bluuebunny/crossref_metadata_2025 huggingface dataset:
The effect of hyperosmotic pressure on antibody production and gene expression in the GS‐NS0 cell line
Authors: Wu Mon‐Han , Dimopoulos George , Mantalaris Athanasios , Varley Julie
Published: July 2004

It has been widely reported that metabolism, cell growth, cell density, product secretion and specific antibody productivity in mammalian cells are strongly affected by osmotic conditions. Previous studies have shown that hyperosmotic pressure suppresses cell growth while enhancing the productivity of individual cells, but the effect of these two changes does not result in an increase in final product concentration in the culture. An improved understanding of the basic cellular processes of a GS‐NS0 mammalian cell culture system would assist in the design of a more efficient mammalian cell culture system and in further optimization of production processes. In this study, various properties of mammalian culture systems, such as productivity, cell viability, metabolism, ion balance and the genes regulated during the culture of the GS‐NS0 system under osmotic pressure of iso‐ (290 mOsm/kg) and hyper‐ (450 mOsm/kg) osmolarity have been investigated, and we demonstrate that there is a decrease in the growth rate and an increase in specific production rate of hyperosmotic cultures as compared with iso‐osmotic cultures. Furthermore, differences between iso‐ and hyper‐osmotic cultures have been identified in calcium accumulation and metabolism of NH4+, glucose and lactate. Analysis of gene expression reveals regulation of over 600 genes that are implicated in processes known to be affected by changes in osmotic pressure, such as ion transport, accumulation of osmolytes, cell cycle distribution, proliferation, cytoskeletal organization and cell metabolism.
Stem‐cell plasticity and therapy for injuries of the peripheral nervous system
Authors: Tohill Mel , Terenghi Giorgio
Published: July 2004

Numerous publications have investigated stem‐cell biology and the possible therapeutic use of stem cells in a wide range of injuries and diseases. This interest has been fuelled by recent reports suggesting that mesenchymal stem cells can show unorthodox plasticity, their being able to transdifferentiate into cells of different lineages, such as neuronal phenotypes. This capability has obvious implications for their potential application in tissue engineering and tissue regeneration. The peripheral nervous system has an inherent capacity for regeneration, but this is limited and not matched by the level of reinnervation of target organs, with a resulting loss of functional recovery. Several approaches have been attempted in order to overcome this deficiency, and transplant of cultured Schwann cells into bioengineered conduits has been shown to improve regeneration. An alternative may be the use of stem‐cell technology, whereby cultured and differentiated stem cells can be transplanted to the site of injury in order to promote enhanced regeneration. The present review discusses the use of stem cells applied to the repair of peripheral nerve injury and their role in the regeneration process.
An improved, inexpensive procedure for the large‐scale purification of recombinant human erythropoietin
Authors: Hu Yunlong , Chen Song , Xu Mei , Zhang Shuangquan
Published: July 2004

A rapid and simple chromatographic procedure has been developed for the large‐scale purification of therapeutic‐grade rHuEPO (recombinant human erythropoietin) from medium‐conditioned cell cultures, which includes ion‐exchange, hydrophobic‐interaction and gel‐filtration chromatography. A combination of these well‐connected steps results in highly purified rHuEPO (>99%), as revealed by SDS/PAGE and HPLC analyses, with a total yield of 38%. The specific activity of purified rHuEPO was 160104 i.u./mg. Immunoblotting studies revealed that the protein possesses native EPO immunity. N‐terminal sequencing of rHuEPO shows that the first 15 amino acids coincide with those of native EPO reported previously.
The biology and engineering of stem‐cell control
Authors: O'Neill Analeah , Schaffer David V.
Published: July 2004

There is significant interest in studying stem cells, both to elucidate their basic biological functions during development and adulthood as well as to learn how to utilize them as new sources of specialized cells for tissue repair. Whether the motivation is basic biology or biomedical application, however, progress will hinge upon learning how to better control stem‐cell function at a quantitative and molecular level. There are several major challenges within the field, including the identification of new signals and conditions that regulate and influence cell function, and the application of this information towards the design of stem‐cell bioprocesses and therapies. Both of these efforts can significantly benefit from the synthesis of biological data into quantitative and increasingly mechanistic models that not only describe, but also predict, how a stem cell's environment can control its fate. This review will briefly summarize the history and current state of the stem‐cell biology field, but will then focus on the development of predictive models for stem‐cell control. Early models formulated on the assumption that cell fate was decided by stochastic, cell‐intrinsic processes have gradually evolved into hybrid deterministic–stochastic models with increasingly finer molecular resolution that accounts for environmental regulation. As our understanding of cellular control mechanisms expands from the cell surface and towards the nucleus, these efforts may culminate in the development of a stem‐cell culture programme, or a series of signals to provide to the cells as a function of time to guide them along a desired developmental trajectory.
Bioprosthetic heart valves: the need for a quantum leap
Authors: Zilla Peter , Human Paul , Bezuidenhout Deon
Published: July 2004

More than 250000 bioprosthetic heart valves are being implanted annually. Although the majority of recipients are elderly developed‐world patients, the most urgent need for tissue valves is in younger patients, where rapid degeneration of contemporary prostheses remains a serious obstacle. After decades of empirical and mostly futile attempts to extend the longevity of tissue valve prostheses, new insights and solutions are on the horizon. Aetiologically, a shift of focus from mineralization to immune responses and inflammation emerges. On the development side, new engineering approaches to both selective extraction of tissue components and cross‐links are increasingly defining the new direction. In order to dramatically improve the performance of bioprosthetic heart valves, these new developments need to lead to a broad consensus for a paradigm shift in a hitherto rather stagnant field of medical research.
Binding of α2-macroglobulin to GRAB (Protein G-related α2-macroglobulin-binding protein), an important virulence factor of group A streptococci, is mediated by two charged motifs in the ΔA region
Authors: GODEHARDT Antonia W. , HAMMERSCHMIDT Sven , FRANK Ronald , CHHATWAL Gursharan S.
Published: July 2004

GRAB (Protein G-related α2M-binding protein) is a surface protein of group A streptococci and exhibits high affinity for α2-macroglobulin (α2M), a broad-range protease inhibitor. It is the sole α2M-binding protein of group A streptococci that has been shown to promote bacterial virulence in a mouse model of skin infection. The binding site for α2M was predicted to be in the N-terminal A domain of GRAB. In the present study, the α2M-binding domain was first narrowed down to 34 amino acids (amino acids 34–67) using variable truncated N-terminal GRAB fusion proteins. The sequence of the identified domain was used to design overlapping synthetic peptides of different sizes, which were then immobilized on a membrane and assayed for their α2M-binding activity. The peptide screening revealed two binding motifs of ten amino acids length, located in the ΔA (N-terminal part of the A domain) region (amino acids 34–67) with the sequences PRIIPNGGTL (amino acids 41–50) and NAPEKLALRN (amino acids 56–65) respectively. These motifs were used for systematic mutational analysis by generating synthetic peptides containing individual amino acid substitutions at every position of the mapped binding regions. The results indicated a critical role for the arginine residue at position 42 in the first binding domain and at position 64 in the second binding region. Validation of arginine residues as the critical amino acids for α2M binding was achieved by site-directed mutagenesis and binding assays. Competitive inhibition assays with GRAB containing amino acid substitutions R42G (Arg42→Gly), R64G and R42G/R64G indicated differential contribution of the arginine residues at positions 42 and 64 to α2M-binding activity and, thus, their involvement in GRAB-induced virulence.
The C-terminus of rat L-histidine decarboxylase specifically inhibits enzymic activity and disrupts pyridoxal phosphate-dependent interactions with L-histidine substrate analogues
Authors: FLEMING John V. , FAJARDO Ignacio , LANGLOIS Michael R. , SÁNCHEZ-JIMÉNEZ Francisca , WANG Timothy C.
Published: July 2004

Full-length rat HDC (L-histidine decarboxylase) translated in reticulocyte cell lysate reactions is inactive, whereas C-terminally truncated isoforms are capable of histamine biosynthesis. C-terminal processing of the ∼74 kDa full-length protein occurs naturally in vivo, with the production of multiple truncated isoforms. The minimal C-terminal truncation required for the acquisition of catalytic competence has yet to be defined, however, and it remains unclear as to why truncation is needed. Here we show that ∼74 kDa HDC monomers can form dimers, which is the conformation in which the enzyme is thought to be catalytically active. Nevertheless, the resulting dimer is unable to establish pyridoxal phosphate-dependent interactions with an L-histidine substrate analogue. Protein sequences localized to between amino acids 617 and 633 specifically mediate this inhibition. Removing this region or replacing the entire C-terminus with non-HDC protein sequences permitted interactions with the substrate analogue to be re-established. This corresponded exactly with the acquisition of catalytic competence, and the ability to decarboxylate natural L-histidine substrate. These studies suggested that the ∼74 kDa full-length isoform is deficient in substrate binding, and demonstrated that C-terminally truncated isoforms with molecular masses between ∼70 kDa and ∼58 kDa have gradually increasing specific activities. The physiological relevance of our results is discussed in the context of differential expression of HDC isoforms in vivo.
Analysis of the ERK1,2 transcriptome in mammary epithelial cells
Authors: GRILL Constance , GHEYAS Ferdous , DAYANANTH Priya , JIN Weihong , DING Wei , QIU Ping , WANG Luquan , DOLL Ronald J. , ENGLISH Jessie M.
Published: July 2004

MAPK (mitogen-activated protein kinase) pathways constitute major regulators of cellular transcriptional programmes. We analysed the ERK1,2 (extracellular-signal-regulated kinase 1,2) transcriptome in a non-transformed MEC (mammary epithelial cell) line, MCF-12A, utilizing rAd MEK1EE, a recombinant adenovirus encoding constitutively active MEK1 (MAPK/ERK kinase 1). rAd MEK1EE infection induced morphological changes and DNA synthesis which were inhibited by the MEK1,2 inhibitor PD184352. Hierarchical clustering of data derived from seven time points over 24 h identified 430 and 305 co-ordinately up-regulated and down-regulated genes respectively. c-Myc binding sites were identified in the promoters of most of these up-regulated genes. A total of 46 candidate effectors of the Raf/MEK/ERK1,2 pathway in MECs were identified by comparing our dataset with previously reported Raf-1-regulated genes. These analyses led to the identification of a suite of growth factors co-ordinately induced by MEK1EE, including multiple ErbB ligands, vascular endothelial growth factor and PHRP (parathyroid hormone-related protein). PHRP is the primary mediator of humoral hypercalcaemia of malignancy, and has been implicated in metastasis to bone. We demonstrate that PHRP is secreted by MEK1EE-expressing cells. This secretion is inhibited by PD184352, but not by ErbB inhibitors. Our results suggest that, in addition to anti-proliferative properties, MEK1,2 inhibitors may be anti-angiogenic and possess therapeutic utility in the treatment of PHRP-positive tumours.
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.
Kinetics of Factor X activation by the membrane-bound complex of Factor IXa and Factor VIIIa
Authors: PANTELEEV Mikhail A. , SAENKO Evgueni L. , ANANYEVA Natalya M. , ATAULLAKHANOV Fazoil I.
Published: July 2004

Intrinsic tenase consists of activated Factors IX (IXa) and VIII (VIIIa) assembled on a negatively charged phospholipid surface. In vivo, this surface is mainly provided by activated platelets. In vitro, phosphatidylcholine/phosphatidylserine vesicles are often used to mimic natural pro-coagulant membranes. In the present study, we developed a quantitative mathematical model of Factor X activation by intrinsic tenase. We considered two situations, when complex assembly occurs on either the membrane of phospholipid vesicles or the surface of activated platelets. On the basis of existing experimental evidence, the following mechanism for the complex assembly on activated platelets was suggested: (i) Factors IXa, VIIIa and X bind to their specific platelet receptors; (ii) bound factors form complexes on the membrane: platelet-bound Factor VIIIa provides a high-affinity site for Factor X and platelet-bound Factor IXa provides a high-affinity site for Factor VIIIa; (iii) the enzyme–cofactor–substrate complex is assembled. This mechanism allowed the explanation of co-operative effects in the binding of Factors IXa, VIIIa and X to platelets. The model was reduced to obtain a single equation for the Factor X activation rate as a function of concentrations of Factors IXa, VIIIa, X and phospholipids (or platelets). The equation had a Michaelis–Menten form, where apparent Vmax and Km were functions of the factors’ concentrations and the internal kinetic constants of the system. The equation obtained can be used in both experimental studies of intrinsic tenase and mathematical modelling of the coagulation cascade. The approach of the present study can be applied to research of other membrane-dependent enzymic reactions.
Proteomic response analysis of a threonine-overproducing mutant of Escherichia coli
Authors: KIM Yang-Hoon , PARK Jin-Seung , CHO Jae-Yong , CHO Kwang Myung , PARK Young-Hoon , LEE Jeewon
Published: July 2004

The proteomic response of a threonine-overproducing mutant of Escherichia coli was quantitatively analysed by two-dimensional electrophoresis. Evidently, 12 metabolic enzymes that are involved in threonine biosynthesis showed a significant difference in intracellular protein level between the mutant and native strain. The level of malate dehydrogenase was more than 30-fold higher in the mutant strain, whereas the synthesis of citrate synthase seemed to be severely inhibited in the mutant. Therefore, in the mutant, it is probable that the conversion of oxaloacetate into citrate was severely inhibited, but the oxidation of malate to oxaloacetate was significantly up-regulated. Accumulation of oxaloacetate may direct the metabolic flow towards the biosynthetic route of aspartate, a key metabolic precursor of threonine. Synthesis of aspartase (aspartate ammonia-lyase) was significantly inhibited in the mutant strain, which might lead to the enhanced synthesis of threonine by avoiding unfavourable degradation of aspartate to fumarate and ammonia. Synthesis of threonine dehydrogenase (catalysing the degradation of threonine finally back to pyruvate) was also significantly down-regulated in the mutant. The far lower level of cystathionine β-lyase synthesis in the mutant seems to result in the accumulation of homoserine, another key precursor of threonine. In the present study, we report that the accumulation of important threonine precursors, such as oxaloacetate, aspartate and homoserine, and the inhibition of the threonine degradation pathway played a critical role in increasing the threonine biosynthesis in the E. coli mutant.
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, <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.
Recruitment of the cross-linked opsonic receptor CD32A (FcγRIIA) to high-density detergent-resistant membrane domains in human neutrophils
Authors: ROLLET-LABELLE Emmanuelle , MAROIS Sébastien , BARBEAU Kathy , MALAWISTA Stephen E. , NACCACHE Paul H.
Published: July 2004

We have previously shown that CD32A (or FcγRIIA), one of the main opsonin receptors, was rapidly insolubilized and degraded in intact neutrophils after its cross-linking. In view of these experimental difficulties, the early signalling steps in response to CD32A activation were studied in purified plasma membranes of neutrophils. After CD32A cross-linking in these fractions, the tyrosine phosphorylation of two major substrates, the receptor itself and the tyrosine kinase Syk, was observed. Phosphorylation of these two proteins was observed only in the presence of orthovanadate, indicating the presence, in the membranes, of one or more tyrosine phosphatases that maintain CD32A dephosphorylation. The tyrosine phosphorylation of these two proteins was inhibited by the Src kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). The ligation of CD32A led to its recruitment to a previously uncharacterized subset of high-density flotillin-1-positive DRMs (detergent-resistant membranes). The changes in the solubility properties of CD32A were observed in the absence of added ATP; therefore, they were probably not secondary to the tyrosine phosphorylation of the receptor, rather they preceded it. Src kinases as well as Syk were constitutively present in DRMs of high and low density and no evident changes in their distribution were detected after cross-linking of CD32A. Pretreatment of plasma membranes with methyl-β-cyclodextrin did not inhibit the recruitment of CD32A to DRMs, although it led to the loss of the Src kinase Lyn from these fractions. In addition, methyl-β-cyclodextrin inhibited the tyrosine phosphorylation of CD32A and Syk induced by cross-linking of CD32A. This membrane model allowed us to observe a movement of CD32A from detergent-soluble regions of the membranes to DRMs, where it joined Src kinases and Syk and became tyrosine-phosphorylated.
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.
ERK5 is targeted to myocyte enhancer factor 2A (MEF2A) through a MAPK docking motif
Authors: BARSYTE-LOVEJOY Dalia , GALANIS Alex , CLANCY Anne , SHARROCKS Andrew D.
Published: July 2004

One critical component in determining the specificity, and efficiency of MAPK (mitogen-activated protein kinase) substrate phophorylation is the presence of distinct docking domains in the substrate proteins. Docking domains have been shown to be important for the activities of members of the ERK (extracellular-signal-regulated kinase), JNK (c-Jun N-terminal kinase) and p38 subfamilies of MAPKs towards their substrates. Here, we demonstrate that docking domains also play an important role in ERK5-mediated substrate phosphorylation. The presence of a docking domain promotes both phosphorylation of myocyte enhancer factor, MEF2A, in vitro and its activation in vivo by ERK5. Mutational analysis of the MEF2A docking domain demonstrates that the specificity determinants for ERK5 are similar to those observed with members of the p38 subfamily. A docking domain recognized by ERK5 can direct ERK5 to activate heterologous substrates. Deletion analysis demonstrates that as with other MAPKs, it is the catalytic domain of ERK5 that recognizes the docking domain. Our data therefore extend previous observations on other MAPKs and demonstrate that the requirement for specific docking domains in promoting MAPK action towards substrates is a general property of MAPKs.
Calmidazolium and arachidonate activate a calcium entry pathway that is distinct from store-operated calcium influx in HeLa cells
Authors: PEPPIATT Claire M. , HOLMES Anthony M. , SEO Jeong T. , BOOTMAN Martin D. , COLLINS Tony J. , McMDONALD Fraser , RODERICK H. Llewelyn
Published: July 2004

Agonists that deplete intracellular Ca2+ stores also activate Ca2+ entry, although the mechanism by which store release and Ca2+ influx are linked is unclear. A potential mechanism involves ‘store-operated channels’ that respond to depletion of the intracellular Ca2+ pool. Although SOCE (store-operated Ca2+ entry) has been considered to be the principal route for Ca2+ entry during hormonal stimulation of non-electrically excitable cells, recent evidence has suggested that alternative pathways activated by metabolites such as arachidonic acid are responsible for physiological Ca2+ influx. It is not clear whether such messenger-activated pathways exist in all cells, whether they are truly distinct from SOCE and which metabolites are involved. In the present study, we demonstrate that HeLa cells express two pharmacologically and mechanistically distinct Ca2+ entry pathways. One is the ubiquitous SOCE route and the other is an arachidonate-sensitive non-SOCE. We show that both these Ca2+ entry pathways can provide long-lasting Ca2+ elevations, but that the channels are not the same, based on their differential sensitivity to 2-aminoethoxydiphenyl borate, LOE-908 {(R,S)-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamid mesylate} and gadolinium. In addition, non-SOCE and not SOCE was permeable to strontium. Furthermore, unlike SOCE, the non-SOCE pathway did not require store depletion and was not sensitive to displacement of the endoplasmic reticulum from the plasma membrane using jasplakinolide or ionomycin pretreatment. These pathways did not conduct Ca2+ simultaneously due to the dominant effect of arachidonate, which rapidly curtails SOCE and promotes Ca2+ influx via non-SOCE. Although non-SOCE could be activated by exogenous application of arachidonate, the most robust method for stimulation of this pathway was application of the widely used calmodulin antagonist calmidazolium, due to its ability to activate phospholipase A2.
Identification of the first archaeal Type 1 RNase H gene from Halobacterium sp. NRC-1: archaeal RNase HI can cleave an RNA–DNA junction
Authors: OHTANI Naoto , YANAGAWA Hiroshi , TOMITA Masaru , ITAYA Mitsuhiro
Published: July 2004

All the archaeal genomes sequenced to date contain a single Type 2 RNase H gene. We found that the genome of a halophilic archaeon, Halobacterium sp. NRC-1, contains an open reading frame with similarity to Type 1 RNase H. The protein encoded by the Vng0255c gene, possessed amino acid sequence identities of 33% with Escherichia coli RNase HI and 34% with a Bacillus subtilis RNase HI homologue. The B. subtilis RNase HI homologue, however, lacks amino acid sequences corresponding to a basic protrusion region of the E. coli RNase HI, and the Vng0255c has the similar deletion. As this deletion apparently conferred a complete loss of RNase H activity on the B. subtilis RNase HI homologue protein, the Vng0255c product was expected to exhibit no RNase H activity. However, the purified recombinant Vng0255c protein specifically cleaved an RNA strand of the RNA/DNA hybrid in vitro, and when the Vng0255c gene was expressed in an E. coli strain MIC2067 it could suppress the temperature-sensitive growth defect associated with the loss of RNase H enzymes of this strain. These results in vitro and in vivo strongly indicate that the Halobacterium Vng0255c is the first archaeal Type 1 RNase H. This enzyme, unlike other Type 1 RNases H, was able to cleave an Okazaki fragment-like substrate at the junction between the 3′-side of ribonucleotide and 5′-side of deoxyribonucleotide. It is likely that the archaeal Type 1 RNase H plays a role in the removal of the last ribonucleotide of the RNA primer from the Okazaki fragment during DNA replication.
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.
Domain structure of bi-functional selenoprotein P
Authors: SAITO Yoshiro , SATO Noriko , HIRASHIMA Masaki , TAKEBE Gen , NAGASAWA Shigeharu , TAKAHASHI Kazuhiko
Published: July 2004

Human selenoprotein P (SeP), a selenium-rich plasma glycoprotein, is presumed to contain ten selenocysteine residues; one of which is located at the 40th residue in the N-terminal region and the remaining nine localized in the C-terminal third part. We have shown that SeP not only catalyses the reduction of phosphatidylcholine hydroperoxide by glutathione [Saito, Hayashi, Tanaka, Watanabe, Suzuki, Saito and Takahashi (1999) J. Biol. Chem. 274, 2866–2871], but also supplies its selenium to proliferating cells [Saito and Takahashi (2002) Eur. J. Biochem. 269, 5746–5751]. Treatment of SeP with plasma kallikrein resulted in a sequential limited proteolysis (Arg-235–Gln-236 and Arg-242–Asp-243). The N-terminal (residues 1–235) and C-terminal (residues 243–361) fragments exhibited enzyme activity and selenium-supply activity respectively. These results confirm that SeP is a bi-functional protein and suggest that the first selenocysteine residue is the active site of the enzyme and the remaining nine residues function as a selenium supplier.
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.
Gene expression analysis in mitochondria from chagasic mice: alterations in specific metabolic pathways
Authors: GARG Nisha , GERSTNER Arpad , BHATIA Vandanajay , DeFORD James , PAPACONSTANTINOU John
Published: July 2004

Cardiac hypertrophy and remodelling in chagasic disease might be associated with mitochondrial dysfunction. In the present study, we characterized the cardiac metabolic responses to Trypanosoma cruzi infection and progressive disease severity using a custom-designed mitoarray (mitochondrial function-related gene array). Mitoarrays consisting of known, well-characterized mitochondrial function-related cDNAs were hybridized with 32P-labelled cDNA probes generated from the myocardium of mice during immediate early, acute and chronic phases of infection and disease development. The mitoarray successfully identified novel aspects of the T. cruzi-induced alterations in the expression of the genes related to mitochondrial function and biogenesis that were further confirmed by real-time reverse transcriptase–PCRs. Of note is the up-regulation of transcripts essential for fatty acid metabolism associated with repression of the mRNAs for pyruvate dehydrogenase complex in infected hearts. We observed no statistically significant changes in mRNAs for the enzymes of tricarboxylic acid cycle. These results suggest that fatty acid metabolism compensates the pyruvate dehydrogenase complex deficiencies for the supply of acetyl-CoA for a tricarboxylic acid cycle, and chagasic hearts may not be limited in reduced energy (NADH and FADH2). The observation of a decrease in mRNA level for several subunits of the respiratory chain complexes by mitoarray as well as global genome analysis suggests a limitation in mitochondrial oxidative phosphorylation-mediated ATP-generation capacity as the probable basis for cardiac homoeostasis in chagasic disease.
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.
Functional analysis of Chinese hamster phosphatidylserine synthase 1 through systematic alanine mutagenesis
Authors: OHSAWA Tomoko , NISHIJIMA Masahiro , KUGE Osamu
Published: July 2004

PtdSer (phosphatidylserine) synthesis in mammalian cells occurs through the exchange of L-serine with the base moieties of phosphatidylcholine and phosphatidylethanolamine, which is catalysed by PSS (PtdSer synthase) 1 and 2 respectively. PtdSer synthesis in intact cells and an isolated membrane fraction was inhibited by exogenous PtdSer, indicating that feedback control is involved in the regulation of PtdSer biosynthesis. PSS 1 and 2 are similar in amino acid sequence, with an identity of 32%; however, due to a lack of homology with other known enzymes, their amino acid sequences do not provide information on their catalytic and regulatory mechanisms. In the present study, to identify amino acid residues crucial for the activity and/or regulation of PSS 1, we systematically introduced mutations into a Chinese hamster PSS 1 cDNA clone; namely, each of the 66 polar amino acid residues common to PSS 2 was replaced with an alanine residue. On analysis of Chinese hamster ovary cells transfected with each of the alanine mutant clones, we identified eight amino acid residues (His-172, Glu-197, Glu-200, Asn-209, Glu-212, Asp-216, Asp-221 and Asn-226) as those crucial for the enzyme reaction or the maintenance of the correct structure required for serine base-exchange activity. Among these residues, Asn-209 was suggested to be involved in the recognition and/or binding of free L-serine. We also identified six amino acid residues (Arg-95, His-97, Cys-189, Arg-262, Gln-266 and Arg-336) as those important for regulation of PSS 1. In addition, we found that the alanine mutations at Tyr-111, Asp-166, Arg-184, Arg-323, and Glu-364 affected the production and/or stability of PSS 1 in Chinese hamster ovary cells.
The CBP/p300 TAZ1 domain in its native state is not a binding partner of MDM2
Authors: MATT Theresia , MARTINEZ-YAMOUT Maria A. , DYSON H. Jane , WRIGHT Peter E.
Published: July 2004

The transcriptional co-activator CBP [CREB (cAMP-response-element-binding protein)-binding protein] and its paralogue p300 play a key role in the regulation of both activity and stability of the tumour suppressor p53. Degradation of p53 is mediated by the ubiquitin ligase MDM2 (mouse double minute protein) and is also reported to be regulated by CBP/p300. Direct protein–protein interaction between a central domain of MDM2 and the TAZ1 (transcriptional adaptor zinc-binding domain) [C/H1 (cysteine/histidine-rich region 1)] domain of p300 and subsequent formation of a ternary complex including p53 have been reported previously. We expressed and purified the proposed binding domains of HDM2 (human homologue of MDM2) and CBP, and examined their interactions using CD spectroscopy. The binding studies were extended by using natively purified GST (glutathione S-transferase)–p300 TAZ1 and GST–p53 fusion proteins, together with in vitro translated HDM2 fragments, under similar solution conditions to those in previous studies, but omitting added EDTA, which causes unfolding and aggregation of the zinc-binding TAZ1 domain. Comparing the binding properties of the known TAZ1 interaction partners HIF-1α (hypoxia-inducible factor 1), CITED2 (CBP/p300-interacting transactivator with glutamic- and aspartic-rich tail) and STAT2 (signal transducer and activator of transcription 2) with HDM2, our data suggest that TAZ1 in its native state does not serve as a specific recognition domain of HDM2. Rather, unfolded TAZ1 and HDM2 proteins have a high tendency to aggregate, and non-specific protein complexes are formed under certain conditions.