bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulated expression and temporal induction of the tail-anchored sarcolemmal-membrane-associated protein is critical for myoblast fusion
Authors: GUZZO Rosa M. , WIGLE Jeffery , SALIH Maysoon , MOORE Edwin D. , TUANA Balwant S. 
Published: July 2004

Sarcolemmal-membrane-associated proteins (SLMAPs) define a new class of coiled-coil tail-anchored membrane proteins generated by alternative splicing mechanisms. An in vivo expression analysis indicated that SLMAPs are present in somites (11 days post-coitum) as well as in fusing myotubes and reside at the level of the sarcoplasmic reticulum and transverse tubules in adult skeletal muscles. Skeletal-muscle myoblasts were found to express a single 5.9 kb transcript, which encodes the full-length ∼91 kDa SLMAP3 isoform. Myoblast differentiation was accompanied by the stable expression of the ∼91 kDa SLMAP protein as well as the appearance of an ∼80 kDa isoform. Deregulation of SLMAPs by ectopic expression in myoblasts resulted in a potent inhibition of fusion without affecting the expression of muscle-specific genes. Membrane targeting of the de-regulated SLMAPs was not critical for the inhibition of myotube development. Protein–protein interaction assays indicated that SLMAPs are capable of self-assembling, and the de-regulated expression of mutants that were not capable of forming SLMAP homodimers also inhibited myotube formation. These results imply that regulated levels and the temporal induction of SLMAP isoforms are important for normal muscle development.
The fidelity of replication of the three-base-pair set adenine/thymine, hypoxanthine/cytosine and 6-thiopurine/5-methyl-2-pyrimidinone with T7 DNA polymerase
Authors: RAPPAPORT Harry P. 
Published: July 2004

With the goal of constructing a genetic alphabet consisting of a set of three base pairs, the fidelity of replication of the three base pairs TH (5-methyl-2-pyrimidinone)/HS (6-thiopurine; thiohypoxanthine), C/H (hypoxanthine) and T/A was evaluated using T7 DNA polymerase, a polymerase with a strong 3′→5′ exonuclease activity. An evaluation of the suitability of a new base pair for replication should include both the contribution of the fidelity of a polymerase activity and the contribution of proofreading by a 3′→5′ exonuclease activity. Using a steady-state kinetics method that included the contribution of the 3′→5′ exonuclease activity, the fidelity of replication was determined. The method determined the ratio of the apparent rate constant for the addition of a deoxynucleotide to the primer across from a template base by the polymerase activity and the rate constant for removal of the added deoxynucleotide from the primer by the 3′→5′ exonuclease activity. This ratio was designated the eni (efficiency of net incorporation). The eni of the base pair C/H was equal to or greater than the eni of T/A. The eni of the base pair TH/HS was 0.1 times that of A/T for TH in the template and 0.01 times that of A/T for HS in the template. The ratio of the eni of a mismatched deoxynucleotide to the eni of a matched deoxynucleotide was a measure of the error frequency. The error frequencies were as follows: thymine or TH opposite a template hypoxanthine, 2×10−6; HS opposite a template cytosine, &lt;3×10−4. The remaining 24 mismatched combinations of bases gave no detectable net incorporation. Two mismatches, hypoxanthine opposite a template thymine or a template TH, showed trace incorporation in the presence of a standard dNTP complementary to the next template base. T7 DNA polymerase extended the primer beyond each of the matched base pairs of the set. The level of fidelity of replication of the three base pairs with T7 DNA polymerase suggests that they are adequate for a three-base-pair alphabet for DNA replication.
The lobster mandibular organ produces soluble and membrane-bound forms of 3-hydroxy-3-methylglutaryl-CoA reductase
Authors: LI Sheng , FRIESEN Jon A. , FEI Hong , DING Xiang , BORST David W. 
Published: July 2004

In a previous study [Li, Wagner, Friesen and Borst (2003) Gen. Comp. Endocrinol. 134, 147–155], we showed that the MO (mandibular organ) of the lobster Homarus americanus has high levels of HMGR (3-hydroxy-3-methylglutaryl-CoA reductase) and that most (approx. 75%) of the enzyme activity is soluble. In the present study, we report the biochemical and molecular characteristics of this enzyme. HMGR had two forms in the MO: a more abundant soluble form (66 kDa) and a less abundant membrane-bound form (72 kDa). Two cDNAs for HMGR were isolated from the MO. A 2.6-kb cDNA encoded HMGR1, a 599-amino-acid protein (63 kDa), and a 3.2-kb cDNA encoded HMGR2, a 655-amino-acid protein (69 kDa). These two cDNAs had identical 3′-ends and appeared to be products of a single gene. The deduced amino acid sequences of these two proteins revealed a high degree of similarity to other class I HMGRs. Hydropathy plots indicated that the N-terminus of HMGR1 lacked a transmembrane region and HMGR2 had a single transmembrane segment. Recombinant HMGR1 expressed in Sf9 insect cells was soluble and had kinetic characteristics similar to native HMGR from the MO. Treatment with phosphatase did not affect HMGR activity, consistent with the observation that neither HMGR1 nor HMGR2 has a serine at position 490 or 546, the position of a conserved phosphorylation site found in class I HMGR from higher eukaryotes. Other lobster tissues (i.e. midgut, brain and muscles) had low HMGR activities and mRNA levels. MO with higher HMGR activities had higher HMGR mRNA levels, implying that HMGR is regulated, in part, at the transcription level.
Kinetic and structural analysis of the increased affinity of enoyl-ACP (acyl-carrier protein) reductase for triclosan in the presence of NAD+
Authors: KAPOOR Mili , MUKHI P. L. Swarna , SUROLIA Namita , SUGUNA K. , SUROLIA Avadhesha 
Published: July 2004

The binding of enoyl-ACP (acyl-carrier protein) reductase from Plasmodium falciparum (PfENR) with its substrates and inhibitors has been analysed by SPR (surface plasmon resonance). The binding of the substrate analogue crotonoyl-CoA and coenzyme NADH to PfENR was monitored in real time by observing changes in response units. The binding constants determined for crotonoyl-CoA and NADH were 1.6×104 M−1 and 1.9×104 M−1 respectively. Triclosan, which has recently been demonstrated as a potent antimalarial agent, bound to the enzyme with a binding constant of 1.08×105 M−1. However, there was a 300-fold increase in the binding constant in the presence of NAD+. The increase in the binding constant was due to a 17 times increase in the association rate constant (k1) from 741 M−1·s−1 to 1.3×104 M−1 ·s−1 and a 16 times decrease in the dissociation rate constant (k−1) from 6.84×10−3 s−1 to 4.2×10−4 s−1. These values are in agreement with those determined by steady-state kinetic analysis of the inhibition reaction [Kapoor, Reddy, Krishnasastry, N. Surolia and A. Surolia (2004) Biochem. J. 381, 719–724]. In SPR experiments, the binding of NAD+ to PfENR was not detected. However, a binding constant of 6.5×104 M−1 was obtained in the presence of triclosan. Further support for these observations was provided by the crystal structures of the binary and ternary complexes of PfENR. Thus the dramatic enhancement in the binding affinity of both triclosan and NAD+ in the ternary complex can be explained by increased van der Waals contacts in the ternary complex, facilitated by the movement of residues 318–324 of the substrate-binding loop and the nicotinamide ring of NAD+. Interestingly, the results of the present study also provide a rationale for the increased affinity of NAD+ for the enzyme in the ternary complex.
Modular organization of a Cdc6-like protein from the crenarchaeon Sulfolobus solfataricus
Authors: DE FELICE Mariarita , ESPOSITO Luca , PUCCI Biagio , DE FALCO Mariarosaria , MANCO Giuseppe , ROSSI Mosè , PISANI Francesca M. 
Published: July 2004

In the present paper, we report that a Cdc6 (cell-division control)-like factor from the hyperthermophilic crenarchaeon Sulfolobus solfataricus (referred to as SsoCdc6-2) has a modular organization of its biological functions. A reliable model of the SsoCdc6-2 three-dimensional structure was built up, based on the significant sequence identity with the Pyrobaculum aerophylum Cdc6 (PaeCdc6), whose crystallographic structure is known. This allowed us to design two truncated forms of SsoCdc6-2: the ΔC (residues 1–297, molecular mass 35 kDa) and the ΔN (residues 298–400, molecular mass 11 kDa) proteins. The ΔC protein contains the nucleotide-binding Rossmann fold and the Sensor-2 motif (Domains I and II in the PaeCdc6 structure), and retains the ability to bind and hydrolyse ATP. On the other hand, the ΔN protein contains the C-terminal WH (winged helix)-fold (Domain III), and is able to bind DNA molecules and to inhibit the DNA helicase activity of the SsoMCM (mini-chromosome maintenance) complex, although with lesser efficiency with respect to the full-sized SsoCdc6-2. These results provide direct biochemical evidence that the Cdc6 WH-domain is responsible for DNA-binding and inhibition of MCM DNA helicase activity.
Copper pathways in Plasmodium falciparum infected erythrocytes indicate an efflux role for the copper P-ATPase
Authors: RASOLOSON Dominique , SHI Lirong , CHONG Curtis R. , KAFSACK Bjorn F. , SULLIVAN David J. 
Published: July 2004

Copper, like iron, is a transition metal that can generate oxygen radicals by the Fenton reaction. The Plasmodium parasite invades an erythrocyte host cell containing 20 μM copper, of which 70% is contained in the Cu/Zn SOD (cuprozinc superoxide dismutase). In the present study, we follow the copper pathways in the Plasmodium-infected erythrocyte. Metal-determination analysis shows that the total copper content of Percoll-purified trophozoite-stage-infected erythrocytes is 66% that of uninfected erythrocytes. This decrease parallels the decrease seen in Cu/Zn SOD levels in parasite-infected erythrocytes. Neocuproine, an intracellular copper chelator, arrests parasites at the ring-to-trophozoite stage transition and also specifically decreases intraparasitic levels of Cu/Zn SOD and catalase. Up to 150 μM BCS (2,9-dimethyl-4,7-diphenyl-1,10-phenanthrolinedisulphonic acid), an extracellular copper chelator, has no effect on parasite growth. We characterized a single copy PfCuP-ATPase (Plasmodium falciparum copper P-ATPase) transporter, which, like the Crypto-sporidium parvum copper P-ATPase, has a single copper-binding domain: ‘Met-Xaa-Cys-Xaa-Xaa-Cys’. Recombinant expression of the N-terminal metal-binding domain reveals that the protein specifically binds reduced copper. Transcription of the PfCuP-ATPase gene is the highest at late ring stage/early trophozoite, and is down-regulated in the presence of neocuproine. Immunofluorescence and electron microscopy indicate the transporter to be both in the parasite and on the erythrocyte membrane. Both the decrease in total copper and the location of the PfCuP-ATPase gene indicate a copper-efflux pathway from the infected erythrocyte.
Regulation of Cyp2a5 transcription in mouse primary hepatocytes: roles of hepatocyte nuclear factor 4 and nuclear factor I
Authors: ULVILA Johanna , ARPIAINEN Satu , PELKONEN Olavi , AIDA Kaoru , SUEYOSHI Tatsuya , NEGISHI Masahiko , HAKKOLA Jukka 
Published: July 2004

The cytochrome P4502a5 (Cyp2a5) gene is expressed principally in liver and olfactory mucosa. In the present study, the transcriptional mechanisms of hepatocyte-specific expression of Cyp2a5 were studied in mouse primary hepatocytes. The Cyp2a5 5′-flanking region −3033 to +10 was cloned in front of a luciferase reporter gene and transfected into hepatocytes. Deletion analysis revealed two major activating promoter regions localized at proximal 271 bp and at a more distal area from −3033 to −2014 bp. The proximal activation region was characterized further by DNase I footprinting, and a single clear footprint was detected in the studied area centred over a sequence similar to the NF-I (nuclear factor I)-binding site. The binding of NF-I was confirmed using an EMSA (electrophoretic mobility-shift assay). A putative HNF-4 (hepatocyte nuclear factor 4)-binding site was localized at the proximal promoter by computer analysis of the sequence, and HNF-4α was shown to interact with the site using an EMSA. The functional significance of HNF-4 and NF-I binding to the Cyp2a5 promoter was evaluated by site-directed mutagenesis of the binding motifs in reporter constructs. Both mutations strongly decreased transcriptional activation by the Cyp2a5 promoter in primary hepatocytes, and double mutation almost completely abolished transcriptional activity. Also, the functionality of the distal activation region was found to be dependent on the intact HNF-4 and NF-I sites at the proximal promoter. In conclusion, these results indicate that HNF-4 and NF-I play major roles in the constitutive regulation of hepatic expression of Cyp2a5.
The stereospecificity and catalytic efficiency of the tryptophan synthase-catalysed exchange of the α-protons of amino acids
Authors: NíBEILLIÚ Máire E. , MALTHOUSE J. Paul G. 
Published: July 2004

13C-NMR has been used to follow the tryptophan synthase (EC 4.2.1.20) catalysed hydrogen–deuterium exchange of the pro-2R and pro-2S protons of [2-13C]glycine at pH 7.8. 1H-NMR has also been used to follow the tryptophan-synthase-catalysed hydrogen–deuterium exchange of the α-protons of a range of L- and D-amino acids at pH 7.8. The pKa values of the α-protons of these amino acids have been estimated and we have determined whether or not their exchange rates can be predicted from their pKa values. With the exception of tryptophan and norleucine, the stereospecificities of the first-order α-proton exchange rates are independent of the size and electronegativity of the amino acid R-group. Similar results are obtained with the second-order α-proton exchange rates, except that both L-tryptophan and L-serine have much higher stereospecificities than all the other amino acids studied.
Characterization of transgenic mice with widespread overexpression of spermine synthase
Authors: IKEGUCHI Yoshihiko , WANG Xiaojing , McCLOSKEY Diane E. , COLEMAN Catherine S. , NELSON Paul , HU Guirong , SHANTZ Lisa M. , PEGG Anthony E. 
Published: July 2004

A widespread increase in SpmS (spermine synthase) activity has been produced in transgenic mice using a construct in which the human SpmS cDNA was placed under the control of a composite CMV-IE (cytomegalovirus immediate early gene) enhancer–chicken β-actin promoter. Four separate founder CAG/SpmS mice were studied. Transgenic expression of SpmS was found in all of the tissues examined, but the relative SpmS activities varied widely according to the founder animal and the tissue studied. Very large increases in SpmS activity were seen in many tissues. SpdS (spermidine synthase) activity was not affected. Although there was a statistically significant decline in spermidine content and increase in spermine, the alterations were small compared with the increase in SpmS activity. These results provide strong support for the concept that the levels of the higher polyamines spermidine and spermine are not determined only by the relative activities of the two aminopropyltransferases. Other factors such as availability of the aminopropyl donor substrate decarboxylated S-adenosylmethionine and possibly degradation or excretion must also influence the spermidine/spermine ratio. No deleterious effects of SpmS overexpression were seen. The mice had normal growth, fertility and behaviour up to the age of 12 months. However, breeding the CAG/SpmS mice with MHC (α-myosin heavy chain)/AdoMetDC (S-adenosylmethionine decarboxylase) mice, which have a large increase in S-adenosylmethionine decarboxylase expression in heart, was lethal. In contrast, breeding the CAG/SpmS mice with MHC/ODC (L-ornithine decarboxylase) mice, which have a large increase in cardiac ornithine decarboxylase expression, had a protective effect in preventing the small decrease in viability of the MHC/ODC mice.
Analysis of Plasmodium vivax hexose transporters and effects of a parasitocidal inhibitor
Authors: JOËT Thierry , CHOTIVANICH Kesinee , SILAMUT Kamolrat , PATEL Asha P. , MORIN Christophe , KRISHNA Sanjeev 
Published: July 2004

Plasmodium vivax is the second most common species of malaria parasite and causes up to 80 million episodes of infection each year. New drug targets are urgently needed because of emerging resistance to current treatments. To study new potential targets, we have functionally characterized two natural variants of the hexose transporter of P. vivax (PvHT) after heterologous expression in Xenopus oocytes. We show that PvHT transports both glucose and fructose. Differences in the affinity for fructose between the two variants of PvHT establishes that sequence variation is associated with phenotypic plasticity. Mutation of a single glutamine residue, Gln167, predicted to lie in transmembrane helix 5, abolishes fructose transport by PvHT, although glucose uptake is preserved. In contrast, the exofacial site located between predicted helices 5 and 6 of PvHT is not an important determinant of substrate specificity, despite exhibiting sequence polymorphisms between hexose transporters of different Plasmodium spp. Indeed, replacement of twelve residues located within this region of PvHT by those found in the orthologous Plasmodium falciparum sequence (PfHT) is functionally silent with respect to affinity for hexoses. All PvHT variants are inhibited by compound 3361, a long-chain O-3 derivative of D-glucose effective against PfHT. Furthermore, compound 3361 kills short term cultures of P. vivax isolated from patients. These data provide unique insights into the function of hexose transporters of Plasmodium spp. as well as further evidence that they could be targeted by drugs.
Syntaxin 5 interacts with presenilin holoproteins, but not with their N- or C-terminal fragments, and affects β-amyloid peptide production
Authors: SUGA Kei , TOMIYAMA Takami , MORI Hiroshi , AKAGAWA Kimio 
Published: July 2004

Mutations in presenilins 1 and 2 (PS1 and PS2) account for the majority of cases of early-onset familial Alzheimer's disease. However, the trafficking and interaction of PSs with other proteins in the early secretory pathways are poorly understood. Using co-immunoprecipitation, we found that PS bound to Syx5 (syntaxin 5), which is a target-soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor involved in endoplasmic reticulum (ER)–Golgi vesicular transport in vivo. Syx5 interacted only with the full-length PS holoproteins and not with the naturally occurring N- or C-terminal fragments. The PS holoproteins co-immunoprecipitated with the mutant Syx5, which localized to the ER and Golgi compartments, despite the substitution of the transmembrane region with that of syntaxin 1A. In contrast, the transmembrane deletion mutant that localized to the cytosol, but not to the ER or Golgi compartments, did not co-immunoprecipitate the PS holoproteins. The PS1 variant linked to familial Alzheimer's disease (PS1ΔE9), lacking the region that contains the endoproteolytic cleavage site in the cytoplasmic loop, showed markedly decreased binding to Syx5. Immunofluorescence and sucrose-density-gradient fractionation analyses showed that the full-length PS holoproteins co-localized with Syx5 to the ER and cis-Golgi compartments. Furthermore, Syx5 overexpression resulted in the accumulation of PS holoproteins and the β-amyloid precursor protein, and reduced the secretion of the Aβ (amyloid β) peptide in COS-7 cells. In summary, these results indicate that Syx5 binds to full-length PSs and affects the processing and trafficking of β-amyloid precursor protein in the early secretory compartments.
6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase: head-to-head with a bifunctional enzyme that controls glycolysis
Authors: RIDER Mark H. , BERTRAND Luc , VERTOMMEN Didier , MICHELS Paul A. , ROUSSEAU Guy G. , HUE Louis 
Published: July 2004

Fru-2,6-P2 (fructose 2,6-bisphosphate) is a signal molecule that controls glycolysis. Since its discovery more than 20 years ago, inroads have been made towards the understanding of the structure–function relationships in PFK-2 (6-phosphofructo-2-kinase)/FBPase-2 (fructose-2,6-bisphosphatase), the homodimeric bifunctional enzyme that catalyses the synthesis and degradation of Fru-2,6-P2. The FBPase-2 domain of the enzyme subunit bears sequence, mechanistic and structural similarity to the histidine phosphatase family of enzymes. The PFK-2 domain was originally thought to resemble bacterial PFK-1 (6-phosphofructo-1-kinase), but this proved not to be correct. Molecular modelling of the PFK-2 domain revealed that, instead, it has the same fold as adenylate kinase. This was confirmed by X-ray crystallography. A PFK-2/FBPase-2 sequence in the genome of one prokaryote, the proteobacterium Desulfovibrio desulfuricans, could be the result of horizontal gene transfer from a eukaryote distantly related to all other organisms, possibly a protist. This, together with the presence of PFK-2/FBPase-2 genes in trypanosomatids (albeit with possibly only one of the domains active), indicates that fusion of genes initially coding for separate PFK-2 and FBPase-2 domains might have occurred early in evolution. In the enzyme homodimer, the PFK-2 domains come together in a head-to-head like fashion, whereas the FBPase-2 domains can function as monomers. There are four PFK-2/FBPase-2 isoenzymes in mammals, each coded by a different gene that expresses several isoforms of each isoenzyme. In these genes, regulatory sequences have been identified which account for their long-term control by hormones and tissue-specific transcription factors. One of these, HNF-6 (hepatocyte nuclear factor-6), was discovered in this way. As to short-term control, the liver isoenzyme is phosphorylated at the N-terminus, adjacent to the PFK-2 domain, by PKA (cAMP-dependent protein kinase), leading to PFK-2 inactivation and FBPase-2 activation. In contrast, the heart isoenzyme is phosphorylated at the C-terminus by several protein kinases in different signalling pathways, resulting in PFK-2 activation.