bluuebunny/crossref_metadata_2025 huggingface dataset:
19F‐MAS NMR on Proteorhodopsin: Enhanced Protocol for Site‐Specific Labeling for General Application to Membrane Proteins†
Authors: Hellmich Ute A. , Pfleger Nicole , Glaubitz Clemens
Published: January 2009

Proteorhodopsin (PR) is a light‐driven proton pump found in near‐surface marine γ‐proteobacteria. The green absorbing variant has three cysteines at positions 107, 156 and 175. We probed the accessibility of these residues by 19F‐MAS NMR. For this purpose, an efficient but simple protocol for chemical fluorine labeling of accessible cysteines in membrane proteins was established. This one‐step reaction was applied to detergent‐solubilized PR before reconstitution into phospholipids. All three cysteines could be labeled and showed distinct 19F chemical shifts with different integral intensities. The accessibility of these cysteines is discussed in the context of a homology model. With the chemical cysteine labeling procedure shown here, an attractive option for site‐directed solid‐state NMR studies on other membrane proteins is offered due to the high intrinsic sensitivity of 19F‐MAS NMR.
Water‐mediated Spectral Shifts in Rhodopsin and Bathorhodopsin†
Authors: Sekharan Sivakumar
Published: January 2009

The role of water molecules in spectral tuning of proteins has been left largely unexplored. This topic is important because changing hydrogen bond patterns during the activation process may lead to spectral shifts which can be of diagnostic value for the underlying structures. Arguments put forward in this article are based on spectral shift calculations of the rhodopsin and bathorhodopsin chromophore due to wat2a and 2b in the presence and absence of the counterion and of the amino acids lining the rhodopsin binding pocket. They show, among others, that a single water molecule can shift the absorbance by up to 0.1 eV or 34 nm depending on the environment of the chromophore.
Highly Efficient Detection of Single Fluorophores in Blood Serum Samples with High Autofluorescence
Authors: Fu Yi , Zhang Jian , Lakowicz Joseph R.
Published: January 2009

Single molecule detection (SMD) is usually performed on surface‐immobilized molecules with diffraction‐limited observation volumes, typically with confocal optics to suppress background from the sample and instrument. In this paper we consider the more difficult task of detecting single fluorophores in the presence of a substantial fluorescence background. We determined that for a readily accessible macroscopic observation volume of 1 pL that the background from undiluted blood serum was approximately equal to 2700 Cy5 molecules, and the background from whole blood equal to about 14 000 Cy5 molecules in whole blood. These high backgrounds appear to preclude the possibility of SMD of Cy5 molecules. However, we show that the signal‐to‐noise ratio (SNR) in high background samples can be increased dramatically by reduction of the observed volume. We were able to detect single surface‐bound Cy5‐labeled DNA (Cy5‐DNA) oligomers in diluted blood serum with an SNR near 40. We also examined freely diffusing Cy5‐DNA in blood serum. The data showed that single Cy5‐DNA molecules could be detected even in the undiluted serum. We further investigated the SNR on silver island films. We found that the fluorescence signal was greatly enhanced in the presence of metallic nanostructures showing a larger SNR in the application tested. These results suggest the possibility of clinical assays based on SMD in blood serum and possibly whole blood. Increased SNR near metallic nanostructure could probably overcome the need for diffraction‐limited volumes and enhance our ability to do in situ SMD.
Illumination with Ultraviolet or Visible Light Induces Chemical Changes in the Water‐soluble Manganese Complex, [Mn4O6(bpea)4]Br4
Authors: Antal Taras K. , Lo Wayne , Armstrong William H. , Tyystjärvi Esa
Published: January 2009

We measured the photosensitivity of an artificial tetranuclear oxo–Mn(IV) complex, [Mn4O6(bpea)4]Br4, which has an adamantane‐shaped {Mn4O6}4+ core. Illumination caused changes in the absorption spectrum of the compound consistent with a one‐electron reduction in the compound. Bromide appears to be the most probable electron donor in the reaction system. Chemical modification of the cluster appears to destabilize it, predisposing it to reductive degradation. UV light was more efficient than visible light in causing the changes. The data support the suggestion that the natural oxygen‐evolving Mn complex is photosensitive and can oxidize components of the oxygen‐evolving complex in its excited state causing photoinhibition, and that photostability is an important issue in designing Mn complexes for artificial photosynthesis. Furthermore, light‐induced oxidation of bromide by [Mn4O6(bpea)4]4+ may suggest that oxidation of chloride is involved in natural water splitting or has been involved during the evolution of the water‐splitting enzyme.
Urea Unfolding of Opsin in Phospholipid Bicelles†
Authors: McKibbin Craig , Farmer Nicola A. , Edwards Patricia C. , Villa Claudio , Booth Paula J.
Published: January 2009

Opsin is the unstable apo‐protein of the light‐activated G protein‐coupled receptor rhodopsin. We investigated the stability of bovine opsin, solubilized in 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC)/detergent bicelles, against urea‐induced unfolding. A single irreversible protein unfolding transition was observed from changes in intrinsic tryptophan fluorescence and far‐UV circular dichroism. This unfolding transition correlated with loss of protein activity. Changes in tertiary structure, as indicated by fluorescence measurements, were concomitant with an approximate 50% reduction in α‐helical content of opsin, indicating that global unfolding had been induced by urea. The urea concentration at the midpoint of unfolding was dependent on the lipid/detergent environment, occurring at approximately 1.2 m urea in DMPC/1,2‐dihexanoyl‐sn‐glycero‐3‐phosphocholine bicelles, while being significantly stabilized to approximately 3.5 m urea in DMPC/3‐[(cholamidopropyl)dimethylammonio]‐1‐propanesulfonate bicelles. These findings demonstrate that interactions with the surrounding lipids and detergent are highly influential in the unfolding of membrane protein structure. The urea/bicelle system offers the possibility for a more detailed understanding of the structural changes that take place upon irreversible unfolding of opsin.
Studies on the Stability of the Human Cone Visual Pigments†
Authors: Ramon Eva , Mao Xiang , Ridge Kevin D.
Published: January 2009

The retina of vertebrates contains two kinds of photoreceptor cells, rods and cones, which contain their specific visual pigments that are responsible for scotopic and photopic vision, respectively. In cone photoreceptor cells, there are three types of color pigments: blue, green and red, each with a distinctive absorption maximum. The goal of this investigation was to identify optimal conditions under which these pigments could be obtained and isolated in a stable form, thereby facilitating structural studies using high‐resolution approaches. For this purpose, all three human cone opsins were initially expressed in mammalian cells, reconstituted with 11‐cis retinal, detergent solubilized, purified and their stability compared with rod rhodopsin. As all three pigments showed dramatically reduced stability relative to rhodopsin, site‐directed mutagenesis was used in an attempt to engineer stability into the green cone pigment. The mutations introduced some structural motifs and sites of posttranslational modification present in rhodopsin, as well as amino acid substitutions that have been found to stabilize the rod opsin apo‐protein. We also modified the hydrophobic environment of the green cone pigment by varying the detergent and detergent/lipid composition used during solubilization and purification, and compared them with the retinal reconstituted pigment in membranes. Our results show that these changes do not significantly improve the inherent instability of the human cone pigments, and in some cases, lead to a decrease in stability and protein aggregation. We conclude that further efforts are required to stabilize the human cone pigments in a form suitable for high‐resolution structural studies.
The Heterotrimeric G‐protein Complex Modulates Light Sensitivity in Arabidopsis thaliana Seed Germination
Authors: Botto Javier F. , Ibarra Silvia , Jones Alan M.
Published: January 2009

Release of dormancy and induction of seed germination are complex traits finely regulated by hormonal signals and environmental cues such as temperature and light. The Red (R):Far‐Red (FR) phytochrome photoreceptors mediate light regulation of seed germination. We investigated the possible involvement of heterotrimeric G‐protein complex in the phytochrome signaling pathways of Arabidopsis thaliana seed germination. Germination rates of null mutants of the alpha (Gα) and beta (Gβ) subunits of the G‐protein (Atgpa1‐4 and agb1‐2, respectively) and the double mutant (agb1‐2/gpa1‐4) are lower than the wildtype (WT) under continuous or pulsed light. The Gα and Gβ subunits play a role in seed germination under hourly pulses of R lower than 0.1 μmol m−2 s−1 whereas the Gβ subunit plays a role in higher R fluences. The germination of double mutants of G‐protein subunits with phyA‐211 and phyB‐9 suggests that AtGPA1 seems to act as a positive regulator of phyA and probably phyB signaling pathways, while the role of AGB1 is ambiguous. The imbibition of seeds at 4°C and 35°C alters the R and FR light responsiveness of WT and G‐protein mutants to a similar magnitude. Thus, Gα and Gβ subunits of the heterotrimeric G‐protein complex modulate light induction of seed germination by phytochromes and are dispensable for the control of dormancy by low and high temperatures prior to irradiation. We discuss the possible indirect role of the G‐protein complex on the phytochrome‐regulated germination through hormonal signaling pathways.
A novel third generation bisphosphonate, minodronate (YM529), prevented proliferation and migration of hepatocellular carcinoma cells through inhibition of mevalonate pathway
Authors: Kogure Takayuki , Ueno Yoshiyuki , Kimura Osamu , Kondo Yasuteru , Inoue Jun , Fukushima Koji , Iwasaki Takao , Shimosegawa Tooru
Published: January 2009

Aim:  Skeletal metastases and bone metasitasis are a common occurrence in patients with advanced hepatocellular carcinoma (HCC). Bisphosphonates (BPs), which are used for the treatment of osteoporosis and tumor‐associated hypercalcemia, have recently been reported to decrease skeletal morbidity in patients with metastatic bone disease. Several studies revealed that nitrogen‐containing BPs (N‐BPs) could inhibit tumor growth and migration, indicating the possibility that N‐BPs have direct inhibitory effects. We aimed to determine the effects of novel a N‐BP (YM529) on human HCC cells in vitro.

Methods:  HCC cells were treated with various concentrations of YM529 and the growth inhibition rate was determined. Apoptosis was evaluated by caspase‐3/7 assay and caspase‐9 cleavage detection. The effects of YM529 on the migration of HCC cells induced by hepatocyte growth factor (HGF) were determined by cell migration assay. To evaluate the involvement of the mevalonate pathway, farnesol (FOH) and geranylgeraniol (GGOH) were added.

Results:  YM529 inhibited the proliferation of HCC cells in a dose‐dependent manner. The activation of caspase‐3/7 and cleavage of caspase‐9 demonstrated the involvement of apoptosis in cytotoxicity. GGOH reduced the growth inhibitory effect of YM529 and suppressed the induction of caspase‐3/7 activities by YM529 on HCC cells. YM529 inhibited tumor cell migration induced by HGF and this effect was reduced by co‐treatment with GGOH.

Conclusion:  YM529 inhibited the cell proliferation and migration of HCC cells, implicating the involvement of the mevalonate pathway. These results suggest that N‐BPs are potential agents for the treatment of HCC skeletal metastases.
UGT1A1 gene polymorphism as a potential factor inducing iron overload in the pathogenesis of type 1 hereditary hemochromatosis
Authors: Romanowski Tomasz , Sikorska Katarzyna , Bielawski Krzysztof Piotr
Published: January 2009

Aim  Hereditary hemochromatosis is a common genetic disorder characterized by iron overload and subsequent organ damage. It is caused in most cases by HFE gene mutations which penetrance can be affected by many factors. The aim of this study was to establish the role of UGT1A1 gene polymorphism and serum bilirubin concentration in the pathogenesis of hereditary hemochromatosis.

Methods  Biochemical, histopathological and genetic data indicating iron excess and serum total bilirubin concentration were determined in 32 patients with the type 1 hereditary hemochromatosis. Fluorescent molecular probes assays were used for genotyping of UGT1A1*28 and UGT1A1*60 mutations in these individuals.

Results  High incidence and a significant correlation of UGT1A1 gene mutations with increased serum bilirubin level and lower grades of liver tissue inflammatory activity were observed in study participants. UGT1A1*28 and UGT1A1*60 mutations were strongly linked together. Two of the subjects presented very rare genotypes of UGT1A1 gene: (TA)5/7 and c.‐64G>C heterozygotes.

Conclusions  UGT1A1 gene polymorphism and as its consequence of high serum bilirubin level may promote iron accumulation in hemochromatosis patients by reducing the activity of inflammation. We proposed a possible mechanism of this interaction.
Three‐dimensional high‐resolution imaging of cardiac proteins to construct models of intracellular Ca2+ signalling in rat ventricular myocytes
Authors: Soeller Christian , Jayasinghe Isuru D. , Li Pan , Holden Arun V. , Cannell Mark B.
Published: January 2009

Quantitative understanding of the Ca2+ handling in cardiac ventricular myocytes requires accurate knowledge of cardiac ultrastructure and protein distribution. We have therefore developed high‐resolution imaging and analysis approaches to measure the three‐dimensional distribution of immunolabelled proteins with confocal microscopy. Labelling of single rat cardiac myocytes with an antibody to the Z‐line marker α‐actinin revealed a complex architecture of sarcomere misalignment across single cells. Double immunolabelling was used to relate the Z‐line structure to the distribution of ryanodine receptors (RyRs, the intracellular Ca2+ release channels) and the transverse tubular system. Both RyR and transverse tubular system distributions exhibited frequent dislocations from the simple planar geometry generally assumed in existing mathematical models. To investigate potential effects of these irregularities on Ca2+ dynamics, we determined the three‐dimensional distribution of RyR clusters within an extended section of a single rat ventricular myocyte to construct a model of stochastic Ca2+ dynamics with a measured Ca2+ release unit (CRU) distribution. Calculations with this model were compared with a second model in which all CRUs were placed on flat planes. The model with a realistic CRU distribution supported Ca2+ waves that spread axially along the cell at velocities of ∼50 μm s−1. By contrast, in the model with planar CRU distribution the axial wave spread was slowed roughly twofold and wave propagation often nearly faltered. These results demonstrate that spatial features of the CRU distribution on multiple length scales may significantly affect intracellular Ca2+ dynamics and must be captured in detailed mechanistic models to achieve quantitative as well as qualitative insight.
A meta‐analysis of cardiac electrophysiology computational models
Authors: Niederer S. A. , Fink M. , Noble D. , Smith N. P.
Published: January 2009

Computational models of cardiac electrophysiology are exemplar demonstrations of the integration of multiple data sets into a consistent biophysical framework. These models encapsulate physiological understanding to provide quantitative predictions of function. The combination or extension of existing models within a common framework allows integrative phenomena in larger systems to be investigated. This methodology is now routinely applied, as demonstrated by the increasing number of studies which use or extend previously developed models. In this study, we present a meta‐analysis of this model re‐use for two leading models of cardiac electrophysiology in the form of parameter inheritance trees, a sensitivity analysis and a comparison of the functional significance of the sodium potassium pump for defining restitution curves. These results indicate that even though the models aim to represent the same physiological system, both the sources of parameter values and the function of equivalent components are significantly different.
Spontaneous baroreflex measures are unable to detect age‐related impairments in cardiac baroreflex function during dynamic exercise in humans
Authors: Fisher James P. , Ogoh Shigehiko , Junor Chelif , Khaja Azamuddin , Northrup Mindy , Fadel Paul J.
Published: January 2009

The dynamic relationship between ‘spontaneous’ fluctuations in arterial blood pressure (BP) and heart rate (HR) is increasingly being used to provide an estimate of resting cardiac baroreflex sensitivity. Given the ease of use and clinical utility, spontaneous methods are now also being used to examine cardiac baroreflex sensitivity in distinct subject groups during various laboratory stressors and tasks encountered during daily life, such as physical activity. However, the utility of such spontaneous measures to estimate cardiac baroreflex function during exercise remains unclear, particularly when comparing groups. Therefore, we tested the ability of spontaneous indices to detect age‐related differences in cardiac baroreflex function during dynamic exercise. Beat‐to‐beat HR and BP were measured in eighteen healthy young subjects (24 ± 1 years) and sixteen healthy middle‐aged subjects (59 ± 1 years) at rest and during steady‐state leg cycling. Estimates of spontaneous cardiac baroreflex sensitivity using the sequence technique (GSEQ) and low‐frequency transfer function gain (GTF) were compared with the operating point (GOP) and maximal gain (GMAX) of the full carotid–cardiac baroreflex function curve. At rest GSEQ, GTF, GOP and GMAX were all significantly lower in older subjects. During moderate‐intensity steady‐state exercise no differences were observed in GSEQ and GTF (older 0.26 ± 0.03 beats min−1 mmHg−1versus younger 0.32 ± 0.04 beats min−1 mmHg−1; P > 0.05), whereas GOP and GMAX (older −0.21 ± 0.02 beats min−1 mmHg−1versus younger −0.39 ± 0.03 beats min−1 mmHg−1; P < 0.05) remained lower in older subjects. These data indicate that spontaneous measures of cardiac baroreflex sensitivity alone provide limited information when comparing age‐groups during exercise, which makes genuine differences in baroreflex function difficult to identify.
Mechanosensitive hyaluronan secretion: stimulus–response curves and role of transcription–translation–translocation in rabbit joints
Authors: Wann A. K. T. , Ingram K. R. , Coleman P. J. , McHale N. , Levick J. R.
Published: January 2009

Joint movement was recently shown to stimulate the secretion of the lubricant hyaluronan (HA); also, exercise therapy and intra‐articular hyaluronan injections are used to treat moderate osteoarthritis. The present study quantifies the stimulus–response curves for HA secretion in vivo and reports a role of transcription–translation–translocation in the secretory response. After washing out endogenous HA from anaesthetized, cannulated rabbit knees, the joints were cycled passively at various frequencies and durations, with or without intra‐articular inhibitors of protein synthesis and Golgi processing. Newly secreted HA was harvested for analysis after 5 h. Joints displayed graded, non‐linear stimulus–response curves to both duration and frequency of movement; 1 min duration per 15 min or a frequency of 0.17 Hz raised HA secretion by 42–54%, while rapid (1.5 Hz) or prolonged cycling (9 min per 15 min) raised it by 110–130%. Movement‐stimulated secretion and phorbol ester‐stimulated secretion were partly inhibited by the translation inhibitor cycloheximide, by the transcription–translation inhibitors actinomycin D and puromycin and by the Golgi translocation inhibitor brefeldin A. There is thus a graded coupling between HA secretion and cyclic joint movement that depends partly on new protein synthesis. This is likely to be important for joint homeostasis, providing protection during repetitive cycling and potentially contributing to exercise therapy for osteoarthritis.
Functional connectivity between motor cortex and globus pallidus in human non‐REM sleep
Authors: Salih F. , Sharott A. , Khatami R. , Trottenberg T. , Schneider G. , Kupsch A. , Brown P. , Grosse P.
Published: January 2009

Recent evidence suggests that the motor system undergoes very specific modulation in its functional state during the different sleep stages. Here we test the hypothesis that changes in the functional organization of the motor system involve both cortical and subcortical levels and that these distributed changes are interrelated in defined frequency bands. To this end we evaluated functional connectivity between motor and non‐motor cortical sites (fronto‐central, parieto‐occipital) and the globus pallidus (GP) in human non‐REM sleep in seven patients undergoing deep brain stimulation (DBS) for dystonia using a variety of spectral measures (power, coherence, partial coherence and directed transfer function (DTF)). We found significant coherence between GP and fronto‐central cortex as well as between GP and parieto‐occipital cortex in circumscribed frequency bands that correlated with sleep specific oscillations in ‘light sleep’ (N2) and ‘slow‐wave sleep’ (N3). These sleep specific oscillations were also reflected in significant coherence between the two cortical sites corroborating previous studies. Importantly, we found two different physiological activities represented within the broad band of significant coherence between 9.5 and 17 Hz. One component occurred in the frequency range of sleep spindles (12.5–17 Hz) and was maximal in the coherence between fronto‐central and parieto‐occipital cortex as well as between GP and both cortical sites during N2. This component was still present between fronto‐central and parieto‐occipital cortex in N3. Functional connectivity in this frequency band may be due to a common input to both GP and cortex. The second component consisted of a spectral peak over 9.5–12.5 Hz. Coherence was elevated in this band for all topographical constellations in both N2 and N3, but especially between GP and fronto‐central cortex. The DTF suggested that the 9.5–12.5 Hz activity consisted of a preferential drive from GP to the fronto‐central cortex in N2, whereas in N3 the DTF between GP and fronto‐central cortex was symmetrical. Partial coherence supported distinctive patterns for the 9.5–12.5 and 12.5 and 17 Hz component, so that only coherence in the 9.5–12.5 Hz band was reduced when the effects of GP were removed from the coherence between the two cortical sites. The data suggest that activities in the GP and fronto‐central cortex are functionally connected over 9.5–12.5 Hz, possibly as a specific signature of the motor system in human non‐REM sleep. This finding is pertinent to the longstanding debate about the nature of alpha–delta sleep as a physiological or pathological feature of non‐REM sleep.
Extra‐ and intracellular unstirred layer effects in measurements of CO2 diffusion across membranes – a novel approach applied to the mass spectrometric 18O technique for red blood cells
Authors: Endeward Volker , Gros Gerolf
Published: January 2009

We have developed an experimental approach that allows us to quantify unstirred layers around cells suspended in stirred solutions. This technique is applicable to all types of transport measurements and was applied here to the 18O technique used to measure CO2 permeability of red cells . We measure in well‐stirred red cell (RBC) suspensions of various viscosities adjusted by adding different amounts of 60 kDa dextran. Plotting vs. viscosity ν gives a linear relation, which can be extrapolated to ν= 0. Theoretical hydrodynamics predicts that extracellular unstirred layers vanish at zero viscosity when stirring is maintained, and thus this extrapolation gives us an estimate of the free from extracellular unstirred layer artifacts. The extrapolated value is found to be 0.16 cm s−1 instead of the experimental value in saline of 0.12 cm s−1 (+30%). This effect corresponds to an unstirred layer thickness of 0.5 μm. In addition, we present a theoretical approach modelling the actual geometrical and physico‐chemical conditions of 18O exchange in our experiments. It confirms the role of an extracellular unstirred layer in the determination of . Also, it allows us to quantify the contribution of the so‐called intracellular unstirred layer, which results from the fact that in these transport measurements – as in all such measurements in general – the intracellular space is not stirred. The apparent thickness of this intracellular unstirred layer is about 1/4–1/3 of the maximal intracellular diffusion distance, and correction for it results in a true of the RBC membrane of 0.20 cm s−1. Thus, the order of magnitude of this is unaltered compared to our previous reports. Discussion of the available evidence in the light of these results confirms that CO2 channels exist in red cell and other membranes, and that of red cell membranes in the absence of these channels is quite low.
In Vitro Comparison of the Efficacies of Natamycin and Silver Nitrate against Ocular Fungi
Authors: Xu Yan , Pang Guangren , Gao Chuanwen , Zhao Dongqing , Zhou Lutan , Sun Shengtao , Wang Bingliang
Published: January 2009

The in vitro activity of the silver nitrat
e
was assessed in comparison with that of natamycin against 128 corneal
Fusarium
isolates and 90 corneal
Aspergillus
isolates. MIC
90
s of silver nitrat
e
were 2 μg/ml for
Fusarium
spp. and 1 μg/ml for
Aspergillus
spp. MIC
90
s of natamycin were 8 μg/ml for
Fusarium
spp. and 32 μg/ml for
Aspergillus
spp. Silver nitrat
e
exhibited potent antifungal activity against ocular fungi in vitro.
Radioimmunotherapy Is Effective against High-Inoculum
Cryptococcus neoformans
Infection in Mice and Does Not Select for Radiation-Resistant Cryptococcal Cells
Authors: Bryan Ruth A. , Jiang Zewei , Huang Xianchun , Morgenstern Alfred , Bruchertseifer Frank , Sellers Rani , Casadevall Arturo , Dadachova Ekaterina
Published: January 2009

We investigated the utility of radioimmunotherapy (RIT) in the treatment of experimental cryptococcal infection with high-inoculum and the possibility of RIT treatment selecting for fungal cells with radiation-resistant phenotypes. RIT reduced mortality in high-burden infections, and we found no evidence for the development of radiation-resistant cells.
Dry Powder Nitroimidazopyran Antibiotic PA-824 Aerosol for Inhalation
Authors: Sung Jean C. , Garcia-Contreras Lucila , VerBerkmoes Jarod L. , Peloquin Charles A. , Elbert Katharina J. , Hickey Anthony J. , Edwards David A.
Published: January 2009

We formulated PA-824, a nitroimidazopyran with promise for the treatment of tuberculosis, for efficient aerosol delivery to the lungs in a dry powder porous particle form. The objectives of this study were to prepare and characterize a particulate form of PA-824, assess the stability of this aerosol formulation under different environmental conditions, and determine the pharmacokinetic parameters for the powder after pulmonary administration. The drug was spray dried into porous particles containing a high drug load and possessing desirable aerosol properties for efficient deposition in the lungs. The physical, aerodynamic, and chemical properties of the dry powder were stable at room temperature for 6 months and under refrigerated conditions for at least 1 year. Pharmacokinetic parameters were determined in guinea pigs after the pulmonary administration of the PA-824 powder formulation at three doses (20, 40, and 60 mg/kg of body weight) and compared to those after the intravenous (20 mg/kg) and oral (40 mg/kg) delivery of the drug. Oral and inhaled delivery of PA-824 achieved equivalent systemic delivery at the same body dose within the first 12 h of dosing. However, animals dosed by the pulmonary route showed drug loads that remained locally in the lungs for 32 h postexposure, whereas those given the drug orally cleared the drug more rapidly. Therefore, we expect from these pharmacokinetic data that pulmonary delivery may achieve the same efficacy as oral delivery at the same body dose, with a potential improvement in efficacy related to pulmonary infection. This may translate into the ability to deliver lower body doses of this drug for the treatment of tuberculosis by aerosol.
Escherichia coli
Populations in Great Lakes Waterfowl Exhibit Spatial Stability and Temporal Shifting
Authors: Hansen Dennis L. , Ishii Satoshi , Sadowsky Michael J. , Hicks Randall E.
Published: January 2009

Populations of
Escherichia coli
from juvenile and adult ring-billed gulls, juvenile common terns, and adult Canada geese were sampled over 6 years at five locations on Lake Superior (Duluth, MN, and Wisconsin) and Lake Michigan (Wisconsin, Illinois, and Indiana) to determine the extent of spatial and temporal variability in
E. coli
strains. Strain identity was determined using horizontal fluorophore-enhanced repetitive element palindromic DNA fingerprinting. Multivariate statistics were used to determine if spatial or temporal changes in
E. coli
populations occurred in waterfowl species. Pairwise multivariate analyses of variance revealed that
E. coli
populations of adult gulls from three regions of Lake Michigan and the Wisconsin shore of Lake Superior were similar to one another but different from an
E. coli
population of gulls from the Duluth region of Lake Superior. Juvenile and adult gulls from the Duluth area harbored different
E. coli
populations. The
E. coli
strains from juvenile gulls, however, were similar to those found in juvenile terns obtained from the same island rookery. Temporal changes in
E. coli
populations from several waterfowl species were also demonstrated for this site. Although portions of source tracking databases might be successfully used in other geographic regions, it is clear that juvenile birds should not be the sole source of
E. coli
strains used for source tracking databases, and multiple-year libraries should be constructed in order to identify the potential sources of
E. coli
in the environment.
Tailoring the P450 Monooxygenase Gene for FR-008/Candicidin Biosynthesis
Authors: Chen Shi , Mao Xiangzhao , Shen Yaling , Zhou Yongjun , Li Jialiang , Wang Lianrong , Tao Xinyi , Yang Liang , Wang Yuxiao , Zhou Xiufen , Deng Zixin , Wei Dongzhi
Published: January 2009

Tailoring steps are often important for the activity of mature antibiotics. Here, we report that novel decarboxylated FR-008/candicidin derivatives were obtained from the P450 monooxygenase gene
fscP
mutant of
Streptomyces
sp. strain FR-008. The toxicity of decarboxylated FR-008/candicidin derivatives has been shown to be greatly reduced compared to that of wild-type FR-008/candicidin.
Isolation of a Human Intestinal Bacterium Capable of Daidzein and Genistein Conversion
Authors: Matthies Anastasia , Blaut Michael , Braune Annett
Published: January 2009

A rod-shaped gram-positive anaerobic bacterium, strain HE8, was isolated from human feces. The isolate was able to convert the isoflavones daidzein and genistein to equol and 5-hydroxy-equol, respectively. Based on phenotypic and phylogenetic analyses, strain HE8 is described as a new species,
Slackia isoflavoniconvertens
.
Variations in Archaeal and Bacterial Diversity Associated with the Sulfate-Methane Transition Zone in Continental Margin Sediments (Santa Barbara Basin, California)
Authors: Harrison Benjamin K. , Zhang Husen , Berelson Will , Orphan Victoria J.
Published: January 2009

The sulfate-methane transition zone (SMTZ) is a widespread feature of continental margins, representing a diffusion-controlled interface where there is enhanced microbial activity. SMTZ microbial activity is commonly associated with the anaerobic oxidation of methane (AOM), which is carried out by syntrophic associations between sulfate-reducing bacteria and methane-oxidizing archaea. While our understanding of the microorganisms catalyzing AOM has advanced, the diversity and ecological role of the greater microbial assemblage associated with the SMTZ have not been well characterized. In this study, the microbial diversity above, within, and beneath the Santa Barbara Basin SMTZ was described. ANME-1-related archaeal phylotypes appear to be the primary methane oxidizers in the Santa Barbara Basin SMTZ, which was independently supported by exclusive recovery of related methyl coenzyme M reductase genes (
mcrA
). Sulfate-reducing
Deltaproteobacteria
phylotypes affiliated with the
Desulfobacterales
and
Desulfosarcina-Desulfococcus
clades were also enriched in the SMTZ, as confirmed by analysis of dissimilatory sulfite reductase (
dsr
) gene diversity. Statistical methods demonstrated that there was a close relationship between the microbial assemblages recovered from the two horizons associated with the geochemically defined SMTZ, which could be distinguished from microbial diversity recovered from the sulfate-replete overlying horizons and methane-rich sediment beneath the transition zone. Comparison of the Santa Barbara Basin SMTZ microbial assemblage to microbial assemblages of methane seeps and other organic matter-rich sedimentary environments suggests that bacterial groups not typically associated with AOM, such as
Planctomycetes
and candidate division JS1, are additionally enriched within the SMTZ and may represent a common bacterial signature of many SMTZ environments worldwide.
Comparison of Loop-Mediated Isothermal Amplification Assay and Conventional Culture Methods for Detection of
Campylobacter jejuni
and
Campylobacter coli
in Naturally Contaminated Chicken Meat Samples
Authors: Yamazaki Wataru , Taguchi Masumi , Kawai Takao , Kawatsu Kentaro , Sakata Junko , Inoue Kiyoshi , Misawa Naoaki
Published: January 2009

We investigated the efficacy of a loop-mediated isothermal amplification (LAMP) assay for detection of chicken meat samples naturally contaminated with
Campylobacter jejuni
and
Campylobacter coli
. A total of 144 Preston enrichment broth cultures from chicken meat samples were assessed by using the LAMP assay and conventional culture methods, which consist of a combination of Preston enrichment culturing and plating onto Butzler and modified charcoal cefoperazone deoxycholate agars. Compared with
C. jejuni
-
C. coli
isolation using the conventional culture test, the LAMP results showed 98.5% (67/68) and 97.4% (74/76) sensitivity and specificity, respectively, and the positive and negative predictive values were 97.1% (67/69) and 98.7% (74/75), respectively. The conventional culture test required more than 3 to 4 days to isolate and identify
C. jejuni
and
C. coli
in the Preston enrichment cultures. In contrast, the LAMP assay was markedly faster, requiring less than 90 min from the beginning of DNA extraction to final detection and differentiation of
C. jejuni
and
C. coli
. In total, the LAMP assay required 23.5 to 25.5 h from the beginning of the enrichment culture to final determination. These results suggest that our LAMP assay is a powerful tool for rapid, sensitive, and practical detection of
C. jejuni
and
C. coli
which may facilitate surveillance and control of
C. jejuni-C. coli
contamination in chicken, as well as investigations of food poisoning incidents caused by these organisms. This is the first report of a highly sensitive and specific LAMP assay to detect and differentiate
C. jejuni
and
C. coli
in chicken meat samples.
Prevalence and Impact of Bacteriophages on the Presence of
Escherichia coli
O157:H7 in Feedlot Cattle and Their Environment
Authors: Niu Y. D. , McAllister T. A. , Xu Y. , Johnson R. P. , Stephens T. P. , Stanford K.
Published: January 2009

The relationship between endemic bacteriophages infecting
E. coli
O157:H7 (referred to as “phage”) and levels of shedding of
E. coli
O157:H7 by cattle was investigated in two commercial feedlots in southern Alberta, Canada. Between May and November 2007, 10 pens of cattle were monitored by collection of pooled fecal pats, water with sediment from troughs, manure slurry from the pen floor, and rectal fecal samples from individual animals (20 per pen) at two separate times. Bacteriophages infecting
E. coli
O157:H7 were detected more frequently (
P
< 0.001) after 18 to 20 h enrichment than by initial screening and were recovered in 239 of 855 samples (26.5% of 411 pooled fecal pats, 23.8% of 320 fecal grab samples, 21.8% of 87 water trough samples, and 94.6% of 37 pen floor slurry samples). Overall, prevalence of phage was highest (
P
< 0.001) in slurry. Recovery of phage from pooled fecal pats was highest (
P
< 0.05) in May. Overall recovery did not differ (
P
> 0.10) between fecal grab samples and pooled fecal pats. A higher prevalence of phage in fecal pats or water trough samples was associated (
P
< 0.01) with reduced prevalence of
E. coli
O157:H7 in rectal fecal samples. There was a weak but significant negative correlation between isolation of phage and
E. coli
O157:H7 in fecal grab samples (
r
= −0.11;
P
< 0.05). These data demonstrate that the prevalence of phage fluctuates in a manner similar to that described for
E. coli
O157:H7. Phage were more prevalent in manure slurry than other environmental sources. The likelihood of fecal shedding of
E. coli
O157:H7 was reduced if cattle in the pen harbored phage.
The Unique Branching Patterns of
Deinococcus
Glycogen Branching Enzymes Are Determined by Their N-Terminal Domains
Authors: Palomo M. , Kralj S. , van der Maarel M. J. E. C. , Dijkhuizen L.
Published: January 2009

Glycogen branching enzymes (GBE) or 1,4-α-glucan branching enzymes (EC 2.4.1.18) introduce α-1,6 branching points in α-glucans, e.g., glycogen. To identify structural features in GBEs that determine their branching pattern specificity, the
Deinococcus geothermalis
and
Deinococcus radiodurans
GBE (GBE
Dg
and GBE
Dr
, respectively) were characterized. Compared to other GBEs described to date, these
Deinococcus
GBEs display unique branching patterns, both transferring relatively short side chains. In spite of their high amino acid sequence similarity (88%) the
D. geothermalis
enzyme had highest activity on amylose while the
D. radiodurans
enzyme preferred amylopectin. The side chain distributions of the products were clearly different: GBE
Dg
transferred a larger number of smaller side chains; specifically, DP5 chains corresponded to 10% of the total amount of transferred chains, versus 6.5% for GBE
Dr
. GH13-type GBEs are composed of a central (β/α) barrel catalytic domain and an N-terminal and a C-terminal domain. Characterization of hybrid
Deinococcus
GBEs revealed that the N2 modules of the N domains largely determined substrate specificity and the product branching pattern. The N2 module has recently been annotated as a carbohydrate binding module (CBM48). It appears likely that the distance between the sugar binding subsites in the active site and the CBM48 subdomain determines the average lengths of side chains transferred.
Novel Cell-Based Method To Detect Shiga Toxin 2 from
Escherichia coli
O157:H7 and Inhibitors of Toxin Activity
Authors: Quiñones Beatriz , Massey Shane , Friedman Mendel , Swimley Michelle S. , Teter Ken
Published: January 2009

Escherichia coli
O157:H7 is a leading cause of food-borne illness. This human pathogen produces Shiga toxins (Stx1 and Stx2) which inhibit protein synthesis by inactivating ribosome function. The present study describes a novel cell-based assay to detect Stx2 and inhibitors of toxin activity. A Vero cell line harboring a destabilized variant (half-life, 2 h) of the enhanced green fluorescent protein (d2EGFP) was used to monitor the toxin-induced inhibition of protein synthesis. This Vero-d2EGFP cell line produced a fluorescent signal which could be detected by microscopy or with a plate reader. However, a greatly attenuated fluorescent signal was detected in Vero-d2EGFP cells that had been incubated overnight with either purified Stx2 or a cell-free culture supernatant from Stx1- and Stx2-producing
E. coli
O157:H7. Dose-response curves demonstrated that the Stx2-induced inhibition of enhanced green fluorescent protein fluorescence mirrored the Stx2-induced inhibition of overall protein synthesis and identified a picogram-per-milliliter threshold for toxin detection. To establish our Vero-d2EGFP assay as a useful tool for the identification of toxin inhibitors, we screened a panel of plant compounds for antitoxin activities. Fluorescent signals were maintained when Vero-d2EGFP cells were exposed to Stx1- and Stx2-containing medium in the presence of either grape seed or grape pomace extract. The antitoxin properties of the grape extracts were confirmed with an independent toxicity assay that monitored the overall level of protein synthesis in cells treated with purified Stx2. These results indicate that the Vero-d2EGFP fluorescence assay is an accurate and sensitive method to detect Stx2 activity and can be utilized to identify toxin inhibitors.
Identification and Characterization of Lactocyclicin Q, a Novel Cyclic Bacteriocin Produced by
Lactococcus
sp. Strain QU 12
Authors: Sawa Naruhiko , Zendo Takeshi , Kiyofuji Junko , Fujita Koji , Himeno Kohei , Nakayama Jiro , Sonomoto Kenji
Published: January 2009

Lactococcus
sp. strain QU 12, which was isolated from cheese, produced a novel cyclic bacteriocin termed lactocyclicin Q. By using cation-exchange chromatography, hydrophobic interaction chromatography, and reverse-phase high-performance liquid chromatography, lactocyclicin Q was purified from culture supernatant, and its molecular mass was determined to be 6,062.8 Da by mass spectrometry. Lactocyclicin Q has been characterized by its unique antimicrobial spectrum, high level of protease resistance, and heat stability compared to other reported bacteriocins of lactic acid bacteria. The amino acid sequence of lactocyclicin Q was determined chemically, and this compound is composed of 61 amino acid residues that have a cyclic structure with linkage between the N and C termini by a peptide bond. It showed no homology to any other antimicrobial peptide, including cyclic bacteriocins. On the basis of the amino acid sequences obtained, the sequence of the gene encoding the prepeptide lactocyclicin Q was obtained. This is the first report of a cyclic bacteriocin purified from a strain belonging to the genus
Lactococcus
.
Sequencing and Analysis of Fungal rRNA Operons for Development of Broad-Range Fungal PCR Assays
Authors: Khot Prasanna D. , Ko Daisy L. , Fredricks David N.
Published: January 2009

rRNA genes are attractive targets for developing PCR assays targeting human fungal pathogens. Most studies have focused on the 18S rRNA gene, internal transcribed spacers, and the 5′ end of the 28S rRNA gene. An approximately 2,900-bp region of the 28S rRNA gene remains largely unexplored because sequences of many medically relevant fungi are either unavailable or undefined in genomic databases. The internal transcribed spacers and 28S rRNA gene of nine medically and phylogenetically important fungi were sequenced. In addition, 42 sequences from this region were acquired from public databases, resulting in an alignment of 51 fungal sequences spanning 30 fungal genera. For the nearly 3,950-bp region from the 3′ end of 18S rRNA gene to the 3′ end of the 28S rRNA gene, 27 broad-range PCR primers were designed such that their sequence homology with the human rRNA gene was minimal. All 62 possible amplicons in the size range from 75 to 400 bp from 27 primers were screened using fungal genomic DNA from 26 species spanning 14 genera. Eleven of the 62 amplicons did not cross-react with 1 μg/PCR human DNA but simultaneously amplified 10 fg of fungal DNA. Phylogenetic distance matrices were calculated for regions covered by these 11 amplicons based on 51 fungi. Two of these 11 amplicons successfully amplified 30 fg of fungal DNA from 25 of 26 fungi and provided the most phylogenetic information for species identification based on the distance matrices. These PCR assays hold promise for detection and identification of fungal pathogens in human tissues.
Campylobacter jejuni
Drives MyD88-Independent Interleukin-6 Secretion via Toll-Like Receptor 2
Authors: Friis Lorna M. , Keelan Monika , Taylor Diane E.
Published: January 2009

Gastrointestinal disease caused by
Campylobacter jejuni
is characterized by localized inflammation and the destruction of the epithelial cell barrier that forms host innate protection against pathogens. This can lead to an imbalance in fluid transport across the gastrointestinal tract, resulting in severe diarrhea. The mechanisms of host cell receptor recognition of
C. jejuni
and downstream immune signaling pathways leading to this inflammatory disease, however, remain unclear. The aim of this study was to analyze the mechanisms involved in
C. jejuni
induction of the acute-phase inflammatory response regulator interleukin-6 (IL-6). Polarized intestinal epithelial Caco-2 monolayers responded to infections with
Salmonella enterica
serovar Typhimurium and eight isolates of
C. jejuni
by an increase in levels of expression and secretion of IL-6. No such IL-6 response, however, was produced upon infection with the human commensal organism
Lactobacillus rhamnosus
GG. The IL-6 signaling pathway was further characterized using short interfering RNA complexes to block gene expression. The inhibition of myeloid differentiation primary response protein 88 (MyD88) expression in this manner did not affect
C. jejuni
-induced IL-6 secretion, suggesting a MyD88-independent route to IL-6 signal transduction in
C. jejuni
-infected human epithelial cells. However, a significant reduction in levels of IL-6 was evident in the absence of Toll-like receptor 2 (TLR-2) expression, implying a requirement for TLR-2 in
C. jejuni
recognition. Caco-2 cells were also treated with heat-inactivated and purified membrane components of
C. jejuni
to isolate the factor responsible for triggering IL-6 signaling. The results demonstrate that
C. jejuni
surface polysaccharides induce IL-6 secretion from intestinal epithelial cells via TLR-2 in a MyD88-independent manner.
Evidence for Multiple B- and T-Cell Epitopes inPlasmodium falciparumLiver-Stage Antigen 3
Authors: Toure-Balde Aissatou , Perlaza Blanca-Liliana , Sauzet Jean-Pierre , Ndiaye Mouhamadou , Aribot Georgette , Tall Adama , Sokhna Cheikh , Rogier Christophe , Corradin Giampietro , Roussilhon Christian , Druilhe Pierre
Published: January 2009

Liver-stage antigen 3 (LSA-3) is a new vaccine candidate that can induce protection againstPlasmodium falciparumsporozoite challenge. Using a series of long synthetic peptides (LSP) encompassing most of the 210-kDa LSA-3 protein, a study of the antigenicity of this protein was carried out in 203 inhabitants from the villages of Dielmo (n= 143) and Ndiop (n= 60) in Senegal (the level of malaria transmission differs in these two villages). Lymphocyte responses to each individual LSA-3 peptide were recorded, some at high prevalences (up to 43%). Antibodies were also detected to each of the 20 peptides, many at high prevalence (up to 84% of responders), and were directed to both nonrepeat and repeat regions. Immune responses to LSA-3 were detectable even in individuals of less than 5 years of age and increased with age and hence exposure to malaria, although they were not directly related to the level of malaria transmission. Thus, several valuable T- and B-cell epitopes were characterized all along the LSA-3 protein, supporting the antigenicity of thisP. falciparumvaccine candidate. Finally, antibodies specific for peptide LSP10 located in a nonrepeat region of LSA-3 were found significantly associated with a lower risk of malaria attack over 1 year of daily clinical follow-up in children between the ages of 7 and 15 years, but not in older individuals.