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Yersinia enterocolitica type III secretion chaperone SycH. Recombinant expression, purification, characterisation, and crystallisation.

All pathogenic Yersinia species (Y. enterocolitica, Y. pestis, and Y. pseudotuberculosis) share a type three secretion system (TTSS) that allows translocation of effector proteins into host cells. Yersinia enterocolitica SycH is a chaperone assisting the transport of the effector YopH and two regulatory components of the TTSS, YscM1 and YscM2. We have recombinantly expressed SycH in Escherichia coli. Purification of tag-free SycH to near homogeneity was achieved by combining ammonium sulfate precipitation, anion exchange chromatography, and gel filtration. Functionality of purified SycH was proven by demonstrating binding to YopH. SycH crystals were grown that diffracted to 2.94A resolution. Preliminary crystallographic data and biochemical findings suggest that SycH forms homotetramers. SycH may therefore represent a novel class of TTSS chaperones. In addition, we found that YopH was enzymatically active in the presence of SycH. This implies that the function of the secretion chaperone SycH is not to keep YopH in a globally unfolded state prior to secretion.

Base Sequence↗

Triterpenoid glycosides from Bacopa monnieri.

Two triterpenoid glycosides have been isolated along with 10 known saponins from Bacopa monnieri. Structures of the compounds have been elucidated as 3-O-[beta-D-glucopyranosyl-(1-->3)-beta-D-glucopyranosyl] jujubogenin (1) and 3-O-[beta-D-glucopyranosyl-(1-->3)-beta-D-glucopyranosyl] pseudojujubogenin (2) by high resolution NMR spectral data and chemical correlations. Further, the chemical compositions of bacosides A and B have been delineated.

Animals↗

Breed classification of Lao People's Democratic Republic (Lao PDR) and Thai native chickens using synchrotron radiation-based Fourier transform infrared spectroscopy and genotyping by sequencing.

Lao PDR harbors substantial genetic diversity in native chicken populations, representing an important resource for sustainable production and long-term food security. This study aimed to classify five Lao native chicken breeds-Ou, Black Bone, Horn Chou, Yolk, and Chae-and to discriminate them from a Thai native breed, Leung Hang Khao (LK), using integrative genotype-based approaches. Blood samples were collected from 50 LK and Lao native chickens (32 Ou, 10 Black Bone, 9 Horn Chou, 121 Yolk, and 41 Chae). Genomic DNA was extracted and analyzed using synchrotron radiation-based Fourier-transform infrared (SR-FTIR) spectroscopy to characterize biochemical composition, while genotyping-by-sequencing (GBS) was employed to identify genome-wide single nucleotide polymorphisms (SNPs). SR-FTIR analysis revealed highly significant differences among breeds in nucleotide-associated functional groups, including thymine, adenine, guanine, cytosine, as well as DNA backbone and deoxyribose components (P < 0.001). Multivariate analyses demonstrated that principal component analysis (PCA) of SR-FTIR spectra effectively discriminated chicken breeds, while hierarchical cluster analysis (HCA) further resolved them into two major clusters with distinct sub-clusters, reflecting variation in DNA biochemical composition. In contrast, GBS analysis identified 1484 common SNPs; however, PCA based on SNP data showed limited resolution in clearly separating breeds, despite revealing similar clustering trends. Overall, the results highlight the strong discriminatory power of SR-FTIR spectroscopy for rapid and effective classification of native chicken breeds at the molecular level, outperforming SNP-based differentiation under the current marker density. This study provides novel insights into the application of synchrotron-based spectroscopic techniques in poultry genetics and contributes valuable baseline information for the conservation and utilization of Lao native chicken genetic resources.

Breed classification↗

Purification and 3D structural analysis of oligomeric human multidrug transporter ABCG2.

ABCG2 is a multidrug efflux pump associated with resistance of cancer cells to a plethora of unrelated drugs. ABCG2 is a "half-transporter," and previous studies have indicated that it forms homodimers and higher oligomeric species. In this manuscript, electron microscopic structural analysis directly addressed this issue. An N-terminal hexahistidine-tagged ABCG2(R482G) isoform was expressed to high levels in insect cells. An extensive detergent screen was employed to effect extraction of ABCG2(R482G) from membranes and identified only the fos-choline detergents as efficient. Soluble protein was purified to >95% homogeneity by a three-step procedure while retaining the ability to bind substrates. Cryonegative stain electron microscopy of purified ABCG2(R482G) provided 3D structural data at a resolution of approximately 18 A. Single-particle analysis revealed that the complex forms a tetrameric complex ( approximately 180 A in diameter x approximately 140 A high) with an aqueous central region. We interpret the tetrameric structure as comprising four homodimeric ABCG2(R482G) complexes.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

Valence electron energy-loss spectroscopy in monochromated scanning transmission electron microscopy.

With the development of monochromators for (scanning) transmission electron microscopes, valence electron energy-loss spectroscopy (VEELS) is developing into a unique technique to study the band structure and optical properties of nanoscale materials. This article discusses practical aspects of spatially resolved VEELS performed in scanning transmission mode and the alignments necessary to achieve the current optimum performance of approximately 0.15 eV energy resolution with an electron probe size of approximately 1 nm. In particular, a collection of basic concepts concerning the acquisition process, the optimization of the energy resolution, the spatial resolution and the data processing are provided. A brief study of planar defects in a Y(1)Ba(2)Cu(3)O(7-)(delta) high-temperature superconductor illustrates these concepts and shows what kind of information can be accessed by VEELS.

Journal Article↗

The use of recombinant methods and molecular engineering in protein crystallization.

Recombinant techniques are routinely used for the preparation of protein samples for structural studies including X-ray crystallography. Among other benefits, these methods allow for a vast increase in the amount of obtained protein as compared to purification from source tissues, ease of purification when fusion proteins containing affinity tags are used, introduction of SeMet for phasing, and the opportunity to modify the protein to enhance its crystallizability. Protein engineering may involve removal of flexible regions including termini and interior loops, as well as replacement of residues that affect solubility. Moreover, modification of the protein surface to induce crystal growth may include rational engineering of surface patches that can readily mediate crystal contacts. The latter approach can be used to obtain proteins of crystals recalcitrant to crystallization or to obtain well-diffracting crystals in lieu of wild-type crystals yielding data to limited resolution. This review discusses recent advances in the field and describes a number of examples of diverse protein engineering techniques used in crystallographic investigations.

Chemistry Techniques, Analytical↗

Geographic information systems as a tool for control program management for schistosomiasis in Egypt.

During a 4-year study a geographic information system (GIS) risk model was constructed for predicting the relative risk of schistosomiasis in Kafr El-Sheikh governorate, Egypt. A 1-year 1990-1991 time series on diurnal temperature difference (dT) prepared from the advanced very high resolution radiometer (AVHRR) sensor on the NOAA-11 satellite was used to develop a regional risk model for the Nile delta based on thermal-hydrological domains. A May 15, 1990 Landsat TM scene (path 177, Row 38) was used to develop a local 'village-scale' environmental risk model based on higher resolution satellite sensor data (30 m picture element size at earth surface). Four of ten classes derived from a tasseled cap (Tcap) transformation of the Landsat TM scene were shown to be significantly related to a 5-year Schistosoma mansoni prevalence database from the Ministry of Health. A risk model was developed based on dT and the proportional area of the four Tcap classes in 5 km(2) buffer zones centered on rural health unit (RHU) reporting units. Available historical data on S. mansoni and its snail host Biomphalaria alexandrina, as well as recent field collected data were gathered and incorporated as separate themes. Model validation was done using data collected on snail population bionomics-infection rates, water quality, underground water table and cercariometry at 13 hydrologically representative sites. The role of soil type, water table and water quality was studied at 79 of 154 rural health unit sites. The model permitted retrieval of relevant data by RHU point location. For the first time in Egypt, the Kafr El-Sheikh GIS schistosoma prediction model can support MOH efforts to make more accurate control program decisions based on environmental predilection sites of endemic Schistosomiasis mansoni.

Animals↗

The structure of the H(+)-ATP synthase from chloroplasts and its subcomplexes as revealed by electron microscopy.

The electron microscopic data available on CF(0)F(1) and its subcomplexes, CF(0), CF(1), subunit III complex are collected and the CF(1) data are compared with the high resolution structure of MF(1). The data are based on electron microscopic investigation of negatively stained isolated CF(1), CF(0)F(1) and subunit III complex. In addition, two-dimensional crystals of CF(0)F(1) and CF(0)F(1) reconstituted liposomes were investigated by cryo-electron microscopy. Progress in the interpretation of electron microscopic data from biological samples has been made with the introduction of image analysis. Multi-reference alignment and classification of images have led to the differentiation between different conformational states and to the detection of a second stalk. Recently, the calculation of three-dimensional maps from the class averages led to the understanding of the spatial organisation of the enzyme. Such three-dimensional maps give evidence of the existence of a third connection between the F(0) part and F(1) part.

Chloroplasts↗

Structure of the photosynthetic reaction centre from Rhodobacter sphaeroides reconstituted with anthraquinone as primary quinone Q(A).

In the photosynthetic reaction centre (RC) from the purple bacterium Rhodobacter sphaeroides, the primary quinone, a ubiquinone-10 (Q(A)), has been substituted by anthraquinone. Three-dimensional crystals have been grown from the modified RC and its structure has been determined by X-ray crystallography to 2.4 A resolution. The bindings of the head-group from ubiquinone-10 and of the anthraquinone ring are very similar. In particular, both rings are parallel to each other and the hydrogen bonds connecting the native ubiquinone-10 molecule to AlaM260 and HisM219 are conserved in the anthraquinone containing RC. The space of the phytyl tail missing in the anthraquinone exchanged RC is occupied by the alkyl chain of a detergent molecule. Other structural changes of the Q(A)-binding site are within the limit of resolution. Our structural data bring strong credit to the very large amount of spectroscopic data previously achieved in anthraquinone-replaced RCs and which have participated in the determination of the energetics of the quinone system in bacterial RCs.

Anthraquinones↗

Separation of DNA sequencing fragments up to 1000 bases by using poly(ethylene oxide)-filled capillary electrophoresis.

We have demonstrated that DNA bases up to 1000 base pairs (bp) in a sequencing ladder can be separated using poly(ethylene oxide)-filled capillary electrophoresis (resolution of raw data = 0.5 at 966 bp). Separation performance of this sieving matrix has been tested under different experimental conditions. It was found that the electric field strength played a critical role in the onset of reptation and thus the separation efficiency. Optimized gel composition and concentration is required for good separation, but the total gel concentration should lie between 2.5 and 3.0%. We observed that the capillary length influences the number of theoretical plates and the maximum readable length of DNA. For sequencing up to 500 bp, relatively nonviscous solutions can be used, greatly facilitating the replacement of the sieving matrix in between runs.

DNA↗

Noncontact mapping of cardiac arrhythmias.

The development of new mapping systems is beginning to overcome some of the limitations of conventional techniques by offering percutaneous deployment, simultaneous acquisitions of data, high-resolution maps, and correlation of anatomy and electrophysiology, in addition to a catheter location system. The noncontact mapping system continues to undergo development and does not completely address all requirements for an ideal mapping system. The limitations of the system include deterioration in the quality of electrogram reconstruction with increasing distance between the MEA and the endocardium, the inability of the noncontact system to identify subendocardial activation, and problems with distinguishing noise from low-amplitude diastolic electrograms.

Arrhythmias, Cardiac↗

X-ray structure of a dihydropyrimidinase from Thermus sp. at 1.3 A resolution.

Dihydropyrimidinases (hydantoinases) catalyse the reversible hydrolytic ring-opening of cyclic diamides such as dihydropyrimidines in the catabolism of pyrimidines. In biotechnology, these enzymes find application in the enantiospecific production of amino acids from racemic hydantoins. The crystal structure of a D-enantio-specific dihydropyrimidinase from Thermus sp. (D-hydantoinase) was solved de novo by multiwavelength anomalous diffraction phasing. In spite of a large unit cell the D-hydantoinase crystals exhibit excellent diffraction properties. The structure was subsequently refined at 1.30 A resolution against native data. The core of D-hydantoinase consists of a (alpha/beta)(8)-barrel, which is flanked by a beta-sheet domain and some additional helices. In the active site, a carboxylated lysine residue and the catalytically active hydroxide ion bridge a binuclear zinc centre. The tertiary structure and shape of the active site show strong homology to that of ureases, dihydroorotases, and phosphotriesterases. The homology of the active site was exploited for in silicio docking of substrates in the active site. This could shed light both on the substrate binding in hydantoinases and on the recently highly discussed origin of the proton in the course of hydantoinase catalysis.

Amidohydrolases↗

Trianthenol: an antifungal tetraterpenoid from Trianthema portulacastrum (Aizoaceae).

An antifungal tetraterpenoid named trianthenol 1 has been isolated from the chloroform extract of Trianthema portulacastrum. Its structure was established as 15-hydroxymethyl-2,6,10,18,22,26,30-heptamethyl-14-methylene- 17-hentriacontene on the basis of spectroscopic data including high resolution mass and two-dimensional NMR techniques. A benzaldehyde derivative 2, a pentacyclic triterpenoid 3 and benzoic acid derivatives 4-5 are also reported for the first time from Trianthema portulacastrum.

Antifungal Agents↗

Quantitative comparison of electrically and acoustically evoked auditory perception: implications for the location of perceptual mechanisms.

Electrical stimulation of the human auditory system produces different patterns of spatial and temporal neural activity than those that occur in the normal, acoustically stimulated system. Quantitative comparison of psychophysical performance measured with acoustic and electrical stimulation may allow us to infer the physiological locus of perceptual mechanisms. In this paper we compare psychophysical data on temporal resolution from normal-hearing listeners, cochlear implant listeners, and patients electrically stimulated on the cochlear nucleus. Measures of gap detection, forward masking, and modulation detection will be compared. These comparisons demonstrate that temporal processing is relatively similar across these three groups once the obvious differences in dynamic range are taken into consideration. In addition, preliminary results with speech processors indicate that implant patients can utilize all temporal information in speech. Thus, implant patients have relatively normal temporal resolution and can integrate temporal cues normally for the recognition of complex acoustic patterns such as speech. These results imply that the central auditory systems of implant patients are able to fully utilize the non-natural patterns of temporal neural information produced by electrical stimulation. The differences in the microstructure of the neural pattern (phase locking, stochastic independence of fibers, spatial distribution of activity, etc.) between electrical and acoustic stimulation are apparently not necessary for temporal processing. Thus, the physiological locus of temporal processing mechanisms must be more central in the auditory system than the cochlea and cochlear nucleus.

Acoustic Stimulation↗

Natural radioactivity in phosphates, phosphogypsum and natural waters in Morocco.

The contents of natural radionuclides (uranium, actinium and thorium series) were measured in sedimentary phosphate rock samples using high-resolution gamma spectrometry. Data obtained for uranium content (ppm) were compared with the results obtained by a method based on the measurements using solid-state nuclear track detectors (SSNTD) in the same samples. The potential leaching of radionuclides from sedimentary phosphate rock during the industrial production of the phosphoric acid was studied. The process of leaching of the radioisotopes from phosphogypsum was discussed. A method for the direct alpha counting of 226Ra thin source, elaborated by the deposition of Ra from aqueous solutions on manganese oxides film deposited on polyvinyl support, have been developed and applied for the determination of 226Ra in natural water samples. The results show that only the water sample from the mine area reveals the presence of 226Ra at a level of about 0.2 Bq l-1.

Actinium↗

Effect of chelation therapy on endothelial function in patients with coronary artery disease: PATCH substudy.

OBJECTIVES: The purpose of this study was to evaluate the effect of chelation therapy with ethylenediamine tetraacetic acid (EDTA) on endothelium-dependent vasomotor responses in patients with documented coronary artery disease (CAD). BACKGROUND: Oxidative stress plays an important role in the dysfunction of endothelium and development of atherosclerosis. Modification of cardiac risk factors and employment of antioxidants have been shown to improve endothelial function. Ethylenediamine tetraacetic acid chelation therapy is considered to be a complementary therapy for patients with CAD and is proposed to have antioxidant properties. METHODS: A total of 47 patients enrolled in the Program to Assess Alternative Treatment Strategies to Achieve Cardiac Health (PATCH) participated in this substudy and had complete data. High-resolution ultrasound was used to assess endothelium-dependent brachial artery flow-mediated vasodilation (FMD) in patients with CAD in a randomized, double-blind, and placebo-controlled fashion. Patients were randomized to chelation therapy or placebo. The primary end point was the absolute difference in FMD after the first and 33rd treatments (6 months) of study groups compared with their baselines. RESULTS: At the baseline, the study population had mild impairment of FMD (7.2 +/- 3.4%). The first chelation treatment did not change FMD as compared with placebo (chelation 6.5 +/- 3.5% vs. placebo 7.4 +/- 2.9%; p value = 0.371). The brachial artery studies at six months did not demonstrate significant differences in FMD between study groups (placebo 7.3 +/- 3.4% vs. chelation 7.3 +/- 3.2%; p value = 0.961). CONCLUSIONS: Our results suggest that EDTA chelation therapy in combination with vitamins and minerals does not provide additional benefits on abnormal vasomotor responses in patients with CAD optimally treated with proven therapies for atherosclerotic risk factors.

Aged↗

Role of gamma knife therapy in the management of pituitary tumors.

1. Gamma knife therapy is an effective method of delivering radiation to pituitary tumors that have failed surgery and may be used as primary treatment in circumstances in which the patient refuses or is unsuitable for a transsphenoidal procedure. 2. Stereotactic radiosurgery with the gamma knife unit is generally administered in a single session unlike fractionated radiotherapy, which is administered four to five times per week over a 6-week period. 3. Preliminary data suggest that resolution of pituitary hypersecretion is faster with gamma knife therapy than with conventional radiotherapy. 4. Because of the nature of the gamma knife therapy and the fact that the radiation dose conforms to the tumor shape, there is a steep fall-off of radiation to surrounding tissue. Accordingly, the radiation dose to extrapituitary brain is substantially less with gamma knife radiosurgery than with conventional radiotherapy. This suggests that the development of second brain tumors and neurocognitive complications, which are significant risks with conventional radiotherapy, is much less likely with gamma knife surgery. 5. Gamma knife radiosurgery can be used to ablate tumors invading the cavernous sinus. 6. Gamma knife radiosurgery is safe as long as the dose of radiation to the optic structures is kept under 10 Gy. 7. Long-term follow-up is required for pituitary tumors treated by gamma knife therapy so as to determine its efficacy as well as its effects on pituitary function and any resultant complications.

Adenoma↗

A client/server system for Internet access to biomedical text/image databanks.

Internet access to mixed text/image databanks is finding application in the medical world. An example is a database of medical X-rays and associated data consisting of demographic, socioeconomic, physician's exam, medical laboratory and other information collected as part of a nationwide health survey conducted by the government. Another example is a collection of digitized cryosection images, CT and MR taken of cadavers as part of the National Library of Medicine's Visible Human Project. In both cases, the challenge is to provide access to both the image and the associated text for a wide end user community to create atlases, conduct epidemiological studies, to develop image-specific algorithms for compression, enhancement and other types of image processing, among many other applications. The databanks mentioned above are being created in prototype form. This paper describes the prototype system developed for the archiving of the data and the client software to enable a broad range of end users to access the archive, retrieve text and image data, display the data and manipulate the images. System design considerations include; data organization in a relational database management system with object-oriented extensions; a hierarchical organization of the image data by different resolution levels for different user classes; client design based on common hardware and software platforms incorporating SQL search capability, X Window, Motif and TAE (a development environment supporting rapid prototyping and management of graphic-oriented user interfaces); potential to include ultra high resolution display monitors as a user option; intuitive user interface paradigm for building complex queries; and contrast enhancement, magnification and mensuration tools for better viewing by the user.

Anatomy, Cross-Sectional↗