NAKAMURA Toshikazu
Published: June 2005

HGF (hepatocyte growth factor), a heterodimeric glycoprotein composed of α- and β-chains, exerts biological activities through the c-Met receptor tyrosine kinase. The α-chain has three glycosylation sites, while the β-chain has two; however, the role of sugar chains on HGF is still unknown. To address the significance of glycosylation of HGF, three different types of glycosylation-deficient HGFs, i.e. non-glycosylated in the α-chain, the β-chain, and in both the α- and β-chains, were respectively expressed in COS-7 cells and then purified from culture supernatants. Unexpectedly, glycosylation-deficient HGFs induced tyrosine phosphorylation of the c-Met receptor and subsequent phosphorylation of ERK (extracellular-signal-regulated kinase) and Akt in rat hepatocytes with the same potency as glycosylated HGF. Consistent with this, glycosylation-deficient HGFs strongly stimulated DNA synthesis of hepatocytes equal to glycosylated HGF. Likewise, glycosylation-deficient HGFs induced cell scattering and branching tubulogenesis in MDCK (Madin–Darby canine kidney) cells, and thus were indistinguishable from glycosylated HGF in biological activities. Glycosylation also did not affect stability, protease sensitivity and tissue distribution, although the plasma clearance of HGF was slightly prolonged by glycosylation deficiency. Glycosylation deficiency resulted in a decrease in post-transcriptional biosynthesis of HGF in the cells, whereas extracellularly secreted HGFs were efficiently activated to a two-chain form. These results indicate that glycosylation influences post-transcriptional biosynthesis of HGF, whereas biological activities and basic physicochemical characteristics are retained, even in completely non-glycosylated HGF. Hence, non-glycosylated HGF is promising as an alternative for glycosylated HGF in clinical applications.
Effect of a membrane-targeted sphingosine kinase 1 on cell proliferation and survival
Authors: SAFADI-CHAMBERLAIN Farida , WANG Li-Ping , PAYNE Shawn G. , LIM Chang-Uk , STRATFORD Suzanne , CHAVEZ Jose Antonio , FOX Michael H. , SPIEGEL Sarah , SUMMERS Scott A.
Published: June 2005

Numerous extracellular stimuli activate SK1 (sphingosine kinase type 1) to catalyse the production of sphingosine 1-phosphate, a bioactive lipid that functions as both an extracellular ligand for a family of G-protein-linked receptors and as a putative intracellular messenger. Phorbol esters, calcium or immunoglobulin receptors stimulate SK1 by promoting its translocation to the plasma membrane, which brings it into proximity both to its substrate (i.e. sphingosine) and to activating acidic phospholipids (e.g. phosphatidylserine). To evaluate the consequence of SK translocation, we generated an SK1-derivative tagged with a myristoylation sequence (Myr-SK1) on its N-terminus and overexpressed the construct in 3T3-L1 fibroblasts using recombinant retrovirus. Myr-SK1 overexpression increased SK activity by more than 50-fold in crude membranes, while only stimulating cytoplasmic SK activity by 4-fold. In contrast, the overexpression of WT-SK1 (wild-type SK1), as well as that of a construct containing a false myristoylation sequence (A2-Myr-SK1), markedly increased SK activity in both membrane and cytoplasmic compartments. Immunofluorescence confirmed that Myr-SK1 preferentially localized at the plasma membrane, whereas WT-SK1 and A2-Myr-SK1 partitioned in cytoplasmic/perinuclear cellular regions. Surprisingly, Myr-SK1 overexpression significantly decreased the rates of cell proliferation by delaying exit from G0/G1 phase. Moreover, expression of Myr-SK1 but not WT-SK1 or A2-Myr-SK1 protected cells from apoptosis induced by serum withdrawal. Collectively, these findings reveal that altering the subcellular location of SK1 has marked effects on cell function, with plasma membrane-associated SK having a potent inhibitory effect on the G1–S phase transition.
Characterization of cis-regulatory elements of the vascular endothelial growth inhibitor gene promoter
Authors: XIAO Qingli , HSU Chung Y. , CHEN Hong , MA Xiucui , XU Jan , LEE Jin-Moo
Published: June 2005

VEGI (vascular endothelial growth inhibitor), a member of the tumour necrosis factor superfamily, has been reported to inhibit endothelial cell proliferation, angiogenesis and tumour growth. We identified and cloned approx. 2.2 kb of the VEGI promoter from mouse cerebral endothelial cells. The promoter contained an atypical TATA-box-binding protein sequence TAAAAAA residing at −32/−26 relative to the transcription initiation site (+1), 83 bp upstream from the ATG start codon. To investigate critical sequences in the VEGI promoter, a series of deleted and truncated segments were constructed from a 2300 bp promoter construct (−2201/+96) linked to a luciferase reporter gene. Transient transfection of cerebral microvascular cells (bEND.3) and rat C6 glioma cells demonstrated that a 1700 bp deletion from the −2201 to −501 did not significantly affect promoter activity; however, a truncated construct (−501/+96) lacking the region between −312 and −57 resulted in nearly 90% loss of promoter activity. A consensus NF-κB (nuclear factor κB) and several SP1 (specificity protein-1)-binding sequences were identified within the deleted segment. Supershift analysis revealed that NF-κB subunits, p50 and p65, interacted with the VEGI promoter. Exposure of cerebral endothermic cells to the pro-inflammatory cytokine, tumour necrosis factor-α, increased VEGI mRNA levels and DNA-binding activities, whereas an NF-κB inhibitor attenuated this increase. In addition, p65 overexpression enhanced, whereas p50 overexpression decreased, the luciferase activity. Furthermore, mutation of the NF-κB DNA binding site blocked this p65- and tumour necrosis factor-α-induced luciferase activity. These findings suggest that the transcription factor NF-κB plays an important role in the regulation of VEGI expression.
Mutational and biochemical analysis of cytochrome c′, a nitric oxide-binding lipoprotein important for adaptation of Neisseria gonorrhoeae to oxygen-limited growth
Authors: TURNER Susan M. , MOIR James W. B. , GRIFFITHS Lesley , OVERTON Timothy W. , SMITH Harry , COLE Jeff A.
Published: June 2005

Neisseria gonorrhoeae is a prolific source of c-type cytochromes. Five of the constitutively expressed cytochromes are predicted, based on in silico analysis of the N. gonorrhoeae genome, to be components of the cytochrome bc1 complex, cytochrome c oxidase cbb3 or periplasmic cytochromes involved in electron transfer reactions typical of a bacterium with a microaerobic physiology. Cytochrome c peroxidase was previously shown to be a lipoprotein expressed only during oxygen-limited growth. The final c-type cytochrome, cytochrome c′, similar to cytochrome c peroxidase, includes a lipobox required for targeting to the outer membrane. Maturation of cytochrome c′ was partially inhibited by globomycin, an antibiotic that specifically inhibits signal peptidase II, resulting in the accumulation of the prolipoprotein in the cytoplasmic membrane. Disruption of the gonococcal cycP gene resulted in an extended lag phase during microaerobic growth in the presence but not in the absence of nitrite, suggesting that cytochrome c′ protects the bacteria from NO generated by nitrite reduction during adaptation to oxygen-limited growth. The cytochrome c′ gene was overexpressed in Escherichia coli and recombinant cytochrome c′ was shown to be targeted to the outer membrane. Spectroscopic evidence is presented showing that gonococcal cytochrome c′ is similar to previously characterized cytochrome c′ proteins and that it binds NO in vitro. The demonstration that two of the seven gonococcal c-type cytochromes fulfil specialized functions and are outer membrane lipoproteins suggests that the localization of these lipoproteins close to the bacterial surface provides effective protection against external assaults from reactive oxygen and reactive nitrogen species.
Stabilization of nucleic acids by unusual polyamines produced by an extreme thermophile, Thermus thermophilus
Authors: TERUI Yusuke , OHNUMA Mio , HIRAGA Kaori , KAWASHIMA Etsuko , OSHIMA Tairo
Published: June 2005

Extreme thermophiles produce two types of unusual polyamine: long linear polyamines such as caldopentamine and caldohexamine, and branched polyamines such as quaternary ammonium compounds [e.g. tetrakis(3-aminopropyl)ammonium]. To clarify the physiological roles of long linear and branched polyamines in thermophiles, we synthesized them chemically and tested their effects on the stability of ds (double-stranded) and ss (single-stranded) DNAs and tRNA in response to thermal denaturation, as measured by differential scanning calorimetry. Linear polyamines stabilized dsDNA in proportion to the number of amino nitrogen atoms within their molecular structure. We used the empirical results to derive formulae that estimate the melting temperature of dsDNA in the presence of polyamines of a particular molecular composition. ssDNA and tRNA were stabilized more effectively by tetrakis(3-aminopropyl)ammonium than any of the other polyamines tested. We propose that long linear polyamines are effective to stabilize DNA, and tetrakis(3-aminopropyl)ammonium plays important roles in stabilizing RNAs in thermophile cells.
Molecular cloning and functional characterization of a high-affinity zinc importer (DrZIP1) from zebrafish (Danio rerio)
Authors: QIU Andong , SHAYEGHI Majid , HOGSTRAND Christer
Published: June 2005

Zinc is a vital micronutrient to all organisms and a potential toxicant to aquatic animals. It is therefore of importance to understand the mechanism of zinc regulation. In the present study, we molecularly cloned and functionally characterized a zinc transporter of the SLC39A family [commonly referred to as the ZIP (Zrt- and Irt-related protein) family] from the gill of zebrafish (Danio rerio) (DrZIP1). DrZIP1 protein was found to localize at the plasma membrane and to function as a zinc uptake transporter when being expressed in either chinook salmon (Oncorhynchus tshawytscha) embryonic 214 cells or Xenopus laevis oocytes. In comparison with pufferfish transporter proteins (FrZIP2 and FrECaC) that are known to facilitate cellular zinc uptake, DrZIP1 appears to have high affinity to bind and transport zinc, suggesting that it may be a high-affinity zinc uptake transporter (Km<0.5 μM) in fish. Orthologues of DrZIP1 were also identified in both freshwater and seawater pufferfish (Tetraodon nigroviridis and Takifugu rubripes), indicating that these proteins may be functionally conserved among different fish species. DrZIP1 mRNA is expressed in all the tissues examined in the present study and thus DrZIP1 may be a constitutive zinc uptake transporter in many cell types of zebrafish.
The trans-Golgi network GRIP-domain proteins form α-helical homodimers
Authors: LUKE Michael R. , HOUGHTON Fiona , PERUGINI Matthew A. , GLEESON Paul A.
Published: June 2005

A recently described family of TGN (trans-Golgi network) proteins, all of which contain a GRIP domain targeting sequence, has been proposed to play a role in membrane transport. On the basis of the high content of heptad repeats, GRIP domain proteins are predicted to contain extensive coiled-coil regions that have the potential to mediate protein–protein interactions. Four mammalian GRIP domain proteins have been identified which are targeted to the TGN through their GRIP domains, namely p230, golgin-97, GCC88 and GCC185. In the present study, we have investigated the ability of the four mammalian GRIP domain proteins to interact. Using a combination of immunoprecipitation experiments of epitope-tagged GRIP domain proteins, cross-linking experiments and yeast two-hybrid interactions, we have established that the GRIP proteins can self-associate to form homodimers exclusively. Two-hybrid analysis indicated that the N- and C-terminal fragments of GCC88 can interact with themselves but not with each other, suggesting that the GRIP domain proteins form parallel coiled-coil dimers. Analysis of purified recombinant golgin-97 by CD spectroscopy indicated a 67% α-helical structure, consistent with a high content of coiled-coil sequences. These results support a model for GRIP domain proteins as extended rod-like homodimeric molecules. The formation of homodimers, but not heterodimers, indicates that each of the four mammalian TGN golgins has the potential to function independently.
Identification of 80K-H as a protein involved in GLUT4 vesicle trafficking
Authors: HODGKINSON Conrad P. , MANDER Ann , SALE Graham J.
Published: June 2005

PKCζ (protein kinase Cζ) is a serine/threonine protein kinase controlled by insulin, various growth factors and phosphoinositide 3-kinase. It has been implicated in controlling glucose transport in response to insulin by the translocation of GLUT4-(glucose transporter 4) containing vesicles to the plasma membrane in stimulated cells. How PKCζ modulates GLUT4 vesicle trafficking remains unknown. A yeast two-hybrid screen using full-length human PKCζ identified 80K-H protein as an interactor with PKCζ. GST (glutathione S-transferase) pull-down assays with GST-tagged 80K-H constructs confirmed the interaction and showed that the N-terminal portion of 80K-H was not required for the interaction. Immunoprecipitates of endogenous PKCζ from Cho cells, 3T3-L1 adipocytes or L6 myotubes contained endogenous 80K-H, demonstrating a physiological interaction. Insulin stimulation enhanced the association 3–5-fold. Immunoprecipitates of endogenous 80K-H contained endogenous munc18c and immunoprecipitates of endogenous munc18c contained endogenous PKCζ, with insulin markedly increasing the amount of co-immunoprecipitated protein in each case. These results show that insulin triggers interactions in vivo between PKCζ, 80K-H and munc18c. Overexpression of 80K-H constructs mimicked the action of insulin in stimulating both glucose uptake and translocation of Myc-tagged GLUT4 in Cho cells, with the level of effect proportional to the ability of the constructs to associate with munc18c. These results identify 80K-H as a new player involved in GLUT4 vesicle transport and identify a link between a kinase involved in the insulin signalling cascade, PKCζ, and a known component of the GLUT4 vesicle trafficking pathway, munc18c. The results suggest a model whereby insulin triggers the formation of a PKCζ–80K-H–munc18c complex that enhances GLUT4 translocation to the plasma membrane.