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1.  Genome Sequence of Luminous Piezophile Photobacterium phosphoreum ANT-2200 
Genome Announcements  2014;2(2):e00096-14.
Bacteria of the genus Photobacterium thrive worldwide in oceans and show substantially varied lifestyles, including free-living, commensal, pathogenic, symbiotic, and piezophilic. Here, we present the genome sequence of a luminous, piezophilic Photobacterium phosphoreum strain, ANT-2200, isolated from a water column at 2,200 m depth in the Mediterranean Sea. It is the first genomic sequence of the P. phosphoreum group. An analysis of the sequence provides insight into the adaptation of bacteria to the deep-sea habitat.
doi:10.1128/genomeA.00096-14
PMCID: PMC3990738  PMID: 24744322
2.  Premature Graying as a Consequence of Compromised Antioxidant Activity in Hair Bulb Melanocytes and Their Precursors 
PLoS ONE  2014;9(4):e93589.
Intricate coordinated mechanisms that govern the synchrony of hair growth and melanin synthesis remain largely unclear. These two events can be uncoupled in prematurely gray hair, probably due to oxidative insults that lead to the death of oxidative stress-sensitive melanocytes. In this study, we examined the gene expression profiles of middle (bulge) and lower (hair bulb) segments that had been micro-dissected from unpigmented and from normally pigmented hair follicles from the same donors using quantitative real-time RT-PCR (qPCR) arrays. We found a significant down-regulation of melanogenesis-related genes (TYR, TYRP1, MITF, PAX3, POMC) in unpigmented hair bulbs and of marker genes typical for melanocyte precursor cells (PAX3, SOX10, DCT) in unpigmented mid-segments compared with their pigmented analogues. qPCR, western blotting and spin trapping assays revealed that catalase protein expression and hydroxyl radical scavenging activities are strongly repressed in unpigmented hair follicles. These data provide the first clear evidence that compromised antioxidant activity in gray hair follicles simultaneously affects mature hair bulb melanocytes and their immature precursor cells in the bulge region.
doi:10.1371/journal.pone.0093589
PMCID: PMC3973559  PMID: 24695442
3.  Double-bundle anatomical versus single-bundle isometric medial patellofemoral ligament reconstruction for patellar dislocation 
International Orthopaedics  2013;37(4):617-624.
Purpose
The purpose of this study was to evaluate reconstruction of the medial patellofemoral ligament (MPFL) using the double-bundle anatomical or single-bundle isometric procedure with respect to the patients’ clinical outcomes.
Methods
In this retrospective study, we evaluated the clinical outcome of double-bundle anatomical versus single-bundle isometric reconstruction of the MPFL for patellar dislocation patients. Sixty-three patients were included in this study from August 2004 to January 2008. From August 2004 to September 2006, MPFL reconstruction using a single-bundle isometric technique was performed in 21 patients (26 knees). Since October 2006, the double-bundle anatomical reconstruction of the MPFL has been used as the routine surgical procedure. It was performed in 37 patients (44 knees). Fifty-eight patients (70 knees) could be followed up. According to the different techniques, we divided the patients into two groups: group D with double-bundle anatomical reconstruction (37 patients) and group S with single-bundle isometric reconstruction (21 patients). Clinical evaluation consisted of the number with a patellar re-dislocation, patellar apprehension sign, Kujala score, subjective questionnaire score, the patella lateral shift rate and patellar tilt angle measured by cross-sectional CT scan.
Results
According to the Kujala score and the subjective questionnaire score, the outcome of the double-bundle group was better than the outcome of the single-bundle group especially in the long-term. Patellar re-dislocation occurred in three patients in the group S, while no re-dislocation occurred in the group D. In total, 26.9 % of group S was considered to have patellar instability, compared to 4.54 % of the group D. After operation, the patellar tilt angle (PTA) and the patella lateral shift rate (PLSR) were restored to the normal range, with statistical significance (P < 0.05) compared to the preoperative state.
Conclusion
Single- and double-bundle reconstruction of the MPFL can both effectively restore patella stability and improve knee function. However, outcomes in the follow-up period showed that the double-bundle surgery procedure was much better than in single-bundle surgery.
doi:10.1007/s00264-013-1788-6
PMCID: PMC3609965  PMID: 23371425
4.  (2-Chloro­pyrimidin-4-yl)ferrocene 
In the title compound, [Fe(C5H5)(C9H6ClN2)], the two cyclo­penta­dienyl rings are almost parallel, subtending a dihedral angle of 3.01 (5)°. The dihedral angle between the substituted cyclo­penta­dienyl ring and the pyrimidinyl ring is 12.02 (1)°. The conformation of the two cyclopentadienyl rings in the ferrocenyl moiety is eclipsed.
doi:10.1107/S1600536814004917
PMCID: PMC3998571  PMID: 24826094
5.  Colonization and plant growth promoting characterization of endophytic Pseudomonas chlororaphis strain Zong1 isolated from Sophora alopecuroides root nodules 
Brazilian Journal of Microbiology  2013;44(2):623-631.
The endophytic strain Zong1 isolated from root nodules of the legume Sophora alopecuroides was characterized by conducting physiological and biochemical tests employing gfp-marking, observing their plant growth promoting characteristics (PGPC) and detecting plant growth parameters of inoculation assays under greenhouse conditions. Results showed that strain Zong1 had an effective growth at 28 ºC after placed at 4–60 ºC for 15 min, had a wide range pH tolerance of 6.0–11.0 and salt tolerance up to 5% of NaCl. Zong1 was resistant to the following antibiotics (μg/mL): Phosphonomycin (100), Penicillin (100) and Ampicillin (100). It could grow in the medium supplemented with 1.2 mmol/L Cu, 0.1% (w/v) methylene blue and 0.1–0.2% (w/v) methyl red, respectively. Zong1 is closely related to Pseudomonas chlororaphis based on analysis the sequence of 16S rRNA gene. Its expression of the gfp gene indicated that strain Zong1 may colonize in root or root nodules and verified by microscopic observation. Furthermore, co-inoculation with Zong1 and SQ1 (Mesorhizobium sp.) showed significant effects compared to single inoculation for the following PGPC parameters: siderophore production, phosphate solubilization, organic acid production, IAA production and antifungal activity in vitro. These results suggest strains P. chlororaphi Zong1 and Mesorhizobium sp. SQ1 have better synergistic or addictive effect. It was noteworthy that each growth index of co-inoculated Zong1+SQ1 in growth assays under greenhouse conditions is higher than those of single inoculation, and showed a significant difference (p < 0.05) when compared to a negative control. Therefore, as an endophyte P. chlororaphis Zong1 may play important roles as a potential plant-growth promoting agent.
doi:10.1590/S1517-83822013000200043
PMCID: PMC3833168  PMID: 24294262
PGPC; endophyte; the gpf-marker; colonization; co-inoculation
6.  Novel Rod-Shaped Magnetotactic Bacteria Belonging to the Class Alphaproteobacteria 
Novel large, rod-shaped magnetotactic bacteria (MTB) were discovered in intertidal sediments of the Yellow Sea, China. They biomineralized more than 300 rectangular magnetite magnetosomes per cell. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that they are affiliated with the Alphaproteobacteria and may represent a new genus of MTB.
doi:10.1128/AEM.03869-12
PMCID: PMC3623124  PMID: 23455351
7.  Valproic acid increases white matter repair and neurogenesis after stroke 
Neuroscience  2012;220:313-321.
Acute treatment of stroke with HDAC inhibitors has been shown to reduce ischemic cell damage; however, it is unclear whether delayed treatment with HDAC inhibitors will contribute to the brain repair and plasticity. In the present study, we investigated the effects of delayed treatment of stroke with a pan HDAC inhibitor, valproic acid (VPA), on white matter injury and neurogenesis during stroke recovery. Administration of VPA at a dose of 100 mg/kg for 7 days starting 24 hours after middle cerebral artery occlusion (MCAo) in rats significantly improved neurological outcome measured 7 to 28 days post-MCAo. In addition, the VPA treatment significantly increased oligodendrocyte survival and newly generated oligodendrocytes, which was associated with elevation of myelinated axonal density in the ischemic boundary 28 days after MCAo. VPA treatment also increased the expression of glutamate transporter 1 (GLT1) in the ischemic boundary after stroke, and increased acetylated histone H4 expression in neuroblasts and the number of new neurons in striatal ischemic boundary region. This study provides new evidence that the delayed VPA treatment enhances white matter repair and neurogenesis in ischemic brain, which may contribute to improved functional outcome.
doi:10.1016/j.neuroscience.2012.06.012
PMCID: PMC3412884  PMID: 22704966
Valproic acid; oligodendrocyte; axon; neural progenitor cells; subventricular zone (SVZ); stroke
8.  A Potential Relationship among Beta-Defensins Haplotype, SOX7 Duplication and Cardiac Defects 
PLoS ONE  2013;8(8):e72515.
Objective
To determine the pathogenesis of a patient born with congenital heart defects, who had appeared normal in prenatal screening.
Methods
In routine prenatal screening, G-banding was performed to analyse the karyotypes of the family and fluorescence in situ hybridization was used to investigate the 22q11.2 deletion in the fetus. After birth, the child was found to be suffering from heart defects by transthoracic echocardiography. In the following study, sequencing was used to search for potential mutations in pivotal genes. SNP-array was employed for fine mapping of the aberrant region and quantitative real-time PCR was used to confirm the results. Furthermore, other patients with a similar phenotype were screened for the same genetic variations. To compare with a control, these variations were also assessed in the general population.
Results
The child and his mother each had a region that was deleted in the beta-defensin repeats, which are usually duplicated in the general population. Besides, the child carried a SOX7-gene duplication. While this duplication was not detected in his mother, it was found in two other patients with cardiac defects who also had the similar deletion in the beta-defensin repeats.
Conclusion
The congenital heart defects of the child were probably caused by a SOX7-gene duplication, which may be a consequence of the partial haplotype of beta-defensin regions at 8p23.1. To our knowledge, this is the first congenital heart defect case found to have the haplotype of beta-defensin and the duplication of SOX7.
doi:10.1371/journal.pone.0072515
PMCID: PMC3757027  PMID: 24009689
9.  An O Island 172 Encoded RNA Helicase Regulates the Motility of Escherichia coli O157:H7 
PLoS ONE  2013;8(6):e64211.
Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a major cause of zoonotic food- and water-borne intestinal infections worldwide with clinical consequences ranging from mild diarrhoea to hemolytic uraemic syndrome. The genome of EHEC O157:H7 contains many regions of unique DNA that are referred to as O islands including the Shiga toxin prophages and pathogenicity islands encoding key virulence factors. However many of these O islands are of unknown function. In this study, genetic analysis was conducted on OI-172 which is a 44,434 bp genomic island with 27 open reading frames. Comparative genome analysis showed that O1-72 is a composite island with progressive gain of genes since O157:H7 evolved from its ancestral O55:H7. A partial OI-172 island was also found in 2 unrelated E. coli strains and 2 Salmonella strains. OI-172 encodes several putative helicases, one of which (Z5898) is a putative DEAH box RNA helicase. To investigate the function of Z5898, a deletion mutant (EDL933ΔZ5898) was constructed in the O157:H7 strain EDL933. Comparative proteomic analysis of the mutant with the wild-type EDL933 found that flagellin was down-regulated in the Z5898 mutant. Motility assay showed that EDL933ΔZ5898 migrated slower than the wild-type EDL933 and electron microscopy found no surface flagella. Quantitative reverse transcription PCR revealed that the fliC expression of EDL933ΔZ5898 was significantly lower while the expression of its upstream regulator gene, fliA, was not affected. Using a fliA and a fliC promoter – green fluorescent protein fusion contruct, Z5898 was found to affect only the fliC promoter activity. Therefore, Z5898 regulates the flagella based motility by exerting its effect on fliC. We conclude that OI-172 is a motility associated O island and hereby name it the MAO island.
doi:10.1371/journal.pone.0064211
PMCID: PMC3681947  PMID: 23785398
10.  Digital Gene Expression Analysis of Corky Split Vein Caused by Boron Deficiency in ‘Newhall’ Navel Orange (Citrus sinensis Osbeck) for Selecting Differentially Expressed Genes Related to Vascular Hypertrophy 
PLoS ONE  2013;8(6):e65737.
Corky split vein caused by boron (B) deficiency in ‘Newhall’ Navel Orange was studied in the present research. The boron-deficient citrus exhibited a symptom of corky split vein in mature leaves. Morphologic and anatomical surveys at four representative phases of corky split veins showed that the symptom was the result of vascular hypertrophy. Digital gene expression (DGE) analysis was performed based on the Illumina HiSeq™ 2000 platform, which was applied to analyze the gene expression profilings of corky split veins at four morphologic phases. Over 5.3 million clean reads per library were successfully mapped to the reference database and more than 22897 mapped genes per library were simultaneously obtained. Analysis of the differentially expressed genes (DEGs) revealed that the expressions of genes associated with cytokinin signal transduction, cell division, vascular development, lignin biosynthesis and photosynthesis in corky split veins were all affected. The expressions of WOL and ARR12 involved in the cytokinin signal transduction pathway were up-regulated at 1st phase of corky split vein development. Furthermore, the expressions of some cell cycle genes, CYCs and CDKB, and vascular development genes, WOX4 and VND7, were up-regulated at the following 2nd and 3rd phases. These findings indicated that the cytokinin signal transduction pathway may play a role in initiating symptom observed in our study.
doi:10.1371/journal.pone.0065737
PMCID: PMC3673917  PMID: 23755275
11.  Two Genera of Magnetococci with Bean-like Morphology from Intertidal Sediments of the Yellow Sea, China 
Applied and Environmental Microbiology  2012;78(16):5606-5611.
Magnetotactic bacteria have the unique capacity of being able to swim along geomagnetic field lines. They are Gram-negative bacteria with diverse morphologies and variable phylogenetic relatedness. Here, we describe a group of uncultivated marine magnetococci collected from intertidal sediments of Huiquan Bay in the Yellow Sea. They were coccoid-ovoid in morphology, with an average size of 2.8 ± 0.3 μm by 2.0 ± 0.2 μm. Differential interference contrast microscopy, fluorescence microscopy, and transmission electron microscopy revealed that each cell was apparently composed of two hemispheres. The cells synthesized iron oxide-type magnetosomes that clustered on one side of the cell at the interface between the two hemispheres. In some cells two chains of magnetosomes were observed across the interface. Each cell had two bundles of flagella enveloped in a sheath and displayed north-seeking helical motion. Two 16S rRNA gene sequences having 91.8% identity were obtained, and their authenticity was confirmed by fluorescence in situ hybridization. Phylogenetic analysis revealed that the magnetococci are affiliated with the Alphaproteobacteria and are most closely related to two uncultured magnetococci with sequence identities of 92.7% and 92.4%, respectively. Because they display a >7% sequence divergence to all bacteria reported, the bean-like magnetococci may represent two novel genera.
doi:10.1128/AEM.00081-12
PMCID: PMC3406164  PMID: 22660708
12.  Genome Sequence of the Marine Bacterium Marinobacter hydrocarbonoclasticus SP17, Which Forms Biofilms on Hydrophobic Organic Compounds 
Journal of Bacteriology  2012;194(13):3539-3540.
Marinobacter hydrocarbonoclasticus SP17 forms biofilms specifically at the interface between water and hydrophobic organic compounds (HOCs) that are used as carbon and energy sources. Biofilm formation at the HOC-water interface has been recognized as a strategy to overcome the low availability of these nearly water-insoluble substrates. Here, we present the genome sequence of SP17, which could provide further insights into the mechanisms of enhancement of HOCs assimilation through biofilm formation.
doi:10.1128/JB.00500-12
PMCID: PMC3434751  PMID: 22689231
13.  Expression and purification of 15N- and 13C-isotope labeled 40-residue human Alzheimer’s β-amyloid peptide for NMR-based structural analysis 
Amyloid fibrils of Alzheimer’s β-amyloid peptide (Aβ) are a primary component of amyloid plaques, a hallmark of Alzheimer’s disease (AD). Enormous attention has been given to the structural features and functions of Aβ in amyloid fibrils and other type of aggregates in associated with development of AD. This report describes an efficient protocol to express and purify high-quality 40-residue Aβ(1–40), the most abundant Aβ in brains, for structural studies by NMR spectroscopy. Over-expression of Aβ(1–40) with glutathione S-transferase (GST) tag connected by a Factor Xa recognition site (IEGR▼) in E. Coli resulted in the formation of insoluble inclusion bodies even with the soluble GST tag. This problem was resolved by efficient recovery of the GST-Aβ fusion protein from the inclusion bodies using 0.5% (w/v) sodium lauroyl sarcosinate as solubilizing agent and subsequent purification by affinity chromatography using a glutathione agarose column. The removal of the GST tag by Factor Xa enzymatic cleavage and purification by HPLC yielded as much as ~7 mg and ~1.5 mg of unlabeled Aβ(1–40) and uniformly 15N- and/or 13C-protein Aβ(1–40) from 1 L of the cell culture, respectively. Mass spectroscopy of unlabeled and labeled Aβ and 1H/15N HSQC solution NMR spectrum of the obtained 15N-labeled Aβ in the monomeric form confirmed the expression of native Aβ(1–40). It was also confirmed by electron micrography and solid-state NMR analysis that the purified Aβ(1–40) self-assembles into β-sheet rich amyloid fibrils. To the best of our knowledge, our protocol offers the highest yields among published protocols for production of recombinant Aβ(1–40) samples that are amendable for an NMR-based structural analysis. The protocol may be applied to efficient preparation of other amyloid-forming proteins and peptides that are 13C- and 15N-labeled for NMR experiments.
doi:10.1016/j.pep.2011.05.012
PMCID: PMC3134129  PMID: 21640828
Amyloid β; GST fusion protein; sodium lauroyl sarcosinate; NMR
14.  Recurrence of inflammatory myofibroblastic tumor in bladder secondary to prostate treated with laparoscopic radical cystectomy 
Summary
Background
Inflammatory myofibroblastic tumor (IMT) is a rare borderline tumor. The nomenclature of this disease is confused in the literature.
Case Report
In this report, the case of a 62-year-old man with IMT recurrence of bladder secondary to prostate is presented. The possible etiology of IMT is discussed, along with its clinical manifestation and pathological features. The patient received a laparoscopic bladder radical resection. The pathology finding demonstrated the diagnosis of IMT and no regional lymph node involvement.
Conclusions
IMT is a borderline tumor and unlikely to metastasize to regional lymph nodes. The patient has been observed for 2 years without recurrence.
doi:10.12659/MSM.883255
PMCID: PMC3560699  PMID: 22847204
inflammatory myofibroblastic tumor; bladder; prostate
15.  Papillae alterations around single-implant restorations in the anterior maxillae: thick versus thin mucosa 
To evaluate the papilla alterations around single-implant restorations in the anterior maxillae after crown attachment and to study the influence of soft tissue thickness on the papilla fill alteration. According to the inclusion criteria, 32 patients subjected to implant-supported single-tooth restorations in anterior maxillae were included. The patients were assigned to two groups according to the mucosal thickness: (i) group 1, 1.5 mm≤mucosal thickness≤3 mm; and (ii) group 2, 3 mm
doi:10.1038/ijos.2012.25
PMCID: PMC3412666  PMID: 22627613
esthetic outcome; papilla fill index; single-implant restoration; soft tissue thickness
Cu2+ binding to Alzheimer’s β (Aβ) peptides in amyloid fibrils has attracted broad attention, as it was shown that Cu ion concentration elevates in Alzheimer’s senile plaque and such association of Aβ with Cu2+ triggers the production of neurotoxic reactive oxygen species (ROS) such as H2O2. However, detailed binding sites and binding structures of Cu2+ to Aβ are still largely unknown for Aβ fibrils or other aggregates of Aβ. In this work, we examined molecular details of Cu2+ binding to amyloid fibrils by detecting paramagnetic signal quenching in 1D and 2D high-resolution 13C SSNMR for full-length 40-residue Aβ(1–40). Selective quenching observed in 13C SSNMR of Cu2+-bound Aβ(1–40) suggested that primary Cu2+ binding sites in Aβ(1–40) fibrils include Nε in His-13 and His-14, and carboxyl groups in Val-40 as well as in Glu side chains (Glu-3, Glu-11, and/or Glu-22). 13C chemical shift analysis demonstrated no major structural changes upon Cu2+ binding in the hydrophobic core regions (residues 18–25 and 30–36). Although the ROS production via oxidization of Met-35 in the presence of Cu2+ has been long suspected, our SSNMR analysis of 13CεH3-S- in M35 showed little changes after Cu2+ binding, excluding the possibility of Met-35 oxidization by Cu2+ alone. Preliminary molecular dynamics (MD) simulations on Cu2+-Aβ complex in amyloid fibrils confirmed binding sites suggested by the SSNMR results and the stabilities of such bindings. The MD simulations also indicate the coexistence of a variety of Cu2+-binding modes unique in Aβ fibril, which are realized by both intra- and inter-molecular contacts and highly concentrated coordination sites due to the in-register parallel β-sheet arrangements.
doi:10.1021/ja1072178
PMCID: PMC3074258  PMID: 21341665
Low-resolution refinement tools implemented in REFMAC5 are described, including the use of external structural restraints, helical restraints and regularized anisotropic map sharpening.
Two aspects of low-resolution macromolecular crystal structure analysis are considered: (i) the use of reference structures and structural units for provision of structural prior information and (ii) map sharpening in the presence of noise and the effects of Fourier series termination. The generation of interatomic distance restraints by ProSMART and their subsequent application in REFMAC5 is described. It is shown that the use of such external structural information can enhance the reliability of derived atomic models and stabilize refinement. The problem of map sharpening is considered as an inverse deblurring problem and is solved using Tikhonov regularizers. It is demonstrated that this type of map sharpening can automatically produce a map with more structural features whilst maintaining connectivity. Tests show that both of these directions are promising, although more work needs to be performed in order to further exploit structural information and to address the problem of reliable electron-density calculation.
doi:10.1107/S090744491105606X
PMCID: PMC3322599  PMID: 22505260
low-resolution refinement; REFMAC5
Alteration of epidermal growth factor receptor (EGFR) is involved in various human cancers and has been intensively investigated. A plethora of evidence demonstrates that posttranslational modifications of EGFR play a pivotal role in controlling its function and metabolism. Here, we show that EGFR can be acetylated by CREB binding protein (CBP) acetyltransferase. Interestingly, EGFR acetylation affects its tyrosine phosphorylation, which may contribute to cancer cell resistance to histone deacetylase inhibitors (HDACIs). Since there is an increasing interest in using HDACIs to treat various cancers in the clinic, our current study provides insights and rationale for selecting effective therapeutic regimen. Consistent with the previous reports, we also show that HDACI combined with EGFR inhibitors achieves better therapeutic outcomes and provides a molecular rationale for the enhanced effect of combination therapy. Our results unveil a critical role of EGFR acetylation that regulates EGFR function, which may have an important clinical implication.
doi:10.1016/j.bbrc.2010.11.064
PMCID: PMC3049249  PMID: 21094134
The automated pipelines for molecular replacement MrBUMP and BALBES are reviewed, with an emphasis on understanding their output. Conclusions are drawn from their performance in extensive trials.
Molecular replacement is one of the key methods used to solve the problem of determining the phases of structure factors in protein structure solution from X-ray image diffraction data. Its success rate has been steadily improving with the development of improved software methods and the increasing number of structures available in the PDB for use as search models. Despite this, in cases where there is low sequence identity between the target-structure sequence and that of its set of possible homologues it can be a difficult and time-consuming chore to isolate and prepare the best search model for molecular replacement. MrBUMP and BALBES are two recent developments from CCP4 that have been designed to automate and speed up the process of determining and preparing the best search models and putting them through molecular replacement. Their intention is to provide the user with a broad set of results using many search models and to highlight the best of these for further processing. An overview of both programs is presented along with a description of how best to use them, citing case studies and the results of large-scale testing of the software.
doi:10.1107/S0907444911007530
PMCID: PMC3069746  PMID: 21460449
MrBUMP; BALBES; molecular replacement
The general principles behind the macromolecular crystal structure refinement program REFMAC5 are described.
This paper describes various components of the macromolecular crystallographic refinement program REFMAC5, which is distributed as part of the CCP4 suite. REFMAC5 utilizes different likelihood functions depending on the diffraction data employed (amplitudes or intensities), the presence of twinning and the availability of SAD/SIRAS experimental diffraction data. To ensure chemical and structural integrity of the refined model, REFMAC5 offers several classes of restraints and choices of model parameterization. Reliable models at resolutions at least as low as 4 Å can be achieved thanks to low-resolution refinement tools such as secondary-structure restraints, restraints to known homologous structures, automatic global and local NCS restraints, ‘jelly-body’ restraints and the use of novel long-range restraints on atomic displacement parameters (ADPs) based on the Kullback–Leibler divergence. REFMAC5 additionally offers TLS parameterization and, when high-resolution data are available, fast refinement of anisotropic ADPs. Refinement in the presence of twinning is performed in a fully automated fashion. REFMAC5 is a flexible and highly optimized refinement package that is ideally suited for refinement across the entire resolution spectrum encountered in macromolecular crystallography.
doi:10.1107/S0907444911001314
PMCID: PMC3069751  PMID: 21460454
REFMAC5; refinement
Biochemistry  2009;48(29):7045-7055.
Glutamate racemase (RacE) is a bacterial enzyme that converts L-glutamate to D-glutamate, an essential precursor for peptidoglycan synthesis. In prior work, we have shown that both isoforms co-crystallize with D-glutamate as dimers, and the enzyme is in a closed conformation with limited access to the active site [May et al., (2007) J. Mol. Biol. 371, 1219–1237]. The active site of RacE2 is especially restricted. We now utilize several computational and experimental approaches to understand the overall conformational dynamics involved during catalysis when the ligand enters and product exits the active site. Our steered molecular dynamics simulations and normal mode analysis results indicate that the monomeric form of the enzyme is more flexible than the native dimeric form. These results suggest that the monomeric enzyme might be more active than the dimeric form. We thus generated site-specific mutations that disrupt dimerization, and find that they exhibit significantly higher catalytic rates in the D-Glu to L-Glu reaction direction than the native enzyme. Low resolution models restored from solution X-ray scattering studies correlate well with the first six normal modes of the dimeric form of the enzyme, obtained from NMA. Thus, along with the local active site residues, global domain motions appear to be implicated in the catalytically relevant structural dynamics of this enzyme, and suggest that increased flexibility may accelerate catalysis. This is a novel observation that residues distant from the catalytic site restrain catalytic activity through formation of the dimer structure.
doi:10.1021/bi9005072
PMCID: PMC2734939  PMID: 19552402
Bacillus anthracis; glutamate racemase; disruption of dimerization; normal modes; x-ray solution scattering; global domain motions; catalytically relevant enzyme structural dynamics
PLoS ONE  2010;5(2):e9151.
Magnetotactic bacteria are able to swim navigating along geomagnetic field lines. They synthesize ferromagnetic nanocrystals that are embedded in cytoplasmic membrane invaginations forming magnetosomes. Regularly aligned in the cytoplasm along cytoskeleton filaments, the magnetosome chain effectively forms a compass needle bestowing on bacteria their magnetotactic behaviour. A large genomic island, conserved among magnetotactic bacteria, contains the genes potentially involved in magnetosome formation. One of the genes, mamK has been described as encoding a prokaryotic actin-like protein which when it polymerizes forms in the cytoplasm filamentous structures that provide the scaffold for magnetosome alignment. Here, we have identified a series of genes highly similar to the mam genes in the genome of Magnetospirillum magneticum AMB-1. The newly annotated genes are clustered in a genomic islet distinct and distant from the known magnetosome genomic island and most probably acquired by lateral gene transfer rather than duplication. We focused on a mamK-like gene whose product shares 54.5% identity with the actin-like MamK. Filament bundles of polymerized MamK-like protein were observed in vitro with electron microscopy and in vivo in E. coli cells expressing MamK-like-Venus fusions by fluorescence microscopy. In addition, we demonstrate that mamK-like is transcribed in AMB-1 wild-type and ΔmamK mutant cells and that the actin-like filamentous structures observed in the ΔmamK strain are probably MamK-like polymers. Thus MamK-like is a new member of the prokaryotic actin-like family. This is the first evidence of a functional mam gene encoded outside the magnetosome genomic island.
doi:10.1371/journal.pone.0009151
PMCID: PMC2818848  PMID: 20161777
Applied and Environmental Microbiology  2009;75(12):3835-3841.
Magnetotactic bacteria have the unique capacity of synthesizing intracellular single-domain magnetic particles called magnetosomes. The magnetosomes are usually organized in a chain that allows the bacteria to align and swim along geomagnetic field lines, a behavior called magnetotaxis. Two mechanisms of magnetotaxis have been described. Axial magnetotactic cells swim in both directions along magnetic field lines. In contrast, polar magnetotactic cells swim either parallel to the geomagnetic field lines toward the North Pole (north seeking) or antiparallel toward the South Pole (south seeking). In this study, we used a magnetospectrophotometry (MSP) assay to characterize both the axial magnetotaxis of “Magnetospirillum magneticum” strain AMB-1 and the polar magnetotaxis of magneto-ovoid strain MO-1. Two pairs of Helmholtz coils were mounted onto the cuvette holder of a common laboratory spectrophotometer to generate two mutually perpendicular homogeneous magnetic fields parallel or perpendicular to the light beam. The application of magnetic fields allowed measurements of the change in light scattering resulting from cell alignment in a magnetic field or in absorbance due to bacteria swimming across the light beam. Our results showed that MSP is a powerful tool for the determination of bacterial magnetism and the analysis of alignment and swimming of magnetotactic bacteria in magnetic fields. Moreover, this assay allowed us to characterize south-seeking derivatives and non-magnetosome-bearing strains obtained from north-seeking MO-1 cultures. Our results suggest that oxygen is a determinant factor that controls magnetotactic behavior.
doi:10.1128/AEM.00165-09
PMCID: PMC2698362  PMID: 19376916
Nature methods  2009;6(3):215-218.
We present an approach that speeds up protein solid-state NMR (SSNMR) by 5–20 fold by using paramagnetic doping to condense data-collection time (to ~0.2 s/scan), overcoming a long-standing limitation on slow recycling due to intrinsic 1H T1 longitudinal spin relaxation. By employing low-power schemes under magic-angle spinning at 40 kHz, we show that two-dimensional 13C/13C and 13C/15N SSNMR spectra can be attained for several to tens of nano-moles of β-amyloid fibrils and ubiquitin in just 1–2 days.
doi:10.1038/nmeth.1300
PMCID: PMC2649701  PMID: 19198596
The twin-arginine translocation (Tat) pathway in Corynebacterium glutamicum has been described previously. The minimal functional Tat system in C. glutamicum required TatA and TatC but did not require TatB, although this component was required for maximal efficiency of Tat-dependent secretion. We previously demonstrated that Chryseobacterium proteolyticum pro-protein glutaminase (pro-PG) and Streptomyces mobaraensis pro-transglutaminase (pro-TG) could be secreted via the Tat pathway in C. glutamicum. Here we report that the amounts of pro-PG secreted were more than threefold larger when TatC or TatAC was overexpressed, and there was a further threefold increase when TatABC was overexpressed. These results show that the amount of TatC protein is the first bottleneck and the amount of TatB protein is the second bottleneck in Tat-dependent protein secretion in C. glutamicum. In addition, the amount of pro-TG that accumulated via the Tat pathway when TatABC was overexpressed with the TorA signal peptide in C. glutamicum was larger than the amount that accumulated via the Sec pathway. We concluded that TatABC overexpression improves Tat-dependent pro-PG and pro-TG secretion in C. glutamicum.
doi:10.1128/AEM.01874-08
PMCID: PMC2632119  PMID: 19074606

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