Biomedical subjects

J E Shea

Publications and source records attributed to J E Shea.

18 recordsLinked to original sources

PubMed2006

Adaptations in cortical and trabecular bone in response to mechanical loading with and without weight bearing.

Exercise that imparts rapid, high-magnitude mechanical loading is considered to be advantageous to bone health. Previous rodent studies have suggested that swimming may also be beneficial to bone. We investigated the differential effects of exercise with and without weight bearing on cortical and trabecular bone. Forty female Sprague-Dawley rats (120 days) were weight-stratified and randomized into four groups: swim control (Cs, n = 10), swim (S, n = 10), treadmill control (Ct, n = 10), and treadmill (T, n = 10). Treadmill speed was adjusted to match the average limb loading frequency used for swimming, and all training progressed to 1 hour/day, 5 days/week, for 12 weeks. Femurs and humeri were assessed for cortical morphometry by peripheral quantitative computed tomography, areal bone mineral density (BMD) by peripheral dual-energy X-ray absorptiometry, mineral content by ashing, strength by three-point bending, and trabecular volume (BV/TV) by micro-computed tomography. Swimming was associated with increases in cortical thickness and BMD in the humerus midshaft and trabecular BV/TV in the distal femur and proximal humerus compared with age-matched controls. Compared to swimming, treadmill training was associated with increases in percent ash of the femur and humerus and Young's modulus of the femur. Swimming appears to engender novel bone strains and osteogenic adaptations in the humerus and femur, which are different from those induced by normal cage activity. In summary, our findings suggest that when limb loading frequency is matched, swimming may afford greater benefits to cortical and trabecular bone than uphill treadmill work in rats.

Absorptiometry, Photon↗

PubMed2003

Effects of confinement in chaperonin assisted protein folding: rate enhancement by decreasing the roughness of the folding energy landscape.

Chaperonins, such as the GroE complex of the bacteria Escherichia coli, assist the folding of proteins under non-permissive folding conditions by providing a cavity in which the newly translated or translocated protein can be encapsulated. Whether the chaperonin cage plays a passive role in protecting the protein from aggregation, or an active role in accelerating folding rates, remains a matter of debate. Here, we investigate the role of confinement in chaperonin mediated folding through molecular dynamics simulations. We designed a substrate protein with an alpha/beta sandwich fold, a common structural motif found in GroE substrate proteins and confined it to a spherical hydrophilic cage which mimicked the interior of the GroEL/ES cavity. The thermodynamics and kinetics of folding were studied over a wide range of temperature and cage radii. Confinement was seen to significantly raise the collapse temperature, T(c), as a result of the associated entropy loss of the unfolded state. The folding temperature, T(f), on the other hand, remained unaffected by encapsulation, a consequence of the folding mechanism of this protein that involves an initial collapse to a compact misfolded state prior to rearranging to the native state. Folding rates were observed to be either accelerated or retarded compared to bulk folding rates, depending on the temperature of the simulation. Rate enhancements due to confinement were observed only at temperatures above the temperature T(m), which corresponds to the temperature at which the protein folds fastest. For this protein, T(m) lies above the folding temperature, T(f), implying that encapsulation alone will not lead to a rate enhancement under conditions where the native state is stable (T<T(f)). For confinement to positively impact folding rates under physiological conditions, it is hence necessary for the protein to exhibit a folding transition above the temperature at which it exhibits its fastest folding rate (T(m)<T(f)). We designed a protein with this property by reducing the energetic frustration in the original alpha/beta sandwich substrate protein. The modified protein exhibited a twofold acceleration in folding rates upon encapsulation. This rate enhancement is due to a mechanistic change in folding involving the elimination, upon encapsulation, of accessible local energy minima corresponding to structures with large radii of gyration. For this protein, confinement hence plays more than the role of a passive cage, but rather adopts an active role, accelerating folding rates by decreasing the roughness of the energy landscape of the protein.

Chaperonin 60↗

PubMed2003

Alterations in skeletal and mineral metabolism following thermal injuries.

Severe burns and other chronic inflammatory diseases are associated with altered skeletal metabolism that result in an increased incidence of osteopenia. In thermally injured children and adults there is a dramatic decrease in bone formation accompanied with an increase or maintenance of bone resorption. Children also exhibit a growth delay and subsequently fail to reach a predicted stature. Animal models, including the thermal injury mouse model, are being used to understand the mechanisms behind the uncoupling of bone formation and resorption that occurs following a major burn. The model has numerous commonalities with the human condition such as reduced bone formation, increased bone resorption, and decreased endochondral growth. The mechanisms that modulate calcium and skeletal metabolism following a thermal injury are complex and likely involve a number of endocrine, cytokine, and immune factors. Specifically, the potential roles of glucocorticoids, growth hormone, insulin-like growth factor-1, parathyroid hormone, interleukin-1 and -6, and tumor necrosis factor alpha are addressed. Subsequent to the increased survival rate of burn victims, there has been a heightened focus on therapeutic interventions that prevent or decrease the impact of thermal injuries on the skeletal system. These include exercise programs, exogenous recombinant human growth hormone, insulin, and oxandrolone.

Journal Article↗

PubMed2001

Evidence of a hypermineralised calcified fibrocartilage on the human femoral neck and lesser trochanter.

Femoral neck fractures are a major cause of morbidity and mortality in elderly humans. In addition to the age-related loss of cancellous bone, changes to the microstructure and morphology of the metaphyseal cortex may be a contributing factor in osteoporotic hip fractures. Recent investigations have identified a hypermineralised tissue on the neck of the femur and trochanteric region that increases in fractional area with advancing age in both males (Boyce & Bloebaum, 1993) and females (Vajda & Bloebaum, 1999). The aim of this study was to determine if the hypermineralised tissue previously observed on the proximal femur is calcified fibrocartilage. Regional variations in the fractional area of hypermineralised tissue, cortical bone, and porosity of the cortical bone along the neck of the femur and lesser trochanter were also quantified. Comparison of back scattered electron and light microscope images of the same area show that regions of hypermineralised tissue correlate with the regions of calcified fibrocartilage from tendon and capsular insertions. The hypermineralised tissue and calcified fibrocartilage had similar morphological features such as the interdigitations of the calcified fibrocartilage into the bone, lacunar spaces, and distinctly shaped pores adjacent to the 2 tissues. Regions of the neck that did not contain insertions were covered with periosteum. There were no regional differences (P > 0.05) on the superior and inferior femoral neck in terms of the percentage area of hypermineralised calcified fibrocartilage, cortical bone, or cortical bone porosity. The lesser trochanter exhibited regional differences in the fractional area of hypermineralised calcified fibrocartilage (P = 0.007) and cortical bone (P = 0.007) but not porosity of the cortical bone (P > 0.05). The effects of calcified fibrocartilage on femoral neck periosteal expansion, repair, and mechanics are unknown, but may play a role in osteoporotic fractures and intracapsular fracture healing.

Cartilage↗

PubMed2001

From folding theories to folding proteins: a review and assessment of simulation studies of protein folding and unfolding.

Beginning with simplified lattice and continuum "minimalist" models and progressing to detailed atomic models, simulation studies have augmented and directed development of the modern landscape perspective of protein folding. In this review we discuss aspects of detailed atomic simulation methods applied to studies of protein folding free energy surfaces, using biased-sampling free energy methods and temperature-induced protein unfolding. We review studies from each on systems of particular experimental interest and assess the strengths and weaknesses of each approach in the context of "exact" results for both free energies and kinetics of a minimalist model for a beta-barrel protein. We illustrate in detail how each approach is implemented and discuss analysis methods that have been developed as components of these studies. We describe key insights into the relationship between protein topology and the folding mechanism emerging from folding free energy surface calculations. We further describe the determination of detailed "pathways" and models of folding transition states that have resulted from unfolding studies. Our assessment of the two methods suggests that both can provide, often complementary, details of folding mechanism and thermodynamics, but this success relies on (a) adequate sampling of diverse conformational regions for the biased-sampling free energy approach and (b) many trajectories at multiple temperatures for unfolding studies. Furthermore, we find that temperature-induced unfolding provides representatives of folding trajectories only when the topology and sequence (energy) provide a relatively funneled landscape and "off-pathway" intermediates do not exist.

Models, Molecular↗

PubMed2001

Combating Gram-positive pathogens: emerging techniques to identify relevant virulence targets.

Recent progress in microbial genome sequencing, along with functional genomics technologies based on gene expression, proteomics and genetics have facilitated the identification of significant numbers of Gram-positive virulence genes. These genes represent a novel and heterogeneous class of targets for antimicrobial drug development. This review will concentrate of the contribution of two functional genomics technologies, in vivo expression technology (IVET) based on gene expression and signature-tagged mutagenesis (STM), a genetics based technology to the identification of virulence genes in Gram-positive pathogens.

Journal Article↗

PubMed2000

Elemental and morphological identification of third-body particulate and calcium stearate inclusions in polyethylene components.

Third-body particulate such as human bone chips, hydroxyapatite, and bone cement are considered contributing factors in accelerated wear in total joint replacement. Particulate wear debris is now considered the major contributing factor in aseptic loosening of total joint replacements. The ability to distinguish between different third-body particulate is necessary to better understand wear mechanisms when conducting implant retrieval analysis. The objective of this investigation is to demonstrate that backscattered electron imaging with correlated energy dispersive X-ray analysis can accurately identify third-body particulate in retrieved polyethylene components. It is important that this technique can also distinguish between third-body particulate and normal inclusions in the polyethylene such as calcium stearate, based on the distinct morphology and elemental composition of each material. Therefore, the ability to distinguish third-body particulate from calcium stearate inclusions is essential in gaining a better understanding of the contributing factors associated with coating separation and accelerated wear observed in clinically retrieved polyethylene components.

Biocompatible Materials↗

PubMed2000

Signature-tagged mutagenesis in the identification of virulence genes in pathogens.

Signature-tagged mutagenesis is a functional genomics technique that identifies microbial genes required for infection within an animal host, or within host cells. The application of this technique to a range of microbial pathogens has resulted in the identification of novel virulence determinants in each screen performed to date, so that cumulatively several hundred genes have been ascribed a role in virulence.

Candida↗

PubMed1999

Exploring the origins of topological frustration: design of a minimally frustrated model of fragment B of protein A.

Topological frustration in an energetically unfrustrated off-lattice model of the helical protein fragment B of protein A from Staphylococcus aureus was investigated. This G-type model exhibited thermodynamic and kinetic signatures of a well-designed two-state folder with concurrent collapse and folding transitions and single exponential kinetics at the transition temperature. Topological frustration is determined in the absence of energetic frustration by the distribution of Fersht phi values. Topologically unfrustrated systems present a unimodal distribution sharply peaked at intermediate phi, whereas highly frustrated systems display a bimodal distribution peaked at low and high phi values. The distribution of phi values in protein A was determined both thermodynamically and kinetically. Both methods yielded a unimodal distribution centered at phi = 0.3 with tails extending to low and high phi values, indicating the presence of a small amount of topological frustration. The contacts with high phi values were located in the turn regions between helices I and II and II and III, intimating that these hairpins are in large part required in the transition state. Our results are in good agreement with all-atom simulations of protein A, as well as lattice simulations of a three- letter code 27-mer (which can be compared with a 60-residue helical protein). The relatively broad unimodal distribution of phi values obtained from the all-atom simulations and that from the minimalist model for the same native fold suggest that the structure of the transition state ensemble is determined mostly by the protein topology and not energetic frustration.

Kinetics↗

PubMed1999

Influence of the Salmonella typhimurium pathogenicity island 2 type III secretion system on bacterial growth in the mouse.

We have investigated the in vivo growth kinetics of a Salmonella typhimurium strain (P11D10) carrying a mutation in ssaJ, a Salmonella pathogenicity island 2 (SPI2) gene encoding a component of a type III secretion system required for systemic growth in mice. Similar numbers of mutant and wild-type cells were recovered from the spleens and livers of BALB/c mice up to 8 h after inoculation by the intraperitoneal route. Thereafter, the numbers of wild-type cells continued to increase logarithmically in these organs, whereas those of P11D10 remained relatively static for several days before being cleared. Gentamicin protection experiments on spleen cell suspensions recovered from infected mice showed that viable intracellular wild-type bacteria accumulated over time but that intracellular P11D10 cells did not. Infection experiments were also performed with wild-type and P11D10 cells carrying the temperature-sensitive plasmid pHSG422 to distinguish between bacterial growth rates and killing in vivo. At 16 h postinoculation there were 10-fold more wild-type cells than mutant cells in the spleens of infected mice, but the numbers of cells of both strains carrying the nonreplicating plasmid were very similar, showing that there was little difference in the degree of killing sustained by the two strains and that the SPI2 secretion system must be required for bacterial replication, rather than survival, in vivo. The SPI2 mutant phenotype in mice is similar to that of strains carrying mutations in the Salmonella virulence plasmid spv genes. To determine if these two sets of genes interact together, a double mutant strain carrying SPI2 and spv mutations was constructed and compared with strains carrying single mutations in terms of virulence attenuation. These experiments failed to provide any evidence showing that the SPI2 and spv gene products interact together as part of the same virulence mechanism.

Animals↗

PubMed1998

Probing the active site of mitogillin, a fungal ribotoxin.

Fungal ribotoxins, such as mitogillin and the related Aspergillus toxins restrictocin and alpha-sarcin, are highly specific ribonucleases, which inactivate the ribosome enzymatically by cleaving the eukaryotic 28S RNA of the large ribosomal subunit at a single phosphodiester bond. The site of cleavage occurs between G4325 and A4326, which are present in a 14-base sequence (the alpha-sarcin loop) conserved among the large subunit rRNAs of all living species. The amino acid residues involved in the cytotoxic activities of mitogillin were investigated by introducing point mutations using hydroxylamine into a recombinant Met-mature mitogillin (mitogillin with a Met codon at the N-terminus and no leader sequence) gene constructed from an Aspergillus fumigatus cDNA clone. These constructs were cloned into a yeast expression vector under the control of the GAL1 promoter and transformed into Saccharomyces cerevisiae. Upon induction of mitogillin expression, surviving transformants revealed that substitutions of certain amino acid residues on mitogillin abolished its cytotoxicity. Non-toxic mutant genes were cloned into an Escherichia coli expression vector, the proteins overexpressed and purified to homogeneity and their activities examined by in vitro ribonucleolytic assays. These studies identified the His-49Tyr, Glu-95Lys, Arg-120Lys and His-136Tyr mutations to have a profound impact on the ribonucleolytic activities of mitogillin. We conclude that these residues are key components of the active site contributing to the catalytic activities of mitogillin.

Allergens↗

PubMed1998

Genes encoding putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2 are required for bacterial virulence and proliferation in macrophages.

The type III secretion system of Salmonella pathogenicity island 2 (SPI-2) is required for systemic infection of this pathogen in mice. Cloning and sequencing of a central region of SPI-2 revealed the presence of genes encoding putative chaperones and effector proteins of the secretion system. The predicted products of the sseB, sseC and sseD genes display weak but significant similarity to amino acid sequences of EspA, EspD and EspB, which are secreted by the type III secretion system encoded by the locus of enterocyte effacement of enteropathogenic Escherichia coli. The transcriptional activity of an sseA::luc fusion gene was shown to be dependent on ssrA, which is required for the expression of genes encoding components of the secretion system apparatus. Strains carrying nonpolar mutations in sseA, sseB or sseC were severely attenuated in virulence, strains carrying mutations in sseF or sseG were weakly attenuated, and a strain with a mutation in sseE had no detectable virulence defect. These phenotypes were reflected in the ability of mutant strains to grow within a variety of macrophage cell types: strains carrying mutations in sseA, sseB or sseC failed to accumulate, whereas the growth rates of strains carrying mutations in sseE, sseF or sseG were only modestly reduced. These data suggest that, in vivo, one of the functions of the SPI-2 secretion system is to enable intracellular bacterial proliferation.

Acetyltransferases↗

PubMed1998

Mutations in Salmonella pathogenicity island 2 (SPI2) genes affecting transcription of SPI1 genes and resistance to antimicrobial agents.

The Salmonella typhimurium genome contains two pathogenicity islands (SPI) with genes encoding type III secretion systems for virulence proteins. SPI1 is required for the penetration of the epithelial layer of the intestine. SPI2 is important for the subsequent proliferation of bacteria in the spleens of infected hosts. Although most mutations in SPI2 lead to a strong reduction of virulence, they have different effects in vitro, with some mutants having significantly increased sensitivity to gentamicin and the antibacterial peptide polymyxin B. Previously we showed that certain mutations in SPI2 affect the ability of S. typhimurium to secrete SPI1 effector proteins and to invade cultured eukaryotic cells. In this study, we show that these SPI2 mutations affect the expression of the SPI1 invasion genes. Analysis of reporter fusions to various SPI1 genes reveals highly reduced expression of sipC, prgK, and hilA, the transcriptional activator of SPI1 genes. These observations indicate that the expression of one type III secretion system can be influenced dramatically by mutations in genes encoding a second type III secretion system in the same cell.

Animals↗

PubMed1997

Functional analysis of ssaJ and the ssaK/U operon, 13 genes encoding components of the type III secretion apparatus of Salmonella Pathogenicity Island 2.

We have investigated the structure and transcriptional organization of 13 genes of Salmonella Pathogenicity Island 2 (SPI2) that encode components of the second type III secretion apparatus of Salmonella typhimurium. ssaK, L, M, V, N, O, P, Q, R, S, T, U constitute one operon of 10 kb. ssaJ lles upstream of ssaK and is the terminal gene of another operon. The deduced products of ssaJ, ssaK, ssaV, ssaN, ssaO, ssaQ, ssaR, ssaS, ssaT, and ssaU show greatest similarity to the Yersinia spp. genes yscJ, yscL, lcrD, yscN, yscO, yscQ, yscR, yscS, yscT, and yscU, respectively. The products of the ssaL, ssaM and ssaP genes do not have significant similarity to products of other type III secretion systems, and might be important for the specific function of the SPI2 type III secretion system. Bacterial strains carrying different ssa mutations display minor alterations in terms of serum sensitivity when compared with the wild-type strain, but none are defective in replication within macrophage-like RAW 264.7 cells. However, some of the ssa mutant strains invade HEp2 cells less efficiently and are less cytotoxic to RAW 264.7 macrophages than the wild-type strain. We show that the invasion defect is correlated with a lack of SipC in culture supernatants of these mutant strains. SipC is a product of the SPI1 type III secretion system of S. typhimurium, and is important for epithelial cell invasion. Therefore, mutations in SPI2 can affect the SPI1 secretion system, which raises the possibility of an interaction between the two type III secretion systems.

Amino Acid Sequence↗

PubMed1997

Analysis of the boundaries of Salmonella pathogenicity island 2 and the corresponding chromosomal region of Escherichia coli K-12.

We recently identified a pathogenicity island (SPI2) located at 30.7 centisomes on the Salmonella typhimurium chromosome. SPI2 contains genes encoding a type III secretion system whose function is distinct from that of the type III secretion system encoded by a pathogenicity island (SPI1) at 63 centisomes which is involved in epithelial cell entry. An analysis of the boundaries of SPI2 and comparison with the corresponding region of the Escherichia coli chromosome revealed that SPI2 inserted adjacent to the tRNA(Val) gene. The E. coli chromosome contains 9 kb of DNA at the region corresponding to the SPI2 insertion point which appears to be absent in S. typhimurium. The distribution of SPI1 and SPI2 was examined in various Salmonella isolates. In contrast to type III secretion system genes of SPI1, those of SPI2 are not present in Salmonella bongori, which diverged at the first branch point in the Salmonella lineage. These and other data indicate that SPI2 was acquired by a Salmonella strain already harboring SPI1 by horizontal transfer from an unknown source.

Base Sequence↗

PubMed1996

Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium.

Mapping the insertion points of 16 signature-tagged transposon mutants on the Salmonella typhimurium chromosome led to the identification of a 40-kb virulence gene cluster at minute 30.7. This locus is conserved among all other Salmonella species examined but is not present in a variety of other pathogenic bacteria or in Escherichia coli K-12. Nucleotide sequencing of a portion of this locus revealed 11 open reading frames whose predicted proteins encode components of a type III secretion system. To distinguish between this and the type III secretion system encoded by the inv/spa invasion locus known to reside on a pathogenicity island, we refer to the inv/spa locus as Salmonella pathogenicity island (SPI) 1 and the new locus as SPI2. SPI2 has a lower G+C content than that of the remainder of the Salmonella genome and is flanked by genes whose products share greater than 90% identity with those of the E. coli ydhE and pykF genes. Thus SPI2 was probably acquired horizontally by insertion into a region corresponding to that between the ydhE and pykF genes of E. coli. Virulence studies of SPI2 mutants have shown them to be attenuated by at least five orders of magnitude compared with the wild-type strain after oral or intraperitoneal inoculation of mice.

Animals↗

PubMed1995

Simultaneous identification of bacterial virulence genes by negative selection.

An insertional mutagenesis system that uses transposons carrying unique DNA sequence tags was developed for the isolation of bacterial virulence genes. The tags from a mixed population of bacterial mutants representing the inoculum and bacteria recovered from infected hosts were detected by amplification, radiolabeling, and hybridization analysis. When applied to a murine model of typhoid fever caused by Salmonella typhimurium, mutants with attenuated virulence were revealed by use of tags that were present in the inoculum but not in bacteria recovered from infected mice. This approach resulted in the identification of new virulence genes, some of which are related to, but functionally distinct from, the inv/spa family of S. typhimurium.

Animals↗

PubMed1994

The budding yeast U5 snRNP Prp8 is a highly conserved protein which links RNA splicing with cell cycle progression.

The dbf3 mutation was originally obtained in a screen for DNA synthesis mutants with a cell cycle phenotype in the budding yeast Saccharomyces cerevisiae. We have now isolated the DBF3 gene and found it to be an essential gene with an ORF of 7239 nucleotides, potentially encoding a large protein of 268 kDa. We also obtained an allele-specific high copy number suppressor of the dbf3-1 allele, encoded by the known SSB1 gene, a member of the Hsp70 family of heat shock proteins. The sequence of the Dbf3 protein is 58% identical over 2300 amino acid residues to a predicted protein from Caenorhabditis elegans. Furthermore, partial sequences with 61% amino acid sequence identity were deduced from two files of human cDNA in the EST nucleotide database so that Dbf3 is a highly conserved protein. The nucleotide sequence of DBF3 turned out to be identical to the yeast gene PRP8, which encodes a U5 snRNP required for pre-mRNA splicing. This surprising result led us to further characterise the phenotype of dbf3 which confirmed its role in the cell cycle and showed it to function early, around the time of S phase. This data suggests a hitherto unexpected link between pre-mRNA splicing and the cell cycle.

Alleles↗