bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genomic organization of human arylamine N-acetyltransferase Type I reveals alternative promoters that generate different 5′-UTR splice variants with altered translational activities
Authors: BUTCHER Neville J. , ARULPRAGASAM Ajanthy , GOH Hui Li , DAVEY Tamara , MINCHIN Rodney F. 
Published: March 2005

In humans, a polymorphic gene encodes the drug-metabolizing enzyme NAT1 (arylamine N-acetyltransferase Type 1), which is widely expressed throughout the body. While the protein-coding region of NAT1 is contained within a single exon, examination of the human EST (expressed sequence tag) database at the NCBI revealed the presence of nine separate exons, eight of which were located in the 5′ non-coding region of NAT1. Differential splicing produced at least eight unique mRNA isoforms that could be grouped according to the location of the first exon, which suggested that NAT1 expression occurs from three alternative promoters. Using RT (reverse transcriptase)-PCR, we identified one major transcript in various epithelial cells derived from different tissues. In contrast, multiple transcripts were observed in blood-derived cell lines (CEM, THP-1 and Jurkat), with a novel variant, not identified in the EST database, found in CEM cells only. The major splice variant increased gene expression 9–11-fold in a luciferase reporter assay, while the other isoforms were similar or slightly greater than the control. We examined the upstream region of the most active splice variant in a promoter-reporter assay, and isolated a 257 bp sequence that produced maximal promoter activity. This sequence lacked a TATA box, but contained a consensus Sp1 site and a CAAT box, as well as several other putative transcription-factor-binding sites. Cell-specific expression of the different NAT1 transcripts may contribute to the variation in NAT1 activity in vivo.
Uptake of denatured collagen into hepatic stellate cells: evidence for the involvement of urokinase plasminogen activator receptor-associated protein/Endo180
Authors: MOUSAVI Seyed Ali , SATO Mitsuru , SPORSTØL Marita , SMEDSRØD Baard , BERG Trond , KOJIMA Naosuke , SENOO Haruki 
Published: March 2005

Tissue remodelling is dependent on the integration of signals that control turnover of ECM (extracellular matrix). Breakdown and endocytosis of collagen, a major component of the ECM, is central to this process. Whereas controlled secretion of matrix-degrading enzymes (such as matrix metalloproteinases) has long been known to mediate ECM breakdown, it is becoming clear that uPARAP/Endo180 (where uPARAP stands for urokinase plasminogen activator receptor-associated protein) serves as a receptor that mediates endocytosis of collagen by several types of cells. In the liver, the stellate cells play a major role in turnover of ECM including collagens. These cells synthesize various collagens and also produce matrix metalloproteinases. In the present study, we investigated the capacity of rat hepatic stellate cells to endocytose and degrade 125I-labelled heat-denatured collagen I. It was found that the collagen is efficiently taken up and degraded by these cells. Degradation was inhibited by inhibitors of lysosomal proteases (leupeptin and E-64d) and the vacuolar proton pump (concanamycin A), indicating that it takes place in lysosomes. Furthermore, endocytosed FITC-labelled collagen was shown to reach late endocytic compartments in which it colocalized with LysoTracker (a marker of late endocytic compartments). Competition experiments showed that uPA and unlabelled collagen are capable of inhibiting binding and uptake of [125I]collagen in a dose-dependent manner. Moreover, Western-blot analysis of cell lysate (using a polyclonal rabbit human-Endo180 antiserum) revealed a single band at 180 kDa. In addition, the antiserum was capable of reducing [125I]collagen binding to the cell surface. Finally, using two primers designed from the human uPARAP/Endo180 mRNA sequence, the expression of uPARAP/Endo180 mRNA was detected by reverse transcriptase–PCR. These results together suggest that uPARAP/Endo180 mediates endocytosis of collagen in rat liver stellate cells.
A novel human complement-related protein, C1r-like protease (C1r-LP), specifically cleaves pro-C1s
Authors: LIGOUDISTIANOU Christina , XU Yuanyuan , GARNIER Gerard , CIRCOLO Antonella , VOLANAKIS John E. 
Published: March 2005

The availability of the human genome sequence allowed us to identify a human complement-related, C1r-like protease gene (c1r-LP) located 2 kb centromeric of the C1r gene (c1r). Compared with c1r, c1r-LP carries a large deletion corresponding to exons 4–8 of c1r. The open reading frame of the C1r-LP cDNA predicts a 50 kDa modular protein displaying 52% amino acid residue identity with the corresponding regions of C1r and 75% identity with a previously described murine C1r-LP. The serine protease domain of C1r-LP, despite an overall similarity with the AGY group of complement serine proteases, has certain structural features characteristic of C2 and factor B, thus raising interesting evolutionary questions. Northern blotting demonstrated the expression of C1r-LP mRNA mainly in the liver and ELISA demonstrated the presence of the protein in human serum at a concentration of 5.5±0.9 μg/ml. Immunoprecipitation experiments failed to demonstrate an association of C1r-LP with the C1 complex in serum. Recombinant C1r-LP exhibits esterolytic activity against peptide thioesters with arginine at the P1 position, but its catalytic efficiency (kcat/Km) is lower than that of C1r and C1s. The enzymic activity of C1r-LP is inhibited by di-isopropyl fluorophosphate and also by C1 inhibitor, which forms stable complexes with the protease. Most importantly, C1r-LP also expresses proteolytic activity, cleaving pro-C1s into two fragments of sizes identical with those of the two chains of active C1s. Thus C1r-LP may provide a novel means for the formation of the classical pathway C3/C5 convertase.
Conformation and concerted dynamics of the integrin-binding site and the C-terminal region of echistatin revealed by homonuclear NMR
Authors: MONLEÓN Daniel , ESTEVE Vicent , KOVACS Helena , CALVETE Juan J. , CELDA Bernardo 
Published: March 2005

Echistatin is a potent antagonist of the integrins αvβ3, α5β1 and αIIbβ3. Its full inhibitory activity depends on an RGD (Arg-Gly-Asp) motif expressed at the tip of the integrin-binding loop and on its C-terminal tail. Previous NMR structures of echistatin showed a poorly defined integrin-recognition sequence and an incomplete C-terminal tail, which left the molecular basis of the functional synergy between the RGD loop and the C-terminal region unresolved. We report a high-resolution structure of echistatin and an analysis of its internal motions by off-resonance ROESY (rotating-frame Overhauser enhancement spectroscopy). The full-length C-terminal polypeptide is visible as a β-hairpin running parallel to the RGD loop and exposing at the tip residues Pro43, His44 and Lys45. The side chains of the amino acids of the RGD motif have well-defined conformations. The integrin-binding loop displays an overall movement with maximal amplitude of 30°. Internal angular motions in the 100–300 ps timescale indicate increased flexibility for the backbone atoms at the base of the integrin-recognition loop. In addition, backbone atoms of the amino acids Ala23 (flanking the R24GD26 tripeptide) and Asp26 of the integrin-binding motif showed increased angular mobility, suggesting the existence of major and minor hinge effects at the base and the tip, respectively, of the RGD loop. A strong network of NOEs (nuclear Overhauser effects) between residues of the RGD loop and the C-terminal tail indicate concerted motions between these two functional regions. A full-length echistatin–αvβ3 docking model suggests that echistatin's C-terminal amino acids may contact αv-subunit residues and provides new insights to delineate structure–function correlations.
Proteolytic processing and glycosylation influence formation of porcine prion protein complexes
Authors: NIEZNANSKI Krzysztof , RUTKOWSKI Marcin , DOMINIK Magdalena , STEPKOWSKI Dariusz 
Published: March 2005

High level of heterogeneity seems to be a ubiquitous feature of mammalian PrPs (prion proteins) and may be relevant to the pathogenesis of prion diseases. In the present study, we describe the heterogeneity of PrPC (cellular form of PrP) from porcine brain. It was disclosed and characterized by a combination of one-dimensional PAGE and two-dimensional PAGE analyses with enzymic deglycosylation and copper-affinity experiments. We found that the identified two main populations of porcine PrPC consist of diglycosylated forms and correspond to the full-length (molecular mass 32–36 kDa) and proteolytically modified protein (molecular mass 25–30 kDa), known as C1. The two populations were fully separated during Cu2+-loaded immobilized metal affinity chromatography, indicating different affinity for copper ions. The more basic forms, migrating as species of higher molecular mass, exhibited stronger affinity for copper ions, whereas those with more acidic pI and of lower molecular mass were low-affinity Cu2+-binding molecules and thus could represent N-terminally truncated PrPC. Size-exclusion chromatography revealed that most of the PrPC molecules in porcine brain extracts exist in the form of high-molecular-mass complexes (probably with other proteins). The heterogeneity of porcine PrPC, resulting from proteolytic modification and glycosylation, influences its ability to assemble into these complexes. N-truncated molecules dominate over full-length PrPC in fractions of molecular mass over the range 65–130 kDa, whereas the full-length species are the major forms of PrPC present in the monomeric fraction and in complexes above 130 kDa. Two-dimensional PAGE analysis indicated that the complexed PrPC differs in the composition of pI forms from the monomers.
Prelamin A endoproteolytic processing in vitro by recombinant Zmpste24
Authors: CORRIGAN Douglas P. , KUSZCZAK Danuta , RUSINOL Antonio E. , THEWKE Douglas P. , HRYCYNA Christine A. , MICHAELIS Susan , SINENSKY Michael S. 
Published: March 2005

The nuclear lamins form a karyoskeleton providing structural rigidity to the nucleus. One member of the lamin family, lamin A, is first synthesized as a 74 kDa precursor, prelamin A. After the endopeptidase and methylation reactions which occur after farnesylation of the CAAX-box cysteine, there is a second endoproteolysis that occurs 15 amino acids upstream from the C-terminal farnesylated cysteine residue. Studies with knockout mice have implicated the enzyme Zmpste24 (Face-1) as a suitable candidate to perform one or both of these proteolytic reactions. Evidence has been presented elsewhere establishing that Zmpste24 possesses a zinc-dependent CAAX endopeptidase activity. In the present study, we confirm this CAAX endopeptidase activity with recombinant, membrane-reconstituted Zmpste24 and show that it can accept a prelamin A farnesylated tetrapeptide as substrate. To monitor the second upstream endoproteolytic cleavage of prelamin A, we expressed a 33 kDa prelamin A C-terminal tail in insect cells. We demonstrate that this purified substrate possesses a C-terminal farnesylated and carboxyl-methylated cysteine and, therefore, constitutes a valid substrate for assaying the second endoproteolytic step in lamin A maturation. With this substrate, we demonstrate that insect cell membranes bearing recombinant Zmpste24 can also catalyse the second upstream endoproteolytic cleavage.
The central part of the 5.8 S rRNA is differently arranged in programmed and free human ribosomes
Authors: GRAIFER Dmitri , MOLOTKOV Maxim , EREMINA Anna , VEN'YAMINOVA Aliya , REPKOVA Marina , KARPOVA Galina 
Published: March 2005

A sequence-specific modification of the human 5.8 S rRNA in isolated 60 S subunits, non-programmed 80 S ribosomes and ribosomes complexed with mRNA and tRNAs was studied with the use of a derivative of the nonaribonucleotide UCUGUGUUU bearing a perfluorophenylazide group on the C-5 atom of the 5′-terminal uridine. Part of the oligonucleotide moiety of the derivative was complementary to the 5.8 S rRNA sequence ACACA in positions 82–86 flanked by two guanines at the 5′-terminus. The target for the cross-linking was identified as nucleotide G89 on the 5.8 S RNA. In addition, several ribosomal proteins were modified by the oligonucleotide derivative bound to the 5.8 S rRNA and proteins L6 and L8 were among them. Application of these results to known cryo-electron microscopy images of eukaryotic 60 S subunits made it possible to suggest that the central part of the 5.8 S rRNA containing the sequence 82–86 and proteins L6 and L8 are located at the base of the L1 stalk of the 60 S subunit. The efficacy of cross-linking in non-programmed 80 S ribosomes was much lower than in isolated 60 S subunits and in programmed 80 S ribosomes. We suggest that the difference in the accessibilities of the central part of the 5.8 S rRNA in the programmed and non-programmed 80 S ribosomes is caused by a conformational switch that seems to be required to dissociate the 80 S ribosomes into the subunits after termination of translation to allow initiation of translation of a new template.
Regulation of human methylthioadenosine phosphorylase gene by the CBF (CCAAT binding factor)/NF-Y (nuclear factor-Y)
Authors: KADARIYA Yuwaraj , NAKATANI Kaname , NISHIOKA Junji , FUJIKAWA Takahiko , KRUGER Warren D. , NOBORI Tsutomu 
Published: March 2005

hMTAP (human 5′-deoxy-5′-methylthioadenosine phosphorylase) is a key enzyme in the methionine salvage pathway and is frequently inactivated in human tumour cells. To understand the mechanism of the transcriptional regulation of the MTAP gene, we have cloned the 1.29 kb fragment of the hMTAP promoter and identified cis-acting regulatory sequences using a luciferase reporter gene assay. Maximal promoter activity was associated with sequences between −446 and −152, where two CCAAT elements were located. Electrophoretic mobility-shift assay reveals binding of specific complexes at both CCAAT motifs within the MTAP promoter, although more prominent bands were associated with the distal motif (−372 to −368). Supershift experiments and chromatin immunoprecipitation assays indicate that both the proximal and distal complexes bind CBF (CCAAT-binding factor; also known as nuclear factor-Y), and that the distal CCAAT motif has increased levels of CBF binding. We have mapped seven different transcriptional start sites between −135 and −58. Our results show that the hMTAP expression is regulated by a CBF and that the distal one of two CCAAT motifs plays a major role in the transcriptional activation of hMTAP gene.
Open form of syntaxin-1A is a more potent inhibitor than wild-type syntaxin-1A of Kv2.1 channels
Authors: LEUNG Yuk M. , KANG Youhou , XIA Fuzhen , SHEU Laura , GAO Xiaodong , XIE Huanli , TSUSHIMA Robert G. , GAISANO Herbert Y. 
Published: March 2005

We have shown that SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins not only participate directly in exocytosis, but also regulate the dominant membrane-repolarizing Kv channels (voltage-gated K+ channels), such as Kv2.1, in pancreatic β-cells. In a recent report, we demonstrated that WT (wild-type) Syn-1A (syntaxin-1A) inhibits Kv2.1 channel trafficking and gating through binding to the cytoplasmic C-terminus of Kv2.1. During β-cell exocytosis, Syn-1A converts from a closed form into an open form which reveals its active H3 domain to bind its SNARE partners SNAP-25 (synaptosome-associated protein of 25 kDa) and synaptobrevin. In the present study, we compared the effects of the WT Syn-1A and a mutant open form Syn-1A (L165A, E166A) on Kv2.1 channel trafficking and gating. When co-expressed in HEK-293 cells (human embryonic kidney-293 cells), the open form Syn-1A decreased Kv2.1 current density more than (P&lt;0.05) the WT Syn-1A (166±35 and 371±93 pA/pF respectively; control=911±91 pA/pF). Confocal microscopy and biotinylation experiments showed that both the WT and open form Syn-1A inhibited Kv2.1 expression at the plasma membrane to a similar extent, suggesting that the stronger reduction of Kv2.1 current density by the open form compared with the WT Syn-1A is probably due to a stronger direct inhibition of channel activity. Consistently, dialysis of the recombinant open form Syn-1A protein into Kv2.1-expressing HEK-293 cells caused stronger inhibition of Kv2.1 current amplitude (P&lt;0.05) than the WT Syn-1A protein (73±2 and 82±3% of the control respectively). We found that the H3 but not HABC domain is the putative active domain of Syn-1A, which bound to and inhibited the Kv2.1 channel. When co-expressed in HEK-293 cells, the open-form Syn-1A slowed down Kv2.1 channel activation (τ=12.3±0.8 ms) much more than (P&lt;0.05) WT Syn-1A (τ=7.9±0.8 ms; control τ=5.5±0.6 ms). In addition, only the open form Syn-1A, but not the WT Syn-1A, caused a significant (P&lt;0.05) left-shift in the steady-state inactivation curve (V1/2=33.1±1.3 and −29.4±1.1 mV respectively; control V1/2=−24.8±2 mV). The present study therefore indicates that the open form of Syn-1A is more potent than the WT Syn-1A in inhibiting the Kv2.1 channel. Such stronger inhibition by the open form of Syn-1A may limit K+ efflux and thus decelerate membrane repolarization during exocytosis, leading to optimization of insulin release.
Novel molecular approaches to cystic fibrosis gene therapy
Authors: LEE Tim W. R. , MATTHEWS David A. , BLAIR G. Eric 
Published: March 2005

Gene therapy holds promise for the treatment of a range of inherited diseases, such as cystic fibrosis. However, efficient delivery and expression of the therapeutic transgene at levels sufficient to result in phenotypic correction of cystic fibrosis pulmonary disease has proved elusive. There are many reasons for this lack of progress, both macroscopically in terms of airway defence mechanisms and at the molecular level with regard to effective cDNA delivery. This review of approaches to cystic fibrosis gene therapy covers these areas in detail and highlights recent progress in the field. For gene therapy to be effective in patients with cystic fibrosis, the cDNA encoding the cystic fibrosis transmembrane conductance regulator protein must be delivered effectively to the nucleus of the epithelial cells lining the bronchial tree within the lungs. Expression of the transgene must be maintained at adequate levels for the lifetime of the patient, either by repeat dosage of the vector or by targeting airway stem cells. Clinical trials of gene therapy for cystic fibrosis have demonstrated proof of principle, but gene expression has been limited to 30 days at best. Results suggest that viral vectors such as adenovirus and adeno-associated virus are unsuited to repeat dosing, as the immune response reduces the effectiveness of each subsequent dose. Nonviral approaches, such as cationic liposomes, appear more suited to repeat dosing, but have been less effective. Current work regarding non-viral gene delivery is now focused on understanding the mechanisms involved in cell entry, endosomal escape and nuclear import of the transgene. There is now increasing evidence to suggest that additional ligands that facilitate endosomal escape or contain a nuclear localization signal may enhance liposome-mediated gene delivery. Much progress in this area has been informed by advances in our understanding of the mechanisms by which viruses deliver their genomes to the nuclei of host cells.
Activated tumour necrosis factor-α shedding process is associated with in-hospital complication in patients with acute myocardial infarction
Authors: SHIMODA Yudai , SATOH Mamoru , NAKAMURA Motoyuki , AKATSU Tomonari , HIRAMORI Katsuhiko 
Published: March 2005

TACE [TNF-α (tumour necrosis factor-α)-converting enzyme] plays an essential role in the shedding of TNF-α, which could affect the outcome of AMI (acute myocardial infarction). To investigate the clinical significance of the TACE–TNF-α system in AMI, we examined TACE-mediated TNF-α synthesis in PBMCs (peripheral blood mononuclear cells), which are a possible source of TNF-α in AMI. Forty-one patients with AMI and 15 healthy subjects (HS) were enrolled in the present study. PBMCs were isolated from peripheral blood on day 1 and 14 after the onset of AMI. TACE and TNF-α mRNA levels and intracellular median fluorescence intensity were measured by real-time RT (reverse transcriptase)–PCR and flow cytometry respectively. TACE-mediated TNF-α production was evaluated in cultured PBMCs with PMA, which is known to activate TACE. Spontaneous TACE and TNF-α levels were higher in AMI patients than in HS (P&lt;0.001). TACE and TNF-α levels in PMA-stimulated PMBCs were markedly increased in AMI patients compared with HS (P&lt;0.001). There was a positive correlation between TACE and TNF-α levels in AMI. Although spontaneous and stimulated levels of TACE and TNF-α decreased 14 days after the onset of AMI, levels in AMI patients were higher than in HS. In AMI patients with in-hospital complications (n=15; pump failure in ten, recurrent myocardial infarction in one, malignant ventricular arrhythmia in three and cardiac death in one), spontaneous and stimulated levels of TACE and TNF-α were higher than in patients without complications (P&lt;0.01). These levels were higher in AMI patients with in-hospital complications 14 days after onset. These results demonstrate that TACE-mediated TNF-α maturation in PBMCs may play an important role in poor outcomes from AMI, suggesting that TACE may be a potential target for the inhibition of cellular TNF-α production in AMI.
Change in β1-adrenergic receptor protein concentration in adipose tissue correlates with diet-induced weight loss
Authors: RASMUSSEN Mads , BELZA Anita , ALMDAL Thomas , TOUBRO Søren , BRATHOLM Palle , ASTRUP Arne , CHRISTENSEN Niels J. 
Published: March 2005

The aim of the present study was to examine gene expression and protein concentrations of β1- and β2-adrenergic receptors in subcutaneous adipose tissue in obese subjects in response to weight loss. Eighteen obese subjects were studied during diet-induced weight loss. β-Adrenergic receptor mRNA levels were quantified by reverse transcription-PCR–HPLC. β-Adrenergic receptor protein concentrations were measured by Western blotting using fluorescence laser scanning for detection. Subjects lost 12.8±0.8 kg (mean±S.E.M.) during diet treatment. There was a 34% decrease in the β1-adrenergic receptor mRNA level (0.92±0.09 compared with 0.61±0.06 amol/μg of DNA; P&lt;0.002). β2-Adrenergic receptor mRNA did not decrease significantly. β2-Adrenergic receptor protein concentration decreased 37% (25.5±7.1 compared with 16.0±5.6 arbitrary units/ng of DNA; P=0.008), whereas β1-adrenergic receptor protein concentration did not decrease significantly. The degree of weight loss was correlated with the concentration of β1-adrenergic receptor protein (r=0.65, P&lt;0.003) and changes in receptor protein concentration (r=0.50, P=0.035) during the very-low-calorie diet. In conclusion, the present study demonstrates a relationship between β1-adrenergic receptor protein concentration in adipose tissue and the degree of weight loss. This relationship is not directly related to energy expenditure and deserves further investigation.