bluuebunny/crossref_metadata_2025 huggingface dataset:
Risk Stratification of Individuals with the Brugada Electrocardiogram: A Meta‐Analysis
Authors: GEHI ANIL K. , DUONG TRUONG D. , METZ LOUISE D. , GOMES J. ANTHONY , MEHTA DAVENDRA
Published: April 2006

Objectives: We performed a meta‐analysis of prognostic studies of patients with a Brugada ECG to assess predictors of events.

Background: The Brugada syndrome is an increasingly recognized cause of idiopathic ventricular fibrillation; however, there is wide variation in the prognosis of patients with the Brugada ECG.

Methods and Results: We retrieved 30 prospective studies of patients with the Brugada ECG, accumulating data on 1,545 patients. Summary estimates of the relative risk (RR) of events (sudden cardiac death [SCD], syncope, or internal defibrillator shock) for a variety of potential predictors were made using a random‐effects model. The overall event rate at an average of 32 months follow‐up was 10.0% (95% CI 8.5%, 11.5%). The RR of an event was increased (P < 0.001) among patients with a history of syncope or SCD (RR 3.24 [95% CI 2.13, 4.93]), men compared with women (RR 3.47 [95% CI 1.58, 7.63]), and patients with a spontaneous compared with sodium‐channel blocker induced Type I Brugada ECG (RR 4.65 [95% CI 2.25, 9.58]). The RR of events was not significantly increased in patients with a family history of SCD (P = 0.97) or a mutation of the SCN5A gene (P = 0.18). The RR of events was also not significantly increased in patients inducible compared with noninducible by electrophysiologic study (EPS) (RR 1.88 [95% CI 0.62, 5.73], P = 0.27); however, there was significant heterogeneity of the studies included.

Conclusions: Our findings suggest that a history of syncope or SCD, the presence of a spontaneous Type I Brugada ECG, and male gender predict a more malignant natural history. Our findings do not support the use of a family history of SCD, the presence of an SCN5A gene mutation, or EPS to guide the management of patients with a Brugada ECG.
Characteristics of Slow Pathway Conduction After Successful AVNRT Ablation
Authors: POSAN EMOKE , GULA LORNE J. , SKANES ALLAN C. , KRAHN ANDREW D. , YEE RAYMOND , PETRELLIS BASILIOS , REDFEARN DAMIAN P. , MOHAMED UWAIS , GOULD PAUL A. , KLEIN GEORGE J.
Published: April 2006

Background: AV node slow pathway conduction can persist following successful ablation for AV node reentrant tachycardia (AVNRT). We hypothesized that careful examination of AV nodal conduction curves before and after effective AVNRT ablation in patients with persistent slow pathway conduction could shed light on this apparent paradox.

Methods and Results: Thirty patients (age 40.9 ± 14.3; 8 male) were included. AV node function curves were created based on pre‐ and postablation atrial extrastimulus testing. Analysis of slow pathway function curves demonstrated significant decrease in AH for any given coupling interval after ablation (mean difference –68.1 [–94.5, –41.7] P < 0.001), graphically indicated by downward displacement of the curve. In addition, mean slow pathway effective refractory period (ERP) increased from 247.9 ± 36.1 msec to 288.6 ± 56.0 msec (P < 0.001); mean maximum AH interval decreased from 361.3 ± 114.2 msec to 306.9 ± 65.2 msec (P = 0.013); mean difference in minimum and maximum AH interval during slow pathway conduction decreased (from 94.5 ± 75.8 msec to 59.6 ± 46.2 msec (P = 0.016). Finally, mean difference between the fast and slow pathway effective refractory periods, the span of coupling intervals over which slow pathway conduction occurred, decreased (from 113.9 ± 61.4 msec to 63.2 ± 41.5 msec, P = 0.001).

Conclusions: Ablation, which successfully eliminates inducible and spontaneous AVNRT in the presence of persistent slow pathway conduction, is associated with significantly altered slow pathway conduction characteristics, indicating the presence of a damaged or different slow pathway after ablation, incapable of sustaining tachycardia.
Two‐Year Follow‐up of Sirolimus‐Eluting Stents for the Treatment of Proximal Left Anterior Descending Coronary Artery Stenosis
Authors: VALENCIA JOSÉ , BERENGUER ALBERTO , MAINAR VICENTE , BORDES PASCUAL , GÓMEZ SILVIA , TELLO ANTONIO , LÓPEZ‐ARANDA MIGUEL ÁNGEL , CATURLA JUAN
Published: April 2006

Introduction: Sirolimus‐eluting stents (SES) have demonstrated low target vessel revascularizations and low incidence of angiographic restenosis in several clinical scenarios. The aim of the present study was to assess the efficacy and safety of SES for the treatment of proximal left anterior descending coronary artery (pLAD) lesions.

Methods: Ninety‐six patients with severe pLAD stenosis were enrolled. Angiographic and clinical follow‐up were performed at 6 and 24 months, respectively. Death, myocardial infarction (MI), new target lesion revascularization (TLR), and target vessel failure (TVF) were registered. Clinical, angiographic, and procedural variables were analyzed to identify predictors of restenosis.

Results: Mean clinical follow‐up was 858 ± 158 days (26.5 ± 8.3 months). Angiographic procedural success was 100%. Angiographic follow‐up showed 8.4% of binary restenosis without edge‐restenosis phenomenon. Late loss was 0.15 ± 0.65 mm; 15.6% of patients had an adverse cardiac event, with 1% of death, 5.2% of MI, 6.3% of TLR, and 9.4% of TVF. At 2 years, the probabilities of cumulative TVF‐ and TLR‐free survival were 90.6% and 93.7%, respectively. Interestingly, no adverse cardiac events were registered between the first and second years. Female gender (OR 10.7 CI 95%[1.7–66.7]) and in‐stent restenosis (OR 8.2, CI 95%[1.2–56.4]) were found as independent predictors of binary restenosis.
Advanced chronic renal failure showed a strong trend toward worse outcome in terms of binary restenosis (P = 0.063).

Conclusions: SES for the treatment of pLAD stenosis proved safe and effective in a long‐term follow‐up with low incidence of adverse cardiac events and restenosis. Female gender and in‐stent restenosis were predictors of binary restenosis.
Usefulness of Fractional Flow Reserve in Multivessel Coronary Artery Disease with Intermediate Lesions
Authors: JIMÉNEZ‐NAVARRO MANUEL F. , ALONSO‐BRIALES JUAN , HERNÁNDEZ‐GARCÍA JOSÉ MARÍA , CURIEL EMILIO , KÜHLMORGEN BIRGIT , GÓMEZ‐DOBLAS JUAN JOSÉ , GARCÍA‐PINILLA JOSÉ MANUEL , ROBLEDO JUAN , DE TERESA EDUARDO
Published: April 2006

Background: Calculation of myocardial fractional flow reserve (FFR) enables coronary stenoses to be evaluated.

Objectives: We determined the usefulness of measuring the FFR in multivessel coronary artery disease, reflected in changes in the therapeutic options for patients with moderate coronary stenosis.

Methods: We studied 38 patients (30 men, 8 women; mean age: 59.8 ± 10 years) with multivessel coronary artery disease with 41 moderate lesions. Indications for coronary angiography were unstable angina in 24 patients (60%), acute myocardial infarction in 10 (27%), and stable angina in 4 (13%). We studied the FFR (in nonactive lesions) in the left anterior descending artery in 23 patients (56%), the left coronary trunk in 8 (19.5%), the circumflex artery in 5 (12.2%), the right coronary artery in 3 (7.3%), and the left internal mammary artery and diagonal branch in 1 patient each.

Results: Twelve patients had a positive FFR, which resulted in no change in the mode of revascularization; 26 patients had a negative FFR, in 20 (77%) of whom the revascularization approach was changed, especially those with moderate lesions of the left coronary trunk or anterior descending artery. No differences were detected in the angiographic characteristics of the lesions examined. Cardiac events during follow‐up were few.

Conclusions: The results of FFR may influence the decision‐making process after diagnostic coronary angiography in multivessel coronary artery disease with moderate lesions, especially in patients with a negative FFR in nonculprit lesions of the left trunk or left anterior descending artery.
Intravascular Misplacement of an Extravascular Closure System: StarClose™
Authors: STOCK ULRICH , FLACH PETER , GROSS MICHAEL , MEYHÖFER JOERG , ALBES JOHANNES , BUTTER CHRISTIAN
Published: April 2006

Closure devices for closing femoral arteries following catheterization procedures are routinely used. They reduce time of immobilization and decrease complications such as bleeding and aneurysm formation. We report of a new vascular closure device (StarClose™, marketed by Abbott) that was misplaced in the femoral artery with subsequent vascular stenosis and need for surgical removal and plastic reconstruction.
Development of the MAGIC Congenital Heart Disease Catheterization Database for Interventional Outcome Studies
Authors: EVERETT ALLEN D. , RINGEL RICHARD , RHODES JOHN F. , DOYLE THOMAS P. , OWADA CARL Y. , HOLZER RALF J. , CHEATHAM JOHN P. , RINGEWALD JEREMY , BANDISODE VARSHA , CHEN YU‐LI , LIM D. SCOTT
Published: April 2006

As the field of catheter‐based therapies for congenital heart disease continues to expand, we lack the evidence‐based data to make appropriate therapeutic decisions in the catheterization laboratory. A stumbling block to the determination of evidence‐based therapies is our inability to simply and reliably share outcome data across multiple centers. We investigated whether a commonly used congenital heart disease catheterization database program (PedCath™) could be used as an automatic catheterization data submission tool to a central database for outcome analysis. To test the feasibility of such a tool for collaborative outcomes research we formed a national group of seven congenital heart disease centers, the Mid‐Atlantic Group of Interventional Cardiology, to warehouse and analyze catheterization data. We successfully modified PedCath™ to transfer the results of catheter‐based therapies on 256 therapeutic procedures for atrial septal defect (ASD) closure, coarctation of the aorta angioplasty and stenting, and pulmonary and aortic balloon valvuloplasties over a 13‐month pilot period. Short‐term follow‐up within the 13‐month period was received on 31 patients. This study demonstrated the successful development of a simple process, requiring minimal data entry for investigators from around the world to share cardiac catheterization data for long‐term outcome determination of catheter‐based therapies for congenital heart disease.
Spontaneous Late Thrombolysis of an Occluded Saphenous Vein Graft Subsequent to Acute Myocardial Infarction Treated with Percutaneous Coronary Intervention to the Native Culprit Vessel
Authors: SHAH RAJESH A. , KHANAL SANJAYA , KUGELMASS AARON
Published: April 2006

A 67‐year‐old male with prior history of myocardial infarction and coronary artery bypass grafting (individual vein grafts to the left anterior descending artery [LAD] and right coronary artery) presented with an acute anterior ST elevation myocardial infarction and cardiogenic shock. The vein graft to the LAD was occluded with heavy thrombus burden and there was severe native CAD. Given the degree of thrombus burden and other anatomic considerations, percutaneous intervention with stenting was performed to the native proximal LAD. Three months later, after complaining of atypical chest pain, repeat angiogram revealed a spontaneous widely patent vein graft to the LAD and occluded proximal LAD.
On the mechanisms of glycolytic oscillations in yeast
Authors: Madsen Mads F. , Danø Sune , Sørensen Preben G
Published: April 2006

This work concerns the cause of glycolytic oscillations in yeast. We analyse experimental data as well as models in two distinct cases: the relaxation‐like oscillations seen in yeast extracts, and the sinusoidal Hopf oscillations seen in intact yeast cells. In the case of yeast extracts, we use flux‐change plots and model analyses to establish that the oscillations are driven by on/off switching of phosphofructokinase. In the case of intact yeast cells, we find that the instability leading to the appearance of oscillations is caused by the stoichiometry of the ATP‐ADP‐AMP system and the allosteric regulation of phosphofructokinase, whereas frequency control is distributed over the reaction network. Notably, the NAD+/NADH ratio modulates the frequency of the oscillations without affecting the instability. This is important for understanding the mutual synchronization of oscillations in the individual yeast cells, as synchronization is believed to occur via acetaldehyde, which in turn affects the frequency of oscillations by changing this ratio.
Adventitious reactions of alkene monooxygenase reveal common reaction pathways and component interactions among bacterial hydrocarbon oxygenases
Authors: Fosdike William L. J. , Smith Thomas J. , Dalton Howard
Published: April 2006

Alkene monooxygenase (AMO) from Rhodococcus rhodochrous (formerly Nocardia corallina) B‐276 belongs to a family of multicomponent nonheme binuclear iron‐centre oxygenases that includes the soluble methane monooxygenases (sMMOs) found in some methane‐oxidizing bacteria. The enzymes catalyse the insertion of oxygen into organic substrates (mostly hydrocarbons) at the expense of O2 and NAD(P)H. AMO is remarkable in its ability to oxidize low molecular‐mass alkenes to their corresponding epoxides with high enantiomeric excess. sMMO and other well‐characterized homologues of AMO exhibit two adventitious activities: (1) turnover‐dependent inhibition by alkynes and (2) activation by hydrogen peroxide in lieu of oxygen and NAD(P)H (the peroxide shunt reaction). Previous studies of the AMO had failed to detect these activities and opened the possibility that the mechanism of AMO might be fundamentally different from that of its homologues. Thanks to improvements in the protocols for cultivation of R. rhodochrous B‐276 and purification and assay of AMO, it has been possible to detect and characterize turnover‐dependent inhibition of AMO by propyne and ethyne and activation of the enzyme by hydrogen peroxide. These results indicate a similar mechanism to that found in sMMO and also, unexpectedly, that the enantiomeric excess of the chiral epoxypropane product is significantly reduced during the peroxide shunt reaction. Inhibition of the oxygen/NADH‐activated reaction, but not the peroxide shunt, by covalent modification of positively charged groups revealed an additional similarity to sMMO and may indicate very similar patterns of intersubunit interactions and/or electron transfer in both enzyme complexes.
Aspartate transcarbamylase from the hyperthermophilic archaeon Pyrococcus abyssi
Authors: Van Boxstael Sigrid , Maes Dominique , Cunin Raymond
Published: April 2006

Aspartate transcarbamylase (ATCase) (EC 2.1.3.2) from the hyperthermophilic archaeon Pyrococcus abyssi was purified from recombinant Escherichia coli cells. The enzyme has the molecular organization of class B microbial aspartate transcarbamylases whose prototype is the E. coli enzyme. P. abyssi ATCase is cooperative towards aspartate. Despite constraints imposed by adaptation to high temperature, the transition between T‐ and R‐states involves significant changes in the quaternary structure, which were detected by analytical ultracentrifugation. The enzyme is allosterically regulated by ATP (activator) and by CTP and UTP (inhibitors). Nucleotide competition experiments showed that these effectors compete for the same sites. At least two regulatory properties distinguish P. abyssi ATCase from E. coli ATCase: (a) UTP by itself is an inhibitor; (b) whereas ATP and UTP act at millimolar concentrations, CTP inhibits at micromolar concentrations, suggesting that in P. abyssi, inhibition by CTP is the major control of enzyme activity. While Vmax increased with temperature, cooperative and allosteric effects were little or not affected, showing that molecular adaptation to high temperature allows the flexibility required to form the appropriate networks of interactions. In contrast to the same enzyme in P. abyssi cellular extracts, the pure enzyme is inhibited by the carbamyl phosphate analogue phosphonacetate; this difference supports the idea that in native cells ATCase interacts with carbamyl phosphate synthetase to channel the highly thermolabile carbamyl phosphate.
Aly/ REF, a factor for mRNA transport, activates RH gene promoter function
Authors: Suganuma Hiroshi , Kumada Maki , Omi Toshinori , Gotoh Takaya , Lkhagvasuren Munkhtulga , Okuda Hiroshi , Kamesaki Toyomi , Kajii Eiji , Iwamoto Sadahiko
Published: April 2006

The rhesus (Rh) blood group antigens are of considerable importance in transfusion medicine as well as in newborn or autoimmune hemolytic diseases due to their high antigenicity. We identified a major DNaseI hypersensitive site at the 5′ flanking regions of both RHD and RHCE exon 1. A 34 bp fragment located at −191 to −158 from a translation start position, and containing the TCCCCTCCC sequence, was involved in enhancing promoter activity, which was assessed by luciferase reporter gene assay. A biotin‐labelled 34 bp probe isolated an mRNA transporter protein, Aly/REF. The specific binding of Aly/REF to RH promoter in erythroid was confirmed by chromatin immunoprecipitation assay. The silencing of Aly/REF by siRNA reduced not only the RH promoter activity of the reporter gene but also transcription from the native genome. These facts provide second proof of Aly/REF as a transcription coactivator, initially identified as a coactivator for the TCRα enhancer function. Aly/REF might be a novel transcription cofactor for erythroid‐specific genes.
Expression profiling reveals differences in metabolic gene expression between exercise‐induced cardiac effects and maladaptive cardiac hypertrophy
Authors: Strøm Claes C. , Aplin Mark , Ploug Thorkil , Christoffersen Tue E. H. , Langfort Jozef , Viese Michael , Galbo Henrik , Haunsø Stig , Sheikh Søren P.
Published: April 2006

While cardiac hypertrophy elicited by pathological stimuli eventually leads to cardiac dysfunction, exercise‐induced hypertrophy does not. This suggests that a beneficial hypertrophic phenotype exists. In search of an underlying molecular substrate we used microarray technology to identify cardiac gene expression in response to exercise. Rats exercised for seven weeks on a treadmill were characterized by invasive blood pressure measurements and echocardiography. RNA was isolated from the left ventricle and analysed on DNA microarrays containing 8740 genes. Selected genes were analysed by quantitative PCR. The exercise program resulted in cardiac hypertrophy without impaired cardiac function. Principal component analysis identified an exercise‐induced change in gene expression that was distinct from the program observed in maladaptive hypertrophy. Statistical analysis identified 267 upregulated genes and 62 downregulated genes in response to exercise. Expression changes in genes encoding extracellular matrix proteins, cytoskeletal elements, signalling factors and ribosomal proteins mimicked changes previously described in maladaptive hypertrophy. Our most striking observation was that expression changes of genes involved in β‐oxidation of fatty acids and glucose metabolism differentiate adaptive from maladaptive hypertrophy. Direct comparison to maladaptive hypertrophy was enabled by quantitative PCR of key metabolic enzymes including uncoupling protein 2 (UCP2) and fatty acid translocase (CD36). DNA microarray analysis of gene expression changes in exercise‐induced cardiac hypertrophy suggests that a set of genes involved in fatty acid and glucose metabolism could be fundamental to the beneficial phenotype of exercise‐induced hypertrophy, as these changes are absent or reversed in maladaptive hypertrophy.
A novel transmembrane topology of presenilin based on reconciling experimental and computational evidence
Authors: Henricson Anna , Käll Lukas , Sonnhammer Erik L. L.
Published: April 2006

The transmembrane topology of presenilins is still the subject of debate despite many experimental topology studies using antibodies or gene fusions. The results from these studies are partly contradictory and consequently several topology models have been proposed. Studies of presenilin‐interacting proteins have produced further contradiction, primarily regarding the location of the C‐terminus. It is thus impossible to produce a topology model that agrees with all published data on presenilin. We have analyzed the presenilin topology through computational sequence analysis of the presenilin family and the homologous presenilin‐like protein family. Members of these families are intramembrane‐cleaving aspartyl proteases. Although the overall sequence homology between the two families is low, they share the conserved putative active site residues and the conserved ‘PAL’ motif. Therefore, the topology model for the presenilin‐like proteins can give some clues about the presenilin topology. Here we propose a novel nine‐transmembrane topology with the C‐terminus in the extracytosolic space. This model has strong support from published data on γ‐secretase function and presenilin topology. Contrary to most presenilin topology models, we show that hydrophobic region X is probably a transmembrane segment. Consequently, the C‐terminus would be located in the extracytosolic space. However, the last C‐terminal amino acids are relatively hydrophobic and in conjunction with existing experimental data we cannot exclude the possibility that the extreme C‐terminus could be buried within the γ‐secretase complex. This might explain the difficulties in obtaining consistent experimental evidence regarding the location of the C‐terminal region of presenilin.
Formation of nucleoprotein RecA filament on single‐stranded DNA
Authors: Bugreeva Irina P. , Bugreev Dmitry V. , Nevinsky Georgy A.
Published: April 2006

RecA protein plays a pivotal role in homologous recombination in Escherichia coli. RecA polymerizes on single‐stranded (ss) DNA forming a nucleoprotein filament. Then double‐stranded (ds) DNA is bound and searched for segments homologous to the ssDNA. Finally, homologous strands are exchanged, a new DNA duplex is formed, and ssDNA is displaced. We report a quantitative analysis of RecA interactions with ss d(pN)n of various structures and lengths using these oligonucleotides as inhibitors of RecA filamentation on d(pT)20. DNA recognition appears to be mediated by weak interactions between its structural elements and RecA monomers within a filament. Orthophosphate and dNMP are minimal inhibitors of RecA filamentation (I50 = 12–20 mm). An increase in homo‐d(pN)2−40 length by one unit improves their affinity for RecA (f factor) approximately twofold through electrostatic contacts of RecA with internucleoside phosphate DNA moieties (f≈ 1.56) and specific interactions with T or C bases (f≈ 1.32); interactions with adenine bases are negligible. RecA affinity for d(pN)n containing normal or modified nucleobases depends on the nature of the base, features of the DNA structure. The affinity considerably increases if exocyclic hydrogen bond acceptor moieties are present in the bases. We analyze possible reasons underlying RecA preferences for DNA sequence and length and propose a model for recognition of ssDNA by RecA.
PKA independent and cell type specific activation of the expression of caudal homeobox gene Cdx‐2 by cyclic AMP
Authors: Chen Liang , Wang Peixiang , Andrade Cristiano F. , Zhao Ian Y. , Dubé Philip E. , Brubaker Patricia L. , Liu Mingyao , Jin Tianru
Published: April 2006

Cdx‐2 is a transactivator for the proglucagon gene in pancreatic and intestinal endocrine cells. Cdx‐2 is also expressed in differentiated intestinal epithelia of nonendocrine origin. Cdx‐2–/– mice are embryonic lethal, while Cdx‐2+/– mutants show multiple malfunctions including the formation of intestinal polyps. Within the polyps, the remaining wild type Cdx‐2 allele ceases its expression, while the expression of both Cdx‐2 and proglucagon in the endocrine cells remains unaltered, indicating that Cdx‐2 could be haplo‐insufficient for nonendocrine cells, but not for proglucagon producing endocrine cells. We propose that mechanisms underlying Cdx‐2 expression and auto‐regulation [Xu F, Li H & Jin T (1999), J Biol Chem274, 34310–34316] differ in these two types of cells. We show here that forskolin and cAMP upregulate Cdx‐2 expression in proglucagon producing cells, but not in colon cancer cells and primary intestinal cell cultures. It is unlikely that the activation is mainly mediated by PKA, because the activation was observed in a PKA deficient cell line. Cotransfecting a dominant negative Ras expression plasmid substantially repressed the Cdx‐2 promoter, in contrast to a previous finding that Ras is a negative factor for Cdx‐2 expression in colon cancer cells. Furthermore, forskolin activated ERK1/2 phosphorylation in the endocrine cells, and attenuation of ERK1/2 phosphorylation by its inhibitor is associated with attenuated Cdx‐2 expression. Finally, an Epac pathway specific cAMP analogue stimulated both ERK1/2 phosphorylation and Cdx‐2 expression. Taken together, our observations suggest that Cdx‐2 expression is regulated by the second messenger cAMP, cell‐type specifically, via the Epac pathway.
The yeast stress response
Authors: Rodrigues‐Pousada Claudina , Nevitt Tracy , Menezes Regina
Published: April 2006

The budding yeast Saccharomyces cerevisiae possesses a very flexible and complex programme of gene expression when exposed to a plethora of environmental insults. Therefore, yeast cell homeostasis control is achieved through a highly coordinated mechanism of transcription regulation involving several factors, each performing specific functions. Here, we present our current knowledge of the function of the yeast activator protein family, formed by eight basic‐leucine zipper trans‐activators, which have been shown to play an important role in stress response.
A natural osmolyte trimethylamine N‐oxide promotes assembly and bundling of the bacterial cell division protein, FtsZ and counteracts the denaturing effects of urea
Authors: Mukherjee Arnab , Santra Manas K. , Beuria Tushar K. , Panda Dulal
Published: April 2006

Assembly of FtsZ was completely inhibited by low concentrations of urea and its unfolding occurred in two steps in the presence of urea, with the formation of an intermediate [Santra MK & Panda D (2003) J Biol Chem278, 21336–21343]. In this study, using the fluorescence of 1‐anilininonaphthalene‐8‐sulfonic acid and far‐UV circular dichroism spectroscopy, we found that a natural osmolyte, trimethylamine N‐oxide (TMAO), counteracted the denaturing effects of urea and guanidium chloride on FtsZ. TMAO also protected assembly and bundling of FtsZ protofilaments from the denaturing effects of urea and guanidium chloride. Furthermore, the standard free energy changes for unfolding of FtsZ were estimated to be 22.5 and 28.4 kJ·mol−1 in the absence and presence of 0.6 m TMAO, respectively. The data are consistent with the view that osmolytes counteract denaturant‐induced unfolding of proteins by destabilizing the unfolded states. Interestingly, TMAO was also found to affect the assembly properties of native FtsZ. TMAO increased the light‐scattering signal of the FtsZ assembly, increased sedimentable polymer mass, enhanced bundling of FtsZ protofilaments and reduced the GTPase activity of FtsZ. Similar to TMAO, monosodium glutamate, a physiological osmolyte in bacteria, which induces assembly and bundling of FtsZ filaments in vitro[Beuria TK, Krishnakumar SS, Sahar S, Singh N, Gupta K, Meshram M & Panda D (2003) J Biol Chem278, 3735–3741], was also found to counteract the deleterious effects of urea on FtsZ. The results together suggested that physiological osmolytes may regulate assembly and bundling of FtsZ in bacteria and that they may protect the functionality of FtsZ under environmental stress conditions.
A pH‐dependent conformational change in EspA, a component of the Escherichia coli O157:H7 type III secretion system
Authors: Kato Tomoaki , Hamada Daizo , Fukui Takashi , Hayashi Makoto , Honda Takeshi , Murooka Yoshikatsu , Yanagihara Itaru
Published: April 2006

pH‐Dependent structural changes for Escherichia coli O157:H7 EspA were characterized by CD, 8‐anilino‐2‐naphthyl sulfonic acid (ANS) fluorescence, and sedimentation equilibrium ultracentrifugation. Far‐ and near‐UV CD spectra, recorded between pH 2.0 and 7.0, indicate that the protein has significant amounts of secondary and tertiary structures. An increase in ANS fluorescence intensity (in the presence of EspA) was observed at acidic pH; whereas, no increased ANS fluorescence was observed at pH 7.0. These results suggest the presence of a partially unfolded state. Interestingly, urea‐induced unfolding transitions, monitored by far‐UV CD spectroscopy, showed that the protein is destabilized at pH 2.0 as compared with EspA at neutral pH. Although increased ANS fluorescence was observed at pH 3.0, the urea‐induced unfolding curve is similar to that found at pH 7.0. This result suggests the presence, at pH 3.0, of an ordered, but partially unfolded state, which differs from typical molten globule. The results of analytical ultracentrifugation and infrared spectroscopy indicate that EspA molecules associate at pH 7.0, suggesting the formation of short filamentous oligomers containing α‐helical structures, whereas the protein tend to form nonspecific aggregates containing intermolecular β‐sheets at pH 2.0. Our experiments indicate that EspA has the potential to spontaneously form filamentous oligomers at neutral pH; whereas the protein is partially unfolded, assuming different conformations, at acidic pH.
Autophosphorylation of Archaeoglobus fulgidus Rio2 and crystal structures of its nucleotide–metal ion complexes
Authors: LaRonde‐LeBlanc Nicole , Guszczynski Tad , Copeland Terry , Wlodawer Alexander
Published: April 2006

The highly conserved, atypical RIO serine protein kinases are found in all organisms, from archaea to man. In yeast, the kinase activity of Rio2 is necessary for the final processing step of maturing the 18S ribosomal rRNA. We have previously shown that the Rio2 protein from Archaeoglobus fulgidus contains both a small kinase domain and an N‐terminal winged helix domain. Previously solved structures using crystals soaked in nucleotides and Mg2+ or Mn2+ showed bound nucleotide but no ordered metal ions, leading us to the conclusion that they did not represent an active conformation of the enzyme. To determine the functional form of Rio2, we crystallized it after incubation with ATP or ADP and Mn2+. Co‐crystal structures of Rio2–ATP–Mn and Rio2–ADP–Mn were solved at 1.84 and 1.75 Å resolution, respectively. The γ‐phosphate of ATP is firmly positioned in a manner clearly distinct from its location in canonical serine kinases. Comparison of the Rio2–ATP–Mn complex with the Rio2 structure with no added nucleotides and with the ADP complex indicates that a flexible portion of the Rio2 molecule becomes ordered through direct interaction between His126 and the γ‐phosphate oxygen of ATP. Phosphopeptide mapping of the autophosphorylation site of Rio2 identified Ser128, within the flexible loop and directly adjacent to the part that becomes ordered in response to ATP, as the target. These results give us further information about the nature of the active site of Rio2 kinase and suggest a mechanism of regulation of its enzymatic activity.
FGF‐2, IL‐1β and TGF‐β regulate fibroblast expression of S100A8
Authors: Rahimi Farid , Hsu Kenneth , Endoh Yasumi , Geczy Carolyn L.
Published: April 2006

Growth factors, including fibroblast growth factor‐2 (FGF‐2) and transforming growth factor‐β (TGF‐β) regulate fibroblast function, differentiation and proliferation. S100A8 and S100A9 are members of the S100 family of Ca2+‐binding proteins and are now accepted as markers of inflammation. They are expressed by keratinocytes and inflammatory cells in human/murine wounds and by appropriately activated macrophages, endothelial cells, epithelial cells and keratinocytes in vitro. In this study, regulation and expression of S100A8 and S100A9 were examined in fibroblasts. Endotoxin (LPS), interferon γ (IFNγ), tumour‐necrosis factor (TNF) and TGF‐β did not induce the S100A8 gene in murine fibroblasts whereas FGF‐2 induced mRNA maximally after 12 h. The FGF‐2 response was strongly enhanced and prolonged by heparin. Interleukin‐1β (IL‐1β) alone, or in synergy with FGF‐2/heparin strongly induced the gene in 3T3 fibroblasts. S100A9 mRNA was not induced under any condition. Induction of S100A8 in the absence of S100A9 was confirmed in primary fibroblasts. S100A8 mRNA induction by FGF‐2 and IL‐1β was partially dependent on the mitogen‐activated‐protein‐kinase pathway and dependent on new protein synthesis. FGF‐2‐responsive elements were distinct from the IL‐1β‐responsive elements in the S100A8 gene promoter. FGF‐2‐/heparin‐induced, but not IL‐1β‐induced responses were significantly suppressed by TGF‐β, possibly mediated by decreased mRNA stability. S100A8 in activated fibroblasts was mainly intracytoplasmic. Rat dermal wounds contained numerous S100A8‐positive fibroblast‐like cells 2 and 4 days post injury; numbers declined by 7 days. Up‐regulation of S100A8 by FGF‐2/IL‐1β, down‐regulation by TGF‐β, and its time‐dependent expression in wound fibroblasts suggest a role in fibroblast differentiation at sites of inflammation and repair.
The SCO2299 gene from Streptomyces coelicolor A3(2) encodes a bifunctional enzyme consisting of an RNase H domain and an acid phosphatase domain
Authors: Ohtani Naoto , Saito Natsumi , Tomita Masaru , Itaya Mitsuhiro , Itoh Aya
Published: April 2006

The SCO2299gene from Streptomyces coelicolor encodes a single peptide consisting of 497 amino acid residues. Its N‐terminal region shows high amino acid sequence similarity to RNase HI, whereas its C‐terminal region bears similarity to the CobC protein, which is involved in the synthesis of cobalamin. The SCO2299 gene suppressed a temperature‐sensitive growth defect of an Escherichia coli RNase H‐deficient strain, and the recombinant SCO2299 protein cleaved an RNA strand of RNA·DNA hybrid in vitro. The N‐terminal domain of the SCO2299 protein, when overproduced independently, exhibited RNase H activity at a similar level to the full length protein. On the other hand, the C‐terminal domain showed no CobC‐like activity but an acid phosphatase activity. The full length protein also exhibited acid phosphatase activity at almost the same level as the C‐terminal domain alone. These results indicate that RNase H and acid phosphatase activities of the full length SCO2299 protein depend on its N‐terminal and C‐terminal domains, respectively. The physiological functions of the SCO2299 gene and the relation between RNase H and acid phosphatase remain to be determined. However, the bifunctional enzyme examined here is a novel style in the Type 1 RNase H family. Additionally, S. coelicolor is the first example of an organism whose genome contains three active RNase H genes.
Mg2+‐modulated KMnO4 reactivity of thymines in the open transcription complex reflects variation in the negative electrostatic potential along the separated DNA strands
Authors: Łoziński Tomasz , Wierzchowski Kazimierz L.
Published: April 2006

There is still a controversy over the mechanism of promoter DNA strand separation upon open transcription complex (RPo) formation by Escherichia coli RNA polymerase: is it a single or a stepwise process controlled by Mg2+ ions and temperature? To resolve this question, the kinetics of pseudo‐first‐order oxidation of thymine residues by KMnO4 in the −11 … +2 DNA region of RPo at the λPR promoter was examined under single‐hit conditions as a function of temperature (13–37 °C) in the absence or presence of 10 mm MgCl2. The reaction was also studied with respect to thymidine and its nucleotides (TMP, TTP and TpT) as a function of temperature and [MgCl2]. The kinetic parameters, oxk and oxEa, and Mg‐induced enhancement of oxk proved to be of the same order of magnitude for RPo–λPR and the nucleotides. Unlike the complex, oxEa for the nucleotides was found to be Mg‐independent. The isothermal increase in oxk with increasing [Mg2+] was thus interpreted in terms of a simple model of screening of the negative charges on phosphate groups by Mg2+ ions, lowering the electrostatic barrier to the diffusion of MnO4– anions to the reactive double bond of thymine. Similar screening isotherms were determined for the oxidation of two groups of thymines in RPo at a consensus‐like Pa promoter, differing in the magnitude of the Mg effect. Together, the findings show that: (a) the two DNA strands in the −11…+2 region of RPo–λPR are completely separated over the whole range of temperatures investigated (13–37 °C) in the absence of Mg2+ (b) Mg2+ ions induce an increase in the rate of the oxidation reaction by screening negatively charged phosphate and carboxylate groups; and (c) the observed thymine reactivity and the magnitude of the Mg effect reflect variation in the strength of the electrostatic potential along the separated DNA strands, in agreement with the current structural model of RPo.
Electron transfer chain reaction of the extracellular flavocytochrome cellobiose dehydrogenase from the basidiomycete Phanerochaete chrysosporium
Authors: Igarashi Kiyohiko , Yoshida Makoto , Matsumura Hirotoshi , Nakamura Nobuhumi , Ohno Hiroyuki , Samejima Masahiro , Nishino Takeshi
Published: April 2006

Cellobiose dehydrogenase (CDH) is an extracellular flavocytochrome containing flavin and b‐type heme, and plays a key role in cellulose degradation by filamentous fungi. To investigate intermolecular electron transfer from CDH to cytochrome c, Phe166, which is located in the cytochrome domain and approaches one of propionates of heme, was mutated to Tyr, and the thermodynamic and kinetic properties of the mutant (F166Y) were compared with those of the wild‐type (WT) enzyme. The mid‐point potential of heme in F166Y was measured by cyclic voltammetry, and was estimated to be 25 mV lower than that of WT at pH 4.0. Although presteady‐state reduction of flavin was not affected by the mutation, the rate of subsequent electron transfer from flavin to heme was halved in F166Y. When WT or F166Y was reduced with cellobiose and then mixed with cytochrome c, heme re‐oxidation and cytochrome c reduction occurred synchronously, suggesting that the initial electron is transferred from reduced heme to cytochrome c. Moreover, in both enzymes the observed rate of the initial phase of cytochrome c reduction was concentration dependent, whereas the second phase of cytochrome c reduction was dependent on the rate of electron transfer from flavin to heme, but not on the cytochrome c concentration. In addition, the electron transfer rate from flavin to heme was identical to the steady‐state reduction rate of cytochrome c in both WT and F166Y. These results clearly indicate that the first and second electrons of two‐electron‐reduced CDH are both transferred via heme, and that the redox reaction of CDH involves an electron‐transfer chain mechanism in cytochrome c reduction.
Extended half‐life upon binding of destabilized intrabodies allows specific detection of antigen in mammalian cells
Authors: Sibler Annie‐Paule , Courtête Jérôme , Muller Christian D. , Zeder‐Lutz Gabrielle , Weiss Etienne
Published: April 2006

The ectopic expression of antibody fragments inside mammalian cells (intrabodies) is a challenging approach for probing and modulating target activities. We previously described the shuttling activity of intracellularly expressed Escherichia coliβ‐galactosidase conferred by the single‐chain Fv (scFv) fragment 13R4 equipped with nuclear import/export signals. Here, by appending to scFvs the proteolytic PEST signal sequence (a protein region rich in proline, glutamic acid, serine and threonine) of mouse ornithine decarboxylase, we tested whether short‐lived or destabilized intrabodies could affect the steady‐state level of target by redirecting it to the proteasomes. In the absence of antigen, the half‐life of the modified scFv 13R4, relative to untagged molecules, was considerably reduced in vivo. However, after coexpression with either cytoplasmic or nuclear antigen, the destabilized 13R4 fragments were readily maintained in the cell and strictly colocalized with β‐galactosidase. Analysis of destabilized site‐directed mutants, that were as soluble as 13R4 in the intracellular context, demonstrated that binding to antigen was essential for survival under these conditions. This unique property allowed specific detection of β‐galactosidase, even when expressed at low level in stably transformed cells, and permitted isolation by flow cytometry from a transfected cell mixture of those living cells specifically labeled with bound intrabody. Altogether, we show that PEST‐tagged intrabodies of sufficient affinity and solubility are powerful tools for imaging the presence and likely the dynamics of protein antigens that are resistant to proteasomal degradation in animal cells.
Experimental proof for a signal peptidase I like activity in Mycoplasma pneumoniae, but absence of a gene encoding a conserved bacterial type I SPase
Authors: Catrein Ina , Herrmann Richard , Bosserhoff Armin , Ruppert Thomas
Published: April 2006

Although the annotation of the complete genome sequence of Mycoplasma pneumoniae did not reveal a bacterial type I signal peptidase (SPase I) we showed experimentally that such an activity must exist in this bacterium, by determining the N‐terminus of the N‐terminal gene product P40 of MPN142, formerly called ORF6 gene. Combining mass spectrometry with a method for sulfonating specifically the free amino terminal group of proteins, the cleavage site for a typical signal peptide was located between amino acids 25 and 26 of the P40 precursor protein. The experimental results were in agreement with the cleavage site predicted by computational methods providing experimental confirmation for these theoretical analyses.
Strength Enhancement of Aluminosilicate Aggregate Through Modified Thermal Treatment
Authors: Luscher Walter G. , Hellmann John R. , Scheetz Barry E. , Wilson Brett A.
Published: April 2006

Commercial aluminosilicate aggregates derived from kaolinite and bauxite were evaluated for various physical and mechanical properties. Suites of thermal treatments based on known phase relationships were performed, resulting in significant modification of microstructural and crystalline phase evolution. These structural changes were evaluated and rationalized via scanning electron microscopy and X‐ray diffraction, respectively. Subsequent measurement of mechanical strengths by diametral compression and density by gas pycnometry indicated increases in overall strengths (up to 24%) at a comparable densities (approximately 2.8 g/cm3) in kaolinite‐derived aggregates. Results of this study are being used to drive processing modifications to achieve higher specific strength in aluminosilicate‐derived aggregates.
Preparation of Silica‐Coated Lanthanum–Strontium Manganite Particles with Designable Curie Point, for Application in Hyperthermia Treatments
Authors: Uskoković Vuk , Kos̆ak Aljos̆a , Drofenik Miha , Drofenik Miha
Published: April 2006

Silica‐coated lanthanum–strontium manganite particles with La0.76Sr0.24MnO3+δ stoichiometric formula, exhibiting Curie temperature at ∼40°C, were prepared by using a traditional solid‐state method of synthesis of magnetic ceramic particles, followed by milling and a low‐temperature coating procedure in an aqueous alcoholic alkali medium. The properties of the obtained material establish it as a potential candidate for self‐regulated power‐absorbing and temperature‐controlling materials in hyperthermia treatments. Moreover, core‐comprising LaSr–manganites with different stoichiometries, ranging from La0.5Sr0.5MnO3+δ to LaMnO3+δ, were synthesized, with magnetic and structural properties examined thereof. Herein reported findings can potentially be used in the preparation of silica‐coated magnetic particles with designable Curie temperature, offering a wide range of possibilities of adapting the material to practical instrumental setups in drug delivery and hyperthermia treatments.
Comparison of the Mechanical Properties of Single Crystal and Polycrystalline Yttrium Aluminum Garnet
Authors: Mezeix Laurent , Green David J.
Published: April 2006

Polycrystalline neodymium‐doped yttrium aluminum garnet (YAG), Nd: Y3Al5O12, has been produced in a transparent form and is being considered as a substitute for single crystal YAG in laser applications. To determine whether such a substitution could impact mechanical reliability, it is critical to compare the baseline mechanical properties of the single crystal and polycrystalline materials. In this study, elastic constants, hardness, fracture toughness and strength properties were measured using standard experimental procedures. In addition, fractography was used to gain insight into the fracture process and the nature of the flaws controlling the strength behavior. Overall, it was determined that the mechanical behavior of the polycrystalline YAG was very similar to that of the single crystal material. The elastic constants of polycrystalline YAG were almost the same as the single crystal and the polycrystalline material showed slight advantages in hardness and fracture toughness. Surface machining flaws were found to control the strength behavior with the single crystal material being the more sensitive to contact damage and, hence exhibiting a lower strength.
Noncontact Methods for Measuring Thermal Barrier Coating Temperatures
Authors: Gentleman Molly M. , Lughi Vanni , Nychka John A. , Clarke David R.
Published: April 2006

Three noncontact, optical methods for measuring temperature are reviewed with an emphasis on their application to the measurement of temperatures of thermal barrier coatings (TBCs). The methods are: infrared pyrometry, Raman spectroscopy, and photo‐stimulated luminescence from lanthanide‐doped coatings. Although each has the capability of measuring temperatures pertinent to monitoring TBCs, the finite thickness of typical coatings together with the optical properties of zirconia place severe restrictions on the depth from which the temperature sensing can be obtained. Some of these limitations can be circumvented using photo‐stimulated luminescence with coatings containing dopants at specific locations. To illustrate this, it is demonstrated that by depositing coatings with a lanthanide dopant, such as Eu3+, at specific locations, for instance in contact with the metallic alloy, temperature sensing can be performed with much higher spatial resolution.
Synthesis and Tribological Behavior of Silicon Oxycarbonitride Thin Films Derived from Poly(Urea)Methyl Vinyl Silazane
Authors: Cross Tsali J. , Raj Rishi , Cross Tsali J. , Prasad Somuri V. , Tallant David R.
Published: April 2006

A process for deposition of silicon oxycarbonitride films from poly(urea)methyl vinyl silazane (PUMVS) by spin coating precursor solutions onto a substrate, followed by polymerization, cross‐linking and pyrolysis has been developed. The cross‐linked polymer films (350 nm thick), deposited on variety substrates (e.g., silicon, sapphire, zirconia), were pyrolyzed in nitrogen or ammonia environments either in a hot isostatic press or in a tube furnace. Their microstructure was characterized using infrared and Raman spectroscopy. The tribological (friction and wear) behavior was evaluated in dry nitrogen and air with 50% relative humidity using a unidirectional linear wear tester in a ball‐on‐disk configuration. Wear surfaces, transfer films and wear debris were analyzed by scanning electron micrograph (SEM)/energy dispersive spectroscopy (EDS).
Durable, Ultraluminous Structure for Incandescent, High‐Power White‐LED
Authors: Ishikawa Toshihiro , Sakata Shin‐ichi , Mitani Atsuyuki
Published: April 2006

A new particle‐ and resin‐free system composed of transparent, binary single crystals entangled with each other continuously has been successfully developed for a light source for illumination. These single crystals exist in a semi‐coherent state like a block copolymer; one of these single crystals exhibits effective photoluminescence and the other plays an important role as a light guide of excitation and emitting lights. This material can have low backscattering and high quantum efficiency, resulting in twice the emission light of conventional particle‐dispersed systems. Furthermore, this system shows outstanding durability, because of its thermostructural properties. This concept will be expanded into wide combinations to create incandescent systems in the future.