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PubMed2002

Construction of a tetR-integrated Salmonella enterica serovar Typhi CVD908 strain that tightly controls expression of the major merozoite surface protein of Plasmodium falciparum for applications in human Vaccine production.

Attenuated Salmonella strains are an attractive live vector for delivery of a foreign antigen to the human immune system. However, the problem with this vector lies with plasmid segregation and the low level of expression of the foreign gene in vivo when constitutive expression is employed, leading to a diminished immune response. We have established inducible expressions of foreign genes in the Salmonella enterica serovar Typhi CVD908 vaccine strain using the tetracycline response regulatory promoter. To set up this system, a tetracycline repressor (tetR) was integrated into a defined Delta aroC locus of the chromosome via suicide plasmid pJG12/tetR-neo. To remove the neo gene conferring kanamycin resistance from the locus, a cre expression vector under the control of the tetracycline response promoter was transformed into the clone; expression of the Cre recombinase excised the neo gene and generated the end strain CVD908-tetR. Expression of the luciferase reporter gene in this strain is dependent on the presence of tetracycline in the medium and can be regulated up to 4,773-fold. Moreover, the tightly controlled expression of major merozoite surface protein 1 (MSP1) and parts of Plasmodium falciparum was achieved, and the product yield was increased when the inducible expression system was employed. Inoculation of bacteria harboring plasmid pZE11/MSP1(42) in mice produced the protein in liver and spleen controlled by the inducer. The persistence of the plasmid-carrying bacteria in mice was determined. Peak colonization of both liver and spleen was detected on the third day postinoculation and was followed by a decline in growth curves. After 14 days postinfection, the majority of the bacteria (>90%) recovered from the liver and spleen of the mice retained the plasmid when expression was induced; this clearly indicated that stability of the expression vector in vivo was improved by inducible expression. Establishment of the regulatory system in the vaccine strain may broaden the range of its use by enhancing plasmid stability and expression levels in vivo. Moreover, the availability of the vaccine strain inducibly expressing the entire MSP1 provides possibilities for examining its immunogenicity, particularly the cellular response in animal models.

Animals↗
PubMed1978

Insertion of bacteriophage lambda into the deo operon of Escherichia coli K-12 and isolation of plaque-forming lambdadeo+ transducing bacteriophages.

A procedure has been devised to isolate plaque-forming lambda cI857S7 transducing bacteriophage which carry the internal promoter, P3, of the deo operon of Escherichia coli and the deoB and deoD genes, while lacking the deoP and cytP promoters of the same operon, in order to study, specifically, regulation at the P3 site. This has been accomplished by selecting for the insertion of bacteriophage lambda into the deoA gene in a strain deleted for the normal lambda attachment site (delta att lambda) and isolating from this lysogen lambda spi- and lambda EDTAr phage. Among these, lambda pdeoB+D+ phage were identified by their transducing abilities. From in vivo enzyme induction experiments performed on a delta deo strain lysogenized with such phage, they were shown to carry the P3 promoter while lacking the deoP and cytP promoters. A lambdapdeo B+D+ phage phage was used to lysogenize a deo+ delta att lambda strain, integration of lambda occurring within the region of homology, and, from a heat-induced lysate of this strain, a plaque-forming lambda+ phage carrying the complete deo operon was obtained. Phage lambda was also inserted into the deoB and deoD genes and into the tdk gene. By isolating lambdaspi- and lambdaEDTAr phage from the deo::(lambda) mutants and determining which bacterial genes they carried and whether they retained the int gene of lambda, it was found that lambda had inserted into deoD with the same orientation as lambda inserted into attlambda, whereas lambda inserted into deoA and deoB had the opposite orientation. Deletions extending from the site of lambda insertion into the bacterial chromosome were isolated by selecting for heat-resistant revertants. These confirmed the order of markers to be deo-serB-trpR-thr and also placed a locus, msp, determining sensitivity or resistance of male strains to male-specific phages, between trpR and thr. For some reason unknown, but which may be related to the orientation of the lambda prophages, short deletions rendering the bacterium Ser- Thr+ were of much lower frequency from the deoD::(lambda) lysogen than from the other two lysogens. From an examination of the residual deoD enzyme levels in deoB::(lambda) mutants, it was deduced that there may be two promoter sites within the deoB::(lambda) mutants, it was deduced that there may be two promoter sites within the deoB gene, transcription from one of these being sufficient to account for the noncoordinate nature of the induction of deoB and deoD gene products.

Chromosome Mapping↗
PubMed1984

Method for determining whether a gene of Escherichia coli is essential: application to the polA gene.

We have developed a general method for determining whether a gene of Escherichia coli is essential for viability. The method requires cloned DNA spanning the gene in question and a reasonably detailed genetic and physical map of the cloned segment. Using this information, one constructs a deletion of the target gene in vitro. For convenience, the deletion can be marked by an antibiotic resistance gene. A DNA segment containing the deletion is then cloned onto an att delta phage lambda vector. Integration of this phage, by homologous recombination at the target locus, and subsequent excision provide an efficient route for crossing the marked deletion onto the bacterial chromosome. Failure to delete the target gene indicates either that the resulting deletion was not viable or that the desired recombinational event did not take place. The use of prophage excision to generate the deletion allows one to estimate the fraction of deletion-producing events by analysis of the other product of the excision, the phage produced on induction of the prophage. In this way one can determine whether failure to recover a particular chromosomal deletion was due to its never having been formed, or, once formed, to its failure to survive. Applying this method to the polA gene, we found that polA is required for growth on rich medium but not on minimal medium. We repeated the experiment in the presence of plasmids carrying functional fragments of the polA gene, corresponding to the 5'-3' exonuclease and the polymerase-3'-5' exonuclease portions of DNA polymerase I. Surprisingly, either of these fragments, in the absence of the other, was sufficient to allow growth on rich medium.

Bacteriophage lambda↗
PubMed1991

A novel transposon trap for mycobacteria: isolation and characterization of IS1096.

In the course of developing strategies to obtain a mutation in the aspartate semialdehyde dehydrogenase (asd) gene of Mycobacterium smegmatis, an efficient transposon trap was constructed which may be generally useful for the identification of transposable elements in mycobacteria. A DNA fragment containing the asd gene was replaced with an aminoglycoside phosphotransferase gene (aph) to generate a delta asd::aph allele. Attempts to replace the wild-type asd gene with the delta asd::aph allele were unsuccessful, suggesting that this deletion was lethal to the growth of M. smegmatis. The plasmid, pYUB215, which contains beta-galactosidase expressed from a mycobacteriophage promoter and delta asd::aph, was integrated into the chromosome of M. smegmatis by a homologous, single-crossover, recombination event. Visual screening for inactivation of the beta-galactosidase gene in the resulting strain allowed the isolation of a novel mycobacterial insertion element from M. smegmatis. This insertion element, which is unique to M. smegmatis, was designated IS1096 and transposes at a frequency of 7.2 x 10(-5) per cell in an apparently random fashion. IS1096 is 2,275 bp in length and contains two open reading frames which are predicted to encode proteins involved in transposition. This insertion element exhibits several characteristics that suggest it may be a useful tool for genetic analysis of mycobacteria, possibly including the study of mechanisms of pathogenesis.

Amino Acid Sequence↗
PubMed2006

Muscle size, neuromuscular activation, and rapid force characteristics in elderly men and women: effects of unilateral long-term disuse due to hip-osteoarthritis.

Substantial evidence exists for the age-related decline in muscle strength and neural function, but the effect of long-term disuse in the elderly is largely unexplored. The present study examined the effect of unilateral long-term limb disuse on maximal voluntary quadriceps contraction (MVC), lean quadriceps muscle cross-sectional area (LCSA), contractile rate of force development (RFD, Delta force/Delta time), impulse (integral force dt), muscle activation deficit (interpolated twitch technique), maximal neuromuscular activity [electromyogram (EMG)], and antagonist muscle coactivation in elderly men (M: 60-86 yr; n = 19) and women (W: 60-86 yr; n = 20) with unilateral chronic hip-osteoarthritis. Both sides were examined to compare the effect of long-term decreased activity on the affected (AF) leg with the unaffected (UN) side. AF had a significant lower MVC (W: 20%; M: 20%), LCSA (W: 8%; M: 10%), contractile RFD (W: 17-26%; M: 15-24%), impulse (W: 10-19%, M: 19-20%), maximal EMG amplitude (W: 22-25%, M: 22-28%), and an increased muscle activation deficit (-18%) compared with UN. Furthermore, women were less strong (AF: 40%; UN: 39%), had less muscle mass (AF: 33%; UN: 34%), and had a lower RFD (AF: 38-50%; UN: 41-48%) compared with men. Similarly, maximum EMG amplitude was smaller for both agonists (AF: 51-63%; UN: 35-61%) and antagonist (AF: 49-64%; UN: 36-56%) muscles in women compared with men. However, when MVC and RFD were normalized to LCSA, there were no differences between genders. The present data demonstrate that disuse leads to a marked loss of muscle strength and muscle mass in elderly individuals. Furthermore, the data indicate that neuromuscular activation and contractile RFD are more affected by long-term disuse than maximal muscle strength, which may increase the future risk for falls.

Aged↗
PubMed1991

Blood pressure changes as predictors of future mortality in the seven countries study.

Twelve cohorts of men aged 40-59 at entry, enrolled in six countries (Finland, The Netherlands, Italy, Yugoslavia, Greece and Japan), giving a total of 8,287 subjects, were examined for the measurement of cardiovascular risk factors and then followed-up for 20 years. Changes of systolic blood pressure (SBP) occurred over the first 10 years in 6,767 men and these values, computed by an integral-like procedure (Delta-SBP), were used as possible predictors of fatal events recorded in the second 10 years of follow-up. Men who had a relative increase of SBP in the first 10 years showed an excess risk of death as compared with those who had a relative decrease of SBP, after adjustment for age and entry levels of SBP. Such relative risks were greater than 1 in the cohorts pooled within each of the six countries, ranging from 1.07 to 1.79 for all causes of death and from 1.12 to 2.15 for atherosclerotic cardiovascular disease deaths (coronary, stroke and peripheral atherosclerotic disease). The Cox model was solved using the same two end-points as dependent variables and, as coviariates, six risk factors measured at entry examination (age, cigarettes smoked per day, body mass index, serum cholesterol, physical activity at work and systolic blood pressure). By adding Delta-SBP the predictive power of the models was significantly improved and the coefficients of Delta-SBP proved to be statistically significant.

Adult↗
PubMed1981

Hepatic capacitance responses to changes in flow and hepatic venous pressure in dogs.

The effect of changes in inflow and hepatic venous pressure (Phv) on the hepatic vascular bed was studied in denervated, pump-perfused canine livers. Hepatic arterial, portal venous, and hepatic venous pressures and flows were continuously monitored as was the hepatic venous hematocrit (Hct). The hepatic venous outflow, which averaged 1179 +/- 356 (SD) ml x min-1 x kg tissue-1 at control conditions, was controlled by a servosystem set to keep Phv at the desired level. Hepatic volume changes (delta V) were determined by integration of the difference between hepatic inflow (Fin) and outflow (Fhv). Over the range of Phv of 0-15 mmHg, the delta V-to-delta Phv ratio (apparent compliance C) was linear; C = 19.8 ml x mmHg-1 x kg tissue-1. These increases in Phv caused transient and plateau increases in Hct of 1.26 and 0.42%/mmHg, respectively. Varying inflow over the range of 0-156% of control by changing hepatic arterial and/or portal venous flow(s) resulted in delta V of 0.066 ml/kg per ml x min-1 x kg tissue-1 change in flow. We conclude that changes in Fin as well as changes in Phv cause changes in liver volume; and that a significant fraction of the volume shift accompanying changes in Phv is due to transsinusoidal plasma movement.

Animals↗
PubMedDate not supplied

The effects of sucrose, fructose, and high-fructose corn syrup meals on plasma glucose and insulin in non-insulin-dependent diabetic subjects.

We have previously shown that fructose and sorbitol given with a standard meal cause less increment in plasma glucose than sucrose and high fructose corn syrup (HFCS) in patients with NIDDM. However, there was no direct comparison of sucrose with HFCS. Sixteen men and one woman aged 54-67) with NIDDM were given either 35 g sucrose, 35 g fructose, or 43.75 g HFCS containing 35 g carbohydrate as part of a 400-calorie test meal. Blood samples were obtained at frequent intervals up to 3 h and were analyzed for glucose and insulin. As compared with a fructose meal, the mean increment in plasma glucose (delta PG) after a sucrose meal was significantly higher at 45 min and after an HFCS meal it was significantly higher at 30 and 45 min, but sucrose and HFCS meals did not differ. When delta PGs were compared in nine patients with basal PG greater than 140 mg/dl and in eight patients with basal PG less than 140 mg/dl, differences in delta PG after sucrose and HFCS versus fructose meals became more significant but still did not differ from each other. The integrated total areas under the delta PG curves after sucrose, HFCS, and fructose meals were not statistically different. However, the areas under the curves up to 90 min after sucrose and HFCS meals, which did not differ, were greater than the fructose meal. The mean delta IRI after sucrose meals was markedly elevated at 45, 60, and 75 min (P less than 0.05) and after HFCS meals at 45 min as compared with fructose meals.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1973

Molecular structure of an R factor, its component drug-resistance determinants and transfer factor.

Plasmid DNA from Escherichia coli strains harboring drug resistance either of the infectious or noninfectious kind has been separated by CsCl centrifugation of crude cell lysates in the presence of ethidium bromide and examined by electron microscopy. Plasmid deoxyribonucleic acid (DNA) from an S(+) strain (which has the property of noninfectious streptomycin-sulfonamide resistance) consists of a monomolecular covalently closed circular species of 2.7 mum in contour length (5.6 x 10(6) atomic mass units; amu). DNA from a strain carrying a transfer factor, termed Delta, but no determinant for drug resistance, is a monomolecular covalently closed circular species of 29.3 mum in contour length (61 x 10(6) amu). DNA from either Delta(+)A(+) or Delta(+)S(+) strains, (which are respectively ampicillin or streptomycin-sulfonamide resistant, and can transfer this drug resistance), shows a bimodal distribution of molecules of contour lengths 2.7 mum and 29.3 mum, whereas DNA from a (Delta-T)(+) strain (showing infectious tetracycline resistance) contains only one species of molecule measuring 32.3 mum (67 x 10(6) amu). We conclude that ampicillin resistance is carried by a DNA molecule (the A determinant) of 2.7 mum, and streptomycin-sulfonamide resistance is carried by an independent molecule (the S determinant) of similar size. These molecules are not able to effect their own transfer, but can be transmitted to other cells due to the simultaneous presence of the transfer factor, Delta, which also constitutes an independent molecule, of size 29.3 mum. In general, there appears to be little recombination or integration of the A or S molecules into that of Delta, although a small proportion (5-10%) of recombinant molecules cannot be excluded. In contrast, the third drug-resistance determinant, that for tetracycline resistance (denoted as T), is integrated in the Delta molecule to form the composite structure Delta-T of size 32.3 mum, which determines infectious tetracycline resistance. The Delta(+)A(+) and Delta(+)S(+) strains are defined as harboring plasmid aggregates, and the (Delta-T)(+) strain is defined as carrying a plasmid cointegrate; the properties of all three strains are characteristic of strains harboring R factors. These results are compatible with the previously published genetic data. The number of Delta molecules per cell appears to be equal to the chromosomal number irrespective of growth phase, and this plasmid can thus be defined as stringently regulated in DNA replication. In contrast, S and A exist as multiple copies, probably in at least a 10-fold excess of chromosomal copy number. S and A can thus be defined as relaxed in the regulation of their DNA replication.

Ampicillin↗
PubMed1994

Targeting of proteins to the thylakoids by bipartite presequences: CFoII is imported by a novel, third pathway.

The CFoII subunit of the ATP synthase is an integral component of the thylakoid membrane which is synthesized in the cytosol with a bipartite, lumen-targeting presequence similar in structural terms to those of imported lumenal proteins such as plastocyanin. This presequence is shown to possess a terminal cleavage site for the thylakoidal processing peptidase, but no intermediate site for the stromal processing peptidase. The integration of CFoII into the thylakoid membrane of Pisum sativum has been analysed using in vitro assays for the import of proteins into intact chloroplasts or isolated thylakoids. Efficient integration into thylakoids is observed in the light and dark, and the integration process does not require the presence of either stromal extracts or nucleoside triphosphates. The uncoupler nigericin inhibits integration only very slightly, indicating that the thylakoidal delta pH does not play a significant role in the integration mechanism. In each of these respects, the requirements for CFoII integration differ notably from those determined for integration of the light-harvesting chlorophyll-binding protein of photosystem II. The integration mechanism also differs significantly from the two mechanisms involved in the translocation of lumenal proteins across the thylakoid membrane, since one of these processes requires the presence of stromal protein factors and ATP, and the other mechanism is dependent on the thylakoidal delta pH. This conclusion is reinforced by the finding that saturation of the translocation system for the precursor to the lumenal 23 kDa oxygen-evolving complex protein does not affect integration of CFoII into thylakoids.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoproteins↗
PubMed1997

Validation of a new and specific intraoperative measurement of vein graft resistance.

PURPOSE: Clinical studies have revealed that the most important predictor of successful bypass grafting is the origin and quality of the bypass conduit. Attempts at intraoperative evaluation of the hemodynamic properties of the conduit, including assessment of blood flow (Q), pressure gradients (delta P), and resistance (R), have not been useful. This is because each of these parameters measures the characteristics of the graft plus the outflow bed. To date, no specific measurement of the resistive properties of the conduit only is available. The purpose of this investigation was to evaluate longitudinal impedance (ZL) as a measure of conduit-specific resistance and to evaluate its potential in predicting the outcome of infrainguinal vascular reconstructions. METHODS: ZL was measured during surgery in 73 infrainguinal autologous vein reconstructions performed in 68 patients in two separate institutions over a 21-month period. Vein graft ultrasonic transit time Q and delta P (from proximal to distal anastomosis) were measured at baseline and after maximal peripheral vasodilatation with an intraarterial injection of papaverine 30 mg. Waveforms were recorded for 10 seconds at 200 Hz using a digital acquisition system. R was calculated as proximal mean pressure divided by mean blood flow (Q). After Fourier transformation, ZL was calculated as delta P/Q at each harmonic and total ZL (integral of ZL) was defined as the integral of moduli from 0 to 4 Hz. RESULTS: All hemodynamic variables were significantly affected by papaverine vasodilatation (delta P, 3.9 +/- 0.5 vs 6.3 +/- 0.8 mm Hg; Q, 78.2 +/- 7.0 vs 126 +/- 11 ml/min; R, 134 +/- 17 vs 72.7 +/- 6.2 x 10(3) dyne.sec.cm-5; p < 0.0001), except integral of ZL, which remained constant (31.1 +/- 2.8 vs 30.8 +/- 2.8 x 10(3) dyne.cm-5; p = NS). After follow-up of 1 week to 17 months (median, 5 months), the 1-year primary, primary-assisted, and secondary patency rates were 72% +/- 7%, 77% +/- 6%, and 81% +/- 6%, respectively. Using Cox analysis, primary patency was significantly associated with decreased integral of ZL (p = 0.0001), but not with baseline or papaverine-stimulated delta P, Q, delta P/Q, or R integral of ZL > 47 x 10(3) dyne.cm-5 predicted primary failure with 90% positive and negative predictive value. CONCLUSIONS: Intraoperative measurement of integral of ZL in infrainguinal vein grafts is independent of outflow conditions (that is, does not change with papaverine), and hence describes the resistive properties of the conduit only. In addition, these preliminary data suggest that integral of ZL is predictive of short-term primary patency. integral of ZL is the first available hemodynamic measurement that is conduit-specific and may therefore be a better predictor of graft patency than currently available methods.

Aged↗
PubMed2000

Water availability and branch length determine delta(13)C in foliage of Pinus pinaster.

The stable carbon isotope composition (delta(13)C) of foliage integrates signals resulting from environmental and hydraulic constraints on water movement and photosynthesis. We used branch length as a simple predictor of hydraulic constraints to water fluxes and determined the response of delta(13)C to varying water availability. Foliage up to 6 years old was taken from Pinus pinaster Ait. trees growing at four sites differing in precipitation (P; 414-984 mm year(-1)) and potential evaporation (ET; 1091-1750 mm year(-1)). Branch length was the principal determinant of temporal trends in delta(13)C. The strong relationship between delta(13)C and branch length was a function of hydraulic conductance, which was negatively correlated with branch length (r(2) = 0.84). Variation in P and ET among sites was reflected in delta(13)C, which was negatively correlated with P/ET (r(2) = 0.66). However, this analysis was confounded by differences in branch length. If the effects of branch length on delta(13)C were first removed, then the 'residual' delta(13)C was more closely related to P/ET (r(2) = 0.99), highlighting the importance of accounting for variation in hydraulic constraints to water flux between sites and years. For plant species that exhibit considerable phenotypic plasticity in response to changes in environment (e.g., variation in leaf area, branch length and number, or stem form), the environmental effects on delta(13)C in foliage can only be reliably assessed if deconvoluted from hydraulic constraints.

Journal Article↗
PubMed2000

Incorporation of protease K into larval insect membrane vesicles does not result in disruption of integrity or function of the pore-forming Bacillus thuringiensis delta-endotoxin.

Bacillus thuringiensis delta-endotoxins insert into the brush border membranes of insect larval cells to form ion channels. A possible interaction of these toxins with a cytoplasmic component was examined by preloading vesicles from insect larval cells with protease K followed by incubation with toxin. There was no evidence for toxin antigens smaller than the intact toxin in extracts of solubilized vesicles, nor was there an effect of the inclusion of protease K on either of two functional properties, the formation of toxin aggregates or of ion pores. These toxins, physically and functionally, appear to be confined to the membrane.

Animals↗
PubMed2005

A versatile and efficient gene-targeting system for Aspergillus nidulans.

Aspergillus nidulans is an important experimental organism, and it is a model organism for the genus Aspergillus that includes serious pathogens as well as commercially important organisms. Gene targeting by homologous recombination during transformation is possible in A. nidulans, but the frequency of correct gene targeting is variable and often low. We have identified the A. nidulans homolog (nkuA) of the human KU70 gene that is essential for nonhomologous end joining of DNA in double-strand break repair. Deletion of nkuA (nkuA delta) greatly reduces the frequency of nonhomologous integration of transforming DNA fragments, leading to dramatically improved gene targeting. We have also developed heterologous markers that are selectable in A. nidulans but do not direct integration at any site in the A. nidulans genome. In combination, nkuA delta and the heterologous selectable markers make up a very efficient gene-targeting system. In experiments involving scores of genes, 90% or more of the transformants carried a single insertion of the transforming DNA at the correct site. The system works with linear and circular transforming molecules and it works for tagging genes with fluorescent moieties, replacing genes, and replacing promoters. This system is efficient enough to make genomewide gene-targeting projects feasible.

Antigens, Nuclear↗
PubMed1998

Deletion and site-directed mutagenesis of the Wolinella succinogenes fumarate reductase operon.

Wolinella succinogenes is known to grow at the expense of fumarate respiration with formate or sulfide as electron donor. A W. succinogenes mutant (delta frdCAB) lacking the fumarate reductase operon did not grow with fumarate as terminal electron acceptor and either formate or sulfide as electron donor. The delta frdCAB mutant grown with formate and nitrate did not contain fumarate reductase activity and did not catalyze electron transport from sulfide to fumarate, in contrast to the nitrate-grown wild-type strain. A mutant constructed by integration of frdCAB into the delta frdCAB mutant genome showed wild-type properties with respect to growth and enzyme activities. The frdC2 gene located downstream of the fumarate reductase operon frdCAB possibly encodes a diheme cytochrome b that is similar to FrdC (41% identical residues). The corresponding transcript differs from that of frdCAB. A delta frdC2 mutant showed wild-type properties with respect to growth and enzyme activities. Using site-directed mutagenesis, each of the four histidine residues that are predicted to serve as the axial heme ligands in FrdC (His44, His93, His143, and His182) was replaced by alanine or other residues. The resulting mutants did not grow with formate and fumarate and did not contain fumarate reductase activity, FrdA or FrdC when grown with formate and nitrate. In contrast, substitution of two histidine residues that are not considered heme ligands, yielded mutants (H114A and H120A) that grew with fumarate. It is concluded that FrdCAB is an obligatory component of fumarate respiration with formate and with sulfide in W. succinogenes. FrdC2 is not involved in fumarate respiration. Replacement of the putative heme ligands of FrdC prevents formation of a functional fumarate reductase.

Alanine↗
PubMed1995

H-NS regulation of virulence gene expression in enteroinvasive Escherichia coli harboring the virulence plasmid integrated into the host chromosome.

We have previously shown that integration of the virulence plasmid pINV into the chromosome of enteroinvasive Escherichia coli and of Shigella flexneri makes these strains noninvasive (C. Zagaglia, M. Casalino, B. Colonna, C. Conti, A. Calconi, and M. Nicoletti, Infect. Immun. 59:792-799, 1991). In this work, we have studied the transcription of the virulence regulatory genes virB, virF, and hns (virR) in wild-type enteroinvasive E. coli HN280 and in its pINV-integrated derivative HN280/32. While transcription of virF and of hns is not affected by pINV integration, transcription of virB is severely reduced even if integration does not occur within the virB locus. This indicates that VirF cannot activate virB transcription when pINV is integrated, and this lack of expression accounts for the noninvasive phenotype of HN280/32. Virulence gene expression in strains HN280 and HN280/32, as well as in derivatives harboring a mxiC::lacZ operon fusion either on the autonomously replicating pINV or on the integrated pINV, was studied. The effect of the introduction of plasmids carrying virB (pBNI) or virF (pHW745 and pMYSH6504), and of a delta hns deletion, in the different strains was evaluated by measuring beta-galactosidase activity, virB transcription, and virB-regulated virulence phenotypes like synthesis of Ipa proteins, contact-mediated hemolysis, and capacity to invade HeLa cells. The introduction of pBN1 or of the delta hns deletion in pINV-integrated strains induces temperature-regulated expression or temperature-independent expression, respectively, of beta-galactosidase activity and of all virulence phenotypes, while an increase in virF gene dosage does not, in spite of a high-level induction of virB transcription. Moreover, a wild-type hns gene placed in trans fully reversed the induction of beta-galactosidase activity due to the delta hns deletion. These results indicate that virB transcription is negatively regulated by H-NS both at 30 and at 37 degrees C in pINV-integrated strains and that there is also a dose-dependent effect of VirF on virB transcription. The negative effect of H-NS on virB transcription at the permissive temperature of 37 degrees C could be due to changes in the DNA topology occurring upon pINV integration that favor more stable binding of H-NS to the virB promoter DNA region. At 30 degrees C, the introduction of the high-copy-number plasmid pMYSH6504 (but not of the low-copy-number pHW745) or of the deltahns deletion induces, in strains harboring an autonomously replicating pINV, beta-galactosidase activity, virB transcription, and expression of the virulence phenotypes, indicating that, as for HN280/32, the increase in virF gene dosage overcomes the negative regulatory effect of H-NS on virB transcription. Moreover, we have found that virF transcription is finely modulated by temperature and, with E. coli K-12 strains containing a virF-lacZ gene fusion, by H-NS. This leads us to speculate that, in enteroinvasive bacteria, the level of Virf inside the cell controls the temperature-regulated expression of invasion genes.

Animals↗
PubMed2003

Quantitative assessment of myocardial ischemia by electrocardiographic and scintigraphic imaging.

We calculated distributions of epicardial potentials from body-surface electrocardiograms (ECGs) recorded during controlled myocardial ischemia and compared them with scintigraphic estimates of ischemia's extent/severity. The study population consisted of patients suffering from single-vessel coronary artery disease, referred for elective percutaneous transluminal coronary angioplasty of either the left anterior descending (n=7), the right coronary (n=9), or the left circumflex (n=2) artery. After the target vessel had been dilated, a 1960s "study" inflation was performed with a non-perfusion-type balloon catheter; at its commencement, technetium-99m sestamibi was injected via a femoral-vein catheter, and ECGs were recorded throughout the inflation from 120 leads. Single photon emission computed tomographic imaging was performed one hour after the injection of radionuclide to obtain an "occlusion image", and again one hour after a repeat injection 24 hours later to obtain a "control image"; the latter image was subtracted from the former, to derive a scintigraphic difference map (Delta map). The ECGs were signal-averaged over a 10-s window at preinflation and peak-inflation states, the preinflation averaged complexes were subtracted from the peak-inflation ones to produce body-surface Delta maps, and the corresponding Delta maps of epicardial potentials were calculated by applying the electrocardiographic inverse solution; this procedure is referred to as electrocardiographic imaging. The ECG-derived epicardial Delta maps related spatially to the scintigraphic Delta maps in all patients. The percent areas and surface integrals of positive values in ECG-derived Delta maps were found to be very good single-variable predictors of the extent (r=0.73; p=0.0006) and severity (r=0.72; p=0.0008) of the scintigraphically-estimated perfusion defect; a regression equation using two ECG-derived predictors further improved the agreement with scintigraphic estimates (r=0.81; p=0.0004 for estimates of severity). These findings suggest that noninvasive electrocardiographic imaging might provide quantitative estimates of the extent/severity of myocardial ischemia that agree closely with those provided by scintigraphic techniques.

Adult↗
PubMed1986

Visco-elastic properties of dental elastomeric impression materials.

The visco-elastic properties of a number of set elastomeric impression materials were determined using a torsional pendulum apparatus, i.e. the real (G1) and imaginary (G2) parts of the complex shear modulus and the mechanical loss tangent (Tan delta). Comparison with published data on tension and compression set for these materials showed that whilst there was no correlation between Tan delta and tension set, there was significant (greater than 99%) correlation with compression set. Treating a permanent set test as the application of a rectangular stress pulse of duration tau, and assuming (i) linear visco-elasticity theory and (ii) that G1 and G2 are independent of frequency. Fourier integral analysis predicts that Tan delta should be proportional to permanent set. Tension set does not conform to this prediction because the deformation is nonlinear. The compression set test is shown to involve shear strains and elastomers generally are Hookean in shear. Whilst the theoretical treatment applies qualitatively, there are major quantitative disparities, the reasons for which are discussed.

Dental Impression Materials↗