Miracle-dz/UniFAR-FTUs huggingface dataset:
Flavin motion in p-hydroxybenzoate hydroxylase. Substrate and effector specificity of the Tyr22-->Ala mutant.

The side chain of Tyr222 in p-hydroxybenzoate hydroxylase interacts with the carboxy moiety of the substrate. Studies on the Tyr222-->Phe mutant, [F222]p-hydroxybenzoate hydroxylase, have shown that disruption of this interaction hampers the hydroxylation of 4-hydroxybenzoate. Tyr222 is possibly involved in flavin motion, which may facilitate the exchange of substrate and product during catalysis. To elucidate the function of Tyr222 in more detail, in the present study the substrate and effector specificity of the Tyr222-->Ala mutant, [A222]p-hydroxybenzoate hydroxylase, was investigated. Replacement of Tyr222 by Ala impairs the binding of the physiological substrate 4-hydroxybenzoate and the substrate analog 4-aminobenzoate. With these compounds, [A222]p-hydroxybenzoate hydroxylase mainly acts as a NADPH oxidase. [A222]p-hydroxybenzoate hydroxylase tightly interacts with 2,4-dihydroxybenzoate and 2-hydroxy-4-aminobenzoate. Crystallographic data [Schreuder, H.A. , Mattevi, A. , Oblomova, G. , Kalk, K.H. , Hol, W.G. J., van der Bolt, F.J.T. & van Berkel, W.J.H. (1994) Biochemistry 33, 10161-10170] suggest that this is due to motion of the flavin ring out of the active site, allowing hydrogen-bond interaction between the 2-hydroxy group of the substrate analogs and N3 of the flavin. [A222]p-Hydroxybenzoate hydroxylase produces about 0.6 mol 2,3,4-trihydroxybenzoate from 2,4-dihydroxybenzoate/mol NADPH oxidized. This indicates that reduction of the Tyr222-->Ala mutant shifts the equilibrium of flavin conformers towards the productive "in' position. [A222]p-Hydroxybenzoate hydroxylase converts 2-fluoro-4-hydroxybenzoate to 2-fluoro-3,4-dihydroxybenzoate. The regioselectivity of hydroxylation suggests that [A222]p-hydroxybenzoate hydroxylase binds the fluorinated substrate in the same orientation as wild-type. Spectral studies suggest that wild-type and [A222]p-hydroxybenzoate hydroxylase bind 2-fluoro-4-hydroxybenzoate in the phenolate form with the flavin ring preferring the "out' conformation. Despite activation of the fluorinated substrate and in contrast to the wild-type enzyme, [A222]p-hydroxybenzoate hydroxylase largely produces hydrogen peroxide. The effector specificity of p-hydroxybenzoate hydroxylase is not changed by the Tyr222-->Ala replacement. This supports the idea that the effector specificity is mainly dictated by the protein-substrate interactions at the re-side of the flavin ring.
NADPH Oxidase-dependent Acid Production in Airway Epithelial Cells*

The purpose of this study was to determine the role of NADPH oxidase in H+ secretion by airway epithelia. In whole cell patch clamp recordings primary human tracheal epithelial cells (hTE) and the human serous gland cell line Calu-3 expressed a functionally similar zincblockable plasma membrane H+ conductance. However, the rate of H+ secretion of confluent epithelial monolayers measured in Ussing chambers was 9-fold larger in hTE compared with Calu-3. In hTE H+ secretion was blocked by mucosal ZnCl2 and the NADPH oxidase blockers acetovanillone and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), whereas these same blockers had no effect in Calu-3. We determined levels of transcripts for the NADPH oxidase transmembrane isoforms (Nox1 through -5, Duox1 and -2, and p22phox) and found Duox1, -2, and p22phox to be highly expressed in hTE, as well as the intracellular subunits p40phox, p47phox, and p67phox. In contrast, Calu-3 lacked transcripts for Duox1, p40phox, and p47phox. Anti-Duox antibody staining resulted in prominent apical staining in hTE but no significant staining in Calu-3. When treated with amiloride to block the Na+/H+ exchanger, intracellular pH in hTE acidified at significantly higher rates than in Calu-3, and treatment with AEBSF blocked acidification. These data suggest a role for an apically located Duox-based NADPH oxidase during intracellular H+ production and H+ secretion, but not in H+ conduction.
Identification of Bacillus subtilis genes expressed early during sporulation

Labelled cDNA transcribed in vitro from early‐sporulation RNA was enriched for sporulation‐specific sequences by subtractive hybridization to an excess of vegetative RNA and used to probe libraries of Bacillus subtilis chromosomal DNA. From the initial collection of clones that coded for RNAs transcribed preferentially during sporutation, several were subcloned and studied in more detail. It was found that two clones contained sequences (dciA and dciB) that had an undetectable level of transcription during vegetative growth but had transcripts that started to appear no later than eight minutes after induction of sporulation. A third DNA segment (dciC) was expressed at a low level in vegetative cells and increased within four minutes after induction of sporulation.
LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing

Little is known about the protein constituents of autophagosome membranes in mammalian cells. Here we demonstrate that the rat microtubule‐associated protein 1 light chain 3 (LC3), a homologue of Apg8p essential for autophagy in yeast, is associated to the autophagosome membranes after processing. Two forms of LC3, called LC3‐I and ‐II, were produced post‐translationally in various cells. LC3‐I is cytosolic, whereas LC3‐II is membrane bound. The autophagic vacuole fraction prepared from starved rat liver was enriched with LC3‐II. Immunoelectron microscopy on LC3 revealed specific labelling of autophagosome membranes in addition to the cytoplasmic labelling. LC3‐II was present both inside and outside of autophagosomes. Mutational analyses suggest that LC3‐I is formed by the removal of the C‐terminal 22 amino acids from newly synthesized LC3, followed by the conversion of a fraction of LC3‐I into LC3‐II. The amount of LC3‐II is correlated with the extent of autophagosome formation. LC3‐II is the first mammalian protein identified that specifically associates with autophagosome membranes.
Preparation and Characterization of Camptothecin Solid Lipid Nanoparticles

ABSTRACT Camptothecin (CA), an antitumor drug, was incorporated into solid lipid nanoparticles (SLNs) prepared by high-pressure homogenization. A Taguchi orthogonal experimental design was used to study the influence of four different variables, with each variable having three value levels on nanoparticle size. Analysis of variance (ANOVA) has been used to evaluate the preparation of CA-SLNs and perform product optimization. The optimized CA-SLNs suspension was lyophilized using mannitol and glucose as cryoprotectants. The physicochemical characteristics of CA-SLNs were evaluated using transmission electron microscopy (TEM), electrophoresis, and differential scanning calorimetry (DSC). The release of camptothecin from CA-SLNs in various media was evaluated using a high-performance liquid chromatography (HPLC) method. The results showed that the concentration of emulsifier and the homogenization pressure had a significant influence on the particle size. The optimized CA-SLNs had an average diameter of about 200 nm, exhibited monodispersity with Dw/Dn of 1.06, and carried a negative charge. The optimal cryoprotectants consisted of 10% mannitol and 5% glucose in nanoparticle suspension. Lyophilized product was reconstituted in distilled water within 0.5 min without change of nanoparticle size. Camptothecin might exist in an amorphous state in SLNs. In vitro results showed that drug release was achieved for up to one week, and the released camptothecin quickly changed to open carboxylate form in the biological pH phosphate buffer. The results indicate that SLNs might be good potential sustained-release delivery vehicles for camptothecin or other lipophilic drugs.
Crystal structures of bis[2-(pyridin-2-yl)phenyl-κ2 N,C 1]rhodium(III) complexes containing an acetonitrile or monodentate thyminate(1−) ligand

Bis[2-(pyridin-2-yl)phenyl]rhodium(III) complexes bearing acetonitrile or monodeprotonated thyminate (Hthym−) were characterized by X-ray analysis, and in the latter complexes it was revealed that Hthym− coordinated to an RhIII centre through the N1 atom together with a hydrogen-bonded methanol or ethanol co-ligand in the cis position.
Archaeology of the Russian Far East: Essays in Stone Age Prehistory

Arranged in chronological order and by region, each of these studies is written by a specialist who has participated in some or all of the archaeological expeditions reported here. They show not just the unanticipated richness of the archaeology of the Russian Far East but, more important, the contributions these sites can make to the archaeology of the region and of the world.
Screening, Nucleotide Sequence, and Biochemical Characterization of an Esterase from Pseudomonas fluorescens with High Activity towards Lactones

ABSTRACT A genomic library of Pseudomonas fluorescens DSM 50106 in a λRESIII phage vector was screened in Escherichia coli K-12 for esterase activity by using α-naphthyl acetate and Fast Blue RR. A 3.2-kb DNA fragment was subcloned from an esterase-positive clone and completely sequenced. Esterase EstF1 was encoded by a 999-bp open reading frame (ORF) and exhibited significant amino acid sequence identity with members of the serine hydrolase family. The deduced amino acid sequences of two other C-terminal truncated ORFs exhibited homology to a cyclohexanone monooxygenase and an alkane hydroxylase. However, esterase activity was not induced by growing of P. fluorescens DSM 50106 in the presence of several cyclic ketones. The esterase gene was fused to a His tag and expressed in E. coli. The gene product was purified by zinc ion affinity chromatography and characterized. Detergents had to be added for purification, indicating that the enzyme was membrane bound or membrane associated. The optimum pH of the purified enzyme was 7.5, and the optimum temperature was 43°C. The showed highest purified enzyme activities towards lactones. The activity increased from γ-butyrolactone (18.1 U/mg) to ɛ-caprolactone (21.8 U/mg) to δ-valerolactone (36.5 U/mg). The activities towards the aliphatic esters were significantly lower; the only exception was the activity toward ethyl caprylate, which was the preferred substrate.
Two Complementary Mechanisms Underpin Cell Wall Patterning during Xylem Vessel Development[OPEN]

The CELLULOSE SYNTHASE INTERACTING1 protein directs secondary wall patterning during the early phases of xylem vessel development. The evolution of the plant vasculature was essential for the emergence of terrestrial life. Xylem vessels are solute-transporting elements in the vasculature that possess secondary wall thickenings deposited in intricate patterns. Evenly dispersed microtubule (MT) bands support the formation of these wall thickenings, but how the MTs direct cell wall synthesis during this process remains largely unknown. Cellulose is the major secondary wall constituent and is synthesized by plasma membrane-localized cellulose synthases (CesAs) whose catalytic activity propels them through the membrane. We show that the protein CELLULOSE SYNTHASE INTERACTING1 (CSI1)/POM2 is necessary to align the secondary wall CesAs and MTs during the initial phase of xylem vessel development in Arabidopsis thaliana and rice (Oryza sativa). Surprisingly, these MT-driven patterns successively become imprinted and sufficient to sustain the continued progression of wall thickening in the absence of MTs and CSI1/POM2 function. Hence, two complementary principles underpin wall patterning during xylem vessel development.
Influences of semantic and prosodic cues on word repetition and categorization in autism.

PURPOSE To investigate sensitivity to prosodic and semantic cues to emotion in individuals with high-functioning autism (HFA). METHOD Emotional prosody and semantics were independently manipulated to assess the relative influence of prosody versus semantics on speech processing. A sample of 10-year-old typically developing children (n = 10) and children with HFA (n = 10) were asked to repeat words that were either emotionally congruent or incongruent in form and content (Experiment 1A). In a second task (Experiment 1B), the same participants were asked to classify stimuli on the basis of emotional prosody. A final experiment (Experiment 2) focused on sensitivity to congruence in a non-emotional source of variation: talker gender. RESULTS The results revealed a selective impairment in spontaneous integration of prosodic and semantic cues to emotion in HFA; however, the same participants were able to categorize emotions on the basis of prosody under reduced task demands. Individuals with HFA were highly sensitive to another surface characteristic in speech: talker gender. CONCLUSIONS The study reveals impairment in the spontaneous integration of prosodic and semantic cues to emotion in HFA; however, insensitivity to surface detail, such as prosody, in HFA appears to be highly task dependent and selective to the domain of emotion.
Genome Properties of the Diatom Phaeodactylum tricornutum 212

Diatoms are a ubiquitous class of microalgae of extreme importance for global primary productivity and for the biogeochemical cycling of minerals such as silica. However, very little is known about diatom cell biology or about their genome structure. For diatom researchers to take advantage of genomics and post-genomics technologies, it is necessary to establish a model diatom species.Phaeodactylum tricornutum is an obvious candidate because of its ease of culture and because it can be genetically transformed. Therefore, we have examined its genome composition by the generation of approximately 1,000 expressed sequence tags. Although more than 60% of the sequences could not be unequivocally identified by similarity to sequences in the databases, approximately 20% had high similarity with a range of genes defined functionally at the protein level. It is interesting that many of these sequences are more similar to animal rather than plant counterparts. Base composition at each codon position and GC content of the genome were compared with Arabidopsis, maize (Zea mays), andChlamydomonas reinhardtii. It was found that distribution of GC within the coding sequences is as homogeneous inP. tricornutum as in Arabidopsis, but with a slightly higher GC content. Furthermore, we present evidence that the P. tricornutum genome is likely to be small (less than 20 Mb). Therefore, this combined information supports the development of this species as a model system for molecular-based studies of diatom biology. The nucleotide sequence data reported has been deposited in GenBank Nucleotide Sequence Database (dbEST section) under accession nos. BI306757 through BI307753.
In Vivo Immune Evasion Mediated by the Herpes Simplex Virus Type 1 Immunoglobulin G Fc Receptor

ABSTRACT Herpes simplex virus (HSV) glycoproteins gE and gI form an immunoglobulin G (IgG) Fc receptor (FcγR) that binds the Fc domain of human anti-HSV IgG and inhibits Fc-mediated immune functions in vitro. gE or gI deletion mutant viruses are avirulent, probably because gE and gI are also involved in cell-to-cell spread. In an effort to modify FcγR activity without affecting other gE functions, we constructed a mutant virus, NS-gE339, that has four amino acids inserted into gE within the domain homologous to mammalian IgG FcγRs. NS-gE339 expresses gE and gI, is FcγR−, and does not participate in antibody bipolar bridging since it does not block activities mediated by the Fc domain of anti-HSV IgG. In vivo studies were performed with mice because the HSV-1 FcγR does not bind murine IgG; therefore, the absence of an FcγR should not affect virulence in mice. NS-gE339 causes disease at the skin inoculation site comparably to wild-type and rescued viruses, indicating that the FcγR− mutant virus is pathogenic in animals. Mice were passively immunized with human anti-HSV IgG and then infected with mutant or wild-type virus. We postulated that the HSV-1 FcγR should protect wild-type virus from antibody attack. Human anti-HSV IgG greatly reduced viral titers and disease severity in NS-gE339-infected animals while having little effect on wild-type or rescued virus. We conclude that the HSV-1 FcγR enables the virus to evade antibody attack in vivo, which likely explains why antibodies are relatively ineffective against HSV infection.
Drosophila Rolling Blackout Displays Lipase Domain‐Dependent and ‐Independent Endocytic Functions Downstream of Dynamin

Drosophila temperature‐sensitive rolling blackout (rbots) mutants display a total block of endocytosis in non‐neuronal cells and a weaker, partial defect at neuronal synapses. RBO is an integral plasma membrane protein and is predicted to be a serine esterase. To determine if lipase activity is required for RBO function, we mutated the catalytic serine 358 to alanine in the G‐X‐S‐X‐G active site, and assayed genomic rescue of rbo mutant non‐neuronal and neuronal phenotypes. The rboS358A mutant is unable to rescue rbo null 100% embryonic lethality, indicating that the lipase domain is critical for RBO essential function. Likewise, the rboS358A mutant cannot provide any rescue of endocytic blockade in rbots Garland cells, showing that the lipase domain is indispensable for non‐neuronal endocytosis. In contrast, rbots conditional paralysis, synaptic transmission block and synapse endocytic defects are all fully rescued by the rboS358A mutant, showing that the RBO lipase domain is dispensable in neuronal contexts. We identified a synthetic lethal interaction between rbots and the well‐characterized dynamin GTPase conditional shibire (shits1) mutant. In both non‐neuronal cells and neuronal synapses, shits1; rbots phenocopies shits1 endocytic defects, indicating that dynamin and RBO act in the same pathway, with dynamin functioning upstream of RBO. We conclude that RBO possesses both lipase domain‐dependent and scaffolding functions with differential requirements in non‐neuronal versus neuronal endocytosis mechanisms downstream of dynamin GTPase activity.
External DDR2-constrained NOC-based 24-processors MPSOC design and implementation on single FPGA

Network on chip (NOC) has been proposed for the connection substrate of multiprocessor system on chip (SoC) due to limited bandwidth of bus based solutions. Although some designs are emerging actual design experiences of NOC based multiprocessor system on chip remain scarce contrary to simulation based studies. However, implementation constraints clearly affects th design and modelling of a complex multiprocessor. In this paper we present the design and implementation of a 24-processors multiprocessor system with 24 processors under the constraints of limited access to 4 external DDR2 memory banks. All the processors and DDR2 memories are connected to a network on chip through open core protocol (OCP) interface. Multiple clock domains result ing from various IP complexities requires global asynchronous local synchronous (GALS) design methodlogy which adds some extra area. The multiprocessor system is fully implemented on Xilinx Virtex-4 FX140 FPGA based board and uses about 90 % of the chip area.
Validity and Reliability of the Western Ontario Rotator Cuff Index (WORC) for Use in Brazil

Objective:To evaluate the validity and reliability of the Brazilian Portuguese version of the Western Ontario Rotator Cuff Index (WORC). Design and Setting:A cross-sectional survey at the Physiotherapy Unit and Outpatient Orthopedic and Traumatology Clinic, Universidade Federal de São Paulo. Methods:To test validity, 100 patients with rotator cuff disorders were evaluated using the WORC, clinically relevant outcomes for patients with shoulder disorders (pain, range of motion, and strength), and the Disabilities of Arm, Shoulder, and Hand (DASH), University of California Los Angeles Shoulder Rating Scale (UCLA), and Short Form-36 (SF-36) self-report measures. The WORC was repeated on 50 patients on the same day (more than 1 hour later) and after a mean interval of 7 days to evaluate the test-retest reliability. Concurrent validity was tested by correlating the WORC to the other outcome measures using Pearson's correlation coefficient. Test-retest reliability and internal consistency were determined by the intraclass correlation coefficient and Cronbach's alpha coefficient, respectively. The scores were used to assess the standard error measurement (SEM) and minimal detectable change (MDC). Results:Analysis between the WORC and clinically relevant outcomes for patients with shoulder disorders revealed weak to strong correlations; the weakest for active internal rotation (r = -0.22) and the strongest for pain during movement (r = -0.75). Strong correlations were found among the WORC and the DASH and UCLA (r = -0.86 and r = 0.80, respectively). There were moderate correlations between the WORC and SF-36 domains (0.37 to 0.69); the best correlations related to the physical domains. Reliability analysis revealed excellent results, with the intraclass correlation coefficient ranging from 0.95 to 0.99 and Cronbach's alpha ranging from 0.88 to 0.97. The SEM was 5.2 and 3.0 for time 0 and after a mean interval of 7 days, respectively. The MDC was 7.1 over this mean time interval (90% confidence interval). Conclusions:The Brazilian version of the WORC proved to be a valid and reliable measurement tool for assessing health-related quality of life in patients with rotator cuff diseases.
Comparative proteomics of seed maturation in oilseeds reveals differences in intermediary metabolism

Proteomics is increasingly being used to understand enzyme expression and regulatory mechanisms involved in the accumulation of storage reserves in crops with sequenced genomes. During the past six years, considerable progress has been made to characterize proteomes of both mature and developing seeds, particularly oilseeds – plants which accumulate principally oil and protein as storage reserves. This review summarizes the emerging proteomics data, with emphasis on seed filling in soy, rapeseed, castor and Arabidopsis as each of these oilseeds were analyzed using very similar proteomic strategies. These parallel studies provide a comprehensive view of source–sink relationships, specifically sucrose assimilation into organic acid intermediates for de novo amino acid and fatty acid synthesis. We map these biochemical processes for seed maturation and illustrate the differences and similarities among the four oilseeds. For example, while the four oilseeds appear capable of producing cytosolic phosphoenolpyruvate as the principal carbon intermediate, soybean and castor also express malic enzymes and malate dehydrogenase, together capable of producing malate that has been previously proposed to be the major intermediate for fatty acid synthesis in castor. We discuss these and other differences in the context of intermediary metabolism for developing oilseeds.