bluuebunny/crossref_metadata_2025 huggingface dataset:
A simple cellular automaton model for coaggregation
Authors: Wimpenny J. , Colasanti R.
Published: April 2005

Aggregation is a common phenomenon in microbiology. It manifests itself in different forms ranging from loose irregular structures such as effluent floc and marine snow to the ordered aggregates that show the reproducible patterns seen in many microbial colonies. Aggregation conveys advantages to microorganisms. These include transfer of chemical signals, exchange of genetic information, protection from adverse environmental conditions, metabolic cooperation between different species, as well as cell differentiation in some populations. Coaggregation is now recognized as a mechanism for allowing specific association between collaborating bacterial species. We describe a simple cellular automaton that illustrates structures that could form when different species interact. In particular the importance of neighbours is investigated.
Bacteria harnessing complexity
Authors: Jacob E. Ben , Aharonov Y. , Shapira Y.
Published: April 2005

The study of bacterial colonies is a crucial step towards understanding biofilms. We review some of the exciting discoveries about bacterial self-organization that might shed new light on biocomplexity in general and biofilms in particular. This review is aimed at researchers from different disciplines – microbiology, biology, chemistry, physics, mathematics and computer science. To make the presentation comprehensible we have avoided the use of specialized terminology of the different disciplines and limited the experimental and computational details.

Bacteria can self-organize into hierarchically structured colonies of 109 to 1012 bacteria, each utilizing a great variety of biochemical communication agents, such as simple molecules, polymers, peptides, complex proteins, genetic material and also “cassettes of genetic information” such as plasmids and viruses. Bacteria use their intracellular flexibility, involving signal transduction networks and genomic plasticity, to collectively maintain self and shared interpretations of chemical cues, exchange of meaning-bearing chemical messages, and dialogues. The meaning-based communication permits the formation of colonial intentional behavior, purposeful alteration of colony structure and decision-making – features we might begin to associate with bacterial social intelligence. Such social intelligence, should it exist, would require going beyond communication to encompass additional intracellular processes, as yet unknown, for generating inheritable colonial memory and commonly shared genomic context.
Colony formation in bacteria: experiments and modeling
Authors: Matsushita M. , Hiramatsu F. , Kobayashi N. , Ozawa T. , Yamazaki Y. , Matsuyama T.
Published: April 2005

We present experimental results of colony formation in bacteria as an example of pattern formation resulting from reproduction and movement in biological populations. The bacterium Bacillus subtilis is known to exhibit at least five distinct types of colony pattern, depending on the substrate softness and nutrient concentration: diffusion-limited aggregation (DLA), compact Eden-like, dense branching morphology (DBM), concentric ring-like, and disk. We established a morphological diagram of the colony patterns, and then examined and characterized both macroscopically and microscopically how the the colonies grow. There seem to be two kinds of bacterial cells – active and inactive – and the active form drives the colony interfaces outwards. The active cells may be clearly distinguished from the inactive ones as they form the characteristic fingernail-like structure at the tips of growing branches of the DBM colony. The concentric ring-like colony is formed as a consequence of repeated alternate migration and resting of the growing interface, the cycle time for which seems to be independent of the substrate softness and nutrient concentration. So far there have been several phenomenological models proposed to qualitatively explain or reproduce the patterns observed in bacterial colonies. A few of them are reviewed here, systematically and critically, in light of our experimental results.
Novel type of Gq/11 protein‐coupled neurosteroid receptor sensitive to endocrine disrupting chemicals in mast cell line (RBL‐2H3)
Authors: Mizota Kaori , Yoshida Akira , Uchida Hitoshi , Fujita Ryousuke , Ueda Hiroshi
Published: April 2005

Agonistic neurosteroids, including pregnenolone, dehydroepiandrosterone and its sulfate (DHEAS), caused rapid degranulation in measurements of β‐hexosaminidase (β‐HEX) release from a mast cell line, RBL‐2H3. This degranulation was blocked by BSA‐conjugated progesterone (PROG‐BSA) or 17β‐estradiol, both of which are antagonistic neurosteroids.

DHEAS‐induced β‐HEX release was blocked by U‐73122 or xestospongin C, but not by PTX or EGTA. DHEAS‐induced β‐HEX release was also abolished by Gq/11‐AS, but not by Gq/11‐MS. Pharmacological analyses revealed that the neurosteroids stimulated a putative membrane receptor through activation of the novel Gq/11 and phospholipase C.

While representative endocrine‐disrupting chemicals (EDCs) did not show any degranulation or nocifensive actions by themselves, they blocked the DHEAS‐induced degranulation.

The binding of a PROG‐BSA‐fluorescein isothiocyanate conjugate (PROG‐BSA‐FITC) to cells was inhibited by neurosteroids and EDCs.

In the algogenic‐induced biting and licking responses test, DHEAS caused agonistic nocifensive actions in a dose‐dependent manner between 1 and 10 fmol (i.pl.). DHEAS‐induced nocifensive actions were abolished by PROG‐BSA or nonylphenol.

Taken together, these results suggest that a Gq/11‐coupled neurosteroid receptor may regulate the neuroimmunological activity related to sensory stimulation and that some EDCs have antagonistic actions for this receptor.

British Journal of Pharmacology (2005) 145, 545–550. doi:10.1038/sj.bjp.0706213
Methylation: lost in hydroxylation?
Authors: Trewick Sarah C. , McLaughlin Paul J. , Allshire Robin C.
Published: April 2005

Methylation of histone tails is a key determinant in forming active and silent states of chromatin. Histone methylation was regarded as irreversible until the recent identification of a lysine‐specific histone demethylase (LSD1), which acts specifically on mono‐ and dimethylated histone H3 lysine 4. Here, we propose that the fission yeast protein Epe1 is a putative histone demethylase that could act by oxidative demethylation. Epe1 modulates the stability of silent chromatin and contains a JmjC domain. The Epe1 protein can be modelled onto the structure of the 2‐oxoglutarate‐Fe(II)‐dependent dioxygenase, factor inhibiting hypoxia inducible factor (FIH), which is a protein hydroxylase that also contains a JmjC domain. Thus, Epe1 and certain other chromatin‐associated JmjC‐domain proteins may be protein hydroxylases that catalyse a novel histone modification. Another intriguing possibility is that, by hydroxylating the methyl groups, Epe1 and certain other JmjC‐domain proteins may be able to demethylate mono‐, di‐ or trimethylated histones.
Lutein transport by Caco-2 TC-7 cells occurs partly by a facilitated process involving the scavenger receptor class B type I (SR-BI)
Authors: REBOUL Emmanuelle , ABOU Lydia , MIKAIL Céline , GHIRINGHELLI Odette , ANDRÉ Marc , PORTUGAL Henri , JOURDHEUIL-RAHMANI Dominique , AMIOT Marie-Josèphe , LAIRON Denis , BOREL Patrick
Published: April 2005

The carotenoid lutein is thought to play a role in the human eye and to protect against age-related macular degeneration. Lutein transport in the human intestine has not been characterized. We examined lutein transport processes using Caco-2 TC-7 monolayers as a model for human intestinal epithelium. Purified lutein was mixed with phospholipids, lysophospholipids, cholesterol, mono-olein, oleic acid and taurocholate to obtain lutein-rich mixed micelles that mimicked those found under physiological conditions. The micelles were added to the apical side of Caco-2 TC-7 cell monolayers for 30 min or 3 h at 37 °C. Absorbed lutein, i.e. the sum of lutein recovered in the scraped cells and in the basolateral chamber, was quantified by HPLC. Transport rate was measured (i) as a function of time (from 15 to 60 min), (ii) as a function of micellar lutein concentration (from 1.5 to 15 μM), (iii) at 4 °C, (iv) in the basolateral to apical direction, (v) after trypsin pretreatment, (vi) in the presence of β-carotene and/or lycopene, (vii) in the presence of increasing concentrations of antibody against SR-BI (scavenger receptor class B type 1) and (viii) in the presence of increasing concentrations of a chemical inhibitor of the selective transfer of lipids mediated by SR-BI, i.e. BLT1 (blocks lipid transport 1). The rate of transport of lutein as a function of time and as a function of concentration was saturable. It was significantly lower at 4 °C than at 37 °C (approx. 50%), in the basal to apical direction than in the opposite direction (approx. 85%), and after trypsin pretreatment (up to 45%). Co-incubation with β-carotene, but not lycopene, decreased the lutein absorption rate (approx. 20%) significantly. Anti-SR-BI antibody and BLT1 significantly impaired the absorption rate (approx. 30% and 57% respectively). Overall, these results indicate that lutein absorption is, at least partly, protein-mediated and that some lutein is taken up through SR-BI.
An eleven nucleotide section of the 3′-untranslated region is required for perinuclear localization of rat metallothionein-1 mRNA
Authors: NURY David , CHABANON Hervé , LEVADOUX-MARTIN Marilyne , HESKETH John
Published: April 2005

Localization of mRNAs provides a novel mechanism for synthesis of proteins close to their site of function. MT1 (metallothionein-1) is a small, metal-binding protein that is largely cytoplasmic but which can be found in the nucleus. The localization of rat MT1 requires the perinuclear localization of its mRNA by a mechanism dependent on the 3′-UTR (3′-untranslated region). The present study investigates the nature of this mRNA localization signal using Chinese-hamster ovary cells transfected with gene constructs in which either MT1 or the globin coding region is linked to different sequences from the MT1 3′-UTR. Deletion, mutagenesis and antisense oligonucleotide approaches indicate that nt 45–76 of the 3′-UTR, in particular nt 66–76, are required for the localization of either MT1 mRNA or chimaeric transcripts in which a β-globin coding region is linked to sequences from the MT1 3′-UTR. This section of the 3′-UTR contains a CACC repeat. Two mutations that are predicted to alter the secondary structure of this region also impair localization. Our hypothesis is that the perinuclear localization signal in MT1 mRNA is formed by a combination of the CACC repeat and its structural context.
Functional studies of the 5′-untranslated region of human 5-HT4 receptor mRNA
Authors: MAILLET Marjorie , GASTINEAU Monique , BOCHET Pascal , ASSELIN-LABAT Marie-Liesse , MOREL Eric , LAVERRIÈRE Jean-Noël , LOMPRÉ Anne-Marie , FISCHMEISTER Rodolphe , LEZOUALC'H Frank
Published: April 2005

The serotonin 5-HT4 receptor (where 5-HT stands for 5-hydroxy-tryptamine) is a member of the seven transmembrane-spanning G-protein-coupled family of receptors and mediates many cellular functions both in the central nervous system and at the periphery. In the present study, we isolated and characterized the 5′-flanking region of the h5-HT4 (human 5-HT4) receptor. We demonstrate the existence of a novel exon that corresponds to the 5′-untranslated region of the h5-HT4 receptor gene. RNase protection analysis and reverse transcriptase–PCR experiments performed on human atrial RNA demonstrated that the major transcription start site of the h5-HT4 receptor gene is located at −3185 bp relative to the first ATG codon. In addition, a 1.2 kb promoter fragment which drives the transcription of the 5-HT4 receptor was characterized. The promoter region lacks TATA and CAAT canonical motifs in the appropriate location, but contains putative binding sites for several transcription factors. Transient transfection assays revealed that the (−3299/−3050) gene fragment possesses the ability to promote the expression of the luciferase reporter gene in human cell lines. In contrast, the promoter was silent in monkey COS-7 cells, indicating the requirement of specific factors to initiate transcription in human cells. In addition to the promoter element, enhancer activity was found in a region (−220/−61) located in the long 5′-untranslated region. Mutational analysis, gel shift and transfection assays identified an Nkx2.5 (NK2-transcription-factor-related 5)-like binding site as a regulatory sequence of this enhancer. Our results suggest a complex regulation of the h5-HT4 receptor gene expression involving distinct promoters and non-coding exons.
Regulation of skeletal ryanodine receptors by dihydropyridine receptor II–III loop C-region peptides: relief of Mg2+ inhibition
Authors: HAARMANN Claudia S. , DULHUNTY Angela F. , LAVER Derek R.
Published: April 2005

The aim of the present study was to explore interactions between surface-membrane DHPR (dihydropyridine receptor) Ca2+ channels and RyR (ryanodine receptor) Ca2+ channels in skeletal-muscle sarcoplasmic reticulum. The C region (725Phe-Pro742) of the linker between the 2nd and 3rd repeats (II–III loop) of the α1 subunit of skeletal DHPRs is essential for skeletal excitation–contraction coupling, which requires a physical interaction between the DHPR and RyR and is independent of external Ca2+. Little is known about the regulatory processes that might take place when the two Ca2+ channels interact. Indeed, interactions between C fragments of the DHPR (C peptides) and RyR have different reported effects on Ca2+ release from the sarcoplasmic reticulum and on RyR channels in lipid bilayers. To gain insight into functional interactions between the proteins and to explore different reported effects, we examined the actions of C peptides on RyR1 channels in lipid bilayers with three key RyR regulators, Ca2+, Mg2+ and ATP. We identified four discrete actions: two novel, low-affinity (>10 μM), rapidly reversible effects (fast inhibition and decreased sensitivity to Mg2+ inhibition) and two slowly reversible effects (high-affinity activation and a slow-onset, low-affinity inhibition). Fast inhibition and high-affinity activation were decreased by ATP. Therefore peptide activation in the presence of ATP and Mg2+, used with Ca2+ release assays, depends on a mechanism different from that seen when Ca2+ is the sole agonist. The relief of Mg2+ inhibition was particularly important since RyR activation during excitation–contraction coupling depends on a similar decrease in Mg2+ inhibition.
Pharmacokinetics of 1-nitrosomelatonin and detection by EPR using iron dithiocarbamate complex in mice
Authors: PEYROT Fabienne , GRILLON Catherine , VERGELY Catherine , ROCHETTE Luc , DUCROCQ Claire
Published: April 2005

The N-nitroso-derivative of melatonin, NOM (1-nitrosomelatonin), which has been demonstrated to be a NO• [oxidonitrogen(•)] donor in buffered solutions, is a new potential drug particularly in neurological diseases. The advantage of NOM, a very lipophilic drug, is its ability to release both melatonin and NO•, an easily diffusible free radical. In order to evaluate the distribution and the pharmacokinetics of NOM, [O-methyl-3H]NOM was administered to and followed in mice. A complementary method for monitoring NOM, EPR, was performed in vitro and ex vivo with (MGD)2–Fe2+ (iron–N-methyl-D-glucamine dithiocarbamate) complex as a spin trap. The behaviour of NOM was compared with that of GSNO (S-nitrosoglutathione), a hydrophilic NO• donor. In the first minutes following [O-methyl-3H]NOM intraperitoneal injection, the radioactivity was found in organs (6% in the liver, 1% in the kidney and 0.6% in the brain), but not in the blood. In both liver and brain, the radioactivity content decreased over time with similar kinetics reflecting the diffusion and metabolism of NOM and of its metabolites. Based on the characterization and the quantification of the EPR signal in vitro with NOM or GSNO using (MGD)2–Fe2+ complex in phosphate-buffered solutions, the detection of these nitroso compounds was realized ex vivo in mouse tissue extracts. (MGD)2–Fe2+–NO was observed in the brain of NOM-treated mice in the first 10 min following injection, revealing that NOM was able to cross the blood–brain barrier, while GSNO was not.
Nuclear envelope assembly is promoted by phosphoinositide-specific phospholipase C with selective recruitment of phosphatidylinositol-enriched membranes
Authors: BYRNE Richard D. , BARONA Teresa M. , GARNIER Marie , KOSTER Grielof , KATAN Matilda , POCCIA Dominic L. , LARIJANI Banafshé
Published: April 2005

Nuclear envelope (NE) formation in a cell-free egg extract proceeds by precursor membrane vesicle binding to chromatin in an ATP-dependent manner, followed by a GTP-induced NE assembly step. The requirement for GTP in the latter step of this process can be mimicked by addition of bacterial PI-PLC [phosphoinositide (PtdIns)-specific phospholipase C]. The NE assembly process is here dissected in relation to the requirement for endogenous phosphoinositide metabolism, employing recombinant eukaryotic PI-PLC, inhibitors and direct phospholipid analysis using ESI-MS (electrospray ionization mass spectrometry). PtdIns (phosphatidylinositol) species analysis by ESI-MS indicates that the chromatin-bound NE precursor vesicles are enriched for specific PtdIns species. Moreover, during GTP-induced precursor vesicle fusion, the membrane vesicles become partially depleted of the PtdIns 18:0/20:4 species. These data indicate that eukaryotic PI-PLC can support NE formation, and the sensitivity to exogenous recombinant PtdIns-5-phosphatases shows that the endogenous PLC hydrolyses a 5-phosphorylated species. It is shown further that the downstream target of this DAG (diacylglycerol) pathway does not involve PKC (protein kinase C) catalytic function, but is mimicked by phorbol esters, indicating a possible engagement of one of the non-PKC phorbol ester receptors. The results show that ESI-MS can be used as a sensitive means to measure the lipid composition of biological membranes and their changes during, for example, membrane fusogenic events. We have exploited this and the intervention studies to illustrate a pivotal role for PI-PLC and its product DAG in the formation of NEs.
Peptide mimotopes of Mycobacterium tuberculosis carbohydrate immunodeterminants
Authors: GEVORKIAN Goar , SEGURA Erika , ACERO Gonzalo , PALMA José P. , ESPITIA Clara , MANOUTCHARIAN Karen , LÓPEZ-MARÍN Luz M.
Published: April 2005

Cell-surface saccharides of Mycobacterium tuberculosis appear to be crucial factors in tuberculosis pathogenicity and could be useful antigens in tuberculosis immunodiagnosis. In the present study, we report the successful antigenic and immunogenic mimicry of mannose-containing cell-wall compounds of M. tuberculosis by dodecamer peptides identified by phage-display technology. Using a rabbit antiserum raised against M. tuberculosis cell-surface saccharides as a target for biopanning, peptides with three different consensus sequences were identified. Phage-displayed and chemically synthesized peptides bound to the anticarbohydrate antiserum. Rabbit antibodies elicited against the peptide QEPLMGTVPIRAGGGS recognize the mannosylated M. tuberculosis cell-wall antigens arabinomannan and lipoarabinomannan, and the glycosylated recombinant protein alanine/proline-rich antigen. Furthermore, antibodies were also able to react with mannan from Saccharomyces cerevisiae, but not with phosphatidylinositol dimannosides or arabinogalactan from mycobacteria. These results suggest that the immunogenic peptide mimics oligomannosidic epitopes. Interestingly, this report provides evidence that, in contrast with previously known carbohydrate mimotopes, no aromatic residues are necessary in a peptide sequence for mimicking unusual glycoconjugates synthesized by mycobacteria. The possible usefulness of the identified peptide mimotopes as surrogate reagents for immunodiagnosis and for the study of functional roles of the native non-peptide epitopes is discussed.
Accumulation of partly folded states in the equilibrium unfolding of the pneumococcal choline-binding module C-LytA
Authors: MAESTRO Beatriz , SANZ Jesús M.
Published: April 2005

Choline-binding modules are present in some virulence factors and many other proteins of Streptococcus pneumoniae (Pneumococcus). The most extensively studied choline-binding module is C-LytA, the C-terminal moiety of the pneumococcal cell-wall amidase LytA. The three-dimensional structure of C-LytA is built up from six loop-hairpin structures forming a left-handed β-solenoid with four choline-binding sites. The affinity of C-LytA for choline and other structural analogues allows its use as an efficient fusion tag for single-step purification of hybrid proteins. In the present study, we characterize the folding and stability of C-LytA by chemical and thermal equilibrium denaturation experiments. Unfolding experiments using guanidinium chloride at pH 7.0 and 20 °C suggest the existence of two partly folded states (I1 and I2) in the following model: N (native)→I1⇆I2. The N→I1 transition is non-co-operative and irreversible, and is significant even in the absence of a denaturant. In contrast, the I1⇆I2 transition is co-operative and reversible, with an associated freeenergy change (ΔG0) of 30.9±0.8 kJ·mol−1. The residual structure in the I2 state is unusually stable even in 7.4 M guanidinium chloride. Binding of choline stabilizes the structure of the native state, induces its dimerization and prevents the accumulation of the I1 species ([N]2⇆[I2]2, ΔG0=50.1±0.8 kJ·mol−1). Fluorescence and CD measurements, gel-filtration chromatography and limited proteolysis suggest that I1 differs from N in the local unfolding of the N-terminal β-hairpins, and that I2 has a residual structure in the C-terminal region. Thermal denaturation of C-LytA suggests the accumulation of at least the I1 species. These results might pave the way for an effective improvement of its biotechnological applications by protein engineering.
The prototype γ-2 herpesvirus nucleocytoplasmic shuttling protein, ORF 57, transports viral RNA through the cellular mRNA export pathway
Authors: WILLIAMS Ben J. L. , BOYNE James R. , GOODWIN Delyth J. , ROADEN Louise , HAUTBERGUE Guillaume M. , WILSON Stuart A. , WHITEHOUSE Adrian
Published: April 2005

HVS (herpesvirus saimiri) is the prototype γ-2 herpesvirus. This is a subfamily of herpesviruses gaining importance since the identification of the first human γ-2 herpesvirus, Kaposi's sarcoma-associated herpesvirus. The HVS ORF 57 (open reading frame 57) protein is a multifunctional transregulatory protein homologous with genes identified in all classes of herpesviruses. Recent work has demonstrated that ORF 57 has the ability to bind viral RNA, shuttles between the nucleus and cytoplasm and promotes the nuclear export of viral transcripts. In the present study, we show that ORF 57 shuttles between the nucleus and cytoplasm in a CRM-1 (chromosomal region maintenance 1)-independent manner. ORF 57 interacts with the mRNA export factor REF (RNA export factor) and two other components of the exon junction complex, Y14 and Magoh. The association of ORF 57 with REF stimulates recruitment of the cellular mRNA export factor TAP (Tip-associated protein), and HVS infection triggers the relocalization of REF and TAP from the nuclear speckles to several large clumps within the cell. Using a dominant-negative form of TAP and RNA interference to deplete TAP, we show that it is essential for bulk mRNA export in mammalian cells and is required for ORF 57-mediated viral RNA export. Furthermore, we show that the disruption of TAP reduces viral replication. These results indicate that HVS utilizes ORF 57 to recruit components of the exon junction complex and subsequently TAP to promote viral RNA export through the cellular mRNA export pathway.
Critical role for Ets, AP-1 and GATA-like transcription factors in regulating mouse Toll-like receptor 4 (Tlr4) gene expression
Authors: ROGER Thierry , MICONNET Isabelle , SCHIESSER Anne-Laure , KAI Hirofumi , MIYAKE Kensuke , CALANDRA Thierry
Published: April 2005

TLR4 (Toll-like receptor 4) is essential for sensing the endotoxin of Gram-negative bacteria. Mutations or deletion of the TLR4 gene in humans or mice have been associated with altered predisposition to or outcome of Gram-negative sepsis. In the present work, we studied the expression and regulation of the Tlr4 gene of mouse. In vivo, TLR4 levels were higher in macrophages compared with B, T or natural killer cells. High basal TLR4 promoter activity was observed in RAW 264.7, J774 and P388D1 macrophages transfected with a TLR4 promoter reporter vector. Analysis of truncated and mutated promoter constructs identified several positive [two Ets (E twenty-six) and one AP-1 (activator protein-1) sites] and negative (a GATA-like site and an octamer site) regulatory elements within 350 bp upstream of the transcriptional start site. The myeloid and B-cell-specific transcription factor PU.1 bound to the proximal Ets site. In contrast, none among PU.1, Ets-1, Ets-2 and Elk-1, but possibly one member of the ESE (epithelium-specific Ets) subfamily of Ets transcription factors, bound to the distal Ets site, which was indispensable for Tlr4 gene transcription. Endotoxin did not affect macrophage TLR4 promoter activity, but it decreased TLR4 steady-state mRNA levels by increasing the turnover of TLR4 transcripts. TLR4 expression was modestly altered by other pro- and anti-inflammatory stimuli, except for PMA plus ionomycin which strongly increased promoter activity and TLR4 mRNA levels. The mouse and human TLR4 genes were highly conserved. Yet, notable differences exist with respect to the elements implicated in gene regulation, which may account for species differences in terms of tissue expression and modulation by microbial and inflammatory stimuli.
Perturbation of actin dynamics induces NF-κB activation in myelomonocytic cells through an NADPH oxidase-dependent pathway
Authors: KUSTERMANS Gaelle , BENNA Jamel EL , PIETTE Jacques , LEGRAND-POELS Sylvie
Published: April 2005

Although several reports showed the effect of compounds disrupting microtubules on NF-κB (nuclear factor κB) activation, nothing is known about agents perturbing actin dynamics. In the present study, we have shown that actin cytoskeleton disruption induced by actin-depolymerizing agents such as cytochalasin D and latrunculin B and actin-polymerizing compounds such as jasplakinolide induced NF-κB activation in myelomonocytic cells. The transduction pathway involved the IκB (inhibitory κB) kinase complex and a degradation of IκBα. We have shown that NF-κB activation in response to the perturbation of actin dynamics required reactive oxygen species, as demonstrated by the effect of antioxidants. Actin cytoskeleton disruption by cytochalasin D induced O2− release from human monocytes, through the activation of the NADPH oxidase, as confirmed by the phosphorylation and by the membrane translocation of p47phox. NF-κB activation after actin cytoskeleton disruption could be physiologically relevant during monocyte activation and/or recruitment into injured tissues, where cellular attachment, migration and phagocytosis result in cyclic shifts in cytoskeletal organization and disorganization.
Cleavage at the stem region releases an active ectodomain of the membrane type 1 matrix metalloproteinase
Authors: TOTH Marta , OSENKOWSKI Pamela , HESEK Dusan , BROWN Stephen , MEROUEH Samy , SAKR Wael , MOBASHERY Shahriar , FRIDMAN Rafael
Published: April 2005

MT1-MMP (membrane type 1 matrix metalloproteinase) is a membrane-anchored MMP that can be shed to the extracellular milieu. In the present study we report the primary structure and activity of the major soluble form of MT1-MMP. MS analysis of the purified 50-kDa soluble MT1-MMP form shows that the enzyme extends from Tyr112 to Val524, indicating that formation of this species requires a proteolytic cleavage within the stem region. In agreement, deletion of the entire stem region of MT1-MMP inhibited shedding of the 50-kDa species. A recombinant 50-kDa species (Tyr112–Val524) expressed in cells exhibited enzymatic activity against pro-MMP-2 and galectin-3, and thus this species is a competent protease. The recombinant 50-kDa soluble form also decreased the level of surface-associated TIMP-2 (tissue inhibitor of metalloproteinase 2) when administered to cells expressing wild-type membrane-anchored MT1-MMP, suggesting that ectodomain shedding of MT1-MMP can alter the MMP/TIMP balance on the cell surface. A ∼53-kDa species of MT1-MMP was also isolated from a non-detergent extract of human breast carcinoma tissue and was found to lack the cytosolic tail, as determined with specific MT1-MMP domain antibodies. Together, these data show that MT1-MMP ectodomain shedding is a physiological process that may broaden MT1-MMP activity to the pericellular space.
Domain architecture of the smooth-muscle plasma membrane: regulation by annexins
Authors: DRAEGER Annette , WRAY Susan , BABIYCHUK Eduard B.
Published: April 2005

Individual signalling events are processed in distinct, spatially segregated domains of the plasma membrane. In a smooth muscle, the sarcolemma is divided into domains of focal adhesions alternating with caveolae-rich zones, both harbouring a specific subset of membrane-associated proteins. Recently, we have demonstrated that the sarcolemmal lipids are similarly segregated into domains of cholesterol-rich lipid rafts and glycerophospholipid-rich non-raft regions. In the present study, we provide a detailed structural analysis of the relationship between these proteinaceous and lipid domains. We demonstrate that the segregation of plasmalemmal protein constituents is intimately linked to that of the membrane lipids. Our results imply that lipid segregation is critical for the preservation of membrane protein architecture and essential for directional translocation of proteins to the sarcolemma. We show that the membrane lipid segregation is supported by the annexin protein family in a Ca2+-dependent manner. Eukaryotic cells harbour numerous, tissue-specific subsets of annexins. By examining the significance of this variety in a smooth muscle, we demonstrate that four different annexins target membrane sites of distinct lipid composition and that each annexin requires a different [Ca2+] for its translocation to the sarcolemma. Our results suggest that the interactions of annexins with distinct plasma membrane regions promote membrane segregation and, in combination with their individual Ca2+ sensitivity, might allow a spatially confined, graded response to a multitude of extra- or intracellular stimuli.
Transcriptional modulation of the pre-implantation embryo-specific Rnf35 gene by the Y-box protein NF-Y/CBF
Authors: HUANG Chiu-Jung , WU Shinn-Chih , CHOO Kong-Bung
Published: April 2005

Maternal-to-zygotic transition of a fertilized egg and the subsequent pre-implantation development of the embryo involve zygotic genome activation and reprogramming of gene expression. The goal of the present study is to establish a model suitable for the characterization of transcriptional modulation of mammalian pre-implantation development. Rnf35 is a mouse RING-finger protein gene that is temporally transcribed in the early embryo, but is permanently silenced before the blastocyst stage of development. We first show that the Chinese-hamster ovary-K1 cells are unique in supporting Rnf35 promoter activities in transient transfection assays. Using the permissive Chinese-hamster ovary-K1 cell line, we show that Rnf35 transcription is driven by an Inr (initiator) core promoter element in the absence of a TATA box; the Inr promoter function is confirmed by direct microinjection of mouse one-cell embryos. This is the first demonstration of the involvement of an Inr core promoter element in transcription in pre-implantation development. We show that the Rnf35 promoter is regulated by three obligatory Y-box (CCAAT-box) elements: two Y boxes (YI and YII) located at −81 are coupled in a palindrome and act synergistically in contributing to Rnf35 transcription; the third Y box (YIII) is situated at −13, just upstream of the Inr element, and may be an integral part of the Inr function. Electrophoretic mobility-shift assays and competition experiments further reveal that the YI box is bound by the ubiquitous NF-Y (nuclear factor-Y)/CBF (CCAAT-binding factor) and that YII is targeted by an unidentified protein(s) that acts synergistically with the NF-Y. We suggest that the NF-Y, targeting at a Y-box sequence, may function as an important activator in transcriptional regulation of the Rnf35 gene in the pre-implantation embryo.
Semenogelins I and II bind zinc and regulate the activity of prostate-specific antigen
Authors: JONSSON Magnus , LINSE Sara , FROHM Birgitta , LUNDWALL Åke , MALM Johan
Published: April 2005

In semen, the gel proteins SgI and SgII (semenogelins I and II) are digested by PSA (prostate-specific antigen), resulting in liquefaction and release of motile spermatozoa. Semen contains a high concentration of Zn2+, which is known to inhibit the protease activity of PSA. We characterized the binding of Zn2+ to SgI and SgII and found evidence that these proteins are involved in regulating the activity of PSA. Intact SgI and SgII and synthetic semenogelin peptides were used in the experiments. Binding of Zn2+ was studied by radioligand blotting, titration with a zinc (II) fluorophore chelator and NMR analysis. A chromogenic substrate was used to measure the enzymatic activity of PSA. SgI and SgII bound Zn2+ with a stoichiometry of at least 10 mol (mol of protein)−1 and with an average dissociation constant of approx. 5 μM per site. Moreover, Zn2+-inhibited PSA was activated by exposure to SgI or SgII. Since both proteins have high affinity for Zn2+ and are the dominating proteins in semen, they probably represent the major Zn2+ binders in semen, one function of which may be to regulate the activity of PSA. The system is self-regulating, and PSA is maintained in an active state by its substrate.
MSK1 activity is controlled by multiple phosphorylation sites
Authors: McCOY Claire E. , CAMPBELL David G. , DEAK Maria , BLOOMBERG Graham B. , ARTHUR J. Simon C.
Published: April 2005

MSK1 (mitogen- and stress-activated protein kinase) is a kinase activated in cells downstream of both the ERK1/2 (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) cascades. In the present study, we show that, in addition to being phosphorylated on Thr-581 and Ser-360 by ERK1/2 or p38, MSK1 can autophosphorylate on at least six sites: Ser-212, Ser-376, Ser-381, Ser-750, Ser-752 and Ser-758. Of these sites, the N-terminal T-loop residue Ser-212 and the ‘hydrophobic motif’ Ser-376 are phosphorylated by the C-terminal kinase domain of MSK1, and their phosphorylation is essential for the catalytic activity of the N-terminal kinase domain of MSK1 and therefore for the phosphorylation of MSK1 substrates in vitro. Ser-381 is also phosphorylated by the C-terminal kinase domain, and mutation of Ser-381 decreases MSK1 activity, probably through the inhibition of Ser-376 phosphorylation. Ser-750, Ser-752 and Ser-758 are phosphorylated by the N-terminal kinase domain; however, their function is not known. The activation of MSK1 in cells therefore requires the activation of the ERK1/2 or p38 MAPK cascades and does not appear to require additional signalling inputs. This is in contrast with the closely related RSK (p90 ribosomal S6 kinase) proteins, whose activity requires phosphorylation by PDK1 (3-phosphoinositide-dependent protein kinase 1) in addition to phosphorylation by ERK1/2.
Catalytic mechanism of α-retaining glucosyl transfer by Corynebacterium callunae starch phosphorylase: the role of histidine-334 examined through kinetic characterization of site-directed mutants
Authors: SCHWARZ Alexandra , PIERFEDERICI Francesco Maria , NIDETZKY Bernd
Published: April 2005

Purified site-directed mutants of Corynebacterium callunae starch phosphorylase in which His-334 was replaced by an alanine, glutamine or asparagine residue were characterized by steady-state kinetic analysis of enzymic glycosyl transfer to and from phosphate and studies of ligand binding to the active site. Compared with wild-type, the catalytic efficiencies for phosphorolysis of starch at 30 °C and pH 7.0 decreased approx. 150- and 50-fold in H334Q (His334→Gln) and H334N mutants, and that of H334A was unchanged. In the direction of α-glucan synthesis, selectivity for the reaction with G1P (α-D-glucose 1-phosphate) compared with the selectivity for reaction with α-D-xylose 1-phosphate decreased from a wild-type value of ∼20000 to 2600 and 100 in H334N and H334Q respectively. Binding of G1P to the free enzyme was weakened between 10-fold (H334N, H334Q) and 50-fold (H334A) in the mutants, whereas binding to the complex of enzyme and α-glucan was not affected. Quenching of fluorescence of the pyridoxal 5′-phosphate cofactor was used to examine interactions of the inhibitor GL (D-gluconic acid 1,5-lactone) with wild-type and mutant enzymes in transient and steady-state experiments. GL binding to the free enzyme and the enzyme–phosphate complex occurred in a single step. The 50-fold higher constant (Kd) for GL dissociation from H334Q bound to phosphate resulted from an increased off-rate for the ligand in the mutant, compared with wild-type. A log-log correlation of catalytic-centre activity for phosphorolysis of starch with a reciprocal Kd value established a linear free-energy relationship (slope=1.19±0.07; r2=0.991) across the series of wild-type and mutant enzymes. It reveals that GL in combination with phosphate has properties of a transition state analogue and that the His-334 side chain has a role in selectively stabilizing the transition state of the reaction.
Molecular basis of N-acetylglucosaminyltransferase I deficiency in Arabidopsis thaliana plants lacking complex N-glycans
Authors: STRASSER Richard , STADLMANN Johannes , SVOBODA Barbara , ALTMANN Friedrich , GLÖSSL Josef , MACH Lukas
Published: April 2005

GnTI (N-acetylglucosaminyltransferase I) is a Golgi-resident enzyme essential for the processing of high-mannose to hybrid and complex N-glycans. The Arabidopsis thaliana cgl mutant lacks GnTI activity and as a consequence accumulates oligomannosidic structures. Molecular cloning of cgl GnTI cDNA revealed a point mutation, which causes a critical amino acid substitution (Asp144→Asn), thereby creating an additional N-glycosylation site. Heterologous expression of cgl GnTI in insect cells confirmed its lack of activity and the use of the N-glycosylation site. Remarkably, introduction of the Asp144→Asn mutation into rabbit GnTI, which does not result in the formation of a new N-glycosylation site, led to a protein with strongly reduced, but still detectable enzymic activity. Expression of Asn144 rabbit GnTI in cgl plants could partially restore complex N-glycan formation. These results indicate that the complete deficiency of GnTI activity in cgl plants is mainly due to the additional N-glycan, which appears to interfere with the proper folding of the enzyme.
Identification of adipocyte adhesion molecule (ACAM), a novel CTX gene family, implicated in adipocyte maturation and development of obesity
Authors: EGUCHI Jun , WADA Jun , HIDA Kazuyuki , ZHANG Hong , MATSUOKA Takashi , BABA Masako , HASHIMOTO Izumi , SHIKATA Kenichi , OGAWA Norio , MAKINO Hirofumi
Published: April 2005

Few cell adhesion molecules have been reported to be expressed in mature adipocytes, and the significance of cell adhesion process in adipocyte biology is also unknown. In the present study, we identified ACAM (adipocyte adhesion molecule), a novel homologue of the CTX (cortical thymocyte marker in Xenopus) gene family. ACAM cDNA was isolated during PCR-based cDNA subtraction, and its mRNA was shown to be up-regulated in WATs (white adipose tissues) of OLETF (Otsuka Long–Evans Tokushima fatty) rats, an animal model for Type II diabetes and obesity. ACAM, 372 amino acids in total, has a signal peptide, V-type (variable) and C2-type (constant) Ig domains, a single transmembrane segment and a cytoplasmic tail. The amino acid sequence in rat is highly homologous to mouse (94%) and human (87%). ACAM mRNA was predominantly expressed in WATs in OLETF rats, and increased with the development of obesity until 30 weeks of age, which is when the peak of body mass is reached. Western blot analysis revealed that ACAM protein, approx. 45 kDa, was associated with plasma membrane fractions of mature adipocytes isolated from mesenteric and subdermal adipose deposits of OLETF rats. Up-regulation of ACAM mRNAs in obesity was also shown in WATs of genetically obese db/db mice, diet-induced obese ICR mice and human obese subjects. In primary cultured mouse and human adipocytes, ACAM mRNA expression was progressively up-regulated during differentiation. Several stably transfected Chinese-hamster ovary K1 cell lines were established, and the quantification of ACAM mRNA and cell aggregation assay revealed that the degree of homophilic aggregation correlated well with ACAM mRNA expression. In summary, ACAM may be the critical adhesion molecule in adipocyte differentiation and development of obesity.
Vanishin is a novel ubiquitinylated death-effector domain protein that blocks ERK activation
Authors: SUR Runa , RAMOS Joe W.
Published: April 2005

The ERK (extracellular-signal regulated-kinase)/MAPK (mitogen-activated protein kinase) pathway can regulate transcription, proliferation, migration and apoptosis. The small DED (death-effector domain) protein PEA-15 (phosphoprotein enriched in astrocytes-15) binds ERK and targets it to the cytoplasm. Other DED-containing proteins including cFLIP and DEDD can also regulate signal transduction events and transcription in addition to apoptosis. In the present study, we report the identification of a novel DED-containing protein called Vanishin. The amino acid sequence of Vanishin is closest in similarly to PEA-15 (61% identical). Vanishin mRNA is expressed in several mouse tissues and in both mouse and human cell lines. Interestingly, Vanishin is regulated by ubiquitinylation and subsequent degradation by the 26 S proteasome. The ubiquitinylation is complex and occurs at both the internal lysine residues and the N-terminus. We further show that Vanishin binds ERK/MAPK but not the DED proteins Fas-associated death domain, caspase 8 or PEA-15. Vanishin is present in both the nucleus and Golgi on overexpression and forces increased ERK accumulation in the nucleus in the absence of ERK stimulation. Moreover, Vanishin expression inhibits ERK activation and ERK-dependent transcription in cells, but does not alter MAPK/ERK activity. Therefore Vanishin is a novel regulator of ERK that is controlled by ubiquitinylation.
Integral membrane lipid phosphatases/phosphotransferases: common structure and diverse functions
Authors: SIGAL Yury J. , McDERMOTT Mark I. , MORRIS Andrew J.
Published: April 2005

Phospholipids and sphingolipids play critical roles in signal transduction, intracellular membrane trafficking, and control of cell growth and survival. We discuss recent progress in the identification and characterization of a family of integral membrane proteins with central roles in bioactive lipid metabolism and signalling. These five groups of homologous proteins, which we collectively term LPTs (lipid phosphatases/phosphotransferases), are characterized by a core domain containing six transmembrane-spanning α-helices connected by extramembrane loops, two of which interact to form the catalytic site. LPT family members are localized to all major membrane compartments of the cell. The transmembrane topology of these proteins places their active site facing the lumen of endomembrane compartments or the extracellular face of the plasma membrane. Sequence conservation between the active site of the LPPs (lipid phosphate phosphatases), SPPs (sphingosine phosphate phosphatases) and the recently identified SMSs (sphingomyelin synthases) with vanadium-dependent fungal oxidases provides a framework for understanding their common catalytic mechanism. LPPs hydrolyse LPA (lysophosphatidic acid), S1P (sphingosine 1-phosphate) and structurally-related substrates. Although LPPs can dephosphorylate intracellularly generated substrates to control intracellular lipid metabolism and signalling, their best understood function is to regulate cell surface receptor-mediated signalling by LPA and S1P by inactivating these lipids at the plasma membrane or in the extracellular space. SPPs are intracellularly localized S1P-selective phosphatases, with key roles in the pathways of sphingolipid metabolism linked to control of cell growth and survival. The SMS enzymes catalyse the interconversion of phosphatidylcholine and ceramide with sphingomyelin and diacylglycerol, suggesting a pivotal role in both housekeeping lipid synthesis and regulation of bioactive lipid mediators. The remaining members of the LPT family, the LPR/PRGs (lipid phosphatase-related proteins/plasticity-related genes) and CSS2s (type 2 candidate sphingomyelin synthases), are presently much less well studied. These two groups include proteins that lack critical amino acids within the catalytic site, and could therefore not use the conserved LPT reaction mechanism to catalyse lipid phosphatase or phosphotransferase reactions. In this review, we discuss recent ideas about their possible biological activities and functions, which appear to involve regulation of cellular morphology and, possibly, lipid metabolism and signalling in the nuclear envelope.
A genomic view of the complexity of mammalian proteolytic systems
Authors: Puente X.S. , Sánchez L.M. , Gutiérrez-Fernández A. , Velasco G. , López-Otín C.
Published: April 2005

Proteolytic enzymes play an essential role in different physiological processes, including development, reproduction and host defence, as well as in numerous pathologies, like inflammatory diseases, neurological disorders or cancer. The completion of the human genome sequence allowed us to determine that more than 2% of all human genes are proteases or protease inhibitors, reflecting the importance of proteolysis in human biology. To understand better the complexity of proteases in human and other model organisms, we have used the available genome sequences of different mammalian organisms, including mouse, rat and chimpanzee, to identify and compare their degradomes, the complete set of protease genes in these species. Surprisingly, the rodent protease complement is more complex when compared with that of primates, mainly due to the expansion of protease families implicated in reproduction and host defence. Similarly, most differences between human and chimpanzee proteases are found in genes implicated in the immune system, which might explain some of the differences between both organisms. We have also found several genes implicated in reproduction, nutrition and the immune system, which are functional in rat, mouse or chimpanzee, but have been inactivated by mutations in the human lineage. These findings suggest that pseudogenization of specific protease genes has been a mechanism contributing to the evolution of the human genome. Finally, we found that proteases implicated in human hereditary diseases, and especially in neurodegenerative disorders, are highly conserved among mammals.