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Biomedical subjects

G Leonard

Publications and source records attributed to G Leonard.

At least 19 recordsLinked to original sources
PubMed2002

The 2.4-A crystal structure of the penicillin-resistant penicillin-binding protein PBP5fm from Enterococcus faecium in complex with benzylpenicillin.

Penicillin-binding proteins (PBPs) are membrane proteins involved in the final stages of peptidoglycan synthesis and represent the targets of beta-lactam antibiotics. Enterococci are naturally resistant to these antibiotics because they produce a PBP, named PBP5fm in Enterococcus faecium, with low-level affinity for beta-lactams. We report here the crystal structure of the acyl-enzyme complex of PBP5fm with benzylpenicillin at a resolution of 2.4 A. A characteristic of the active site, which distinguishes PBP5fm from other PBPs of known structure, is the topology of the loop 451-465 defining the left edge of the cavity. The residue Arg464, involved in a salt bridge with the residue Asp481, confers a greater rigidity to the PBP5fm active site. In addition, the presence of the Val465 residue, which points into the active site, reducing its accessibility, could account for the low affinity of PBP5fm for beta-lactam. This loop is common to PBPs of low affinity, such as PBP2a from Staphylococcus aureus and PBP3 from Bacillus subtilis. Moreover, the insertion of a serine after residue 466 in the most resistant strains underlines even more the determining role of this loop in the recognition of the substrates.

Amino Acid Sequence↗
PubMed2001

Crystal structure of GerE, the ultimate transcriptional regulator of spore formation in Bacillus subtilis.

The small, DNA-binding protein GerE regulates gene transcription in the terminally differentiated mother-cell compartment during late stages of sporulation in Bacillus subtilis. This versatile transcription factor shares sequence homology with the LuxR/FixJ/UhpA family of activators and modulates the expression of a number of genes, in particular those encoding the components of the coat that surrounds the mature spore. GerE orchestrates the final stages of coat deposition and maturation that lead to a spore with remarkable resistance properties but that must be responsive to low levels of germination signals. As this germination process is largely passive and can occur in the absence of de novo protein synthesis, the correct assembly of germination machinery, including germinant receptors and energy storage compounds, is crucial to the survival of the cell. The crystal structure of GerE has been solved at 2.05 A resolution using multi-wavelength anomalous dispersion techniques and reveals the nature of the GerE dimer. Each monomer comprises four alpha-helices, of which the central pair forms a helix-turn-helix DNA-binding motif. Implications for DNA-binding and the structural organisation of the LuxR/FixJ/UhpA family of transcription activator domains are discussed.

Amino Acid Sequence↗
PubMed2001

Maturation of white matter in the human brain: a review of magnetic resonance studies.

This review focuses on the maturation of brain white-matter, as revealed by magnetic resonance (MR) imaging carried out in healthy subjects. The review begins with a brief description of the nature of the MR signal and its possible biological underpinnings, and proceeds with a description of MR findings obtained in newborns, infants, children and adolescents. On MR images, a significant decrease in water content leads to a decrease of longitudinal relaxation times (T1) and transverse relaxation times (T2) and consequent "adult-like" appearance of T1-weighted and T2-weighted images becomes evident towards the end of the first year of life. Owing to the onset of myelination and the related increase of lipid content, MR images gradually acquire an exquisite grey-white matter contrast in a temporal sequence reflecting the time course of myelination. Albeit less pronounced, age-related changes in white matter continue during childhood and adolescence; white matter increases its overall volume and becomes more myelinated in a region-specific fashion. Detection of more subtle changes during this "late" phase of brain development is greatly aided by computational analyses of MR images. The review also briefly outlines future directions, including the use of novel MR techniques such as diffusion tensor imaging and magnetization transfer, as well as the suggestion for the concurrent use of experimental behavioral test-batteries, with structural MR imaging, to study developmental changes in structure-function relationships.

Adolescent↗
PubMed2000

Domain swapping in the sporulation response regulator Spo0A.

Adaptive responses of micro-organisms, such as chemotaxis and sporulation, are governed by two-component systems consisting of sensor kinases, that interpret environmental signals, and response regulators which activate the appropriate physiological responses. Signal transduction via response regulator proteins is mediated through transient phosphorylation of aspartic acid residues. In Spo0A, the key regulator of development (sporulation) in Bacillus, phosphorylation of the N-terminal receiver domain (N-Spo0A) at aspartate-55 switches on the transcription activation functions residing in the C-terminal effector domain. Here we report the crystal structure of N-Spo0A from Bacillus stearothermophilus at 1.6 A spacing, revealing a dimer formed by an alpha-helix swap. Comparison of this structure with the recently described structure of phosphorylated N-Spo0A shows that dimer formation results from a cis-trans isomerization of the Lys106--Pro107 peptide bond. The quaternary reorganization is associated with alterations in the active site stereochemistry which may have implications for signalling. Remarkably, this 3-D domain swapped N-Spo0A dimer has an identical topology to a hypothetical CheY-like dimer, recently proposed as an intermediate in the evolution of the family of periplasmic substrate binding proteins.

Amino Acid Sequence↗
PubMed2000

The high-resolution structure of the NADP(H)-binding component (dIII) of proton-translocating transhydrogenase from human heart mitochondria.

BACKGROUND: Transhydrogenase, located in the inner membranes of animal mitochondria and the cytoplasmic membranes of bacteria, couples the transfer of reducing equivalents between NAD(H) and NADP(H) to proton pumping. The protein comprises three subunits termed dI, dII and dIII. The dII component spans the membrane. The dI component, which contains the binding site for NAD(+)/NADH, and the dIII component, which has the binding site for NADP(+)/NADPH, protrude from the membrane. Proton pumping is probably coupled to changes in the binding affinities of dIII for NADP(+) and NADPH. RESULTS: The first X-ray structure of the NADP(H)-binding component, dIII, of human heart transhydrogenase is described here at 2.0 A resolution. It comprises a single domain resembling the classical Rossmann fold, but NADP(+) binds to dIII with a reversed orientation. The first betaalphabetaalphabeta motif of dIII contains a Gly-X-Gly-X-X-Ala/Val 'fingerprint', but it has a different function to that in the classical Rossmann structure. The nicotinamide ring of NADP(+) is located on a ridge where it is exposed to interaction with NADH on the dI subunit. Two distinctive features of the dIII structure are helix D/loop D, which projects from the beta sheet, and loop E, which forms a 'lid' over the bound NADP(+). CONCLUSIONS: Helix D/loop D interacts with the bound nucleotide and loop E, and probably interacts with the membrane-spanning dII. Changes in ionisation and conformation in helix D/loop D, resulting from proton translocation through dII, are thought to be responsible for the changes in affinity of dIII for NADP(+) and NADPH that drive the reaction.

Amino Acid Sequence↗
PubMed2000

Molecular detection of Gluconacetobacter sacchari associated with the pink sugarcane mealybug Saccharicoccus sacchari (Cockerell) and the sugarcane leaf sheath microenvironment by FISH and PCR.

Molecular tools for the detection of the newly described acetic acid bacterium Gluconacetobacter sacchari from the pink sugarcane mealybug, Saccharicoccus sacchari Cockerell (Homiptera: Pseudococcidae), and in the sugarcane leaf sheath microenvironment were developed. G. sacchari specific 16S rRNA-targeted oligonucleotide primers were designed and used in PCR amplification of G. sacchari DNA directly from mealybugs, and in a nested PCR to detect low numbers of the bacteria from sugarcane leaf sheath fluid and cane internode scrapings. A sensitivity level of detection of 40-400 cells/reaction was obtained using PCR from exponentially grown bacterial cultures and of 1-10 cells in cane internode scrapings and leaf sheath fluid samples using nested PCR. The specificity of the primer set was demonstrated by the lack of amplification product formation in PCR by closely related acetic acid bacteria, including Gluconacetobacter liquefaciens, and Gluconacetobacter diazotrophicus. A Cy3 labeled probe for G. sacchari was designed and shown to be specific for the species. Investigation of the mealybug microenvironment by whole cell fluorescent in situ hybridization revealed that G. sacchari appears to represent only a minor proportion of the population of the microbiota in the mealybugs tested. This study has shown the usefulness of 16S rRNA-based molecular tools in the identification and detection of G. sacchari from environmental samples and will allow these tools to be used in further ecological research.

Journal Article↗
PubMed2000

Structure of the AAA ATPase p97.

p97, an abundant hexameric ATPase of the AAA family, is involved in homotypic membrane fusion. It is thought to disassemble SNARE complexes formed during the process of membrane fusion. Here, we report two structures: a crystal structure of the N-terminal and D1 ATPase domains of murine p97 at 2.9 A resolution, and a cryoelectron microscopy structure of full-length rat p97 at 18 A resolution. Together, these structures show that the D1 and D2 hexamers pack in a tail-to-tail arrangement, and that the N domain is flexible. A comparison with NSF D2 (ATP complex) reveals possible conformational changes induced by ATP hydrolysis. Given the D1 and D2 packing arrangement, we propose a ratchet mechanism for p97 during its ATP hydrolysis cycle.

Adenosine Diphosphate↗
PubMed2000

Growth-related oncogene-alpha expression in human nasal polyps.

The cytokine growth-related oncogene-alpha (GRO-alpha) is a potent mediator of leukocyte recruitment and proliferation in inflammatory diseases. We hypothesized that GRO-alpha is produced in the inflammatory nasal polyp microenvironment. Evaluation of nasal polyps from 27 patients for distribution and content of GRO-alpha antigen, by use of immunohistochemical techniques and ELISA, revealed its presence in all 27 tissue samples. It was found predominantly within the eosinophils and neutrophils, with tissue levels ranging from 34 pg/mg total protein (TP) to 1746 pg/mg TP, with a mean value of 631 +/-98 pg/mg TP. Control tissues contained between 82 pg/mg TP and 316 pg/mg TP (mean 176+/-38 pg/mg TP). These results were statistically significant (P<0.03). Clinical correlations and statistical comparisons were calculated. These data suggest that GRO-alpha may be an important factor in the recruitment and activation of leukocytes in nasal polyposis, making it a potential target for therapeutic intervention.

Adult↗
PubMed2000

The purification, crystallization and preliminary structural characterization of glucose-1-phosphate thymidylyltransferase (RmlA), the first enzyme of the dTDP-L-rhamnose synthesis pathway from Pseudomonas aeruginosa.

Glucose-1-phosphate thymidylyltransferase (RmlA; E.C. 2.7.7.24) is the first of four enzymes involved in the biosynthesis of dTDP-L-rhamnose, the precursor of L-rhamnose, a key component of the cell wall of many pathogenic bacteria. RmlA catalyses the condensation of thymidine triphosphate (dTTP) and alpha-D-glucose-1-phosphate (G1P), yielding dTDP-D-glucose. RmlA from Pseudomonas aeruginosa has been overexpressed and purified. Crystals of the enzyme have been grown using the sitting-drop vapour-diffusion technique with PEG 6000 and lithium sulfate as precipitant. Several diffraction data sets of single frozen crystals were collected to a resolution of 1.66 A. Crystals belonged to space group P1, with unit-cell parameters a = 71.5, b = 73.1, c = 134.7 A, alpha = 89.9, beta = 80.9, gamma = 81.1 degrees. The asymmetric unit contains eight monomers in the form of two RmlA tetramers with a solvent content of 51%. Selenomethionine-labelled protein has been obtained and crystallized.

Chromatography, High Pressure Liquid↗
PubMed1999

Crystal structure of intact elongation factor EF-Tu from Escherichia coli in GDP conformation at 2.05 A resolution.

The crystal structure of intact elongation factor Tu (EF-Tu) from Escherichia coli in GDP-bound conformation has been determined using a combination of multiple isomorphous replacement (MIR) and multiwavelength anomalous diffraction (MAD) methods. The current atomic model has been refined to a crystallographic R factor of 20.3 % and free R-factor of 26.8 % in the resolution range of 10-2.05 A. The protein consists of three domains: domain 1 has an alpha/beta structure; while domain 2 and domain 3 are beta-barrel structures. Although the global fold of the current model is similar to those of published structures, the secondary structural assignment has been improved due to the high quality of the current model. The switch I region (residues 40-62) is well ordered in this structure. Comparison with the structure of EF-Tu in GDP-bound form from Thermus aquaticus shows that although the individual domain structures are similar in these two structures, the orientation of domains changes significantly. Interactions between domains 1 and 3 in our E. coli EF-Tu-GDP complex are quite different from those of EF-Tu with bound GTP from T. aquaticus, due to the domain rearrangement upon GTP binding. The binding sites of the Mg2+ and guanine nucleotide are revealed in detail. Two water molecules that co-ordinate the Mg2+ have been identified to be well conserved in the GDP and GTP-bound forms of EF-Tu structures, as well as in the structure of Ras p21 with bound GDP. Comparisons of the Mg2+ binding site with other guanine nucleotide binding proteins in GDP-bound forms show that the Mg2+ co-ordination patterns are well preserved among these structures.

Bacterial Proteins↗
PubMed1999

Description of Gluconacetobacter sacchari sp. nov., a new species of acetic acid bacterium isolated from the leaf sheath of sugar cane and from the pink sugar-cane mealy bug.

A new species of the genus Gluconacetobacter, for which the name Gluconacetobacter sacchari sp. nov. is proposed, was isolated from the leaf sheath of sugar cane and from the pink sugar-cane mealy bug, Saccharicoccus sacchari, found on sugar cane growing in Queensland and northern New South Wales, Australia. The nearest phylogenetic relatives in the alpha-subclass of the Proteobacteria are Gluconacetobacter liquefaciens and Gluconacetobacter diazotrophicus, which have 98.8-99.3% and 97.9-98.5% 16S rDNA sequence similarity, respectively, to members of Gluconacetobacter sacchari. On the basis of the phylogenetic positioning of the strains, DNA reassociation studies, phenotypic tests and the presence of the Q10 ubiquinone, this new species was assigned to the genus Gluconacetobacter. No single phenotypic characteristic is unique to the species, but the species can be differentiated phenotypically from closely related members of the acetic acid bacteria by growth in the presence of 0.01% malachite green, growth on 30% glucose, an inability to fix nitrogen and an inability to grow with the L-amino acids asparagine, glycine, glutamine, threonine and tryptophan when D-mannitol was supplied as the sole carbon and energy source. The type strain of this species is strain SRI 1794T (= DSM 12717T).

Acetobacteraceae↗
PubMed1998

Transforming growth factor-beta expression in otitis media with effusion.

OBJECTIVE: To characterize the existence and role of transforming growth factor-beta (TGF-beta) in otitis media with effusion (OME). STUDY DESIGN: Retrospective. METHODS: The levels of two major TGF-beta isoforms, TGF-beta1 and TGF-beta2, in the middle ear effusions (MEEs) of 44 children were evaluated using enzyme-linked immunospecific assays (ELISAs). Forty-eight MEEs were separated into three clinically relevant groups (i.e., serous, mucoid, and purulent), and TGF-beta levels were correlated with clinical parameters of disease for these MEEs. RESULTS: Both TGF-beta1 and TGF-beta2 were present in the samples. Mean levels of TGF-beta1 (920.36 +/- 437.75 pg/mg total protein) were generally 100-fold greater than those of TGF-beta2 (9.65 +/- 11.19 pg/mg total protein). TGF-beta1 levels were elevated in association with a history of previous tympanostomy tube placements (TTPs) (P = .029) and mucoid effusions (P = .042). TGF-beta2 levels were elevated in association with a history of previous TTPs (P = .100) and chronic (i.e., serous or mucoid) effusions (P = .003). CONCLUSIONS: TGF-beta1 is present in the MEEs of children with OME. Furthermore, TGF-beta1 and TGF-beta2 levels were elevated differentially in the presence of chronic disease indicators in OME, suggesting that these isoforms may have differing roles in the inflammatory processes that characterize OME.

Child↗
PubMed1998

Characterization of the eosinophil chemokine RANTES in nasal polyps.

Previous studies have demonstrated that the cytokine RANTES (Regulated And Normal T cell Expressed and Secreted) has been shown to be a potent mediator of eosinophil chemotaxis in vitro and of leukocyte recruitment. Because eosinophils are the hallmark cells in nasal polyposis, we hypothesize that RANTES is locally produced within the nasal polyp microenvironment and is responsible for the eosinophil recruitment seen in nasal polyposis. To begin to test this hypothesis, we evaluated nasal polyps from 17 patients and 3 control specimens for distribution and content of RANTES using immunohistochemical techniques and enzyme-linked immunosorbent assay technology. Our immunohistochemical studies demonstrated that in nasal polyposis, RANTES antigen staining occurred predominantly within eosinophils and epithelial cells. To quantify the relative levels of RANTES in normal and nasal polyp specimens, tissue homogenates were prepared, quantified, and normalized to protein levels. We detected RANTES in all 17 nasal polyp tissue homogenates (566 +/- 16 pg/mg total protein). The RANTES levels in nasal polyp homogenates were nearly 40-fold higher than the RANTES levels in normal tissue (15.7 +/- 28.2 pg/mg total protein). Thus, it appears that increased expression of RANTES by eosinophils and epithelial cells within the nasal polyp microenvironment promotes eosinophil recruitment and activation within nasal polyps. We hypothesize that RANTES induces increased recruitment and activation of eosinophils, presumably contributing to the increased tissue changes associated with nasal polyposis.

Adult↗
PubMed1997

Topical corticosteroid treatment of anosmia associated with nasal and sinus disease.

OBJECTIVE: To establish the efficacy of topical corticosteroid nasal spray treatment of severe olfactory loss associated with severe nasal and sinus disease. DESIGN: Efficacy before and after open-label trial of topical corticosteroid nasal spray used exclusively in the head-down-forward position. SETTING: Taste and smell clinic of a university teaching hospital and research facility. PATIENTS: Taste and smell clinic patients with anosmia or severe hyposmia associated with paranasal sinus disease and nasal polyposis including 39 of 45 patients recruited from 1988 to 1994 who completed the topical corticosteroid treatment course and returned for subsequent testing. INTERVENTION: At least 8 weeks of treatment with flunisolide (Nasalide), 2 sprays in each nostril twice a day, with concurrent antibiotic treatment of any bacterial infection. MAIN OUTCOME MEASURES: Subjective olfactory symptoms, objective olfactory function tests, and otolaryngological evaluation (including endoscopic examination). RESULTS: Olfactory scores significantly improved following treatment (P < .001); signs of nasal and sinus disease significantly decreased (P < .001); and 26 (66%) of the patients reported a subjective improvement in their sense of smell. CONCLUSION: Topical corticosteroid nasal spray administered in a head-down-forward position is an effective treatment of severe olfactory loss associated with severe nasal and sinus disease.

Administration, Topical↗
PubMed1997

Cytokine expression in otitis media with effusion. Tumor necrosis factor soluble receptor.

BACKGROUND: A variety of cytokines, such as interleukin 1 and tumor necrosis factor (TNF), appear to play a key role in the initiation and maintenance of the inflammatory response in a variety of diseases, including chronic otitis media with effusion (COME). Recently, cytokine inhibitors have been isolated and are presumed to regulate the proinflammatory effects of these cytokines. This has lead us to hypothesize that the chronic inflammation seen in COME is the result of an imbalance in the ratio of proinflammatory cytokines and inhibitors that favors the proinflammatory cytokines. DESIGN: Middle ear effusions were evaluated for the cytokine TNF-alpha and the inhibitor TNF soluble receptor (TNFsolR) using a combination of enzyme-linked immunosorbent assays and radioactive immunoassays. We determined the presence and quantified the amounts of TNF-alpha and TNFsolR and correlated this result with clinical parameters in patients with COME. SETTING: University hospital. PATIENTS: To test this hypothesis, 35 middle ear effusions were obtained from 29 children aged 2 to 102 months (mean, 39.6 months) undergoing tympanostomy tube insertion for COME. OUTCOME: Children were followed up for at least 12 months postoperatively and the number of subsequent episodes of otitis media correlated with cytokine levels. RESULTS: The TNF-alpha and TNFsolR were present in 83% and 91% of effusions, respectively. The TNF-alpha mean (+/-SEM) cytokine values were 110.6 +/- 32.1 pg/mg of total protein, and 12886 +/- 3108 pg/mg total protein for TNFsolR. Analysis of correlations in a univariate model revealed a statistically significant relationship for TNF-alpha correlated with TNFsolR (R2 = 0.463; P < .001). The TNF-alpha levels were significantly lower for children with multiple tube insertions (P = .02). Higher levels of TNF-alpha were noted in those children who subsequently developed episodes of otitis media after tube placement (P = .02). The ratio of TNF-alpha to TNFsolR, the mean (+/-SEM) inhibitor index was 430 +/- 220 U. CONCLUSIONS: Our data support the hypothesis that cytokines and their inhibitors are present in a large number of middle ear effusions and in part are likely important in the regulation of inflammatory processes in COME.

Analysis of Variance↗
PubMed1997

Interleukin-8 expression in human nasal polyps.

The cytokine interleukin 8 (IL-8) has been shown to be a potent mediator of leukocyte recruitment and neovascularization in inflammatory and neoplastic diseases. In this study we hypothesize that IL-8 produced in the nasal polyp microenvironment is responsible for the leukocyte recruitment seen in nasal polyposis. To test this hypothesis we evaluated nasal polyps for distribution and content of IL-8 antigen with immunohistochemical techniques and radioimmunoassay to determine tissue levels of IL-8. The immunohistochemical results demonstrated that IL-8 antigen staining occurred predominantly within inflammatory cells and epithelium. IL-8 was detected in all nasal polyp tissue homogenates (a mean value of 1767 +/- 1633 pg/mg total protein (TP) with a range of 134 to 3668 pg/mg TP vs control specimens with a mean value of 77 pg/mg TP with a range of 0.09 to 255 pg/mg TP). These data demonstrate the presence and distribution and levels of IL-8 antigen in nasal polyps in vivo, supporting our hypothesis that local production of IL-8 could be an important factor in the sustained recruitment of leukocytes in nasal polyposis. Thus IL-8 likely plays a significant role in the pathogenesis of this disease process and therefore is a potential target for therapeutic intervention.

Adult↗
PubMed1997

A new method of measuring distortion product otoacoustic emissions using multiple tone pairs: study of human adults.

OBJECTIVE: To increase the speed of a distortion product otoacoustic emissions (DPOE) test of cochlear function by employing a new multiple-tone-pair method and to validate the method. DESIGN: We used a new method of employing multiple tone pairs in measuring DPOEs proposed by Zurek and Rabinowitz and implemented in a modified Grason-Stadler Model GSI-60. We investigated the applicability of the multiple-pair method by comparing the 2f1-f2 DPOEs obtained with a 3-pair method with the conventional 1-pair results in human adults; f1 and f2 represent two frequencies of each tone pair, f1 < f2, f2/f1 = 1.2. We used two sets of 3-pair stimuli: 1) f2 at 1.5, 3, and 6 kHz; and 2) f2 at 2, 4, and 8 kHz. The 1-pair stimuli had f2 at each of the above six frequencies. The primary tone levels were L1 = 65 and L2 = 50 dB SPL re 20 microPa. RESULTS: We obtained DPOEs from 98 ears with normal hearing and 94 ears with sensorineural hearing loss in 103 human adults. We found: 1) that the DPOE levels obtained with the 3-pair method were strongly correlated with those of the 1-pair method, with correlation coefficients of 0.83 to 0.96; 2) that the mean DPOE level versus frequency functions for the normal ears were similar between the 3-pair and 1-pair methods; a 2-way analysis of variance indicated no significant difference between the two methods; 3) that the mean difference between DPOE levels obtained with the 3-pair and 1-pair methods was small (less than 1.3 dB in 11 of the 12 conditions examined, i.e., six frequencies each for normal ears and hearing-impaired ears) although the differences were significant in 6 of the 12 conditions as determined by the paired t-test; and 4) that the DPOE test performances of distinguishing normal from impaired ears were similar between the 3-pair and 1-pair tests; areas under the receiver operating characteristic curves were not significantly different between the two tests for four of the six frequencies tested as determined by a statistical procedure of Hanley and McNeil; for the remaining two frequencies where the differences were significant, each of the two tests was better than the other at one frequency. CONCLUSIONS: The 3-pair DPOE method yielded results generally in good agreement with those of the conventional 1-pair DPOE method. To our knowledge, this is the first systematic study of a multiple-pair DPOE method. Because a 3-pair method can be conducted approximately two to three times as fast as a 1-pair method, the former is advantageous, particularly for an auditory screening test.

Adolescent↗