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PubMed1998

In vitro activities of co-amoxiclav at concentrations achieved in human serum against the resistant subpopulation of heteroresistant Staphylococcus aureus: a controlled study with vancomycin.

The effects of concentrations that simulated those in human serum after a single intravenous dose of amoxicillin (2 g), amoxicillin-clavulanic acid (2,000 and 200 mg, respectively), or vancomycin (500 mg), on the viability and beta-lactamase activity of two isogenic (beta-lactamase and non-beta-lactamase producer) heteroresistant Staphylococcus aureus strains were studied in an in vitro pharmacodynamic model. A reduction of > or = 97% of the initial inoculum was obtained with vancomycin and amoxicillin-clavulanic acid against both strains, with respect to the total bacterial population and the oxacillin-resistant subpopulation. The same pattern was observed with amoxicillin and the beta-lactamase-negative strain. beta-Lactamase activity in the beta-lactamase-positive strain changed over time parallel to viability, decreasing with amoxicillin-clavulanic acid or vancomycin and increasing in the amoxicillin and control groups. Clavulanic acid concentrations achievable in serum that changed over time allowed amoxicillin to act against the beta-lactamase-producing methicillin-resistant S. aureus to a similar extent as vancomycin.

Amoxicillin↗
PubMed1980

Enhancement of carcinogenesis in cultured respiratory tract epithelium by 12-0-tetradecanoylphorbol-13-acetate.

The enhancement of chemical carcinogenesis was demonstrated in vitro in rat tracheal epithelium exposed in organ culture to N-methyl-N'-nitro- N-nitrosoguanidine (MNNG) followed by multiple exposures to 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The explants were exposed to 0 or 0.0001 mu g MNNG per ml for 6 h on days 3 and 6 of culture, and then to 0 or 1.0 mu g TPA per ml for I h on days 9, 15, 21, and 27 of culture. Primary cell cultures were then established from epithelial outgrowths of these explants. Morphologically altered cells were seen in the primary cell cultures derived from MNNG + TPA-exposed explants on an average of 130 days after MNNG exposure. In primary cultures derived from explants exposed to either TPA or MNNG alone, morphologically altered cells were seen at 190 days and 210 days, respectively. No morphologically altered cells were seen in solvent control primary cell cultures. Only cultures containing morphologically altered cells could be repeatedly subcultured. Cell lines established from these cultures were inoculated i.m. into immunosuppressed isogeneic hosts at 150, 200, 250, 310, and 365 days following carcinogen exposure. All five explants exposed to both MNNG and TPA produced at least one cell line which formed carcinomas when inoculated 365 days following exposure, while only two of the five explants produced tumorigenic cell lines in the MNNG-only group. Cell lines from explants exposed to both MNNG and TPA were tumorigenic an average of 90 days earlier than cell lines from explants exposed to MNNG alone. None of the cell lines obtained from the explants exposed only to TPA were tumorigenic. These studies show that, following carcinogen exposure, TPA can enhance the oncogenesis of cultured respiratory tract epithelium in terms of increasing the frequency of transformation and decreasing the time at which transformation could first be observed.

Animals↗
PubMed1981

A differential killing assay for mutagens and carcinogens based on an improved repair-deficient strain of Escherichia coli.

The lexA gene suppresses the spontaneous inviability of recA strains of Escherichia coli without affecting their repair deficiency. We have taken advantage of this to construct a uvrA recA lexA triple mutant CM871 that combines extreme repair deficiency with near wild-type growth. We have used this strain in conjunction with an isogenic strain WP67 uvrA polA and isogenic wild-type strain WP2 in a differential killing test. Dilute suspensions in buffer of the repair-deficient strains and repair-proficient control are incubated for 2 h at 37 degrees C with test compound in the presence or absence of a S9 metabolising system. Survival is determined by Miles Misra plating. For each strain three 10 microliter spots of each of three concentrations of test compound and of the untreated control are placed on a nutrient agar plate. Following overnight incubation survivors are counted. Results with reference compounds are given and the advantages and disadvantages of the test are discussed.

Animals↗
PubMed2004

Assessment of experimental Bt events against fall armyworm and corn earworm in field corn.

Performance of experimental Bacillus thuringiensis (Bt) MON events alone and pyramided with MON810 were evaluated over 3 yr in Georgia and Alabama. Ability of events to prevent whorl defoliation by the fall armyworm, Spodoptera frugiperda (J. E. Smith), and natural ear feeding damage by the corn earworm, Helicoverpa zea (Boddie) was assessed. In each year, near-isogenic hybrids with novel single transformation events and crosses pyramided with the MON810 event were compared with the standard single MON810 event and nontransformed susceptible control. Events were tested for resistance to whorl damage by manual infestations of fall armyworm and ear damage by natural infestations of corn earworm. All Bt events tested reduced fall armyworm whorl damage ratings per plant compared with the susceptible hybrid. All Bt treatments also had considerably less ear infestation and damage by corn earworm compared with the nontransgenic isoline. The MON841, MION849, and MON851 events reduced ear damage by H. zea but were not as effective as other novel events and were not advanced for further testing after the 1999 season. Pyramiding events compared with single events did not improve control of fall armyworm whorl damage, but they generally did prevent more ear damage by corn earworm. The MON84006 event singly and pyramided with MON810 had superior control of whorl-stage damage by S. frugiperda and ear damage by H. zea compared with MON810. Deployment of new events and genes could provide additional tools for managing the potential for insect resistance to Bt toxins. Furthermore, improved control of whorl and ear infestations by H. zea and S. frugiperda would increase the flexibility of planting corn, Zea mays L., and permit double cropping of corn in areas where these pests perennially reach damaging levels.

Animals↗
PubMed2017

Rapid DNA replication origin licensing protects stem cell pluripotency.

Complete and robust human genome duplication requires loading minichromosome maintenance (MCM) helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single-cell analyses, we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation toward all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance.

Cell Cycle↗
PubMed2000

Immunogenicity and safety of a hexavalent meningococcal outer-membrane-vesicle vaccine in children of 2-3 and 7-8 years of age.

To study the reactogenicity and immunogenicity of a hexavalent meningococcal outer-membrane-vesicle vaccine (OMV), two different dosages of this vaccine (7.5 and 15 microg of individual PorA proteins) consisting of vesicles expressing class 1 outer-membrane proteins (OMPs) of subtypes P1.7,16; P1.5,2; P1.19,15 and P1.5(c), 10; P1.12,13; P1.7(h),4 were administered to a group of 7-8 year (n=165) and a group of 2-3 year old children (n=172). Control groups of children with similar ages were vaccinated against hepatitis B. All participants received three injections. Pre- and postimmunisation sera were tested for bactericidal antibodies against six isogenic meningococcal vaccine strains expressing different PorA proteins. Antibody titres against OMP of the two different vesicles (PL16215 and PL10124) were measured by ELISA. The meningococcal hexavalent OMV vaccine was well tolerated. No statistically significant differences were seen between the high and low dose of hexavalent meningococcal OMV vaccine. The percentage of children showing a fourfold increase of bactericidal antibody titres against the specific serosubtype varied in toddlers from 28 to 98% and in older children from 16 to 100%. Both ELISA antibody titres and bactericidal activity showed the highest level in the youngest age-group.

Antibodies, Bacterial↗
PubMed2000

In vivo adenovirus-mediated endothelial nitric oxide synthase gene transfer ameliorates lung allograft ischemia-reperfusion injury.

OBJECTIVE: Nitric oxide regulates vascular tone, inhibits platelet aggregation, and inhibits leukocyte adhesion, all of which are important modulators of ischemia-reperfusion injury. This study aimed to determine the effects of endothelial constitutive nitric oxide synthase gene transfer on ischemia-reperfusion injury in a rat lung transplant model. METHODS: In group I, donor animals were injected intravenously with 5 x 10(9) pfu of adenovirus-encoding endothelial constitutive nitric oxide synthase. Groups II and III served as controls, whereby donor animals were injected with either 5 x 10(9) pfu of adenovirus encoding beta-galactosidase or saline solution, respectively. Twenty-four hours after injection, left lungs were harvested and preserved for 18 hours at 4 degrees C, then implanted into isogeneic recipients, which were put to death 24 hours later. Recombinant endothelial constitutive nitric oxide synthase gene expression was evaluated by Western blotting and immunohistochemistry. Lung grafts were assessed by measuring arterial oxygenation, myeloperoxidase activity, and wet/dry weight ratios. RESULTS: Western blotting confirmed the overexpression of endothelial constitutive nitric oxide synthase in lungs so transfected compared with controls. Twenty-four hours after reperfusion, mean arterial oxygenation was significantly improved in group I compared with group II and III controls (189.4 +/- 47.1 mm Hg vs 71.7 +/- 8.9 mm Hg and 67.8 +/- 12.2 mm Hg, P =.02, P =.01, respectively). Myeloperoxidase activity, a reflection of tissue neutrophil sequestration, was also significantly reduced in group I compared with groups II and III (0.136 +/- 0.038 DeltaOD/mg/min vs 0. 587 +/- 0.077 and 0.489 +/- 0.126 DeltaOD/mg/min, P =.001, P =.01, respectively). CONCLUSION: Adenovirus-mediated gene transfer with endothelial constitutive nitric oxide synthase ameliorates ischemia-reperfusion injury as manifested by significantly improved oxygenation and decreased neutrophil sequestration in transplanted lung isografts. Endothelial constitutive nitric oxide synthase gene transfer may reduce acute lung dysfunction after lung transplantation.

Adenoviridae↗
PubMed1986

Opacity genes in Neisseria gonorrhoeae: control of phase and antigenic variation.

The chromosome of N. gonorrhoeae contains several complete expression genes coding for variant opacity proteins. DNA sequence analysis of two opacity genes derived from the same locus (opaE1) of two isogenic gonococcal variants reveals common and variable regions in these genes. Genomic blotting experiments using synthetic probes suggest gene conversion as a principle for the assembly of variant sequence information in opacity genes. The 5' region of opacity genes is composed of identical pentameric pyrimidine units (CTCTT) encoding the hydrophobic portion of the opacity leader peptide. This coding repeat is variable in a given locus with respect of the number of pentameric units. While all expression loci in a single cell are constitutively transcribed, the production of opacity proteins is determined by the coding repeat sequence on the translational level.

Amino Acid Sequence↗
PubMed2003

Identification of quorum-sensing regulated proteins in the opportunistic pathogen Pseudomonas aeruginosa by proteomics.

The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen which is responsible for severe nosocomial infections in immunocompromised patients and is the major pathogen in cystic fibrosis. The bacterium utilizes two interrelated quorum-sensing (QS) systems, which rely on N-acyl-homoserine lactone (AHL) signal molecules, to control the expression of virulence factors and biofilm development. In this study, we compared the protein patterns of the intracellular, extracellular and surface protein fractions of the PAO1 parent strain with those of an isogenic lasI rhlI double mutant by means of two-dimensional gel electrophoresis (2-DE). This analysis showed that the intensities of 23.7% of all detected protein spots differed more than 2.5-fold between the two strains. We only considered those protein spots truly QS regulated that were changed in the mutant in the absence of signal molecules but were rescued to the wild-type situation when the medium was supplemented with AHLs. These protein spots were characterized by MALDI-TOF peptide mapping. Twenty-seven proteins were identified that were previously reported to be AHL controlled, among them several well-characterized virulence factors. For one of the identified proteins, the serine protease PrpL, a biochemical assay was established to verify that expression of this factor is indeed QS regulated. Furthermore, it is shown that the quorum-sensing blocker C-30 specifically interferes with the expression of 67% of the AHL-controlled protein spots of the surface fraction, confirming the high specificity of the compound. Importantly, 20 novel QS-regulated proteins were identified, many of which are involved in iron utilization, suggesting a link between quorum sensing and the iron regulatory system. Two of these proteins, PhuR and HasAp, are components of the two distinct haem-uptake systems present in P. aeruginosa. In agreement with the finding that both proteins are positively regulated by the QS cascade, we show that the lasI rhlI double mutant grows poorly with haemoglobin as the only iron source when compared with the wild type. These results add haemoglobin utilization to the list of phenotypes controlled through QS in P. aeruginosa. The surprisingly high number of AHL-regulated proteins relative to the number of regulated genes suggests that quorum-sensing control also operates via post-transcriptional mechanisms. To strengthen this hypothesis we investigated the role of quorum sensing in the post-translational modification of HasAp, an extracellular protein required for the uptake of free and haemoglobin-bound haem.

4-Butyrolactone↗
PubMed2005

Laboratory and field evaluations of transgenic soybean exhibiting high-dose expression of a synthetic Bacillus thuringiensis cry1A gene for control of Lepidoptera.

Transgenic lines of soybean, Glycine max (L.) Merrill, expressing a synthetic cry1A gene (tic107) from Bacillus thuringiensis (Bt), were evaluated in screenhouse and conventional field trials for efficacy against lepidopteran pests. In screenhouse trials, Bt soybean and negative checks (isogenic segregants and parental lines) were evaluated against Anticarsia gemmatalis Hübner and Pseudoplusia includens (Walker) in the United States and against A. gemmatalis, Epinotia aporema (Walsingham), Rachiplusia nu (Guenée), and Spilosoma virginica (F.) in Argentina. Bt soybean exhibited virtually complete efficacy against each of these pests, whereas negative checks suffered significant damage. Bt soybean and negative checks also were evaluated in conventional trials against native populations of A. gemmatalis and P. includens in the southeastern United States. Each of these insects caused significant damage to negative checks in one or more locations, whereas Bt soybean exhibited virtually complete efficacy against these pests. In the laboratory, lyophilized leaf tissues from Bt soybean incorporated in artificial diet at a concentration representing a 25-fold dilution of fresh tissue caused complete mortality of A. gemmatalis and near complete mortality of P. includens neonates after 11 d, whereas mortality on negative checks did not exceed 10% for either insect. Average TIC107 expression approached or exceeded 50 microg/g fresh weight at V3 stage of growth and 200 microg/g by R6 stage of growth. These results demonstrate that expression of TIC107 in soybean can not only achieve highly efficacious control of several lepidopterans under field conditions but also provide a high dose for effective insect resistance management.

Animals↗
PubMed1990

Genes of the Escherichia coli pur regulon are negatively controlled by a repressor-operator interaction.

Fusions of lacZ were constructed to genes in each of the loci involved in de novo synthesis of IMP. The expression of each pur-lacZ fusion was determined in isogenic purR and purR+ strains. These measurements indicated 5- to 17-fold coregulation of genes purF, purHD, purC, purMN, purL, and purEK and thus confirm the existence of a pur regulon. Gene purB, which encodes an enzyme involved in synthesis of IMP and in the AMP branch of the pathway, was not regulated by purR. Each locus of the pur regulon contains a 16-base-pair conserved operator sequence that overlaps with the promoter. The purR product, purine repressor, was shown to bind specifically to each operator. Thus, binding of repressor to each operator of pur regulon genes negatively coregulates expression.

Base Sequence↗
PubMed1995

Accessory gene regulator controls Staphylococcus aureus virulence in endophthalmitis.

PURPOSE: To evaluate the contribution of toxins to the severity of Staphylococcus aureus endophthalmitis. METHODS: Experimental endophthalmitis was established by injecting rabbit eyes with wild type S. aureus ISP479 and the isogenic attenuated strain, ISP546, defective in expression of the global regulator locus agr. agr regulates expression of at least 19 exoproteins that are potentially important in the pathogenesis of endophthalmitis. Infections were evaluated using electroretinography, slit lamp biomicroscopy, and histology. Two concentrations (approximately 10 and 1000 organisms) of bacteria were injected. RESULTS: The agr- strain consistently resulted in slower loss of b-wave response when compared to the wild type strain, irrespective of inoculum size. Clinical signs were less severe among the agr- group at 24 and 48 hours when 10 organisms were injected. However, when the number of bacteria injected was increased to 1000, earlier onset of clinical signs was observed, with both groups showing maximum cell and flare and a white fundal reflex at 48 hours after infection. Histologic examination of eyes enucleated 36 hours after inoculation revealed that the wild type strain induced focal retinal destruction and mild vitritis, whereas eyes infected with the agr- strain remained completely normal. Histologic examination carried out when loss of B-wave response was 100% revealed that retinal changes for both groups could not be distinguished. CONCLUSIONS: These data indicate that toxin production by S. aureus contributes to severity of endophthalmitis by accelerating the rate of onset of retinal damage. Therefore, toxin-targeting therapies instituted early in the course of infection could preserve retinal function.

Animals↗
PubMed1976

Increased growth rate of a benzo(a)pyrene-induced transplantable tumor in bursectomized chickens.

A benzo(a)pyrene-induced transplantable tumor was used to study the effects of bursectomy and thymectomy on tumor development in recipients that were isogenic with the donor for the major histocompatibility locus. Surgical bursectomies and thymectomies were done in different groups of chickens on the day of hatching, with no adjunct treatment. The following day, the experimental and control animals were given 10(3) live tumor cells s.c. Tumor frequency and size were followed weekly. With the experimental protocol used, thymectomy did not significantly affect tumor frequency or growth. Bursectomy did not affect tumor frequency, but the transplanted tumors grew significantly more rapidly in bursectomized than in control animals. Thus, bursectomy and, consequently, the antibody-forming system influence not only virus-induced tumors, but also the development of a chemical carcinogen-induced transplantable tumor line. The data underscore the host-protective role of the antibody-forming system in oncogenesis.

Animals↗
PubMed1997

Nifedipine protects small intestine from cyclosporine-induced hemodynamic and functional impairment.

We have previously shown that cyclosporine (CsA) causes intestinal hemodynamic and functional impairments. In this study, we evaluated whether nifedipine protects the small intestine from such toxic side effects. Isogeneic small intestinal transplantation was performed in rats which then received one of the following two-week treatments: olive oil, 0.15 ml/kg/day i.m. as vehicle controls in group 1; nifedipine, 1 mg/kg/day i.m. in group 2; CsA, 15 mg/kg/day i.m. in group 3; and both nifedipine and CsA in group 4. Vascular resistance, whole tissue blood flow and its mucosal and serosal/muscularis distributions in both graft and recipient residual native intestines, and absorptive function were determined. The data showed that two-week treatment with CsA resulted in a marked elevation of vascular resistance from 51.0 +/- 6.8 to 72.4 +/- 11.1 U/g in the native whole tissue and from 53.7 +/- 7.2 to 78.2 +/- 12.1 U/g in the graft whole tissue, and decreases in blood flow from 1.59 +/- 0.26 to 1.11 +/- 0.17 ml/g/min in the native whole tissue and from 1.50 +/- 0.21 to 1.03 +/- 0.18 ml/g/min in the graft whole tissue and absorption from 227 +/- 36 to 166 +/- 26 mg glucose/dl. Mucosa was preferentially affected, while serosal/muscularis layers remained relatively unchanged. When nifedipine was concomitantly used with CsA, vascular resistance and blood flow values in the mucosal layer and whole intestinal tissue as well as absorptive function showed no significant differences from the baseline data. The changes observed in denervated grafts and recipient native intestines were similar. We conclude that nifedipine is effective in protecting both graft and native small intestines from CsA-induced toxicity in the rat.

Animals↗
PubMed1992

CDC25-dependent induction of inositol 1,4,5-trisphosphate and diacylglycerol in Saccharomyces cerevisiae by nitrogen.

The addition of ammonium sulfate to starved yeast cells leads to a 3- to 4-fold rapid increase of the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), the products of phosphoinositide-specific phospholipase C (PI-PLC). This response is reduced by dissecting the RAS-activating Cdc25 protein, and is completely abolished by the cdc25-1 mutation even at permissive temperature. Starved cdc25-1 mutant cells have a strongly reduced IP3 content, but an at least 10-fold increased DAG level compared to the isogenic wild-type strain. NH4 does not stimulate cAMP synthesis, and glucose does not induce IP3 and DAG. Our data suggest that the Cdc25 protein controls a nitrogen-specific signalling pathway involving the effector PI-PLC, in addition to the glucose-induced activation of adenylyl cyclase (AC).

Cell Cycle Proteins↗
PubMed2004

Role of Helicobacter pylori rfaJ genes (HP0159 and HP1416) in lipopolysaccharide synthesis.

The genome of Helicobacter pylori 26695 has been sequenced and the lipopolysaccharide (LPS) O sidechain of this strain has been shown to express both Lewis x and Lewis y units. To determine the role of HP0159 and HP1416, genes recognized as rfaJ homologs and implicated in LPS synthesis, isogenic mutants of H. pylori 26695 were generated. The LPS of mutant 26695::HP0159Kan did not express either Lewis epitope as detected by immunoblotting, whereas the control strain and 26695::HP1416Kan produced both epitopes. Structural analysis of the LPS of the mutants showed that HP0159 encodes an alpha(1,2/3)-glucosyltransferase whereas HP1416 encodes an alpha(1,2/4)-glucosyltransferase.

Genes, Bacterial↗
PubMed2000

An experimental model of cutaneous infection induced by superantigen-producing Staphylococcus aureus.

Skin infections caused by Staphylococcus aureus, such as erysipelas, are commonly occurring, painful, and costly for society. Despite the high prevalence of this condition, little is known about the host immune responsiveness and bacterial virulence factors during S. aureus dermatitis. We present here a mouse model of infectious dermatitis in which S. aureus is inoculated by an intracutaneous injection to the shaved back of NMRI mice. Visible skin inflammation, characterized by redness and swelling, was noted 48 h after inoculation of staphylococci in mice that received 2 x 108 colony-forming units of S. aureus. Microscopic evaluation revealed a dermal and subcutaneous infiltrate rich in macrophages and neutrophilic granulocytes already within 6 h after inoculation. A sparse influx of T lymphocytes was noted somewhat later. Bacterial cultures from skin revealed high numbers of staphylococci early after inoculation, with a successive decline during 2 wk follow-up. Total white blood cell count as well as the number of polymorphonuclear leukocytes peaked 2 d after bacterial inoculation. Also, serum interleukin-6 levels peaked within 2 d, with a 10-fold increase compared to non-infected control mice, indicating a systemic reaction to skin infection. The role of toxic shock syndrome toxin 1 in the pathogenesis of the dermatitis was assessed using isogenic S. aureus strains. Even though the gross inflammatory skin reaction was similar for mice infected with either of the strains, it was apparent that bacteria secreting toxic shock syndrome toxin 1 preferentially triggered influx of T lymphocytes to the skin. In addition, mice inoculated with staphylococci producing toxic shock syndrome toxin 1 showed a weight decrease during the experiment whereas mice inoculated with the isogenic strain showed a weight increase. This model of staphylococcal dermatitis will enable future in-depth studies regarding the host-bacterium relationship.

Animals↗