bluuebunny/crossref_metadata_2025 huggingface dataset:
Down‐regulation of the tumor suppressor gene C‐terminal Src kinase: An early event during premalignant colonic epithelial hyperproliferation
Authors: Kunte Dhananjay P. , Wali Ramesh K. , Koetsier Jennifer L. , Hart John , Kostjukova Maria N. , Kilimnik Anna Y. , Pyatkin Ilia G. , Strelnikova Svetlana R. , Roy Hemant K.
Published: June 2005

Hyperproliferation of the premalignant epithelium is critical for colonic carcinogenesis; however the mechanisms remain largely unexplored. We report herein that prior to occurrence of neoplastic lesions in the azoxymethane‐rat model of colon carcinogenesis; the tumor suppressor gene C‐terminal Src kinase (Csk) was down‐regulated with a concomitant increase in Src activity. Furthermore, pharmacological or genetic (RNA interference) inhibition of Csk resulted in increased proliferation in colon cancer cell lines through the mitogen‐activated protein kinase dependent pathway. Thus, we demonstrate, for the first time, that Csk suppression is an important early event in colorectal cancer pathogenesis.
Adipose tissues as an ancestral immune organ: Site‐specific change in obesity
Authors: Caspar-Bauguil S. , Cousin B. , Galinier A. , Segafredo C. , Nibbelink M. , André M. , Casteilla L. , Pénicaud L.
Published: June 2005

Close relationships have been demonstrated between adipose tissue and the inflammatory/immune system. Furthermore, obesity is increasingly considered as a state of chronic inflammation. Cytofluorometric analysis reveals the presence of significant levels of lymphocytes in the stroma‐vascular fraction of white adipose tissues. In epididymal (EPI) fat, lymphocytes display an “ancestral” immune system phenotype (up to 70% of natural killer (NK), γδ+ T and NKT cells among all lymphocytes) whereas the inguinal (ING) immune system presents more adaptive characteristics (high levels of αβ+ T and B cells). The percentage of NK cells in EPI fat was decreased in obese mice fed with a high‐fat diet, whereas γδ positive cells were significantly increased in ING fat. These data support the notion that adipose tissue may elaborate immunological mechanisms to regulate its functions which might be altered in obesity.
Cry11Aa toxin from Bacillus thuringiensis binds its receptor in Aedes aegypti mosquito larvae through loop α‐8 of domain II
Authors: Fernández Luisa E. , Pérez Claudia , Segovia Lorenzo , Rodríguez Mario H. , Gill Sarjeet S. , Bravo Alejandra , Soberón Mario
Published: June 2005

Bacillus thuringiensis subs israelensis produces Cry toxins active against mosquitoes. Receptor binding is a key determinant for specificity of Cry toxins composed of three domains. We found that exposed loop α‐8 of Cry11Aa toxin, located in domain II, is an important epitope involved in receptor interaction. Synthetic peptides corresponding to exposed regions in domain II (loop α‐8, β‐4 and loop 3) competed binding of Cry11Aa to membrane vesicles from Aedes aegypti midgut microvilli. The role of loop α‐8 of Cry11A in receptor interaction was demonstrated by phage display and site‐directed mutagenesis. We isolated a peptide‐displaying phage (P5.tox), that recognizes loop α‐8 in Cry11Aa, interferes interaction with the midgut receptor and attenuates toxicity in bioassay. Loop α‐8 mutants affected in toxicity and receptor binding were characterized.
Respiratory cytochrome c oxidase can be efficiently reduced by the photosynthetic redox proteins cytochrome c6 and plastocyanin in cyanobacteria
Authors: Navarro José A. , Durán Raúl V. , De la Rosa Miguel A. , Hervás Manuel
Published: June 2005

Plastocyanin and cytochrome c
6 are two small soluble electron carriers located in the intrathylacoidal space of cyanobacteria. Although their role as electron shuttle between the cytochrome b
6
f and photosystem I complexes in the photosynthetic pathway is well established, their participation in the respiratory electron transport chain as donors to the terminal oxidase is still under debate. Here, we present the first time‐resolved analysis showing that both cytochrome c
6 and plastocyanin can be efficiently oxidized by the aa
3 type cytochrome c oxidase in Nostoc sp. PCC 7119. The apparent electron transfer rate constants are ca. 250 and 300 s−1 for cytochrome c
6 and plastocyanin, respectively. These constants are 10 times higher than those obtained for the oxidation of horse cytochrome c by the oxidase, in spite of being a reaction thermodynamically more favourable.
Mutations in the NDUFS4 gene of mitochondrial complex I alter stability of the splice variants
Authors: Petruzzella Vittoria , Panelli Damiano , Torraco Alessandra , Stella Alessandro , Papa Sergio
Published: June 2005

The effect on the stability of alternative transcripts of different mutations of the NDUFS4 gene in patients with Leigh syndrome with complex I deficiency is presented. Normally, two NDUFS4 splice variants are degraded by nonsense mediated mRNA decay (NMD) while a third form does not trigger NMD degradation. In a patient with a premature termination codon in exon 1, all the three splice variants are up‐regulated. The present is the first case of a nonsense mutation leading to the abrogation of NMD, which can represent an additional event to be considered in the evaluation of clinically relevant mutations.
Charge substitution shows that repulsive electrostatic interactions impede the oligomerization of Alzheimer amyloid peptides
Authors: Guo Meng , Gorman Paul M. , Rico Manuel , Chakrabartty Avijit , Laurents Douglas V.
Published: June 2005

The strong pH dependence of Aβ oligomerization could arise from favorable intermolecular charge–charge interactions between His and carboxylate groups, or, alternatively, by mutual electrostatic repulsion of peptide molecules. To test between these two possibilities, the pH dependence of the oligomerization of Aβ and three charge substitution variants with Asp, Glu and His substituted by Ala is measured. All four peptides oligomerize, as detected by thioflavin T fluorescence, turbidity, and amyloid fibril formation; therefore, specific charge–charge interactions are nonessential for oligomerization. The strong negative correlation between net charge and oligomerization indicates that electrostatic repulsion between Aβ monomers impedes their association.
Occurrence of the aminoacyl‐tRNA synthetases in high‐molecular weight complexes correlates with the size of substrate amino acids
Authors: Wolfson Alexey , Knight Rob
Published: June 2005

One of the distinctive and mysterious features of mammalian aminoacyl‐tRNA synthetases (AARSs) is the existence of stable high‐molecular weight complexes containing 10 out of 20 AARSs. The composition and structure of these complexes are conserved among multicellular animals. No specific function associated with these structures has been found, and there is no evident rationale for a particular separation of AARSs in “complex‐bound” and “free” forms. We have demonstrated a strong association between the occurrence of AARSs in the complexes and the volume of their substrate amino acids. The significance of this association is discussed in terms of the structural organization of translation in the living cell.
Cyclin‐dependent kinase 11p58 interacts with HBO1 and enhances its histone acetyltransferase activity
Authors: Zong Hongliang , Li Zejuan , Liu Liyun , Hong Yi , Yun Xiaojing , Jiang Jianhai , Chi Yayun , Wang Hanzhou , Shen Xiaoyun , Hu Yun , Niu Ziyue , Gu Jianxin
Published: June 2005

CDK11p58, a 58 kDa protein of the PITSLRE kinase family, plays an important role in cell cycle progression, and is closely related to cell apoptosis. To gain further insight into the function of CDK11p58, we screened a human fetal liver cDNA library for its interacting proteins using the yeast two‐hybrid system. Here we report that histone acetyltransferase (HAT) HBO1, a MYST family protein, interacts with CDK11p58 in vitro and in vivo. CDK11p58 and HBO1 colocalize in the cell nucleus. Recombinant CDK11p58 enhances the HAT activity of HBO1 significantly in vitro. Meanwhile, overexpression of CDK11p58 in mammalian cells leads to the enhanced HAT activity of HBO1 towards free histones. Thus, we conclude that CDK11p58 is a new interacting protein and a novel regulator of HBO1. Both of the proteins may be involved in the regulation of eukaryotic transcription.
Analysis of prey size preference of North Sea whiting, saithe, and grey gurnard
Authors: Floeter Jens , Temming Axel
Published: June 2005

Size preference for prey fish of North Sea whiting, saithe, and grey gurnard was analysed. The analysis combined size-specific prey abundance estimates derived from bottom-trawl surveys with size frequencies of prey in predator stomachs from the International North Sea Stomach Database. To estimate the abundance of all potential prey fish in the sea, predator-specific length-based number spectra were calculated. Prey spectra were weighted by local predator abundance to take the spatial–temporal overlap between predator and their prey into consideration. Species-specific prey size preference models are presented. Contrary to former results, the preferred predator–prey weight ratio of whiting and grey gurnard is an exponentially increasing function of predator size and an exponentially decreasing function of the slope of the number spectrum. When predators grow, they prefer larger prey in absolute units. However, from a species-specific body size onwards they increasingly shift their prey preference towards relatively smaller prey sizes. From a bioenergetic point of view, this behaviour most likely maximizes the predator's foraging efficiency by reducing the expenditure of costly, anaerobically generated energy expended during burst swimming.
Seasonal cycles in weight and condition in Atlantic cod (Gadus morhua L.) in relation to fisheries
Authors: Mello L.G.S. , Rose G.A.
Published: June 2005

Seasonal cycle in weight and physiological condition of Atlantic cod (Gadus morhua) influenced productivity and economic impacts of the cod fishery in Placentia Bay, Newfoundland. Condition indices (Fulton's K condition factor and hepatosomatic index – HSI) were lowest during the spawning season (spring) and increased rapidly during the postspawning period, reaching maximum values by fall (K and HSI increased on average 24% and 82% between spring and fall, respectively). Somatic weight and condition indices varied seasonally. Condition indices were correlated with an industry index of product yield. Historically, cod fisheries have been prosecuted during all seasons, but simulations of 1997–1999 fisheries indicate that a fall fishery (period of peak physiological condition) resulted in a 8–17% decrease in the number of cod removed from the stock while maintaining the same weight-based quotas, and profiting from maximum yield and better product quality. Spring and summer fisheries resulted in lower yield (6%) and quality (5–26%) of fish products by weight. Seasonal biological cycles could be used as templates for management strategies that promote fisheries conservation and economic benefits by harvesting fish during periods when biological impacts are minimal and economic returns maximal.
Emerging roles for TGF‐β1 in nervous system development
Authors: Gomes Flávia Carvalho Alcantara , Sousa Vivian de Oliveira , Romão Luciana
Published: June 2005

Transforming growth factor betas (TGF‐βs) are known as multifunctional growth factors, which participate in the regulation of key events of development, disease and tissue repair. In central nervous system (CNS), TGF‐β1 has been widely recognized as an injury‐related cytokine, specially associated with astrocyte scar formation in response to brain injury. TGF‐βs family is represented by three isoforms: TGF‐β1, ‐β2 and ‐β3, all produced by both glial and neuronal cells. They are involved in essential tissue functions, including cell‐cycle control, regulation of early development and differentiation, neuron survival and astrocyte differentiation. TGF‐β signaling is mediated mainly by two serine threonine kinase receptors, TGFRI and TGFRII, which activate Smad 2/3 and Smad 4 transcription factors. Phosphorylation and activation of these proteins is followed by formation of Smad 2/3–4 complex, which translocates to the nucleus regulating transcriptional responses to TGF‐β. Very few data are available concerning the intracellular pathway required for the effect of TGF‐β in brain cells. Recently, emerging data on TGF‐β1 and its signaling molecules have been suggesting that besides its role in brain injury, TGF‐β1 might be a crucial regulator of CNS development. In this review, we will focus on TGF‐βs members, specially TGF‐β1, in neuron and astrocyte development. We will discuss some advances concerning the emerging scenario of TGF‐β1 and its signaling pathways as putative modulators of astrocyte biology and their implications as a novel mediator of cellular interactions in the CNS.
Intrajoint comparisons of gene expression patterns in human osteoarthritis suggest a change in chondrocyte phenotype
Authors: Yagi Rieko , McBurney Denise , Laverty David , Weiner Scott , Horton Walter E.
Published: June 2005

Osteoarthritis (OA) is a degenerative cartilage disease with varying degrees of severity within a given joint. The purpose of this study was to define a sampling procedure for comparing human minimal and advanced OA cartilage in the same patient and to determine basic patterns of gene expression in these regions. A specific hypothesis under study was that the expression level of Bcl‐2 would correlate with Sox9 and aggrecan mRNA expression in vivo as has been demonstrated in vitro. Femoral condylar advanced OA cartilage was located within 1 cm of overt lesions, and minimal cartilage was taken from areas with no obvious surface defects. Histological sections were scored for disease severity and chondroitin sulfate and hydroxyproline content was determined. The expression level of nine specific genes (aggrecan, collagen type II, Bcl‐2, Sox9, Link protein, osteopontin, and MMP‐13, ‐3, and ‐9) was determined by quantitative real time PCR. The scores for fibrillation, chondrocyte cloning, and proteoglycan depletion were significantly different between advanced and minimal OA cartilage. The advanced OA cartilage had significantly less chondroitin sulfate than the minimal OA cartilage. Osteopontin mRNA expression showed a 3.6‐fold increase in advanced compared to minimal OA cartilage. In contrast, the level of mRNA coding for aggrecan, link protein, Bcl‐2, Sox9 and MMP‐3, ‐9, ‐13 were all decreased in advanced compared to minimal cartilage in the majority of the patients studied. Collagen type II mRNA expression displayed a wide‐range of variation. A statistically significant correlation was observed both between Bcl‐2 and Sox9 mRNA level, and between Bcl‐2 and aggrecan mRNA expression. The patient matched comparison of minimal and advanced OA cartilage revealed differences in cellular and tissue characteristics, and changes in gene expression that may be involved in OA progression. In addition, Bcl‐2 may also play a role in regulating the expression of aggrecan through Sox9 in vivo as well as in vitro. © 2005 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.
Molecular signaling in intervertebral disk development
Authors: Dipaola Christian P. , Farmer James C. , Manova Katia , Niswander Lee A.
Published: June 2005

The purpose of this investigation is to identify and study the expression pattern of pertinent molecular factors involved in the differentiation of the intervertebral disk (IVD). It is likely that hedgehog genes and the BMP inhibitors are key factors involved in spinal joint formation. Radioactive in situ hybridization with mRNA probes for pax‐1, SHH, IHH and Noggin gene was performed on mouse embryo and adult tissue. Immunohistochemistry was performed to localize hedgehog receptor, “patched” (ptc). From 14.5 dpc until birth pax‐1 mRNA was expressed in the developing anulus fibrosus (AF). During the same developmental period Noggin mRNA is highly expressed throughout the spine, in the developing AF, while ptc protein and SHH mRNA were expressed in the developing nucleus pulposus (NP). IHH mRNA was expressed by condensing chondrocytes of the vertebral bodies and later becomes confined to the vertebral endplate. We show for the first time that pax‐1 is expressed in the adult intervertebral disk. Ptc expression in the NP is an indicator of hedgehog protein signaling in the developing IVD. The expression pattern of the BMP inhibitor Noggin appears to be important for the normal formation of the IVD and may prove to play a role in its segmental pattern formation. © 2005 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.
Nitrogen fixation by Trichodesmium spp.: An important source of new nitrogen to the tropical and subtropical North Atlantic Ocean
Authors: Capone Douglas G. , Burns James A. , Montoya Joseph P. , Subramaniam Ajit , Mahaffey Claire , Gunderson Troy , Michaels Anthony F. , Carpenter Edward J.
Published: June 2005

The broad distribution and often high densities of the cyanobacterium Trichodesmium spp. in oligotrophic waters imply a substantial role for this one taxon in the oceanic N cycle of the marine tropics and subtropics. New results from 154 stations on six research cruises in the North Atlantic Ocean show depth‐integrated N2 fixation by Trichodesmium spp. at many stations that equalled or exceeded the estimated vertical flux of NO3− into the euphotic zone by diapycnal mixing. Areal rates are consistent with those derived from several indirect geochemical analyses. Direct measurements of N2 fixation rates by Trichodesmium are also congruent with upper water column N budgets derived from parallel determinations of stable isotope distributions, clearly showing that N2 fixation by Trichodesmium is a major source of new nitrogen in the tropical North Atlantic. We project a conservative estimate of the annual input of new N into the tropical North Atlantic of at least 1.6 × 1012 mol N by Trichodesmium N2 fixation alone. This input can account for a substantial fraction of the N2 fixation in the North Atlantic inferred by several of the geochemical approaches.
Influence of small‐ and large‐scale variables on the chemical and isotopic compositions of urban rainwater, as illustrated by a case study in Ashdod, Israel
Authors: Asaf L. , Nativ R. , Hassan M. A. , Shain D. , Geyer S. , Ziv B.
Published: June 2005

The source of air pollution must be identified to select the appropriate monitoring activities and remedial measures. The present case study illustrates how analyses of (1) regional‐scale synoptics and air mass trajectories, (2) local‐scale atmospheric measurements (wind direction and speed, air temperature, dust load, sulfur and nitrogen oxide concentrations in the air, rain amount, and intensity) and (3) chemical and isotopic compositions of rainwater from 46 rain events, collected in the coastal city of Ashdod, Israel, were used to identify the various sources contributing to rainwater salts and contaminants and their timing. Rainwater affected by the Mediterranean Sea was characterized by a large marine fraction of salts, high chloride concentrations, and low values of δ18O and δD. These rain events were associated with the Cyprus Low system, which typically prevails in midwinter. Rainwater affected by continental sources had a small contribution of marine salts, high bicarbonate and calcium concentrations, and high values of δ18O and δD. These rains were related to continental trajectories from the Red Sea and north African coast prevailing during the fall and spring. Rainwater affected by anthropogenic sources was characterized by low marine fraction, high sulfate concentrations, and low values of δ34S. Whereas remote anthropogenic source was associated with the Red Sea trough system and were characterized by high nitrate concentrations (representing desert dust), the local anthropogenic source was characterized by high air concentrations of sulfur oxides at the local monitoring stations and high potassium and low nitrate concentrations. The identification of local anthropogenic source suggests that measures taken to reduce emissions from local oil refineries and power stations are likely to reduce the overall air pollution in the study area.
Application of dynamical error estimation for statistical optimization of radio occultation bending angles
Authors: Lohmann Martin S.
Published: June 2005

Errors in stratospheric bending angles retrieved from GPS radio occultations can be significantly reduced by using statistical optimization. In order for this technique to work optimally, the error covariance of the observations and the error covariance of the first guess must be known. This is generally not the case, and it is therefore common practice to assume that each of these errors is uncorrelated. In this study, it is shown that when this assumption is applied together with dynamical error estimation, it is important to account for the fact that first guess and the observation bending angle errors are not damped equally when refractivity profiles are computed through the Abel transform. It is demonstrated that this difference in damping can be accounted for by scaling the ratio of observation to first‐guess bending angle error variances. It is shown that the scaling factor can be related to the ratio between the error correlation lengths of the observation errors and the first‐guess errors. We present a simple procedure where both correlation lengths and variances are estimated dynamically and are scaled as described above. It is found that the relative errors can be reduced by up to 30% when compared to a standard statistical optimization scheme where the relative error in the first‐guess bending angle profile is assumed to be 20%. However, it was also found that if the first guess is properly adjusted, the need for corrections is greatly reduced.
Estimation of the rupture length and velocity of the Great Sumatra earthquake of Dec 26, 2004 using hydroacoustic signals
Authors: de Groot‐Hedlin Catherine D.
Published: June 2005

Unprecedented hydroacoustic observations of the megathrust earthquake of 26 Dec, 2004 were afforded by a network of 5 small hydroacoustic arrays located in the Indian Ocean, at distances of 2800 to 7000 km from the epicenter. Each array recorded acoustic waves, called T waves, generated by this event. Analysis of a series of short time windows within the T wave coda shows that the receiver to source azimuth varies smoothly as a function of time, indicating that the apparent T wave source is not stationary. The apparent T wave source moves northward along the Sunda trench at an average velocity of 2 km/s, closely tracking event rupture. The hydroacoustic data suggest that the rupture proceeded in two distinct phases; initially it progressed northwest along the Sunda trench with a velocity of approximately 2.4 km/s. At 600 km from the epicenter the rupture slowed to approximately 1.5 km/s, as it continued to propagate to the northwest.
Effects of the 2nd order ionospheric terms on VLBI measurements
Authors: Hawarey Mosab , Hobiger Thomas , Schuh Harald
Published: June 2005

Current VLBI software packages take into account ionospheric terms of 1st order only. This research investigates the effects of considering additionally the 2nd order terms on geodetic VLBI measurements. The mathematical algorithm follows the framework that has been presented for GPS in the literature. However, the approximation of the Earth's magnetic field and the assumption of a 400‐km high ionospheric shell are avoided here by using the IGRF and PIM models to calculate the geomagnetic field vectors and electron densities at 100 representative points along the paths of incoming rays. The 2nd order effects are at the level of 10−12seconds, which we introduced to the VLBI input files to determine the geodetic impact. Results of the VLBI software OCCAM reveal a maximum difference between baseline lengths with and without 2nd order ionospheric terms equivalent to 0.5 mm. Thus, the effect might become visible in VLBI within a few years.
On the anisotropy of the aperture correlation and effective transmissivity in fractures generated by sliding between identical self‐affine surfaces
Authors: Mallikamas Wasin , Rajaram Harihar
Published: June 2005

We present a theoretical analysis of the anisotropy of the aperture correlation and effective transmissivity in fractures generated by sliding/shear between identical self‐affine surfaces. Expressions are derived for the aperture correlation function in different directions, highlighting longer persistence and a larger integral scale normal to the shear than parallel to it. A stochastic perturbation analysis of the Reynolds equation leads to an effective transmissivity tensor with higher transmissivity normal to the shear. The hydraulic anisotropy depends on the coefficient of variation of the aperture field and the Hurst exponent. Numerical simulations are presented to evaluate the above theoretical predictions.
Identification of three critical acidic residues of poly(ADP-ribose) glycohydrolase involved in catalysis: determining the PARG catalytic domain
Authors: PATEL Chandra N. , KOH David W. , JACOBSON Myron K. , OLIVEIRA Marcos A.
Published: June 2005

PARG [poly(ADP-ribose) glycohydrolase] catalyses the hydrolysis of α(1″→2′) or α(1‴→2″) O-glycosidic linkages of ADP-ribose polymers to produce free ADP-ribose. We investigated possible mechanistic similarities between PARG and glycosidases, which also cleave O-glycosidic linkages. Glycosidases typically utilize two acidic residues for catalysis, thus we targeted acidic residues within a conserved region of bovine PARG that has been shown to contain an inhibitor-binding site. The targeted glutamate and aspartate residues were changed to asparagine in order to minimize structural alterations. Mutants were purified and assayed for catalytic activity, as well as binding, to an immobilized PARG inhibitor to determine ability to recognize substrate. Our investigation revealed residues essential for PARG catalytic activity. Two adjacent glutamic acid residues are found in the conserved sequence Gln755-Glu-Glu757, and a third residue found in the conserved sequence Val737-Asp-Phe-Ala-Asn741. Our functional characterization of PARG residues, along with recent identification of an inhibitor-binding residue Tyr796 and a glycine-rich region Gly745-Gly-Gly747 important for PARG function, allowed us to define a PARG ‘signature sequence’ [vDFA-X3-GGg-X6–8-vQEEIRF-X3-PE-X14-E-X12-YTGYa], which we used to identify putative PARG sequences across a range of organisms. Sequence alignments, along with our mapping of PARG functional residues, suggest the presence of a conserved catalytic domain of approx. 185 residues which spans residues 610–795 in bovine PARG.
TEB4 is a C4HC3 RING finger-containing ubiquitin ligase of the endoplasmic reticulum
Authors: HASSINK Gerco , KIKKERT Marjolein , VOORDEN Sjaak van , LEE Shiow-Ju , SPAAPEN Robbert , LAAR Theo van , COLEMAN Catherine S. , BARTEE Eric , FRÜH Klaus , CHAU Vincent , WIERTZ Emmanuel
Published: June 2005

In the present study, the human TEB4 is identified as a novel ER (endoplasmic reticulum)-resident ubiquitin ligase. TEB4 has homologues in many species and has a number of remarkable properties. TEB4 contains a conserved RING (really interesting new gene) finger and 13 predicted transmembrane domains. The RING finger of TEB4 and its homologues is situated at the N-terminus and has the unconventional C4HC3 configuration. The N-terminus of TEB4 is located in the cytosol. We show that the isolated TEB4 RING domain catalyses ubiquitin ligation in vitro in a reaction that is ubiquitin Lys48-specific and involves UBC7 (ubiquitin-conjugating enzyme 7). These properties are reminiscent of E3 enzymes, which are involved in ER-associated protein degradation. TEB4 is an ER degradation substrate itself, promoting its own degradation in a RING finger- and proteasome-dependent manner.
Evidence for co-operativity in coenzyme binding to tetrameric Sulfolobus solfataricus alcohol dehydrogenase and its structural basis: fluorescence, kinetic and structural studies of the wild-type enzyme and non-co-operative N249Y mutant
Authors: GIORDANO Antonietta , FEBBRAIO Ferdinando , RUSSO Consiglia , ROSSI Mosè , RAIA Carlo A.
Published: June 2005

The interaction of coenzyme with thermostable homotetrameric NAD(H)-dependent alcohol dehydrogenase from the thermoacidophilic sulphur-dependent crenarchaeon Sulfolobus solfataricus (SsADH) and its N249Y (Asn-249→Tyr) mutant was studied using the high fluorescence sensitivity of its tryptophan residues Trp-95 and Trp-117 to the binding of coenzyme moieties. Fluorescence quenching studies performed at 25 °C show that SsADH exhibits linearity in the NAD(H) binding [the Hill coefficient (h)∼1) at pH 9.8 and at moderate ionic strength, in addition to positive co-operativity (h=2.0–2.4) at pH 7.8 and 6.8, and at pH 9.8 in the presence of salt. Furthermore, NADH binding is positively co-operative below 20 °C (h∼3) and negatively co-operative at 40–50 °C (h∼0.7), as determined at moderate ionic strength and pH 9.8. Steady-state kinetic measurements show that SsADH displays standard Michaelis–Menten kinetics between 35 and 45 °C, but exhibits positive and negative co-operativity for NADH oxidation below (h=3.3 at 20 °C) and above (h=0.7 at 70–80 °C) this range of temperatures respectively. However, N249Y SsADH displays non-co-operative behaviour in coenzyme binding under the same experimental conditions used for the wild-type enzyme. In loop 270–275 of the coenzyme domain and segments at the interface of dimer A–B, analyses of the wild-type and mutant SsADH structures identified the structural elements involved in the intersubunit communication and suggested a possible structural basis for co-operativity. This is the first report of co-operativity in a tetrameric ADH and of temperature-induced co-operativity in a thermophilic enzyme.
The CYP2B2 phenobarbital response unit contains binding sites for hepatocyte nuclear factor 4, PBX–PREP1, the thyroid hormone receptor β and the liver X receptor
Authors: BEAUDET Marie-Josée , DESROCHERS Marc , LACHAUD Antoine Amaury , ANDERSON Alan
Published: June 2005

A 163 bp enhancer in the CYP2B2 5′ flank confers PB (phenobarbital) inducibility and constitutes a PBRU (PB response unit). The PBRU contains several transcription factor binding sites, including NR1, NR2 and NR3, which are direct repeats separated by 4 bp of the nuclear receptor consensus half-site AGGTCA, as well as an ER (everted repeat) separated by 7 bp (ER-7). Constitutive androstane receptor (CAR)–RXR (retinoic X receptor) heterodimers are known to bind to NR1, NR2 and NR3. Electrophoretic mobility-shift analysis using nuclear extracts from livers of untreated or PB-treated rats revealed binding of several other proteins to different PBRU elements. Using supershift analysis and in vitro coupled transcription and translation, the proteins present in four retarded complexes were identified as TRβ (thyroid hormone receptor β), LXR (liver X receptor), HNF-4 (hepatocyte nuclear factor 4) and heterodimers of PBX–PREP1 (pre-B cell homoeobox–Pbx regulatory protein 1). LXR–RXR heterodimers bound to NR3 and TRβ bound to NR3, NR1 and ER-7, whereas the PBX–PREP1 site is contained within NR2. The HNF-4 site overlaps with NR1. A mutation described previously, GRE1m1, which decreases PB responsiveness, increased the affinity of this site for HNF-4. The PBRU also contains a site for nuclear factor 1. The PBRU thus contains a plethora of transcription factor binding sites. The profiles of transcription factor binding to NR1 and NR3 were quite similar, although strikingly different from, and more complex than, that of NR2. This parallels the functional differences in conferring PB responsiveness between NR1 and NR3 on the one hand, and NR2 on the other.
Thr176 regulates the activity of the mouse nuclear receptor CAR and is conserved in the NR1I subfamily members PXR and VDR
Authors: UEDA Akiko , MATSUI Kenji , YAMAMOTO Yukio , PEDERSEN Lars C. , SUEYOSHI Tatsuya , NEGISHI Masahiko
Published: June 2005

The mouse nuclear receptor CAR (constitutively active receptor) is a transcription factor that is activated by phenobarbital-type inducers such as TCPOBOP {1,4 bis[2-(3,5-dichloropyridyloxy)]benzene} in liver in vivo. However, CAR is constitutively active in cell-based transfection assays, the molecular mechanism for which has not been elucidated yet. In the model structure of CAR, Thr176 constitutes a part of the ligand-binding surface, but its side chain is not directed toward the surface, instead it forms a hydrogen bond with Thr350 in the AF2 (activation function 2) domain of CAR. Thr350 is known to regulate CAR activity [Ueda, Kakizaki, Negishi, and Sueyoshi (2002) Mol. Pharmacol. 61, 1284–1288]. Thr176 was mutated to various amino acids to examine whether this interaction played a role in conferring the constitutive activity. Hydrophobic and positively charged amino acids at position 176 abrogated the constitutive activity, whereas polar and negatively charged amino acids retained it. When one of the small hydrophobic amino acids, such as alanine or valine, was substituted for threonine, the mutants were fully activated by TCPOBOP. The co-activator SRC-1 (steroid receptor co-activator-1) regulated the activity changes associated with the mutations. Thr248 and Ser230 are the Thr176-corresponding residues in human pregnane X receptor and mouse vitamin D3 receptor respectively, interacting directly with the conserved threonine in the AF2 domains. Thr248 and Ser230 also regulated the ligand-dependent activity of these receptors by augmenting binding of the receptors to SRC-1. Thr176, Thr248 and Ser230 are conserved residues in the NR1I (nuclear receptor 1I) subfamily members and determine their activity.
Cross down-regulation of leptin and insulin receptor expression and signalling in a human neuronal cell line
Authors: BENOMAR Yacir , ROY Anne-France , AUBOURG Alain , DJIANE Jean , TAOUIS Mohammed
Published: June 2005

Leptin and insulin are major signals to the hypothalamus to regulate energy homoeostasis and body adiposity. IR (insulin receptors) and leptin receptors (long isoform, ObRb) share a number of signalling cascades, such as JAK2/STAT-3 (Janus kinase 2/signal transduction and activator of transcription 3) and PI3K (phosphoinositide 3-kinase); the cross-talk between IR and ObRb have been described previously in non-neuronal cells. Differentiated human neuroblastoma (SH-SY5Y) cells express endogenous ObR and IR, and respond to leptin and insulin with stimulation of STAT-3 and MAPK (mitogen-activated protein kinase) phosphorylation, and PI3K activity. Insulin or leptin pre-treatment of SH-SY5Y cells increased basal STAT-3 phosphorylation, but abolished the acute effect of these hormones, and, interestingly, leptin pre-treatment abolished insulin effect and vice versa. Similar results were obtained for MAPK phosphorylation, but leptin or insulin pre-treatment did not completely abolish the acute effect of insulin or leptin. We have also showed that insulin and leptin are able to activate PI3K through IRS-1 (insulin receptor substrate 1) and IRS-2 respectively. Furthermore, leptin or insulin pre-treatment increased basal PI3K activity and IRS-1 or IRS-2 association with p85 and abolished acute insulin or leptin effect, in addition to the down-regulation of IRS-1 and IRS-2. Finally, insulin pre-treatment reduced leptin binding by approx. 60%, and leptin pre-treatment reduced the expression of insulin receptor by 40% in SH-SY5Y cells, which most likely accounts for the cross down-regulation of leptin and insulin receptors. These results provide evidence to suggest cross down-regulation of leptin and insulin receptors at both receptor and downstream signalling levels. This finding may contribute to the understanding of the complex relationship between leptin resistance and insulin resistance at the neuronal level.
Treatment of the mouse model of mucopolysaccharidosis type IIIB with lentiviral-NAGLU vector
Authors: Di NATALE Paola , Di DOMENICO Carmela , GARGIULO Nadia , CASTALDO Sigismondo , REYERO Enrico GONZALEZ Y , MITHBAOKAR Pratibha , De FELICE Mario , FOLLENZI Antonia , NALDINI Luigi , VILLANI Guglielmo R. D.
Published: June 2005

The Sanfilippo syndrome type B (mucopolysaccharidosis IIIB) is an autosomal recessive disorder due to mutations in the gene encoding NAGLU (α-N-acetylglucosaminidase), one of the enzymes required for the degradation of the GAG (glycosaminoglycan) heparan sulphate. No therapy exists for affected patients. We have shown previously the efficacy of lentiviral-NAGLU-mediated gene transfer in correcting in vitro the defect on fibroblasts of patients. In the present study, we tested the therapy in vivo on a knockout mouse model using intravenous injections. Mice (8–10 weeks old) were injected with one of the lentiviral doses through the tail vein and analysed 1 month after treatment. A single injection of lentiviral-NAGLU vector resulted in transgene expression in liver, spleen, lung and heart of treated mice, with the highest level reached in liver and spleen. Expression of 1% normal NAGLU activity in liver resulted in a 77% decrease in the GAG content; more remarkably, an expression of 0.16% normal activity in lung was capable of decreasing the GAG level by 29%. Long-term (6 months) follow up of the gene therapy revealed that the viral genome integration persisted in the target tissues, although the real-time PCR analysis showed a decrease in the vector DNA content with time. Interestingly, the decrease in GAG levels was maintained in liver, spleen, lung and heart of treated mice. These results show the promising potential and the limitations of lentiviral-NAGLU vector to deliver the human NAGLU gene in vivo.
Homodimerization of calpain 3 penta-EF-hand domain
Authors: RAVULAPALLI Ravikiran , DIAZ Beatriz GARCIA , CAMPBELL Robert L. , DAVIES Peter L.
Published: June 2005

Calpains 1 and 2 are heterodimeric proteases in which large (relative molecular mass Mr 80000) and small (Mr 28000) subunits are linked through their respective PEF (penta-EF-hand) domains. The skeletal muscle-specific calpain 3 is believed not to form a heterodimer with the small subunit but might homodimerize through its PEF domain. Size-exclusion chromatography and analytical ultracentrifugation of the recombinant PEF domain of calpain 3 show that it forms a stable homodimer that does not dissociate on dilution. Molecular modelling suggests that there would be no barriers to the dimerization of the whole enzyme through the PEF domains. This orientation would place the catalytic centres at opposite ends of the dimer.
Effects of troponin C isoform on the action of the cardiotonic agent EMD 57033
Authors: LIPSCOMB Simon , PRESTON Laura C. , ROBINSON Paul , REDWOOD Charles S. , MULLIGAN Ian. P. , ASHLEY Christopher C.
Published: June 2005

The effects of the cardiotonic potentiator EMD 57033 on different TnC (troponin C) isoforms were investigated. Endogenous skeletal TnC was extracted from glycerinated, permeabilized rabbit psoas fibres and replaced with either purified native rabbit psoas TnC (fast TnC) or human recombinant cTnC (cardiac TnC) (3 mg/ml in relaxing solution for 30 min). In both conditions, 10 μM EMD 57033 increased maximal calcium-activated force (Pmax) and gave a leftward shift in the pCa–tension curve. With cTnC, the increase in Pmax was much greater (228%) compared with the effect seen for fast TnC (137%), which was the same as that in unextracted control fibres. When the whole troponin was replaced rather than just TnC, the effects of EMD 57033 on fibres replaced with cTn were the same as with the cTnC subunit alone, except that the force at low Ca2+ concentrations was not increased as much. If TnC was only partially extracted, it was found that the degree of extraction did not influence the effect of EMD 57033, except when force was decreased to below 10% of the pre-extraction Pmax. Dynamic stiffness was not altered by EMD 57033 in any of the preparations. The rate of tension recovery following a release–restretch method (ktr) was decreased by EMD 57033. We conclude that EMD 57033 acts by a rate-modulating effect, and that the quantitative response of this effect is dependent on the TnC isoform present.
Voltage- and NADPH-dependence of electron currents generated by the phagocytic NADPH oxidase
Authors: PETHEŐ Gábor L. , DEMAUREX Nicolas
Published: June 2005

The phagocytic NADPH oxidase generates superoxide by transferring electrons from cytosolic NADPH to extracellular O2. The activity of the oxidase at the plasma membrane can be measured as electron current (Ie), and the voltage dependence of Ie was recently reported to exhibit a strong rectification in human eosinophils, with the currents being nearly voltage independent at negative potentials. To investigate the underlying mechanism, we performed voltage-clamp experiments on inside-out patches from human eosinophils activated with PMA. Electron current was evoked by bath application of different concentrations of NADPH, whereas slow voltage ramps (0.8 mV/ms), ranging from −120 to 200 mV, were applied to obtain ‘steady-state’ current–voltage relationships (I–V). The amplitude of Ie recorded at −40 mV was minimal at 8 μM NADPH and saturated above 1 mM, with half-maximal activity (Km) observed at approx. 110 μM NADPH. Comparison of I–V values obtained at different NADPH concentrations revealed that the voltage-dependence of Ie is strongly influenced by the substrate concentration. Above 0.1 mM NADPH, Ie was markedly voltage-dependent and steeply decreased with depolarization within the physiological membrane potential range (−60 to 60 mV), the I–V curve strongly rectifying only below −100 mV. At lower NADPH concentrations the I–V curve was progressively shifted to more positive potentials and Ie became voltage-independent also within the physiological range. Consequently, the Km of the oxidase decreased by approx. 40% (from 100 to 60 μM) when the membrane potential increased from −60 to 60 mV. We concluded that the oxidase activity depends on both membrane potential and [NADPH], and that the shape of the Ie–V curve is influenced by the concentration of NADPH in the submillimolar range. The surprising voltage-independence of Ie reported in whole-cell perforated patch recordings was most likely due to substrate limitation and is not an intrinsic property of the oxidase.
Direct comparison of the specificity of gene silencing using antisense oligonucleotides and RNAi
Authors: BILANGES Benoit , STOKOE David
Published: June 2005

RNAi (RNA interference) and ASO (antisense oligonucleotide) technologies are the most commonly used approaches for silencing gene expression. However, the specificity of such powerful tools is an important factor to correctly interpret the biological consequences of gene silencing. In the present study, we examined the effects of acute loss of Ser/Thr kinase PDK1 (3-phosphoinositide-dependent kinase 1) expression using ASO and RNAi, and compared, for the first time, these two techniques using Affymetrix microarrays. We show that both ASO- and siRNA (small interfering RNA)-mediated knock-down of PDK1 expression strongly inhibited cell proliferation, although by different mechanisms, thereby questioning the specificity of these reagents. Using microarray analysis, we characterized the specificity of the ASO- and siRNA-mediated gene silencing of PDK1 by examining expression profiles 48 and 72 h following oligonucleotide transfection. At 48 h, a PDK1-dependent pattern of gene alterations was detectable, despite a large number of non-specific changes due to transfection of control nucleic acids. These non-specific alterations became more apparent at the 72 h time point, and obscured any PDK1-specific pattern. This study underscores the importance of defining appropriate control ASOs and siRNAs, using multiple oligonucleotides for each target and preferably short time points following transfection to avoid misinterpretation of the phenotype observed.
The Cys214→Ser mutation in peripherin/rds causes a loss-of-function phenotype in transgenic mice
Authors: STRICKER Heidi M. , DING Xi-Qin , QUIAMBAO Alexander , FLIESLER Steven J. , NAASH Muna I.
Published: June 2005

P/rds (peripherin/retinal degeneration slow) is a photoreceptor-specific membrane glycoprotein necessary for outer segment disc morphogenesis. Mutations in P/rds are associated with different blinding diseases. A C214S (Cys214→Ser) missense mutation has been shown to be the cause for a late-onset form of ADRP (autosomal dominant retinitis pigmentosa) in humans. In the present study, we generated transgenic mice expressing P/rds with the C214S mutation and crossed them into rds mutant mice to elucidate the mechanism underlying the pathology of ADRP. Although an ample amount of transgene message was formed in C214S retinas from all transgenic lines, only a trace amount of the mutant protein was detected by Western blotting and immunoprecipitation. C214S mice on the wild-type or rds+/− backgrounds exhibited no signs of negative effects of the mutation on retinal structure or function, suggesting a loss-of-function phenotype. This phenotype is further supported by the absence of outer segment formation in the C214S mice on the rds−/− background. In contrast, expression of C214S protein in the inner retinal cells of transgenic mice or in COS cells resulted in the formation of a substantial amount of mutant protein, signifying a possible photoreceptor-specific regulation of P/rds. These results provide evidence that the loss-of-function phenotype seen in C214S transgenic mice shows a disease progression that correlates with ADRP patients carrying the same mutation, indicating that the C214S mutation on one allele of P/rds results in haploinsufficiency.
Characterization of the inactivation of rat fatty acid synthase by C75: inhibition of partial reactions and protection by substrates
Authors: RENDINA Alan R. , CHENG Dong
Published: June 2005

C75, a synthetic inhibitor of FAS (fatty acid synthase), has both anti-tumour and anti-obesity properties. In this study we provide a detailed kinetic characterization of the mechanism of in vitro inhibition of rat liver FAS. At room temperature, C75 is a competitive irreversible inhibitor of the overall reaction with regard to all three substrates, i.e. acetyl-CoA, malonyl-CoA and NADPH, exhibiting pseudo-first-order kinetics of the complexing type, i.e. a weak non-covalent enzyme–inhibitor complex is formed before irreversible enzyme modification. C75 is a relatively inefficient inactivator of FAS, with a maximal rate of inactivation of 1 min−1 and an extrapolated KI (dissociation constant for the initial complex) of approx. 16 mM. The apparent second-order rate constants calculated from these values are 0.06 mM−1·min−1 at room temperature and 0.21 mM−1·min−1 at 37 °C. We also provide experimental evidence that C75 inactivates the β-ketoacyl synthase (3-oxoacyl synthase) partial activity of FAS. Unexpectedly, C75 also inactivates the enoyl reductase and thioesterase partial activities of FAS with about the same rates as for inactivation of the β-ketoacyl synthase. In contrast with the overall reaction, the β-ketoacyl synthase activity and the enoyl reductase activity, substrates do not protect the thioesterase activity of rat liver FAS from inactivation by C75. These results differentiate inactivation by C75 from that by cerulenin, which only inactivates the β-ketoacyl synthase activity of FAS, by forming an adduct with an active-site cysteine. Interference by dithiothreitol and protection by the substrates, acetyl-CoA, malonyl-CoA and NADPH, further distinguish the mechanism of C75-mediated inactivation from that of cerulenin. The most likely explanation for the multiple effects observed with C75 on rat liver FAS and its partial reactions is that there are multiple sites of interaction between C75 and FAS.