bluuebunny/crossref_metadata_2025 huggingface dataset:
Geology and Genesis of the Ta'ergou Skarn ‐ Quartz Vein Tungsten Deposit in the North Qilian Caledonian Orogenic Belt, Northwest China
Authors: Zhang Zuoheng , Mao Jingwen , Yang Jianmin , Wang Zhiliang , Zhang Zhaochong
Published: November 2008

Abstract. The Ta'ergou tungsten deposit in Gansu province, northwestern China, is located in the western part of the North Qilian Caledonian orogen, and consists of scheelite skarn bodies and wolframite quartz veins. The tungsten‐bearing skarn developed by the replacement of carbonate layers intercalated in the Precambrian schist and amphibolite whereas wolframite‐quartz ore veins developed along a group of fractures that cut through horizontal skarns. The Ta'ergou tungsten deposit is genetically related to the Caledonian Yeniutan granodiorite intrusion and occurs ca. 500 m wide in the exo‐contact zone 300 ∼ 500 m apart from the intrusion. The granodiorite displays a lower grade of differentiation, low content of SiO2 and high contents of mafic components.

There are three types of fluid inclusions in the wolframite‐quartz vein systems, i. e. aqueous, CO2‐H2O and CO2‐rich. The homogenization temperature of aqueous inclusion ranges from 140 to 380d̀C and their salinities from 6.4 to 17.4 equivalent wt% NaCl. Laser Raman spectroscopy shows that the inclusions contain a relatively high content of CO2. The δ34S values of skarn type sulfides range from +8.1 to +12.7 per mil and those of quartz vein sulfides from +9.3 to +14.9 per mil, similar to sulfides of the granodiorite with from +6.0 to +11.7 per mil. The δ18O values of quartz are between +10.5 and +13.3 per mil and those of wolframite between +3.4 and +5.1 per mil. The δ18O water values of ore forming fluids range from +0.6 to +6.4 per mil and suggest the mixture of magmatic fluids with meteoric water formed the ore‐forming fluids.

It has been proved that Precambrian strata in the west sector of North Qilian region are enriched in tungsten. We propose the strata were remelted to be tungsten‐granitoid during subduction. The polymetallic tungsten was gradually accumulated into the roof pendants of the granite intrusion by fractional crystallization and then was deposited by hydrothermal fluids during metasomatism and infilling along fractures. On the other hand, the granite intrusion also acted as “heating machine” to make hydrothermal fluids leach out the metals from Precambrian strata and these metals joined the ore‐forming hydrothermal system.
Magnetotelluric Investigation of the Hydrothermal System and Heat Source in the Muine‐Toyoha Geothermal Area, Hokkaido, Japan
Authors: Takakura Shinichi , Matsushima Nobuo
Published: November 2008

Abstract. Magnetotelluric (MT) surveys were carried out around the Muine volcano, Hokkaido, Japan, where it is expected that the heat and metal source forming the polymetallic Ag‐Pb‐Zn‐Cu‐In Toyoha deposit is present at depth. Measurements were performed at 20 sites, 18 of which were located along a WSW‐ENE profile traversing the north ridge of Mt. Muine. A resistivity model obtained from 2D inversion of the MT data shows subsurface specific conductive and resistive features. Conductive layers are present at the surface of Mt. Muine. The low resistivity is probably due to the clay‐rich rocks associated with the hydrothermal alteration. A high resistivity layer, which corresponds to the pre‐Tertiary Usubetsu Formation, crops out east of Mt. Muine and dips westward. At the west foot of Mt. Muine, relatively high resistive layers are widely exposed. The resistivity increases with depth and exceeds 1000 ohm‐m. This fact indicates that this region is not influenced by the recent hydrothermal activity. An extremely conductive zone about 3–6 km wide and 6–9 km thick exists at a depth of 2 km below Mt. Muine. This zone mostly corresponds to an elastic wave attenuation zone detected by a seismic survey. It is interpreted as a large hydrothermal reservoir or melted magma, which is a heat source of the hydrothermal system in this area.
Chemical Composition of Ores from the Takara Volcanogenic Massive Sulfide Deposit, Central Japan
Authors: Yakushi Daigoro , Enjoji Mamoru
Published: November 2008

Abstract. The Takara volcanogenic massive sulfide (VMS) deposit occurs in Miocene formation of the Misaka Mountain, the South Fossa Magna region, central Japan. The tectonic setting of the Misaka Mountain is reconstructed to be a part of the paleo Izu‐Ogasawara arc which collided with the Honshu arc and to form accreted body in the present position. The Takara deposit, therefore, is considered to have formed in the paleo Izu‐Ogasawara arc.

The ores from the Takara deposit are classified into pyrite‐type ore, chalcopyrite‐type ore, and sphalerite‐type ore on the basis of chemical composition and their mineral assemblages. Some pyrite‐type ores are characterized by their high Au content. The Au content is hardly recognized in the chalcopyrite‐type and sphalerite‐type ores.

The ores from the Takara deposit have intermediate bulk chemical composition between those from the Besshi‐type deposits and the Kuroko‐type deposits that are two representative VMS deposits. However, the bulk chemical composition is closer to that from the Kuroko‐type deposits. And moreover, chemical composition of tetrahedrite‐tennantite series minerals (tetrahedrite) is similar to that from the Kuroko‐type deposits. The bulk chemical composition (Cu, Zn, Co, Pb, and As contents) of ores is affected by the chemical composition of volcanic rocks associated with VMS deposits.
Der Opa‐Boom — Eine historische Chance für das öffentliche Krankenhauswesen Frankreichs
Authors: Holcman Robert
Published: November 2008

Auszug  In Frankreich entfällt auf das öffentliche Krankenhauswesen ein Großteil der Ausgaben für soziale Sicherheit, und es wird zurzeit verschiedenen Reformen unterzogen, die sein finanzielles Gleichgewicht und die interne Arbeitsorganisation tief greifend verändern. Dennoch konnte es die ihm auferlegten Produktivitätsanforderungen nicht erfüllen: Bezeichnenderweise beanspruchen die Löhne drei Viertel der Ausgaben. Die meisten Angestellten haben Beamtenstatus und können nicht einfach entlassen werden. Einzig die derzeitige demografische Entwicklung in der französischen Bevölkerung, die zur Folge haben wird, dass die Hälfte des Personalbestandes der öffentlichen Krankenhäuser im kommenden Jahrzehnt aus dem Dienst ausscheiden wird, kann zu einer deutlichen Verminderung des Personalbestandes führen.
Gpnmb is a melanoblast‐expressed, MITF‐dependent gene
Authors: Loftus Stacie K. , Antonellis Anthony , Matera Ivana , Renaud Gabriel , Baxter Laura L. , Reid Duncan , Wolfsberg Tyra G. , Chen Yidong , Wang ChenWei ,   NISC Comparative Sequencing Program, Prasad Megana K. , Bessling Seneca L. , McCallion Andrew S. , Green Eric D. , Bennett Dorothy C. , Pavan William J.
Published: November 2008

Expression profile analysis clusters Gpnmb with known pigment genes, Tyrp1, Dct, and Si. During development, Gpnmb is expressed in a pattern similar to Mitf, Dct and Si with expression vastly reduced in Mitf mutant animals. Unlike Dct and Si, Gpnmb remains expressed in a discrete population of caudal melanoblasts in Sox10‐deficient embryos. To understand the transcriptional regulation of Gpnmb we performed a whole genome annotation of 2,460,048 consensus MITF binding sites, and cross‐referenced this with evolutionarily conserved genomic sequences at the GPNMB locus. One conserved element, GPNMB‐MCS3, contained two MITF consensus sites, significantly increased luciferase activity in melanocytes and was sufficient to drive expression in melanoblasts in vivo. Deletion of the 5′‐most MITF consensus site dramatically reduced enhancer activity indicating a significant role for this site in Gpnmb transcriptional regulation. Future analysis of the Gpnmb locus will provide insight into the transcriptional regulation of melanocytes, and Gpnmb expression can be used as a marker for analyzing melanocyte development and disease progression.
Stem cell factor induces ERM proteins phosphorylation through PI3K activation to mediate melanocyte proliferation and migration
Authors: Jeon Songhee , Kim Nan‐Hyung , Kim Ji‐Young , Lee Ai‐Young
Published: November 2008

Stem cell factor (SCF) activates a variety of signals associated with stimulation of proliferation, differentiation, migration, and survival in melanocytes. However, the molecular mechanisms by which SCF and its receptor Kit activates these signaling pathways simultaneously and independently are still poorly defined. Here, we examined whether SCF induces ezrin/radixin/moesin (ERM) proteins phosphorylation as a downstream target of PI3K in melanocytes. ERM proteins are cross‐linkers between the plasma membrane and the actin cytoskeleton and are activated by phosphorylation of a C‐terminal threonine residue. Our results demonstrated that SCF‐induced ERM proteins phosphorylation on threonine residue and Rac1 activation in cultured normal human melanocytes through the activation of PI3K. The functional role of phosphorylated‐ERM proteins was examined using melanocytes infected with adenovirus carrying a dominant negative mutant (Ala‐558, TA) or wild type of moesin. In the TA moesin‐overexpressing melanocytes, SCF‐induced cell proliferation and migration were inhibited. Thus, our results indicate that phosphorylation of ERM proteins plays an important role in the regulation of SCF‐induced melanocyte proliferation and migration.
Positive crosstalk between ERK and p38 in melanoma stimulates migration and in vivo proliferation
Authors: Estrada Yeriel , Dong Jianli , Ossowski Liliana
Published: November 2008

Melanoma is one of the most therapy‐resistant cancers. Activating mutations in BRAF and NRAS are the source of extracellular signal regulated protein kinase (ERK) pathway activation. We show that melanoma cell lines, originating in different metastatic sites, with BRAF or NRAS mutations, in addition to active mitogen activated protein kinase (MAPK)–ERK, also have highly activated stress activated protein kinase (SAPK)‐p38. This is in direct contrast to carcinoma cells in which the activity of the two kinases appears to be mutually exclusive; high level of p38 activity inhibits, through a negative feedback, ERK activity and prevents tumorigenesis. Melanomas are insensitive to ERK inhibition by p38 and utilize p38‐signaling pathway for migration and growth in vivo. We found a positive functional loop linking the high ERK activity to surface expression of αVβ3‐integrin. This integrin, by interacting with vitronectin, induces p38 activity and increases IL‐8 production, enhancing cell migration. Because the negative loop from p38 to ERK is lost, the two kinases can remain simultaneously activated. Inhibition of ERK and p38 activities is more effective in blocking in vivo growth than inhibition of each kinase individually. Future therapies might have to consider targeting of both pathways.
Arrhythmogenic actions of the Ca2+channel agonist FPL‐64716 in Langendorff‐perfused murine hearts
Authors: Ghais Nina S. , Zhang Yanmin , Grace Andrew A. , Huang Christopher L.‐H.
Published: November 2008

The experiments explored the extent to which alterations in L‐type Ca2+channel‐mediated Ca2+entry triggers Ca2+‐mediated arrhythmogenesis in Langendorff‐perfused murine hearts through use of the specific L‐type Ca2+channel modulator FPL‐64716 (FPL). Introduction of FPL (1 μm) resulted in a gradual development (>10 min) of diastolic electrical events and alternans in spontaneously beating hearts from which monophasic action potentials were recorded. In regularly paced hearts, they additionally led to non‐sustained and sustained ventricular tachycardia (nsVT and sVT). Programmed electrical stimulation (PES) resulted in nsVT and sVT after 5–10 and >10 min perfusion, respectively. Pretreatments with nifedipine, diltiazem and cyclopiazonic acid abolished arrhythmogenic tendency induced by subsequent introduction of FPL, consistent with its dependence upon both extracellular Ca2+entry and the degree of filling of the sarcoplasmic reticular Ca2+store. Values for action potential duration at 90% repolarization when any of these agents were applied to FPL‐treated hearts became indistinguishable from those shown by untreated control hearts, in contrast to earlier reports of their altering in long QT syndrome type 3 and hypokalaemic murine models for re‐entrant arrhythmogenesis. These arrhythmic effects instead correlated with alterations in Ca2+homeostasis at the single‐cell level found in investigations of the effects of both FPL and the same agents in regularly stimulated fluo−3 loaded myocytes. These findings are compatible with a prolonged extracellular Ca2+entry that potentially results in an intracellular Ca2+overload and produces the cardiac arrhythmogenecity following addition of FPL.
The action of high K+ and aglycaemia on the electrical properties and synaptic transmission in rat intracardiac ganglion neurones in vitro
Authors: Dyavanapalli Jhansi , Rimmer Katrina , Harper Alexander A.
Published: November 2008

We have investigated the action of two elements of acute ischaemia, high potassium and aglycaemia, on the electrophysiological properties and ganglionic transmission of adult rat intracardiac ganglion (ICG) neurones. We used a whole‐mount ganglion preparation of the right atrial ganglion plexus and sharp microelectrode recording techniques. Increasing extracellular K+ from its normal value of 4.7 mm to 10 mm decreased membrane potential and action potential after‐hyperpolarization amplitude but otherwise had no effect on postganglionic membrane properties. It did, however, reduce the ability of synaptically evoked action potentials to follow high‐frequency (100 Hz) repetitive stimulation. A further increase in K+ changed both the passive and the active membrane properties of the postganglionic neurone: time constant, membrane resistance and action potential overshoot were all decreased in high K+ (20 mm). The ICG neurones display a predominantly phasic discharge in response to prolonged depolarizing current pulses. High K+ had no impact on this behaviour but reduced the time‐dependent rectification response to hyperpolarizing currents. At 20 mm, K+ practically blocked ganglionic transmission in most neurones at all frequencies tested. Aglycaemia, nominally glucose‐free physiological saline solution (PSS), increased the time constant and membrane resistance of ICG neurones but otherwise had no action on their passive or active properties or ganglionic transmission. However, the combination of aglycaemia and 20 mm K+ displayed an improvement in passive properties and ganglionic transmission when compared with 20 mm K+ PSS. These data indicate that the presynaptic terminal is the primary target of high extracellular potassium and that aglycaemia may have protective actions against this challenge.
Specific Detection of Viable
Legionella
Cells by Combined Use of Photoactivated Ethidium Monoazide and PCR/Real-Time PCR
Authors: Chang Bin , Sugiyama Kanji , Taguri Toshitsugu , Amemura-Maekawa Junko , Kura Fumiaki , Watanabe Haruo
Published: November 2008

Legionella
organisms are prevalent in manmade water systems and cause legionellosis in humans. A rapid detection method for viable
Legionella
cells combining ethidium monoazide (EMA) and PCR/real-time PCR was assessed. EMA could specifically intercalate and cleave the genomic DNA of heat- and chlorine-treated dead
Legionella
cells. The EMA-PCR assay clearly showed an amplified fragment specific for
Legionella
DNA from viable cells, but it could not do so for DNA from dead cells. The number of EMA-treated dead
Legionella
cells estimated by real-time PCR exhibited a 10
4
- to 10
5
-fold decrease compared to the number of dead
Legionella
cells without EMA treatment. Conversely, no significant difference in the numbers of EMA-treated and untreated viable
Legionella
cells was detected by the real-time PCR assay. The combined assay was also confirmed to be useful for specific detection of culturable
Legionella
cells from water samples obtained from spas. Therefore, the combined use of EMA and PCR/real-time PCR detects viable
Legionella
cells rapidly and specifically and may be useful in environmental surveillance for
Legionella
.
Biodiversity of Thermophilic Prokaryotes with Hydrolytic Activities in Hot Springs of Uzon Caldera, Kamchatka (Russia)
Authors: Kublanov Ilya V. , Perevalova Anna A. , Slobodkina Galina B. , Lebedinsky Aleksander V. , Bidzhieva Salima K. , Kolganova Tatyana V. , Kaliberda Elena N. , Rumsh Lev D. , Haertlé Thomas , Bonch-Osmolovskaya Elizaveta A.
Published: November 2008

Samples of water from the hot springs of Uzon Caldera with temperatures from 68 to 87°C and pHs of 4.1 to 7.0, supplemented with proteinaceous (albumin, casein, or α- or β-keratin) or carbohydrate (cellulose, carboxymethyl cellulose, chitin, or agarose) biological polymers, were filled with thermal water and incubated at the same sites, with the contents of the tubes freely accessible to the hydrothermal fluid. As a result, several enrichment cultures growing in situ on different polymeric substrates were obtained. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene fragments obtained after PCR withBacteria-specific primers showed that the bacterial communities developing on carbohydrates included the generaCaldicellulosiruptorandDictyoglomusand that those developing on proteins contained members of theThermotogalesorder. DGGE analysis performed after PCR withArchaea- andCrenarchaeota-specific primers showed that archaea related to uncultured environmental clones, particularly those of theCrenarchaeotaphylum, were present in both carbohydrate- and protein-degrading communities. Five isolates obtained from in situ enrichments or corresponding natural samples of water and sediments represented the bacterial generaDictyoglomusandCaldanaerobacteras well as new archaea of theCrenarchaeotaphylum. Thus, in situ enrichment and consequent isolation showed the diversity of thermophilic prokaryotes competing for biopolymers in microbial communities of terrestrial hot springs.
Improved Thermostability and Acetic Acid Tolerance of
Escherichia coli
via Directed Evolution of Homoserine
o
-Succinyltransferase
Authors: Mordukhova Elena A. , Lee Hee-Soon , Pan Jae-Gu
Published: November 2008

In
Escherichia coli
, growth is limited at elevated temperatures mainly because of the instability of a single enzyme, homoserine
o
-succinyltransferase (MetA), the first enzyme in the methionine biosynthesis pathway. The
metA
gene from the thermophile
Geobacillus kaustophilus
cloned into the
E. coli
chromosome was found to enhance the growth of the host strain at elevated temperature (44°C), thus confirming the limited growth of
E. coli
due to MetA instability. In order to improve
E. coli
growth at higher temperatures, we used random mutagenesis to obtain a thermostable MetA

E. coli

protein. Sequencing of the thermotolerant mutant showed five amino acid substitutions: S61T, E213V, I229T, N267D, and N271K. An
E. coli
strain with the mutated
metA
gene chromosomally inserted showed accelerated growth over a temperature range of 34 to 44°C. We used the site-directed
metA
mutants to identify two amino acid residues responsible for the sensitivity of MetA

E. coli

to both heat and acids. Replacement of isoleucine 229 with threonine and asparagine 267 with aspartic acid stabilized the protein. The thermostable MetA

E. coli

enzymes showed less aggregation in vivo at higher temperature, as well as upon acetic acid treatment. The data presented here are the first to show improved
E. coli
growth at higher temperatures solely due to MetA stabilization and provide new knowledge for designing
E. coli
strains that grow at higher temperatures, thus reducing the cooling cost of bioprocesses.
Binding Specificity of the Lantibiotic-Binding Immunity Protein NukH
Authors: Okuda Ken-ichi , Yanagihara Sae , Shioya Kouki , Harada Yoshitaka , Nagao Jun-ichi , Aso Yuji , Zendo Takeshi , Nakayama Jiro , Sonomoto Kenji
Published: November 2008

NukH is a lantibiotic-binding immunity protein that shows strong binding activity against type A(II) lantibiotics. In this study, the binding specificity of NukH was analyzed by using derivatives of nukacin ISK-1, which is a type A(II) lantibiotic produced by
Staphylococcus warneri
ISK-1. Interactions between cells of
Lactococcus lactis
transformants expressing
nukH
and nukacin ISK-1 derivatives were analyzed by using a quantitative peptide-binding assay. Differences in the cell-binding rates of each nukacin ISK-1 derivative suggested that three lysine residues at positions 1 to 3 of nukacin ISK-1 contribute to the effective binding of nukacin ISK-1 to
nukH
-expressing cells. The binding levels of mutants with lanthionine and dehydrobutyrine substitutions (S11A nukacin
4-27
and T24A nukacin
4-27
, respectively) to
nukH
-expressing cells were considerably lower than those of nukacin
4-27
, suggesting that unusual amino acids play a decisive role in NukH recognition. Additionally, it was suggested that T9A nukacin
4-27
, a mutant with a 3-methyllanthionine substitution, binds to NukH via an intermolecular disulfide bond after it is weakly recognized by NukH. We succeeded in the detection of specific type A(II) lantibiotics from the culture supernatants of various bacteriocin producers by using the binding specificity of
nukH
-expressing cells.
Novel Family of Carbohydrate Esterases, Based on Identification of the
Hypocrea jecorina
Acetyl Esterase Gene
Authors: Li Xin-Liang , Skory Christopher D. , Cotta Michael A. , Puchart Vladimir , Biely Peter
Published: November 2008

Plant cell walls have been shown to contain acetyl groups in hemicelluloses and pectin. The gene
aes1
, encoding the acetyl esterase (Aes1) of
Hypocrea jecorina
, was identified by amino-terminal sequencing, peptide mass spectrometry, and genomic sequence analyses. The coded polypeptide had 348 amino acid residues with the first 19 serving as a secretion signal peptide. The calculated molecular mass and isoelectric point of the secreted enzyme were 37,088 Da and pH 5.89, respectively. No significant homology was found between the predicated Aes1 and carbohydrate esterases of known families, but putative
aes1
orthologs were found in genomes of many fungi and bacteria that produce cell wall-degrading enzymes. The
aes1
transcript levels were high when the fungal cells were induced with sophorose, cellulose, oat spelt xylan, lactose, and arabinose. The recombinant Aes1 produced by
H. jecorina
transformed with
aes1
under the cellobiohydrolase I promoter displayed properties similar to those reported for the native enzyme. The enzyme hydrolyzed acetate ester bond specifically. Using 4-nitrophenyl acetate as substrate, the activity of the recombinant enzyme was enhanced by
d
-xylose,
d
-glucose, cellobiose,
d
-galactose, and xylooligosaccharides but not by arabinose, mannose, or lactose. With the use of 4-nitrophenyl-β-
d
-xylopyranoside monoacetate as substrate in a β-xylosidase-coupled assay, Aes1 hydrolyzed positions 3 and 4 with the same efficiency while the
H. jecorina
acetylxylan esterase 1 exclusively deacetylated the position 2 acetyl group. Aes1 was capable of transacetylating methylxyloside in aqueous solution. The data presented demonstrate that Aes1 and other homologous microbial proteins may represent a new family of esterases for lignocellulose biodegradation.
Isolation and Characterization of Methicillin-Resistant
Staphylococcus aureus
Strains from Louisiana Retail Meats
Authors: Pu Shuaihua , Han Feifei , Ge Beilei
Published: November 2008

We investigated the prevalence of
Staphylococcus aureus
and methicillin-resistant
S. aureus
(MRSA) in 120 retail meat samples from 30 grocery stores in Baton Rouge, LA.
S. aureus
strains were recovered from 45.6% of pork samples and 20% of beef samples, whereas MRSA strains were isolated from six meat samples (five pork samples and one beef sample). The MRSA isolates were of two strain types (clones), one harboring Panton-Valentine leucocidin and belonging to pulsed-field gel electrophoresis type USA300 and the other one belonging to USA100.
Microbial Community Composition and Denitrifying Enzyme Activities in Salt Marsh Sediments
Authors: Cao Yiping , Green Peter G. , Holden Patricia A.
Published: November 2008

Denitrifying microbial communities and denitrification in salt marsh sediments may be affected by many factors, including environmental conditions, nutrient availability, and levels of pollutants. The objective of this study was to examine how microbial community composition and denitrification enzyme activities (DEA) at a California salt marsh with high nutrient loading vary with such factors. Sediments were sampled from three elevations, each with different inundation and vegetation patterns, across 12 stations representing various salinity and nutrient conditions. Analyses included determination of cell abundance, total and denitrifier community compositions (by terminal restriction fragment length polymorphism), DEA, nutrients, and eluted metals. Total bacterial (16S rRNA) and denitrifier (
nirS
) community compositions and DEA were analyzed for their relationships to environmental variables and metal concentrations via multivariate direct gradient and regression analyses, respectively. Community composition and DEA were highly variable within the dynamic salt marsh system, but each was strongly affected by elevation (i.e., degree of inundation) and carbon content as well as by selected metals. Carbon content was highly related to elevation, and the relationships between DEA and carbon content were found to be elevation specific when evaluated across the entire marsh. There were also lateral gradients in the marsh, as evidenced by an even stronger association between community composition and elevation for a marsh subsystem. Lastly, though correlated with similar environmental factors and selected metals, denitrifier community composition and function appeared uncoupled in the marsh.
Terminal Restriction Fragment Length Polymorphism for Identification of
Cryptosporidium
Species in Human Feces
Authors: Waldron L. S. , Ferrari B. C. , Gillings M. R. , Power M. L.
Published: November 2008

Effective management of human cryptosporidiosis requires efficient methods for detection and identification of the species of
Cryptosporidium
isolates. Identification of isolates to the species level is not routine for diagnostic assessment of cryptosporidiosis, which leads to uncertainty about the epidemiology of the
Cryptosporidium
species that cause human disease. We developed a rapid and reliable method for species identification of
Cryptosporidium
oocysts from human fecal samples using terminal restriction fragment polymorphism (T-RFLP) analysis of the 18S rRNA gene. This method generated diagnostic fragments unique to the species of interest. A panel of previously identified isolates of species was blind tested to validate the method, which determined the correct species identity in every case. The T-RFLP profiles obtained for samples spiked with known amounts of
Cryptosporidium hominis
and
Cryptosporidium parvum
oocysts generated the two expected diagnostic peaks. The detection limit for an individual species was 1% of the total DNA. This is the first application of T-RFLP to protozoa, and the method which we developed is a rapid, repeatable, and cost-effective method for species identification.
Biotechnological Process for Production of β-Dipeptides from Cyanophycin on a Technical Scale and Its Optimization
Authors: Sallam Ahmed , Kast Alene , Przybilla Simon , Meiswinkel Tobias , Steinbüchel Alexander
Published: November 2008

A triphasic process was developed for the production of β dipeptides from cyanophycin (CGP) on a large scale. Phase I comprises an optimized acid extraction method for technical isolation of CGP from biomass. It yielded highly purified CGP consisting of aspartate, arginine, and a little lysine. Phase II comprises the fermentative production of an extracellular CGPase (CphE
al
) from
Pseudomonas alcaligenes
strain DIP1 on a 500-liter scale in mineral salts medium, with citrate as the sole carbon source and CGP as an inductor. During optimization, it was shown that 2 g liter
−1
citrate, pH 6.5, and 37°C are ideal parameters for CphE
al
production. Maximum enzyme yields were obtained after induction in the presence of 50 mg liter
−1
CGP or CGP dipeptides for 5 or 3 h, respectively. Aspartate at a concentration of 4 g liter
−1
induced CphE
al
production with only about 30% efficiency in comparison to that with CGP. CphE
al
was purified utilizing its affinity for the substrate and its specific binding to CGP. CphE
al
turned out to be a serine protease with maximum activity at 50°C and at pH 7 to 8.5. Phase III comprises degradation of CGP to β-aspartate-arginine and β-aspartate-lysine dipeptides with a purity of over 99% (by thin-layer chromatography and high-performance liquid chromatography), employing a crude CphE
al
preparation. Optimum degradation parameters were 100 g liter
−1
CGP, 10 g liter
−1
crude CphE
al
powder, and 4 h of incubation at 50°C. The overall efficiency of phase III was 91%, while 78% (wt/wt) of the used CphE
al
powder with sustained activity toward CGP was recovered. The optimized process was performed with industrial materials and equipment and is applicable to any desired scale.
Abundances of Hyperthermophilic Autotrophic Fe(III) Oxide Reducers and Heterotrophs in Hydrothermal Sulfide Chimneys of the Northeastern Pacific Ocean
Authors: Ver Eecke Helene C. , Kelley Deborah S. , Holden James F.
Published: November 2008

The abundances of hyperthermophilic heterotrophs, methanogens, and autotrophic reducers of amorphous Fe(III) oxide in 18 samples of deep-sea hydrothermal vent sulfide chimneys of the Endeavour Segment were measured. The results indicate that conditions favor the growth of iron reducers toward the interiors of these deposits and that of heterotrophs toward the outer surfaces near high-temperature polychaete worms (Paralvinella sulfincola).
Developmental and Microbiological Analysis of the Inception of Bioluminescent Symbiosis in the Marine FishNuchequula nuchalis(Perciformes: Leiognathidae)
Authors: Dunlap Paul V. , Davis Kimberly M. , Tomiyama Shinichi , Fujino Misato , Fukui Atsushi
Published: November 2008

Many marine fish harbor luminous bacteria as bioluminescent symbionts. Despite the diversity, abundance, and ecological importance of these fish and their apparent dependence on luminous bacteria for survival and reproduction, little is known about developmental and microbiological events surrounding the inception of their symbioses. To gain insight on these issues, we examined wild-caught larvae of the leiognathid fishNuchequula nuchalis, a species that harborsPhotobacterium leiognathias its symbiont, for the presence, developmental state, and microbiological status of the fish's internal, supraesophageal light organ. Nascent light organs were evident in the smallest specimens obtained, flexion larvae of 6.0 to 6.5 mm in notochord length (NL), a developmental stage at which the stomach had not yet differentiated and the nascent gasbladder had not established an interface with the light organ. Light organs of certain of the specimens in this size range apparently lacked bacteria, whereas light organs of other specimens of 6.5 mm in NL and of all larger specimens harbored large populations of bacteria, representatives of which were identified asP. leiognathi. Bacteria identified asVibrio harveyiwere also present in the light organ of one larval specimen. Light organ populations were composed typically of two or three genetically distinct strain types ofP. leiognathi, similar to the situation in adult fish, and the same strain type was only rarely found in light organs of different larval, juvenile, or adult specimens. Light organs of larvae carried a smaller proportion of strains merodiploid for thelux-riboperon, 79 of 249 strains, than those of adults (75 of 91 strains). These results indicate that light organs ofN. nuchalisflexion and postflexion larvae of 6.0 to 6.7 mm in NL are at an early stage of development and that inception of the symbiosis apparently occurs in flexion larvae of 6.0 to 6.5 mm in NL. Ontogeny of the light organ therefore apparently precedes acquisition of the symbiotic bacteria. Furthermore, bacterial populations in larval light organs near inception of the symbiosis are genetically diverse, like those of adult fish.
Prevalence and Genotypes of
Anaplasma
Species and Habitat Suitability for Ticks in a Mediterranean Ecosystem
Authors: Torina Alessandra , Alongi Angelina , Naranjo Victoria , Estrada-Peña Agustín , Vicente Joaquín , Scimeca Salvatore , Marino Anna M. F. , Salina Felice , Caracappa Santo , de la Fuente José
Published: November 2008

Anaplasma
species are tick-transmitted pathogens that impact veterinary and human health. Sicily is one of the locations where these pathogens are endemic. Sicily represents a typical Mediterranean ecosystem to study
Anaplasma
infection and tick habitat suitability. The aims of this study were (i) to characterize by 16S rRNA and species-specific
msp4
gene PCR the prevalence and genotypes of
A. marginale
,
A. phagocytophilum
, and
A. ovis
in the most abundant host species in Sicilian provinces and (ii) to correlate differences between hosts and between western and eastern Sicily with the habitat suitability for ticks in these regions. Differences were found in the prevalence of
Anaplasma
spp. between different hosts and between western and eastern provinces. The differences in
Anaplasma
prevalence between different hosts may be explained by pathogen host tropism. The differences between western and eastern provinces correlated with the tick habitat suitability in these regions. The analysis of
Anaplasma
genotypes suggested a higher host and regional specificity for
A. phagocytophilum
than for
A. marginale
and
A. ovis
strains, a finding probably associated with the broader host range of
A. phagocytophilum
. The presence of identical
A. marginale
genotypes in the two regions may reflect cattle movement. The results for
A. ovis
suggested the possibility of some genotypes being host specific. These results provide information potentially useful for the management of tick-borne diseases caused by
Anaplasma
spp. in Sicily and other Mediterranean regions and may contribute to the development of models to predict the risks for these tick-borne pathogens.
Selective Extraction of Bacterial DNA from the Surfaces of Macroalgae
Authors: Burke Catherine , Kjelleberg Staffan , Thomas Torsten
Published: November 2008

A novel method has been developed for the selective extraction of DNA from surface-associated bacterial communities from the two model marine benthic algaeUlva australisandDelisea pulchra. The extracted DNA had no detectable contamination with host DNA, was recovered in high yield and quality, and was representative of the bacterial community on the algal surfaces. The DNA is suitable for a variety of subsequent applications, including the construction of large-insert clone libraries and metagenomic sequencing.
Use of 4-Sulfophenyl Isothiocyanate Labeling and Mass Spectrometry To Determine the Site of Action of the Streptococcolytic Peptidoglycan Hydrolase Zoocin A
Authors: Gargis Shaw R. , Heath Harry E. , Heath Lucie S. , LeBlanc Paul A. , Simmonds Robin S. , Abbott Brian D. , Timkovich Russell , Sloan Gary L.
Published: November 2008

Zoocin A is a streptococcolytic peptidoglycan hydrolase with an unknown site of action that is produced by
Streptococcus equi
subsp.
zooepidemicus
4881. Zoocin A has now been determined to be a
d
-alanyl-
l
-alanine endopeptidase by digesting susceptible peptidoglycan with a combination of mutanolysin and zoocin A, separating the resulting muropeptides by reverse-phase high-pressure liquid chromatography, and analyzing them by mass spectrometry (MS) in both the positive- and negative-ion modes to determine their compositions. In order to distinguish among possible structures for these muropeptides, they were N-terminally labeled with 4-sulfophenyl isothiocyanate (SPITC) and analyzed by tandem MS in the negative-ion mode. This novel application of SPITC labeling and MS/MS analysis can be used to analyze the structure of peptidoglycans and to determine the sites of action of other peptidoglycan hydrolases.
Effect of Tree Species and Mycorrhizal Colonization on the Archaeal Population of Boreal Forest Rhizospheres
Authors: Bomberg Malin , Timonen Sari
Published: November 2008

Group 1.1c
Crenarchaeota
are the predominating archaeal group in acidic boreal forest soils. In this study, we show that the detection frequency of 1.1c crenarchaeotal 16S rRNA genes in the rhizospheres of the boreal forest trees increased following colonization by the ectomycorrhizal fungus
Paxillus involutus
. This effect was very clear in the fine roots of
Pinus sylvestris
,
Picea abies
, and
Betula pendula
, the most common forest trees in Finland. The nonmycorrhizal fine roots had a clearly different composition of archaeal 16S rRNA genes in comparison to the mycorrhizal fine roots. In the phylogenetic analysis, the 1.1c crenarchaeotal 16S rRNA gene sequences obtained from the fine roots formed a well-defined cluster separate from the mycorrhizal ones.
Alnus glutinosa
differed from the other trees by having high diversity and detection levels of
Crenarchaeota
both on fine roots and on mycorrhizas as well as by harboring a distinct archaeal flora. The similarity of the archaeal populations in rhizospheres of the different tree species was increased upon colonization by the ectomycorrhizal fungus. A minority of the sequences obtained from the mycorrhizas belonged to
Euryarchaeota
(order
Halobacteriales
).
Identification and Characterization of the Asperthecin Gene Cluster of
Aspergillus nidulans
Authors: Szewczyk Edyta , Chiang Yi-Ming , Oakley C. Elizabeth , Davidson Ashley D. , Wang Clay C. C. , Oakley Berl R.
Published: November 2008

The sequencing of
Aspergillus
genomes has revealed that the products of a large number of secondary metabolism pathways have not yet been identified. This is probably because many secondary metabolite gene clusters are not expressed under normal laboratory culture conditions. It is, therefore, important to discover conditions or regulatory factors that can induce the expression of these genes. We report that the deletion of
sumO
, the gene that encodes the small ubiquitin-like protein SUMO in
A. nidulans
, caused a dramatic increase in the production of the secondary metabolite asperthecin and a decrease in the synthesis of austinol/dehydroaustinol and sterigmatocystin. The overproduction of asperthecin in the
sumO
deletion mutant has allowed us, through a series of targeted deletions, to identify the genes required for asperthecin synthesis. The asperthecin biosynthesis genes are clustered and include genes encoding an iterative type I polyketide synthase, a hydrolase, and a monooxygenase. The identification of these genes allows us to propose a biosynthetic pathway for asperthecin.
Cloning of the
Trichoderma reesei
cDNA Encoding a Glucuronan Lyase Belonging to a Novel Polysaccharide Lyase Family
Authors: Konno Naotake , Igarashi Kiyohiko , Habu Naoto , Samejima Masahiro , Isogai Akira
Published: November 2008

The filamentous fungus
Trichoderma reesei
produces glucuronan lyase (TrGL) when it is grown on β-(1→4)-polyglucuronate (cellouronate) as a sole carbon source. The cDNA encoding TrGL was cloned, and the recombinant enzyme was heterologously expressed in
Pichia pastoris
. The cDNA of TrGL includes a 777-bp open reading frame encoding a 20-amino-acid signal peptide and the 238-amino-acid mature protein. The amino acid sequence showed no similarity to the amino acid sequences of previously described functional proteins, indicating that the enzyme should be classified in a novel polysaccharide lyase (PL) family. Recombinant TrGL catalyzed depolymerization of cellouronate endolytically by β-elimination and was highly specific for cellouronate. The enzyme was most active at pH 6.5 and 50°C, and its activity and thermostability increased in the presence of Ca
2+
, suggesting that its calcium dependence is similar to that of other PLs, such as pectate lyases.
Case-Control Study To Determine whether River Water Can Spread Tetracycline Resistance to Unexposed Impala (
Aepyceros melampus
) in Kruger National Park (South Africa)
Authors: Mariano V. , McCrindle C. M. E. , Cenci-Goga B. , Picard J. A.
Published: November 2008

A case-control study was performed in the Kruger National Park (KNP), South Africa, to find out whether impala (
Aepyceros melampus
) were more likely to harbor tetracycline-resistant
Escherichia coli
(TREC) in their feces when they drank from rivers that contained these bacteria than when they drank from rivers that were uncontaminated with TREC. The following five perennial rivers were selected: the Crocodile, the Letaba, the Olifants, the Sabie, and the Sand. Samples of river water (
n
= 33) and feces (
n
= 209), collected at 11 different sites, were cultured for
E. coli.
The resulting colonies were screened for tetracycline resistance by use of the Lederberg replica plating method (breakpoint, 4 mg/liter). A resistant and/or a susceptible isolate was then selected from each sample and subjected to the CLSI MIC broth microdilution test for tetracyclines. Among the 21 water specimens contaminated by
E. coli
, 19.05% (
n
= 4) were found to be resistant by the MIC method (breakpoint, ≥8 mg/liter). This led to the Crocodile, Olifants, and Letaba rivers being classified as TREC positive. Among the 209 impala feces sampled, 191 were positive for the presence of
E. coli
(91.38%). Within these (
n
= 191), 9.95% (
n
= 19) of the isolates were shown to be TREC by the MIC method. It was found that 1.11% (
n
= 1) of the
E. coli
isolates cultured from the feces of the control group (
n
= 90) were TREC, in comparison with 17.82% (
n
= 18) of those in feces from the exposed group (
n
= 101). The calculation of the odds ratio showed that impala drinking from TREC-contaminated rivers were 19.3 (2.63 to 141.69) times more likely to be infected with TREC than were unexposed impala. This is a significant finding, indicating that surface water could be a possible source of antimicrobial resistance in naïve animal populations and that impala could act as sentinels for antimicrobial resistance.
Detection and Characterization of the Fimbrial
sfp
Cluster in Enterohemorrhagic
Escherichia coli
O165:H25/NM Isolates from Humans and Cattle
Authors: Bielaszewska Martina , Prager Rita , Vandivinit Liz , Müsken Anne , Mellmann Alexander , Holt Nicholas J. , Tarr Phillip I. , Karch Helge , Zhang Wenlan
Published: November 2008

The
sfp
cluster, encoding Sfp fimbriae and located in the large plasmid of sorbitol-fermenting (SF) enterohemorrhagic
Escherichia coli
(EHEC) O157 (pSFO157), has been considered a unique characteristic of this organism. We discovered and then characterized the
sfp
cluster in EHEC O165:H25/NM (nonmotile) isolates of human and bovine origin. All seven strains investigated harbored a complete
sfp
cluster (carrying
sfpA
,
sfpH
,
sfpC
,
sfpD
,
sfpJ
,
sfpF
, and
sfpG
) of 6,838 bp with >99% nucleotide sequence homology to the
sfp
cluster of SF EHEC O157:NM. The
sfp
cluster in EHEC O165:H25/NM strains was located in an ∼80-kb (six strains) or ∼120-kb (one strain) plasmid which differed in structure, virulence genes, and
sfp
flanks from pSFO157. All O165:H25/NM strains belonged to the same multilocus sequence type (ST119) and were only distantly phylogenetically related to SF EHEC O157:NM (ST11). The highly conserved
sfp
cluster in different clonal backgrounds suggests that this segment was acquired independently by EHEC O165:H25 and SF EHEC O157:NM. Its presence in an additional EHEC serotype extends the diagnostic utility of PCR targeting
sfpA
as an easy and efficient approach to seek EHEC in patients' stools. The reasons for the convergence of pathogenic EHEC strains on a suite of virulence loci remain unknown.
Genetic Diversity and Ecological Success of
Staphylococcus aureus
Strains Colonizing Humans
Authors: Sakwinska Olga , Kuhn Gerrit , Balmelli Carlo , Francioli Patrick , Giddey Marlyse , Perreten Vincent , Riesen Andrea , Zysset Frédéric , Blanc Dominique S. , Moreillon Philippe
Published: November 2008

The genetic determinants and phenotypic traits which make a
Staphylococcus aureus
strain a successful colonizer are largely unknown. The genetic diversity and population structure of 133
S. aureus
isolates from healthy, generally risk-free adult carriers were investigated using four different typing methods: multilocus sequence typing (MLST), amplified fragment length polymorphism analysis (AFLP), double-locus sequence typing (DLST), and
spa
typing were compared. Carriage isolates displayed great genetic diversity which could only be revealed fully by DLST. Results of AFLP and MLST were highly concordant in the delineation of genotypic clusters of closely related isolates, roughly equivalent to clonal complexes.
spa
typing and DLST provided considerably less phylogenetic information. The resolution of
spa
typing was similar to that of AFLP and inferior to that of DLST. AFLP proved to be the most universal method, combining a phylogeny-building capacity similar to that of MLST with a much higher resolution. However, it had a lower reproducibility than sequencing-based MLST, DLST, and
spa
typing. We found two cases of methicillin-resistant
S. aureus
colonization, both of which were most likely associated with employment at a health service. Of 21 genotypic clusters detected, 2 were most prevalent: cluster 45 and cluster 30 each colonized 24% of the carrier population. The number of bacteria found in nasal samples varied significantly among the clusters, but the most prevalent clusters were not particularly numerous in the nasal samples. We did not find much evidence that genotypic clusters were associated with different carrier characteristics, such as age, sex, medical conditions, or antibiotic use. This may provide empirical support for the idea that genetic clusters in bacteria are maintained in the absence of adaptation to different niches. Alternatively, carrier characteristics other than those evaluated here or factors other than human hosts may exert selective pressure maintaining genotypic clusters.
UV Disinfection of Adenoviruses: Molecular Indications of DNA Damage Efficiency
Authors: Eischeid Anne C. , Meyer Joel N. , Linden Karl G.
Published: November 2008

Adenovirus is a focus of the water treatment community because of its resistance to standard, monochromatic low-pressure (LP) UV irradiation. Recent research has shown that polychromatic, medium-pressure (MP) UV sources are more effective than LP UV for disinfection of adenovirus when viral inactivation is measured using cell culture infectivity assays; however, UV-induced DNA damage may be repaired during cell culture infectivity assays, and this confounds interpretation of these results. Objectives of this work were to study adenoviral response to both LP and MP UV using (i) standard cell culture infectivity assays and (ii) a PCR assay to directly assess damage to the adenoviral genome without introducing the virus into cell culture. LP and MP UV dose response curves were determined for (i) log inactivation of the virus in cell culture and (ii) UV-induced lesions per kilobase of viral DNA as measured by the PCR assay. Results show that LP and MP UV are equally effective at damaging the genome; MP UV is more effective at inactivating adenovirus in cell culture. This work suggests that the higher disinfection efficacy of MP UV cannot be attributed to a difference in DNA damage induction. These results enhance our understanding of the fundamental mechanisms of UV disinfection of viruses—especially double-stranded DNA viruses that infect humans—and improve the ability of the water treatment community to protect public health.