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PubMed2001

Genome composition, stability and fertility of hexaploid alloploids between Triticum turgidum var. carthlicum and Leymus racemosus.

Hexaploid alloploids between the tetraploid wheat Triticum carthlicum and the perennial tetraploid Leymus racemosus were analysed for chromosome composition and cytogenetic stability. GISH analysis showed different lines to have from 11 to 16 Leymus chromosomes. The alloploids showed a relatively high frequency of univalents in meiotic metaphase and of aneuploid plants and hence they are not stable. The seedset is lower than in wheat, but high enough to secure a safe propagation and preservation. The alloploids are discussed in relation to widening the genetic variation of breadwheat and wheat breeding.

Chromosomes↗
PubMed1979

Selective association of the chromosome with membrane in a stable L-form of Bacillus subtilis.

A stalbe L-form (Sal-1) of Bacillus subtilis was found to have retained a markedly modified chromosome-membrane association when compared to intact cells. The membrane-deoxyribonucleic acid complex of the L-form was similar to that of its parental strain in quantity and stability. Genetic analysis of the L-form membrane-deoxyribonucleic acid complex revealed enrichment for markers close to the replication origin, but not for internal markers, indicating preferential attachment of the origin of chromosomal replication to the membrane. These results are in close agreement with those found for the parental bacterial form. In contrast, the replication termius region was not preferentially attached to the membrane of the L-form, even though it is enriched in the bacterial form. The association of the chromosome with the membrane at the replication terminus does not appear to be necessary for cell growth and separation, but because the L-form divides aberrantly, it may be one of the factors required for normal deoxyribonucleic acid segregation and septation.

Bacillus subtilis↗
PubMed1993

DNA fingerprinting of Streptococcus pneumoniae strains by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis of genomic DNA was carried out on Streptococcus pneumoniae strains to determine its value in the epidemiological survey of pneumococcal infections. Twenty-one clinical strains were chosen to cover a broad range of diversity according to geographic location, penicillin susceptibility, serotype, and multilocus enzyme electrophoresis (MLEE) pattern. The restriction endonucleases ApaI and SmaI were used to digest intact chromosomes, and the fragments were resolved by field inversion gel electrophoresis (FIGE). Each digest produced 10 to 19 fragments for comparison between strains. All the strains, including strains of the same serotype and strains with the same MLEE profile, had different FIGE patterns. In some cases, the restriction patterns differed by only a few fragment bands, and two isolates differed only in the location of a single DNA fragment. The polymorphism obtained with FIGE was greater than those obtained with serotyping and MLEE analysis. The stability of the FIGE profiles was established by testing of two independent clones derived from pneumococcus strain R36A. These results indicated that pulsed-field gel electrophoresis should be an effective tool for the typing of S. pneumoniae strains, capable of subdividing serotypes or MLEE types and of tracing the origin of pneumococcal strains.

DNA Fingerprinting↗
PubMed1999

A lysine-to-arginine change found in natural alleles of the human T-cell lymphotropic/leukemia virus type 1 p12(I) protein greatly influences its stability.

The HTLV-1 singly spliced open reading frame I protein, p12(I), is highly unstable and appears to be necessary for persistent infection in rabbits. Here we demonstrate that p12(I) forms dimers through two putative leucine zipper domains and that its stability is augmented by specific proteasome inhibitors. p12(I) is ubiquitylated, and mutations of its unique carboxy-terminus lysine residue to an arginine greatly enhance its stability. Interestingly, analysis of 53 independent HTLV-1 strains revealed that the natural p12(I) alleles found in ex vivo samples of tropical spastic paraparesis-HTLV-1-associated myelopathy patients contain a Lys at position 88 in some cases, whereas arginine is consistently found at position 88 in HTLV-1 strains from all adult T-cell leukemia-lymphoma (ATLL) cases and healthy carriers studied. This apparent segregation of different alleles in tropical spastic paraparesis-HTLV-associated myelopathy and ATLL or healthy carriers may be relevant in vivo, since p12(I) binds the interleukin-2 receptor beta and gammac chains, raising the possibility that the two natural alleles might affect differently the regulation of these molecules.

Adult↗
PubMed2005

Scavenger decapping activity facilitates 5' to 3' mRNA decay.

mRNA degradation occurs through distinct pathways, one primarily from the 5' end of the mRNA and the second from the 3' end. Decay from the 3' end generates the m7GpppN cap dinucleotide, which is subsequently hydrolyzed to m7Gp and ppN in Saccharomyces cerevisiae by a scavenger decapping activity termed Dcs1p. Although Dcs1p functions in the last step of mRNA turnover, we demonstrate that its activity modulates earlier steps of mRNA decay. Disruption of the DCS1 gene manifests a threefold increase of the TIF51A mRNA half-life. Interestingly, the hydrolytic activity of Dcs1p was essential for the altered mRNA turnover, as Dcs1p, but not a catalytically inactive Dcs1p mutant, complemented the increased mRNA stability. Mechanistic analysis revealed that 5' to 3' exoribonucleolytic activity was impeded in the dcs1Delta strain, resulting in the accumulation of uncapped mRNA. These data define a new role for the Dcs1p scavenger decapping enzyme and demonstrate a novel mechanism whereby the final step in the 3' mRNA decay pathway can influence 5' to 3' exoribonucleolytic activity.

Blotting, Northern↗
PubMed2006

Shear stress induces hepatocyte PAI-1 gene expression through cooperative Sp1/Ets-1 activation of transcription.

Partial hepatectomy causes hemodynamic changes that increase portal blood flow in the remaining lobe, where the expression of immediate-early genes, including plasminogen activator inhibitor-1 (PAI-1), is induced. We hypothesized that a hyperdynamic circulatory state occurring in the remaining lobe induces immediate-early gene expression. In this study, we investigated whether the mechanical force generated by flowing blood, shear stress, induces PAI-1 expression in hepatocytes. When cultured rat hepatocytes were exposed to flow, PAI-1 mRNA levels began to increase within 3 h, peaked at levels significantly higher than the static control levels, and then gradually decreased. The flow-induced PAI-1 expression was shear stress dependent rather than shear rate dependent and accompanied by increased hepatocyte production of PAI-1 protein. Shear stress increased PAI-1 transcription but did not affect PAI-1 mRNA stability. Functional analysis of the 2.1-kb PAI-1 5'-promoter indicated that a 278-bp segment containing transcription factor Sp1 and Ets-1 consensus sequences was critical to the shear stress-dependent increase of PAI-1 transcription. Mutations of both the Sp1 and Ets-1 consensus sequences, but not of either one alone, markedly prevented basal PAI-1 transcription and abolished the response of the PAI-1 promoter to shear stress. EMSA and chromatin immunoprecipitation assays showed binding of Sp1 and Ets-1 to each consensus sequence under static conditions, which increased in response to shear stress. In conclusion, hepatocyte PAI-1 expression is flow sensitive and transcriptionally regulated by shear stress via cooperative interactions between Sp1 and Ets-1.

Animals↗
PubMed1995

Lung tissue resistance and hysteretic moduli of lung parenchyma.

Lung tissue resistance (Rti) represents a large and labile component of total pulmonary resistance, but the mechanism is unknown. One hypothesis that has received some support in the literature is that on exposure to contractile agonists airway smooth muscle shortens and then, by the agency of elastic interdependence, induces distortion in surrounding parenchyma. Parenchymal distortion induced in the vicinity of a constricted airway is a pure shear deformation, but currently there are no data available for shear hysteresivity. Guided by a microstructural model, we have assigned stiffness and hysteresivity to microstructural elements and then computed how those properties are expressed at the macroscale in bulk hysteresivities for both shear and volumetric expansion. Hysteresivity for volumetric expansion is shown to be a stiffness-weighted average of hysteresivities of all microstructural components. But as the hysteresivity of microstructural elements increases, that for shear deformation increases to some degree but eventually attains a plateau. Blunted hysteretic response in shear seems to be an intrinsic property of pressure-supported structures, like the lung, that require an inflating pressure to ensure mechanical stability. The analysis indicates that that part of Rti attributable to parenchymal distortion can be at most a small fraction of that attributable to volumetric expansion. These results are purely theoretical in nature, and this suggests that caution is necessary in their interpretation. However, the mechanical basis of the results is sufficiently general to conclude that the hypothesis that parenchymal distortion secondary to bronchoconstriction can account for Rti and its changes seems to be implausible.

Airway Resistance↗
PubMed2005

Analysis of ankle-hindfoot stability in multiple planes: an in vitro study.

BACKGROUND: It is necessary to have an understanding of ankle and hindfoot motion and stability to accurately diagnosis and treat ankle-hindfoot disorders. METHODS: We devised an ankle ligament testing apparatus to more critically determine ankle stability in all planes with a constant rotational force applied (inversion, eversion, internal rotation, external rotation) throughout the range of sagittal plane motion in 13 cadaver specimens. Three-dimensional kinematics were determined with a magnetic tracking device. RESULTS: With inversion force applied, calcaneal-tibial inversion was greatest in maximal plantarflexion (mean 22.1 +/- 6.0 degrees) and gradually decreased with dorsiflexion, which indicated that the ankle had the most inversion instability in plantarflexion. With eversion force applied, calcaneal-tibial eversion gradually increased with increasing dorsiflexion to 12.7 +/- 7.4 degrees indicating that the most eversion instability was in dorsiflexion. With internal rotation force applied, calcaneal-tibial internal rotation from plantarflexion to neutral ankle position increased. With external rotation force application, external rotation from neutral to maximal dorsiflexion increased. CONCLUSIONS: Ankle laxity was not constant but varied depending on the plantarflexion-dorsiflexion position and the direction of the applied force. The degree of ankle laxity was greater with inversion and internal rotation torque. Variation in laxity between specimens was observed, consistent with previous reports. These data indicate that the ankle is less stable in plantarflexion when inversion and internal rotation forces are applied. This may explain why the lateral ankle ligaments are most prone to injury in this position. The ankle was less stable in dorsiflexion when eversion and external rotation forces were applied. This is consistent with the observation that deltoid ligament injuries occur in the neutral to dorsiflexion position. The study demonstrates the importance of examining patients with suspected ankle ligament injuries in several ankle positions. The ankle testing device has potential application for in vivo testing of patients with suspected ankle ligament instability.

Adult↗
PubMed2003

Gene expression in mature neutrophils: early responses to inflammatory stimuli.

Neutrophils provide an essential defense against bacterial and fungal infection and play a major role in tissue damage during inflammation. Using oligonucleotide microarrays, we have examined the time course of changes in gene expression induced by stimulation with live, opsonized Escherichia coli, soluble lipopolysaccharide, and the chemoattractant formyl-methionyl-leucyl-phenylalanine. The results indicate that activated neutrophils generate a broad and vigorous set of alterations in gene expression. The responses included changes in the levels of transcripts encoding 148 transcription factors and chromatin-remodeling genes and 95 regulators of protein synthesis or stability. Clustering analysis showed distinct temporal patterns with many rapid changes in gene expression within the first hour of exposure. In addition to the temporal clustering of genes, we also observed rather different profiles associated with each stimulus, suggesting that even a nonvirulent organism such as E. coli is able to play a dynamic role in shaping the inflammatory response. Principal component analysis of transcription factor genes demonstrated clear separation of the neutrophil-response clusters from those of resting and stimulated human monocytes. The present study indicates that combinatorial transcriptional regulation including alterations of chromatin structure may play a role in the rapid changes in gene expression that occur in these terminally differentiated cells.

Chromatin Assembly and Disassembly↗
PubMed2001

Interface biomechanics of the Anca Dual fit hip stem: an in vitro experimental study.

The Anca Dual Fit hip stem (Cremascoli Wright, Milan, Italy) is a partially cemented stem developed to overcome the drawbacks of both cemented and uncemented fixations. Its design was based on the hypothesis that partial cementing would ensure the primary stability necessary to allow bone ingrowth on the cement-free stem surfaces. At the same time, the limitation of the cement to the proximal regions would prevent stress-shielding by increasing proximal load transfer. After finite element (FE) simulations and in vitro primary stability assessment, an analysis of the long-term stability of the Anca Dual Fit stem was necessary to conclude the preclinical testing. Three stems were implanted in composite femurs and subjected to testing for 1 x 10(6) cycles, each cycle reproducing the activity of stair climbing. The simulation was designed so as to replicate the physiological loading in a simplified, yet relevant way for this test. Various measurements were collected before, during and after the test in order to give exhaustive information on the response of the implant to long-term, cyclic loading. The present study confirmed the positive results of previous investigations, and proved that the Anca Dual Fit stem has excellent long-term stability; therefore successful clinical outcomes are predicted.

Biomechanical Phenomena↗
PubMed2005

Coarse-grained strategy for modeling protein stability in concentrated solutions.

We present a coarse-grained approach for modeling the thermodynamic stability of single-domain globular proteins in concentrated aqueous solutions. Our treatment derives effective protein-protein interactions from basic structural and energetic characteristics of the native and denatured states. These characteristics, along with the intrinsic (i.e., infinite dilution) thermodynamics of folding, are calculated from elementary sequence information using a heteropolymer collapse theory. We integrate this information into Reactive Canonical Monte Carlo simulations to investigate the connections between protein sequence hydrophobicity, protein-protein interactions, protein concentration, and the thermodynamic stability of the native state. The model predicts that sequence hydrophobicity can affect how protein concentration impacts native-state stability in solution. In particular, low hydrophobicity proteins are primarily stabilized by increases in protein concentration, whereas high hydrophobicity proteins exhibit richer nonmonotonic behavior. These trends appear qualitatively consistent with the available experimental data. Although factors such as pH, salt concentration, and protein charge are also important for protein stability, our analysis suggests that some of the nontrivial experimental trends may be driven by a competition between destabilizing hydrophobic protein-protein attractions and entropic crowding effects.

Binding Sites↗
PubMed2006

The change of protein intradomain mobility on ligand binding: is it a commonly observed phenomenon?

Analysis of changes in the dynamics of protein domains on ligand binding is important in several aspects: for the understanding of the hierarchical nature of protein folding and dynamics at equilibrium; for analysis of signal transduction mechanisms triggered by ligand binding, including allostery; for drug design; and for construction of biosensors reporting on the presence of target ligand in studied media. In this work we use the recently developed HCCP computational technique for the analysis of stabilities of dynamic domains in proteins, their intrinsic motions and of their changes on ligand binding. The work is based on comparative studies of 157 ligand binding proteins, for which several crystal structures (in ligand-free and ligand-bound forms) are available. We demonstrate that the domains of the proteins presented in the Protein DataBank are far more robust than it was thought before: in the majority of the studied proteins (152 out of 157), the ligand binding does not lead to significant change of domain stability. The exceptions from this rule are only four bacterial periplasmic transport proteins and calmodulin. Thus, as a rule, the pattern of correlated motions in dynamic domains, which determines their stability, is insensitive to ligand binding. This rule may be the general feature for a vast majority of proteins.

Biosensing Techniques↗
PubMed2006

Analysis of the immunogenicity and stability of a porcine pulmonary surfactant preparation administered in rabbits.

PURPOSE: To study the immunogenicity and the stability of the porcine pulmonary surfactant preparation produced by the Instituto Butantan. METHOD: Immunogenicity assay: Sixteen New-Zealand-White rabbits (1000 g body weight) were divided into 4 study groups. Each group was assigned to receive either a) Butantan surfactant, b) Survanta (Abbott Laboratories), c) Curosurf (Farmalab Chiesi), or d) no surfactant. The surfactants were administered intratracheally, and the animals were collected immediately before and 60 and 180 days after surfactant administration. Sera were assayed for the presence of antisurfactant antibodies by enzyme-linked immunosorbent assay (ELISA). Stability assay: The Butantan surfactant used in this assay had been stored for one year in the refrigerator (4 to 8 degrees C) and its stability was evaluated in distinct assay conditions using a premature rabbit model. RESULTS: Immunogenicity assay: None of the surfactants analyzed triggered antibody immune responses against their components in any of the animals. Stability assay: The results of this study demonstrate that Butantan surfactant was as effective as Curosurf when both were submitted to the adverse circumstance of short- and long-term storage at room temperature. A similar level of efficacy for the Butantan surfactant, as compared to Curosurf was demonstrated by the pulmonary dynamic compliance, ventilatory pressure, and pressure-volume curve results. CONCLUSION: The results of our study demonstrate that Butantan surfactant may be a suitable alternative for surfactant replacement therapy.

Animals↗
PubMed1986

Seminiferous tubule androgen receptors in experimental cryptorchidism.

As an initial approach to the study of seminiferous tubule androgen receptors in disordered spermatogenesis, cytosol androgen receptors were studied in rats with experimental cryptorchidism. Two weeks after the testis had been repositioned in the abdomen of 6-week-old rats, the animals were hypophysectomized to deplete the testis of androgen, and 1 week later they were killed. Androgen receptor binding was studied in seminiferous tubule cytosol using [3H]methyltrienolone as the radiolabelled probe. The androgen-binding capacity of cryptorchid testis, when expressed as fmol bound/testis, was reduced to 50% of control, in parallel with the decline in testis weight. No change in binding affinity was found. Sucrose density gradient centrifugation using a vertical tube rotor revealed a 9S molybdate-stabilized receptor under low-salt conditions in both cryptorchid and scrotal seminiferous tubule cytosol. Receptor-complex stability studies, analysis by gel filtration and DEAE-cellulose chromatography produced similar results in cryptorchid and scrotal tubules. The mechanism for the reduction in testicular receptor content of an abdominal testis remains to be clarified. The demonstration that testicular androgen receptors can be reduced by cryptorchidism suggests that further studies may indicate the role of receptor binding in testicular function.

Animals↗
PubMed2006

Internal fixation of dorsally displaced fractures of the distal part of the radius. A biomechanical analysis of volar plate fracture stability.

BACKGROUND: Volar plate fixation with use of either a locking plate or a neutralization plate has become increasingly popular among surgeons for the treatment of dorsally comminuted extra-articular distal radial fractures. The purpose of the present study was to compare the relative stability of five distal radial plates (four volar and one dorsal), all of which are commonly used for the treatment of dorsally comminuted extra-articular distal radial fractures, under loading conditions simulating the physiologic forces that are experienced during early active rehabilitation. METHODS: With use of a previously validated Sawbones fracture model, a dorsally comminuted extra-articular distal radial fracture was created. The fracture fixation stability of four volar plates (an AO T-plate, an AO 3.5-mm small-fragment plate, an AO 3.5-mm small-fragment locking plate, and the Hand Innovations DVR locking plate) were compared under axial compression loading and dorsal and volar bending simulating the in vivo stresses that are generated at the fracture site during early unopposed active motion of the wrist and digits. A single dorsal plate (an AO pi plate) was used for comparison, with and without simulated volar cortical comminution. The construct stiffness was measured to assess the resistance to fracture gap motion, and comparisons were made among the implants. RESULTS: The volar AO locking and DVR plates had greater resistance to fracture gap motion (greater stiffness) compared with the volar AO nonlocking and AO T-plates under axial and dorsal loading conditions (p < 0.01), with no significant difference between the AO volar locking and DVR plates. The volar AO locking plate had greater resistance to fracture gap motion than did the volar AO nonlocking plate under axial loading and dorsal bending forces (p < 0.01). The dorsal pi plate had the greatest resistance to fracture gap motion under axial loading and volar and dorsal bending forces (p < 0.01). However, the pi plate was significantly less stable to axial load and dorsal bending forces when the volar cortex was comminuted (p < 0.01). CONCLUSIONS: In this model of dorsally comminuted extra-articular distal radial fractures, dorsal pi-plate fixation demonstrated better resistance to fracture gap motion than did the four types of volar plate fixation. The AO volar locking and DVR plates conferred the greatest resistance to fracture gap motion among the four volar plates tested. Volar locking technology conferred a significant increase in resistance to fracture gap motion as compared with nonlocking plate technology.

Biomechanical Phenomena↗
PubMed1992

Myocardial adenine nucleotide depletion within 1 h of acute coronary artery occlusion.

In anaesthetized open-chest casts with occlusion of the left anterior descending coronary artery (LAD), adenine nucleotides and degradation products were studied in small myocardial tissue samples (10-20 mg) with high-pressure liquid chromatography, and tissue blood flow was measured with radioactive microspheres 5, 10, 20, 40, and 60 min after LAD occlusion. There was a rapid and parallel decrease of myocardial ATP and accumulation of adenosine, inosine, hypoxanthine, and xanthine both in epicardial and endocardial half-layers of the ischaemic myocardium within the first 20 min of coronary occlusion. After 40 and 60 min, myocardial ATP content decreased and degradation products accumulated further in the endocardium but stabilized epicardially. Analysis of covariance showed that the slightly higher blood flow in ischaemic epicardial layers, did not explain the transmural difference in ATP content after 40 and 60 min. Adenosine decreased after 40 min of ischaemia in both wall layers reaching negligible amounts after 60 min. It is concluded that breakdown of energy stores is less severe in epicardial than in endocardial wall layers during the first hour after acute coronary occlusion in the cat heart. This transmural difference cannot be explained entirely by less severe epicardial ischaemia. Therefore, transmural heterogeneity in metabolic function during severe ischaemia may also be important.

Adenine Nucleotides↗
PubMed1994

Early failure of hydroxyapatite-coating in total knee arthroplasty. A case report.

We observed early loosening of a hydroxyapatite-coated tibial component in a total knee arthroplasty in a randomized study comparing HA-coating with cement fixation. Symptoms of loosening started 8 weeks after surgery. Roentgen stereophotogrammetric analysis revealed stability during the initial 6 weeks followed by a pronounced subsidence up to 6 months after surgery when revision was done. At revision, the HA-coating was found to have separated from the medial part of the tibial component. The synovium was abundant with macrophages and multinucleated giant cells containing HA-crystals. Overloading of the prosthesis and suboptimal quality of the HA-coating was believed to have caused the loosening process.

Aged↗