bluuebunny/crossref_metadata_2025_split huggingface dataset:
Emerging roles for TGF‐β1 in nervous system development
Authors: Gomes Flávia Carvalho Alcantara , Sousa Vivian de Oliveira , Romão Luciana 
Published: June 2005

AbstractTransforming 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

AbstractOsteoarthritis (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

AbstractThe 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.
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.
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.
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.
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.
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.