bluuebunny/crossref_metadata_2025 huggingface dataset:
Akt1‐mediated Intracellular Oxidation after UVB Irradiation Suppresses Apoptotic Cell Death Induced by Cell Detachment and Serum Starvation
Authors: Ibuki Yuko , Akaike Marie , Toyooka Tatsushi , Goto Rensuke
Published: October 2007

Apoptosis is an important cell death system that deletes damaged and mutated cells to prevent cancer. We have previously reported that a certain dose of UVB irradiation inhibited the apoptosis induced by serum starvation and cell detachment, leading to cell transformation. This antiapoptotic effect was partially inhibited by phosphatidylinositol 3‐kinase (PI3‐kinase) inhibitors. UVB irradiation is known to cause the phosphorylation of Akt via the activation of PI3‐kinase; however, the Akt isoform‐specific relationship has not yet been clarified. Notably, the role in antiapoptotic effect of UVB has yet to be elucidated. In this study, the role of Akt1 in the UVB‐induced inhibition of apoptosis was examined by Akt1 knockdown using small interfering RNA (siRNA). NIH3T3 cells showed typical apoptotic cell death by serum starvation and cell detachment, which was significantly inhibited by UVB irradiation. Akt1 knockdown decreased the antiapoptotic effect of UVB. Hydrogen peroxide‐induced suppression of cell death was also decreased in Akt1 knockdown cells. An antioxidant, N‐acetylcysteine, inhibited the antiapoptotic effect by UVB irradiation, whereas no inhibition was observed in Akt1 knockdown cells. Furthermore, UVB‐induced intracellular peroxidation was not observed in the knockdown cells, indicating that Akt1 played an important role in mediating the intracellular redox status. Treatment with insulin had a similar antiapoptotic effect as UVB irradiation involving intracellular peroxidation, which was also attenuated in Akt1 knockdown cells. These findings suggest that appropriate intracellular oxidation after UVB irradiation prevented apoptosis, a process which might be partially regulated by the production of reactive oxygen species mediated by Akt1.
Developmental changes in functional characteristics of aortic baroreceptor afferents in rats
Authors: Waki Hidefumi , Yamasaki Masao , Katahira Kiyoaki , Katsuda Shinichiro , Maeda Masanobu , Shimizu Tsuyoshi
Published: October 2007

To investigate postnatal developmental changes in functional characteristics of the afferent pathway of the aortic baroreceptor reflex, the responses of aortic nerve activity (ANA) to blood pressure (BP) changes elicited by phenylephrine and sodium nitroprusside administration were tested in 3‐, 8‐ and 20‐week‐old male rats under chloralose (60 mg kg−1i.p.) and urethane (600 mg kg−1i.p.) anaesthesia. The function curve of ANA in response to BP changes showed a sigmoid shape that shifted to the right from 3 to 8 weeks of age. The maximal activity and maximal gain of the aortic nerve, which were calculated by a logistic function analysis, were significantly higher in 20‐week‐old rats (maximal activity, 532 ± 47% of baseline; maximal gain, 7.9 ± 0.8% of baseline mmHg−1; n= 9) than in 3‐week‐old rats (maximal ANA, 268 ± 25% of baseline, P < 0.001; maximal gain, 4.9 ± 0.5% of baseline mmHg−1, P < 0.01, n= 9) and 8‐week‐old rats (maximal ANA, 309 ± 18% of baseline, P < 0.001; maximal gain, 4.9 ± 0.3% of baseline mmHg−1, P < 0.01, n= 11). These results suggest that the operating point of aortic baroreceptor afferents is reset to the higher pressure level during development from 3 to 8 weeks of age and, thereafter, the afferent gain increases from 8 to 20 weeks of age. This functional change may be an important factor to prevent an excess increase of BP, which would result in pathophysiological problems.
The expression of NFATc1 in adult rat skeletal muscle fibres
Authors: Mutungi Gabriel
Published: October 2007

Although numerous studies have recently implicated the calcineurin–nuclear factor of activated T‐cells (Cn–NFAT) signalling pathway in the regulation of activity‐dependent fibre type switching in adult mammalian skeletal muscles, little is known about the endogenous expression of NFAT proteins in the various fibre types present in these muscles. In this study, the immunolocalization of NFATc1 (also known as NFATc or NFAT2) in the extensor digitorum longus (EDL; a mainly fast‐twitch muscle) and the soleus (a predominantly slow‐twitch muscle) muscles of adult (∼90‐day‐old) Wistar rats was investigated. The results show that NFATc1 is expressed only in oxidative fibres (i.e. type I and type IIA fibres) that stain intensely for succinate dehydrogenase activity irrespective of whether they are from the fast‐ or slow‐twitch muscle. Thus, 99 ± 4% (n= 7 rats) of the muscle fibres in the soleus and 42 ± 2% (n= 7 rats) of those in the EDL expressed NFATc1. In the soleus muscle fibres, NFATc1 was localized mainly in the fibre nuclei, whereas in the EDL fibres it was localized in both the cytoplasm and the nuclei. However, no difference in its localization was observed between type I and type IIA fibres in both muscles. Western blot experiments showed that the soleus expressed more NFATc1 proteins than the EDL. From these results, we suggest that NFATc1 controls the number and distribution of both type I and type IIA fibres, as well as the oxidative capacity of adult mammalian skeletal muscles.
Experimental and Numerical Study of a Stacked Microchannel Heat Sink for Liquid Cooling of Microelectronic Devices
Authors: Wei Xiaojin , Joshi Yogendra , Patterson Michael K.
Published: October 2007

One of the promising liquid cooling techniques for microelectronics is attaching a microchannel heat sink to, or directly fabricating microchannels on, the inactive side of the chip. A stacked microchannel heat sink integrates many layers of microchannels and manifold layers into one stack. Compared with single-layered microchannels, stacked microchannels provide larger flow passages, so that for a fixed heat load the required pressure drop is significantly reduced. Better temperature uniformity can be achieved by arranging counterflow in adjacent microchannel layers. The dedicated manifolds help to distribute coolant uniformly to microchannels. In the present work, a stacked microchannel heat sink is fabricated using silicon micromachining techniques. Thermal performance of the stacked microchannel heat sink is characterized through experimental measurements and numerical simulations. Effects of coolant flow direction, flow rate allocation among layers, and nonuniform heating are studied. Wall temperature profiles are measured using an array of nine platinum thin-film resistive temperature detectors deposited simultaneously with thin-film platinum heaters on the backside of the stacked structure. Excellent overall cooling performance (0.09°C∕Wcm2) for the stacked microchannel heat sink has been shown in the experiments. It has also been identified that over the tested flow rate range, counterflow arrangement provides better temperature uniformity, while parallel flow has the best performance in reducing the peak temperature. Conjugate heat transfer effects for stacked microchannels for different flow conditions are investigated through numerical simulations. Based on the results, some general design guidelines for stacked microchannel heat sinks are provided.
Quantitative Salt-Water Modeling of Fire-Induced Flows for Convective Heat Transfer Model Development
Authors: Yao Xiaobo , Marshall André W.
Published: October 2007

This research provides a detailed analysis of convective heat transfer in ceiling jets by using a quantitative salt-water modeling technique. The methodology of quantitative salt-water modeling builds on the analogy between salt-water flow and fire induced flow, which has been successfully used in the qualitative analysis of fires. Planar laser induced fluorescence and laser doppler velocimetry have been implemented to measure the dimensionless density difference and velocity in salt-water plumes. The quantitative salt-water modeling technique has been validated through comparisons of appropriately scaled salt-water measurements, fire measurements, and theory. This analogy has been exploited to develop an engineering heat transfer model for predicting heat transfer in impinging fire plumes using salt-water measurements along with the adiabatic wall modeling concept. Combining quantitative salt-water modeling and adiabatic wall modeling concepts introduces new opportunities for studying heat transfer issues in basic and complex fire induced flow configurations.
Flow Boiling Heat Transfer in Microchannels
Authors: Liu Dong , Garimella Suresh V.
Published: October 2007

Flow boiling heat transfer to water in microchannels is experimentally investigated. The dimensions of the microchannels considered are 275×636 and 406×1063μm2. The experiments are conducted at inlet water temperatures in the range of 67–95°C and mass fluxes of 221–1283kg∕m2s. The maximum heat flux investigated in the tests is 129W∕cm2 and the maximum exit quality is 0.2. Convective boiling heat transfer coefficients are measured and compared to predictions from existing correlations for larger channels. While an existing correlation was found to provide satisfactory prediction of the heat transfer coefficient in subcooled boiling in microchannels, saturated boiling was not well predicted by the correlations for macrochannels. A new superposition model is developed to correlate the heat transfer data in the saturated boiling regime in microchannel flows. In this model, specific features of flow boiling in microchannels are incorporated while deriving analytical solutions for the convection enhancement factor and nucleate boiling suppression factor. Good agreement with the experimental measurements indicates that this model is suitable for use in analyzing boiling heat transfer in microchannel flows.
Novel Design of a Miniature Loop Heat Pipe Evaporator for Electronic Cooling
Authors: Singh Randeep , Akbarzadeh Aliakbar , Dixon Chris , Mochizuki Masataka
Published: October 2007

Miniature loop heat pipes (mLHPs) are coming up with a promising solution for the thermal management of futuristic electronics systems. In order to implement these devices inside compact electronics, their evaporator has to be developed with small thickness while preserving the unique thermal characteristics and physical concept of the loop scheme. This paper specifically addresses the design and testing of a mLHP with a flat evaporator only 5mm thick for the cooling of high performance microprocessors for electronic devices. A novel concept was used to achieve very small thickness for the mLHP evaporator in which the compensation chamber was positioned on the sides of the wick structure and incorporated in the same plane as the evaporator. This is unlike the conventional design of the flat evaporator for mLHP in which the compensation chamber, as a rule, adds to the overall thickness of the evaporator. The loop was made from copper with water as the heat transfer fluid. For capillary pumping of the working fluid around the loop, a sintered nickel wick with 3–5μm pore radius and 75% porosity was used. In the horizontal orientation, the device was able to transfer heat fluxes of 50W∕cm2 at a distance of up to 150mm by using a transport line with 2mm internal diameter. In the range of applied power, the evaporator was able to achieve steady state without any temperature overshoots or symptoms of capillary structure dryouts. For the evaporator and condenser at the same level and under forced air cooling, the minimum value of 0.62°C∕W for mLHP thermal resistance from evaporator to condenser (Rec) was achieved at a maximum heat load of 50W with the corresponding junction temperature of 98.5°C. The total thermal resistance (Rt) of the mLHP was within 1.5–5.23°C∕W. At low heat loads, the mLHP showed some thermal and hydraulic oscillations in the transport lines, which were predominately due to the flow instabilities imposed by parasitic heat leaks to the compensation chamber. It is concluded form the outcomes of the present investigation that the proposed design of the mLHP evaporator can be effectively used for the thermal control of the compact electronic devices with high heat flux capabilities.
Homogeneous Nucleation of Vapor at Preferred Sites During Rapid Transient Heating of Liquid in Micropassages
Authors: Carey Van P. , Padilla Jorge , Gan Yu
Published: October 2007

Rapid heating of a liquid at the wall of a micropassage may produce homogeneous nucleation of vapor in the liquid in contact with the surface. In such circumstances, nucleation is generally expected to be most likely to occur in the hottest liquid closest to the surface. It is known, however, that in many cases, the liquid molecules closest to the surface will experience long-range attractive forces to molecules in the solid, with the result that the equation of state for the liquid near the surface will differ from that for the bulk liquid. In micro- and nanopassages, this wall-affected region may be a significant fraction of the passage interior volume. Recent investigations of wall force effects on the liquid indicate that these forces increase the spinodal temperature in the near-surface region. The results of these previous investigations suggest that for heated surfaces with nanoscale roughness, protrusion of bulk fluid into crevices in the surface may make them preferred sites for homogeneous nucleation during rapid heating. A detailed model analysis of the heat transfer in a model conical crevice is developed and used to explore the plausibility and apparent mechanisms of preferred-site homogeneous nucleation. The analysis predicts that protrusion of bulk liquid into a conical cavity does, under some conditions, make the cavity a preferred site for the first occurrence of homogeneous nucleation. The analysis is used to examine the range of conditions under which a crevice will be a preferred site. The implications for nucleation near a solid surface during rapid heating are also explored for circumstances similar to those for bubble nucleation adjacent to heaters in microheater reservoirs in inkjet printer heads.
Combined Parameter and Function Estimation With Application to Thermal Conductivity and Surface Heat Flux
Authors: Loulou Tahar
Published: October 2007

This is a numerical and experimental study on the combined parameter and function estimation. The determination of thermal conductivity and the surface heat flux is an illustration of combined estimation of one parameter and one function by means of the conjugate gradient method with vectorial descent parameter. The experimental example developed herein uses one set of good data obtained by Beck and Arnold (1977, Parameter Estimation in Engineering and Science, Wiley, New York). For this case, two measured temperatures in the solid are used to illustrate combined estimation. The unknown boundary condition and thermal conductivity of this solid were satisfactorily reconstructed and a good enough comparison is demonstrated between the known and estimated unknowns. The temperature data of Beck and Arnold are found to be excellent. Also, it is shown that the developed approach is general, stable, powerful, and able to process a wide variety of heat transfer problems where a simultaneous estimation is unavoidable.
Gastroenteritis Outbreak Caused by Waterborne Norovirus at a New Zealand Ski Resort
Authors: Hewitt Joanne , Bell Derek , Simmons Greg C. , Rivera-Aban Malet , Wolf Sandro , Greening Gail E.
Published: October 2007

In July 2006, public health services investigated an outbreak of acute gastroenteritis among staff and visitors of a popular ski resort in southern New Zealand. The source of the outbreak was a drinking water supply contaminated by human sewage. The virological component of the investigation played a major role in confirming the source of the outbreak. Drinking water, source stream water, and 31 fecal specimens from gastroenteritis outbreak cases were analyzed for the presence of norovirus (NoV). Water samples were concentrated by ultrafiltration, and real-time reverse transcription-PCR (RT-PCR) was used for rapid detection of NoV from both water and fecal samples. The implicated NoV strain was further characterized by DNA sequencing. NoV genogroup GI/5 was identified in water samples and linked case fecal specimens, providing clear evidence of the predominant pathogen and route of exposure. A retrospective cohort study demonstrated that staff who consumed drinking water from the resort supply were twice as likely to have gastroenteritis than those who did not. This is the first time that an outbreak of gastroenteritis in New Zealand has been conclusively linked to NoV detected in a community water supply. To our knowledge, this is the first report of the use of ultrafiltration combined with quantitative real-time RT-PCR and DNA sequencing for investigation of a waterborne NoV outbreak.
Anaerobic 1-Alkene Metabolism by the Alkane- and Alkene-Degrading Sulfate Reducer
Desulfatibacillum aliphaticivorans
Strain CV2803
T
Authors: Grossi Vincent , Cravo-Laureau Cristiana , Méou Alain , Raphel Danielle , Garzino Frédéric , Hirschler-Réa Agnès
Published: October 2007

The alkane- and alkene-degrading, marine sulfate-reducing bacterium
Desulfatibacillum aliphaticivorans
strain CV2803
T
, known to oxidize
n
-alkanes anaerobically by fumarate addition at C-2, was investigated for its 1-alkene metabolism. The total cellular fatty acids of this strain were predominantly C-(even number) (C-even) when it was grown on C-even 1-alkenes and predominantly C-(odd number) (C-odd) when it was grown on C-odd 1-alkenes. Detailed analyses of those fatty acids by gas chromatography-mass spectrometry after 6- to 10-week incubations allowed the identification of saturated 2- and 4-ethyl-, 2- and 4-methyl-, and monounsaturated 4-methyl-branched fatty acids with chain lengths that correlated with those of the 1-alkene. The growth of
D. aliphaticivorans
on (per)deuterated 1-alkenes provided direct evidence of the anaerobic transformation of these alkenes into the corresponding 1-alcohols and into linear as well as 10- and 4-methyl-branched fatty acids. Experiments performed with [
13
C]bicarbonate indicated that the initial activation of 1-alkene by the addition of inorganic carbon does not occur. These results demonstrate that
D. aliphaticivorans
metabolizes 1-alkene by the oxidation of the double bond at C-1 and by the subterminal addition of organic carbon at both ends of the molecule [C-2 and C-(ω-1)]. The detection of ethyl-branched fatty acids from unlabeled 1-alkenes further suggests that carbon addition also occurs at C-3. Alkylsuccinates were not observed as potential initial intermediates in alkene metabolism. Based on our observations, the first pathways for anaerobic 1-alkene metabolism in an anaerobic bacterium are proposed. Those pathways indicate that diverse initial reactions of 1-alkene activation can occur simultaneously in the same strain of sulfate-reducing bacterium.
Signature-Tagged Mutagenesis of
Edwardsiella ictaluri
Identifies Virulence-Related Genes, Including a
Salmonella
Pathogenicity Island 2 Class of Type III Secretion Systems
Authors: Thune Ronald L. , Fernandez Denise H. , Benoit Jennifer L. , Kelly-Smith Maria , Rogge Matthew L. , Booth Natha J. , Landry Christie A. , Bologna Rachel A.
Published: October 2007

Edwardsiella ictaluri
is the leading cause of mortality in channel catfish culture, but little is known about its pathogenesis. The use of signature-tagged mutagenesis in a waterborne infection model resulted in the identification of 50 mutants that were unable to infect/survive in catfish. Nineteen had minitransposon insertions in miscellaneous genes in the chromosome, 10 were in genes that matched to hypothetical proteins, and 13 were in genes that had no significant matches in the NCBI databases. Eight insertions were in genes encoding proteins associated with virulence in other pathogens, including three in genes involved in lipopolysaccharide biosynthesis, three in genes involved in type III secretion systems (TTSS), and two in genes involved in urease activity. With the use of a sequence from a lambda clone carrying several TTSS genes, Blastn analysis of the partially completed
E. ictaluri
genome identified a 26,135-bp pathogenicity island containing 33 genes of a TTSS with similarity to the
Salmonella
pathogenicity island 2 class of TTSS. The characterization of a TTSS apparatus mutant indicated that it retained its ability to invade catfish cell lines and macrophages but was defective in intracellular replication. The mutant also invaded catfish tissues in numbers equal to those of invading wild-type
E. ictaluri
bacteria but replicated poorly and was slowly cleared from the tissues, while the wild type increased in number.
Detection of
Salmonella enterica
Subpopulations by Phenotype Microarray Antibiotic Resistance Patterns
Authors: Guard-Bouldin Jean , Morales Cesar A. , Frye Jonathan G. , Gast Richard K. , Musgrove Michael
Published: October 2007

Three strains of
Salmonella enterica
serotype Enteritidis were compared to
Salmonella enterica
serotype Heidelberg,
Salmonella enterica
serotype Newport, and
Salmonella enterica
serovar Typhimurium for growth in the presence of 240 antibiotics arranged within a commercial high-throughput phenotype microarray. The results show that antibiotic resistances were different for subpopulations of serotype Enteritidis separated only by genetic drift.
Development and Implementation of a Single-Chain Fv Antibody for Specific Detection of
Bacillus anthracis
Spores
Authors: Mechaly A. , Zahavy E. , Fisher M.
Published: October 2007

A single-chain Fv (scFv) antibody was developed and applied for efficient and specific detection of
Bacillus anthracis
spores. The antibody was isolated from a phage display library prepared from spleens of mice immunized with a water-soluble extract of the outer membrane of the
B. anthracis
spore (exosporium). The library (7 × 10
6
PFU) was biopanned against live, native
B. anthracis
ATCC Δ14185 spores suspended in solution, resulting in the isolation of a unique soluble scFv antibody. The antibody was affinity purified and its affinity constant (3 × 10
8
± 1 × 10
8
M
−1
) determined via flow cytometry (FCM). Preliminary characterization of scFv specificity indicated that the scFv antibody does not cross-react with representatives of some phylogenetically related
Bacillus
spores. The potential use of scFv antibodies in detection platforms was demonstrated by the successful application of the soluble purified scFv antibody in enzyme-linked immunosorbent assays, immunofluorescence assays, and FCM.
Riboflavin Biosynthesis Is Associated with Assimilatory Ferric Reduction and Iron Acquisition by
Campylobacter jejuni
Authors: Crossley Rachel A. , Gaskin Duncan J. H. , Holmes Kathryn , Mulholland Francis , Wells Jerry M. , Kelly David J. , van Vliet Arnoud H. M. , Walton Nicholas J.
Published: October 2007

One of the pathways involved in the acquisition of the essential metal iron by bacteria involves the reduction of insoluble Fe
3+
to soluble Fe
2+
, followed by transport of Fe
2+
to the cytoplasm. Flavins have been implicated as electron donors in this poorly understood process. Ferrous iron uptake is essential for intestinal colonization by the important pathogen
Campylobacter jejuni
and may be of particular importance under low-oxygen conditions. In this study, the links among riboflavin biosynthesis, ferric reduction, and iron acquisition in
C. jejuni
NCTC11168 have been investigated. A riboflavin auxotroph was generated by inactivation of the
ribB
riboflavin biosynthesis gene (Cj0572), and the resulting isogenic
ribB
mutant only grew in the presence of exogenous riboflavin or the riboflavin precursor diacetyl but not in the presence of the downstream products flavin adenine dinucleotide and flavin mononucleotide. Riboflavin uptake was unaffected in the
ribB
mutant under iron-limited conditions but was lower in both the wild-type strain and the
ribB
mutant under iron-replete conditions. Mutation of the
fur
gene, which encodes an iron uptake regulator of
C. jejuni
, resulted in an increase in riboflavin uptake which was independent of the iron content of the medium, suggesting a role for Fur in the regulation of the as-yet-unknown riboflavin transport system. Finally, ferric reduction activity was independent of iron availability in the growth medium but was lowered in the
ribB
mutant compared to the wild-type strain and, conversely, increased in the
fur
mutant. Taken together, the findings confirm close relationships among iron acquisition, riboflavin production, and riboflavin uptake in
C. jejuni
.
Identification of New Kinetoplast DNA Replication Proteins in Trypanosomatids Based on Predicted S-Phase Expression and Mitochondrial Targeting
Authors: Li Yue , Sun Yu , Hines Jane C. , Ray Dan S.
Published: October 2007

Trypanosomatid parasites contain an unusual form of mitochondrial DNA (kinetoplast DNA [kDNA]) consisting of a catenated network of several thousand minicircles and a smaller number of maxicircles. Many of the proteins involved in the replication and division of kDNA are likely to have no counterparts in other organisms and would not be identified by similarity to known replication proteins in other organisms. A new kDNA replication protein conserved in kinetoplastids has been identified based on the presence of posttranscriptional regulatory sequences associated with S-phase gene expression and predicted mitochondrial targeting. The
Leishmania major
protein P105 (
Lm
P105) and
Trypanosoma brucei
protein P93 (
Tb
P93) localize to antipodal sites flanking the kDNA disk, where several other replication proteins and nascent minicircles have been localized. Like some of these kDNA replication proteins, the
Lm
P105 protein is only present at the antipodal sites during S phase. RNA interference (RNAi) of
Tb
P93 expression resulted in a cessation of cell growth and the loss of kDNA. Nicked/gapped forms of minicircles, the products of minicircle replication, were preferentially lost from the population of free minicircles during RNAi, suggesting involvement of
Tb
P93 in minicircle replication. This approach should allow the identification of other novel proteins involved in the duplication of kDNA.
The Ccr4-Not Complex Regulates Skn7 through Srb10 Kinase
Authors: Lenssen Eve , Azzouz Nowel , Michel Agnès , Landrieux Emilie , Collart Martine A.
Published: October 2007

The Ccr4-Not complex is a multifunctional regulatory platform composed of nine subunits that controls diverse cellular events including mRNA degradation, protein ubiquitination, and transcription. In this study, we identified the yeast
Saccharomyces cerevisiae
osmotic and oxidative stress transcription factor Skn7 as a new target for regulation by the Ccr4-Not complex. Skn7 interacts with Not1 in a two-hybrid assay and coimmunoprecipitates with Not5 in a Not4-dependent manner. Skn7-dependent expression of
OCH1
and Skn7 binding to the
OCH1
promoter are increased in
not4
Δ or
not5
Δ mutants. Skn7 purified from wild-type cells but not from
not4
Δ cells is associated with the Srb10 kinase. This kinase plays a central role in the regulation of Skn7 by Not4, since increased
OCH1
expression in
not4
Δ cells requires Srb10. These results reveal a critical role for the Ccr4-Not complex in the mechanism of activation of Skn7 that is dependent upon the Srb10 kinase.
Involvement of Sensor Kinases in the Stress Tolerance Response ofStreptococcus mutans
Authors: Biswas Indranil , Drake Laura , Erkina Dasha , Biswas Saswati
Published: October 2007

The gram-positive bacteriumStreptococcus mutansis the primary causative agent in the formation of dental caries in humans. The ability ofS. mutansto adapt and to thrive in the hostile environment of the oral cavity suggests that this cariogenic pathogen is capable of sensing and responding to different environmental stimuli. This prompted us to investigate the role of two-component signal transduction systems (TCS), particularly the sensor kinases, in response to environmental stresses. Analysis of the annotated genome sequence ofS. mutansindicates the presence of 13 putative TCS. Further bioinformatics analysis in our laboratory has identified an additional TCS in the genome ofS. mutans. We verified the presence of the 14 sensor kinases by using PCR and Southern hybridization in 13 differentS. mutansstrains and found that not all of the sensor kinases are encoded by each strain. To determine the potential role of each TCS in the stress tolerance ofS. mutansUA159, insertion mutations were introduced into the genes encoding the individual sensor kinases. We were successful in inactivating all of the sensor kinases, indicating that none of the TCS are essential for the viability ofS. mutans. The mutantS. mutansstrains were assessed for their ability to withstand various stresses, including osmotic, thermal, oxidative, and antibiotic stress, as well as the capacity to produce mutacin. We identified three sensor kinases, Smu486, Smu1128, and Smu1516, which play significant roles in stress tolerance ofS. mutansstrain UA159.
Genomic Characterization of
Ralstonia solanacearum
Phage φRSA1 and Its Related Prophage (φRSX) in Strain GMI1000
Authors: Fujiwara Akiko , Kawasaki Takeru , Usami Shoji , Fujie Makoto , Yamada Takashi
Published: October 2007

φRSA1 is a wide-host-range bacteriophage isolated from
Ralstonia solanacearum
. In this study, the complete nucleotide sequence of the φRSA1 genomic DNA was determined. The genome was 38,760 bp of double-stranded DNA (65.3% G+C) with 19-bp 5′-extruding cohesive ends (
cos
) and contained 51 open reading frames (ORFs). Two-thirds of the φRSA1 genomic region encodes the phage structural modules, and they are very similar to those reported for coliphage P2 and P2-like phages. A φRSA1 minireplicon with an 8.2-kbp early-expressing region was constructed. A late-expression promoter sequence motif was predicted for these φRSA1 genes as 5′ TGTTGT-(X)
13
-ACAACA. The genomic sequence similarity between φRSA1 and related phages φ52237 and φCTX was interrupted by three AT islands, one of which contained an insertion sequence element, suggesting that they were recombinational hot spots. φRSA1 was found to be integrated into at least three different strains of
R. solanacearum
, and the chromosomal integration site (
attB
) was identified as the 3′ portion of the arginine tRNA(CCG) gene. In the light of the φRSA1 gene arrangement, one possible prophage sequence previously detected on the chromosome of
R. solanacearum
strain GMI1000 was characterized as a φRSA1-related prophage (designated φRSX). φRSX was found to be integrated at the serine tRNA (GGA) gene as an
att
site, and its size was determined to be 40,713 bp. φRSX ORFs shared very high amino acid identity with their φRSA1 counterparts. The relationships and evolution of these P2-like phages are discussed.
Purification and Characterization of Serine Racemase from a Hyperthermophilic Archaeon,Pyrobaculum islandicum
Authors: Ohnishi Masato , Saito Makoto , Wakabayashi Sadao , Ishizuka Morio , Nishimura Katsushi , Nagata Yoko , Kasai Sabu
Published: October 2007

Pyrobaculum islandicumis an anaerobic hyperthermophilic archaeon that is most active at 100°C. A pyridoxal 5′-phosphate-dependent serine racemase called Srr was purified from the organism. The correspondingsrrgene was cloned, and recombinant Srr was purified fromEscherichia coli. It showed the highest racemase activity towardl-serine, followed byl-threonine,d-serine, andd-threonine. Like rodent and plant serine racemases, Srr is bifunctional, showing highl-serine/l-threonine dehydratase activity. The sequence of Srr is 87% similar to that ofPyrobaculum aerophilumIlvA (a putative threonine dehydratase) but less than 32% similar to any other serine racemases and threonine dehydratases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration analyses revealed that Srr is a homotrimer of a 44,000-molecular-weight subunit. Both racemase and dehydratase activities were highest at 95°C, while racemization and dehydration were maximum at pH 8.2 and 7.8, respectively. Unlike other, related Ilv enzymes, Srr showed no allosteric properties: neither of these enzymatic activities was affected by eitherl-amino acids (isoleucine and valine) or most of the metal ions. Only Fe2+and Cu2+caused 20 to 30% inhibition and 30 to 40% stimulation of both enzyme activities, respectively. ATP inhibited racemase activity by 10 to 20%. TheKmandVmaxvalues of the racemase activity of Srr forl-serine were 185 mM and 20.1 μmol/min/mg, respectively, while the corresponding values of the dehydratase activity ofl-serine were 2.2 mM and 80.4 μmol/min/mg, respectively.
Genetic and Proteomic Analyses of a Proteasome-Activating Nucleotidase A Mutant of the Haloarchaeon
Haloferax volcanii
Authors: Kirkland P. Aaron , Gil Malgorzata A. , Karadzic Ivanka M. , Maupin-Furlow Julie A.
Published: October 2007

The halophilic archaeon
Haloferax volcanii
encodes two related proteasome-activating nucleotidase proteins, PanA and PanB, with PanA levels predominant during all phases of growth. In this study, an isogenic
panA
mutant strain of
H. volcanii
was generated. The growth rate and cell yield of this mutant strain were lower than those of its parent and plasmid-complemented derivatives. In addition, a consistent and discernible 2.1-fold increase in the number of phosphorylated proteins was detected when the
panA
gene was disrupted, based on phosphospecific fluorescent staining of proteins separated by 2-dimensional gel electrophoresis. Subsequent enrichment of phosphoproteins by immobilized metal ion and metal oxide affinity chromatography (in parallel and sequentially) followed by tandem mass spectrometry was employed to identify key differences in the proteomes of these strains as well as to add to the restricted numbers of known phosphoproteins within the
Archaea
. In total, 625 proteins (approximately 15% of the deduced proteome) and 9 phosphosites were identified by these approaches, and 31% (195) of the proteins were identified by multiple phosphoanalytical methods. In agreement with the phosphostaining results, the number of identified proteins that were reproducibly exclusive or notably more abundant in one strain was nearly twofold greater for the
panA
mutant than for the parental strain. Enriched proteins exclusive to or more abundant in the
panA
mutant (versus the wild type) included cell division (FtsZ, Cdc48), dihydroxyacetone kinase-linked phosphoenolpyruvate phosphotransferase system (EI, DhaK), and oxidoreductase homologs. Differences in transcriptional regulation and signal transduction proteins were also observed, including those differences (e.g., OsmC and BolA) which suggest that proteasome deficiency caused an up-regulation of stress responses (e.g., OsmC versus BolA). Consistent with this, components of the Fe-S cluster assembly, protein-folding, DNA binding and repair, oxidative and osmotic stress, phosphorus assimilation, and polyphosphate synthesis systems were enriched and identified as unique to the
panA
mutant. The cumulative proteomic data not only furthered our understanding of the archaeal proteasome system but also facilitated the assembly of the first subproteome map of
H. volcanii
.
Transcriptome Analysis of
Pseudomonas putida
in Response to Nitrogen Availability
Authors: Hervás Ana B. , Canosa Inés , Santero Eduardo
Published: October 2007

This work describes a regulatory network of
Pseudomonas putida
controlled in response to nitrogen availability. We define NtrC as the master nitrogen regulator and suggest that it not only activates pathways for the assimilation of alternative nitrogen sources but also represses carbon catabolism under nitrogen-limited conditions, possibly to prevent excessive carbon and energy flow in the cell.
The Major Magnetosome Proteins MamGFDC Are Not Essential for Magnetite Biomineralization inMagnetospirillum gryphiswaldensebut Regulate the Size of Magnetosome Crystals
Authors: Scheffel André , Gärdes Astrid , Grünberg Karen , Wanner Gerhard , Schüler Dirk
Published: October 2007

Magnetospirillum gryphiswaldenseand related magnetotactic bacteria form magnetosomes, which are membrane-enclosed organelles containing crystals of magnetite (Fe3O4) that cause the cells to orient in magnetic fields. The characteristic sizes, morphologies, and patterns of alignment of magnetite crystals are controlled by vesicles formed of the magnetosome membrane (MM), which contains a number of specific proteins whose precise roles in magnetosome formation have remained largely elusive. Here, we report on a functional analysis of the small hydrophobic MamGFDC proteins, which altogether account for nearly 35% of all proteins associated with the MM. Although their high levels of abundance and conservation among magnetotactic bacteria had suggested a major role in magnetosome formation, we found that the MamGFDC proteins are not essential for biomineralization, as the deletion of neithermamC, encoding the most abundant magnetosome protein, nor the entiremamGFDCoperon abolished the formation of magnetite crystals. However, cells lackingmamGFDCproduced crystals that were only 75% of the wild-type size and were less regular than wild-type crystals with respect to morphology and chain-like organization. The inhibition of crystal formation could not be eliminated by increased iron concentrations. The growth of mutant crystals apparently was not spatially constrained by the sizes of MM vesicles, as cells lackingmamGFDCformed vesicles with sizes and shapes nearly identical to those formed by wild-type cells. However, the formation of wild-type-size magnetite crystals could be gradually restored by in-transcomplementation with one, two, and three genes of themamGFDCoperon, regardless of the combination, whereas the expression of all four genes resulted in crystals exceeding the wild-type size. Our data suggest that the MamGFDC proteins have partially redundant functions and, in a cumulative manner, control the growth of magnetite crystals by an as-yet-unknown mechanism.
Substitutions at Auxiliary Operator O3 Enhance Repression by Nitrate-Responsive Regulator NarL at Synthetic
lac
Control Regions in
Escherichia coli
K-12
Authors: Stewart Valley , Bledsoe Peggy J.
Published: October 2007

We constructed monocopy
lac
operon control regions in which the operators O1-
lac
and O3-
lac
were replaced by NarL and NarP binding sites from the
nirB
or
napF
operon control regions. The results support the hypothesis that DNA-bound dimers of phospho-NarL can participate in higher-order cooperative interactions.
c-KIT overexpression, without gene amplification and mutation, in paediatric renal tumours
Authors: Jones Chris , Rodriguez-Pinilla Maria , Lambros Maryou , Bax Dorine , Messahel Boo , Vujanic Gordan M , Reis-Filho Jorge S , Pritchard-Jones Kathy
Published: October 2007

Aims:

To investigate the presence and prognostic relevance of KIT expression in paediatric renal tumours, and to determine whether receptor overexpression is associated with gene amplification and/or mutation.

Methods:

Immunohistochemistry without antigen retrieval for CD117 was carried out on tissue microarrays consisting of 274 Wilms’ tumours, 13 clear cell sarcomas of the kidney (CCSK), 10 mesoblastic nephromas (MN), and 7 rhabdoid tumours of the kidney (RTK). In addition, gene copy number was investigated by chromogenic in situ hybridisation (CISH), and overexpressing tumours were sequenced forKITmutations in exons 9, 11, 13 and 17.

Results:

Only 8/200 (4.0%) Wilms’ tumours exhibited any degree of moderate–strong KIT staining in any of their assessable cell types. This small group of KIT-positive tumours had a shorter time to relapse (p = 0.0044, log-rank test). There were no positive MNs or RTKs; however 3/11 (27.3%) CCSKs were strongly positive, with an additional two cases weakly reactive. No cases exhibited gene amplification or mutation.

Conclusions:

KIT overexpression in rare in Wilms’ tumours, although does appear to confer a worse prognosis, in particular for patients primarily treated with preoperative chemotherapy. CCSKs are associated with an increased expression of KIT, however, in the absence of gene amplification and/or activating mutation. The potential of anti-KIT therapeutic strategies in the treatment of paediatric renal tumours appears to be limited.
Implications of a congenitally abnormal valve: a study of 1025 consecutively excised aortic valves
Authors: Collins M J , Butany J , Borger M A , Strauss B H , David T E
Published: October 2007

Background:

An increasing proportion of patients with congenitally abnormal aortic valves (AV) present for AV replacement.

Aims:

To review morphological changes in a large contemporary patient population undergoing AV replacement.

Methods:

A detailed review was conducted for all 1025 patients who underwent AV replacement from 2002 to 2005, including the clinical indication for surgery, the type of native AV disease, the pathological changes observed in each valve and the need for related surgery.

Results:

Tricuspid (TAV), bicuspid (BAV) and unicuspid (UAV) aortic valves were observed in 64.5%, 31.9% and 3.0% of all patients respectively. A decreased number of cusps was associated with increasing predilection for male gender (83.9%, 73.4%, 59.2% for UAV, BAV, TAV respectively), a younger patient age at surgery (41.6 (14.3), 61.3 (12.8), 67.5 (12.9) years), and an increased occurrence of pathological changes in the cusps, including calcification of both the cusp and the base, ossification and ulceration. UAV and BAV were also associated with increasing replacement of the ascending aorta due to dilatation and aneurysm formation (54.8, 38.8%, 16.6%). The incidence of infective endocarditis and rheumatic heart disease was 3.8% and 11.2% of all excised valves respectively.

Conclusion:

UAV and BAV were increasingly likely to affect men, fail at an earlier age, and show an increasing incidence of pathological changes in the cusps and ascending aorta than TAV. These results suggest that TAV, BAV and UAV may represent a phenotypic continuum of a similar disease process.
Hepatic fibrosis and cirrhosis in the Fontan circulation: a detailed morphological study
Authors: Kendall T J , Stedman B , Hacking N , Haw M , Vettukattill J J , Salmon A P , Cope R , Sheron N , Millward-Sadler H , Veldtman G R , Iredale J P
Published: October 2007

Aims:

To describe the histological features of the liver in patients with a Fontan circulation.

Methods:

Specimens from liver biopsies carried out as part of preoperative assessment prior to extracardiac cavopulmonary conversion of an older style Fontan were examined and scored semi-quantitatively for pertinent histological features. To support the use of the scoring, biopsy specimens were also ranked by eye for severity to allow correlation with assigned scores.

Results:

Liver biopsy specimens from 18 patients with a Fontan circulation were assessed. All specimens showed sinusoidal fibrosis. In 17 cases there was at least fibrous spur formation, with 14 showing bridging fibrosis and 2 showing frank cirrhosis. In 17 cases at least some of the dense or sinusoidal fibrosis was orcein positive, although a larger proportion of the dense fibrous bands were orcein positive compared with the sinusoidal component. All specimens showed marked sinusoidal dilatation, and 14 showed bile ductular proliferation; 1 showed minimal iron deposition, and 1 showed mild lobular lymphocytic inflammation. There was no cholestasis or evidence of hepatocellular damage. Similar appearances were observed in 2 patients with severe tricuspid regurgitation.

Discussion:

The histological features of the liver in patients with a Fontan circulation are similar to those described in cardiac sclerosis. Sinusoidal dilatation and sinusoidal fibrosis are marked in the Fontan series. The presence of a significant amount of orcein negative sinusoidal fibrosis suggests there may be a remediable component, although the dense fibrous bands are predominantly orcein positive, suggesting chronicity and permanence. No inflammation or hepatocellular damage is evident, suggesting that fibrosis may be mediated by a non-inflammatory mechanism.
Internal consistency in the Odin stratospheric ozone products
Authors: Brohede S , Jones A , Jégou F
Published: October 2007

The two independent instruments on the Odin satellite, the Optical Spectrograph and Infrared Imaging System (OSIRIS) and the Sub-Millimetre Radiometer (SMR) produce atmospheric profiles of various atmospheric species including stratospheric ozone. Comparisons are made between OSIRIS version 3.0 and SMR version 2.1 ozone data to evaluate the consistency of the Odin ozone data sets. Results show good agreement between OSIRIS and SMR in the range 25–40 km, where systematic differences are less than 15% for all latitudes and seasons. Larger systematic differences are seen below 25 km, which can be explained by the increase of various error sources and lower signals. The random differences are between 20–30% in the middle stratosphere. Differences between Odin up-scans and down-scans or AM and PM are insignificant in the middle stratosphere. Furthermore, there is little variation from year to year, but a slight positive trend in the differences (OSIRIS minus SMR) of 0.045 ppmv/year at 30 km over validation period (2002–2006). The fact that the two fundamentally different measurement techniques, (absorption spectroscopy of scattering sunlight and emission measurements in the sub-millimetre region) agree so well, provides confidence in the robustness of both techniques.PACS Nos.: 92.60.Hd, 92.75.Rs, 95.55.Fw, 95.55.Jz
Norcantharidin analogues — Synthesis and evaluation of growth inhibition in a panel of selected tumor-cell lines
Authors: Deng Liping , Shen Li , Zhang Jing , Yang Bo , He Qiaojun , Hu Yongzhou
Published: October 2007

A series of norcantharidin (NCTD) analogues have been synthesized by [3+2]1,3-dipolar cycloaddition reaction of norcantharidin derivatives of substituted aromatic amines with four nitrile oximes. All analogues have been screened for their antiproliferative activity in vitro against a panel of tumor cell lines: KB, SGC-7901, HL60, Bel-7402, HO-8910, and ECA109, producing IC50 values from 0.36 µmol/L to >100 µmol/L. Compound 9d showed potency for the treatment of hepatoma, with IC50 value to Bel-7402 cell line comparable to that of norcantharidin.Key words: norcantharidin analogues, isoxazoline, growth inhibition.
Nucleophilic substitution reactions of N-alkoxyimidoyl fluorides by carbon nucleophiles
Authors: Dolliver Debra D , Delatte David B , Linder Derek B , Johnson James E , Canesco Diana C , Rowe Jeffrey E
Published: October 2007

Nucleophilic substitutions of N-alkoxybenzoimidoyl fluorides [p-ClArC(F)=NOR; R = CH3, i-Pr] by enolate-type ions have been performed to produce compounds that can exist in two tautomeric forms: the imine form{p-ClArC(Y)=NOR [Y = CH(CN)2, CH(CN)(CO2Et), CH(CO2Et)2]}or the enamine form {p-ClArC(NHOR)=C(R1)(R2) [R1, R2 = CN, CO2Et]}. These compounds display varying ratios of imine–enamine tautomerizm in chloroform: the diester compound exists almost solely in the imine form, the dicyano compound exists solely in the enamine form, and the cyano-ester compound exists in both tautomeric forms. Comparison of the imine–enamine equilibrium in chloroform of the cyano-ester compound to an analogue where the p-ClAr group has been replaced by a methyl group reveals that the aromatic ring has a significant effect on the imine–enamine distribution. The former exists in both the imine and enamine form, while the latter exists solely in the enamine form. Complete NMR characterization data for the enamine form of CH3C(NHOCH3)=C(CN)(CO2Et) is also reported. The acidity of p-ClArC(NHOR)=C(CN)2 is demonstrated by a high degree of dissociation of the enamine proton in DMSO. Substitution reactions of p-ClArC(F)=NOi-Pr by Grignard reagents (PhMgBr, CH3MgBr, and PhCCMgBr) have also been performed to produce single geometric isomers of p-ClArC(Ph)=NOi-Pr, p-ClArC(CCPh)=NOi-Pr, and p-ClArC(CH3)=NOi-Pr. A modified synthesis of N-alkoxyimidoyl fluorides, which prevents a competing reaction of the product with the solvent, is reported.Key words: N-alkoxyimidoyl fluoride, enolate ion substitution, Grignard substitution, imine–enamine tautomerizm, carbon acid.