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PubMed1991

Molecular cloning of a glutathione S-transferase overproduced in an insecticide-resistant strain of the housefly (Musca domestica).

We report the cloning and sequencing of a glutathione S-transferase (GST) gene from the housefly Musca domestica. A cDNA lambda gt11 library was prepared from the organophosphate insecticide-resistant housefly strain Cornell-R--a variant that has elevated GST activity. The lambda phage GST clone was identified on the basis of its ability to cross-hybridize to a GST DNA probe from Drosophila melanogaster. Based on amino acid homology to other GSTs and expression of GST activity in Escherichia coli, the Musca GST gene (MdGST-1) belongs to the GST gene family. Although organophosphate resistance in Cornell-R is largely due to one of the GSTs, MdGST-1 is probably not the enzyme responsible for resistance. The mutation that controls resistance to organophosphate insecticides in Cornell-R is highly unstable and we isolated spontaneous variants to both insecticide sensitivity and to even higher levels of resistance. This provided us with an isogenic set of three strains. We found that MdGST-1 transcript levels as measured by Northern assays are higher in all three Cornell-R strains relative to the sensitive wild type, but that the sensitive Cornell-R strain has more MdGST-1 transcript than does the highly resistant Cornell-R strain. These data as well as Southern analysis of genomic DNA allow us to conclude: (1) there are multiple GST genes in M. domestica; (2) the natural variant Cornell-R overproduces excess transcript from two and probably more of these genes; and (3) the unstable mutation in Cornell-R influences the levels of multiple GSTs.

Amino Acid Sequence↗
PubMed2005

Quantitative PCR analysis of two molluscan metallothionein genes unveils differential expression and regulation.

The mRNA levels of two components of the mussel (Mytilus galloprovincialis) metallothionein (MT) gene families, MT10 and MT20, were evaluated using real-time quantitative-PCR and Sybr Green I chemistry in animals exposed to heavy metals in vivo and in primary cell cultures. This method was highly specific in detecting the expression of the two genes over a widely dynamic range of starting DNA amounts, showing that the basal level of MT expression is mostly due to MT10 mRNA. Basal MT expression reflected the intracellular concentration of heavy metal as indicated by the use of the heavy metal chelator TPEN on primary cells. MT10 was observed to be inducible by Cd, Zn, and Cu ions, and to a lesser extent by Hg. By contrast, the MT20 expression level was very low under basal conditions, while its mRNA increased dramatically in response to Cd exposure, and to a lesser extend to Hg, leading to levels of expression similar to those of the MT10 gene. The essential metals Cu and Zn had a very small effect on the MT20 gene, whereas the concomitant exposure to Cu and H(2)O(2) produced a rapid rise of expression. In summary, data indicate that the MT isogenes are differentially regulated by heavy metals, while hydroxyl radicals may have a role in MT20 gene activation. Also, protein expression showed metal inducibility only after Cd exposure, suggesting the occurrence of posttranscriptional control mechanisms.

Animals↗
PubMed1996

The influence of A-band and B-band lipopolysaccharide on the surface characteristics and adhesion of Pseudomonas aeruginosa to surfaces.

Pseudomonas aeruginosa PAO1 possesses two distinct lipopolysaccharide (LPS) O-polysaccharide species, A- and B-band LPS, the relative expression of which appears to be under environmental control. In an attempt to identify the influence these LPS types have on surface characteristics and adhesion, we examined the surface hydrophobicity and surface charge of P. aeruginosa PAO1 (O5 serotype) and its isogenic LPS derivatives which possessed A+B-, A-B+ and A-B- LPS. The surface characteristics of the strains affected their ability to adhere to hydrophilic (glass) and hydrophobic (polystyrene) surfaces. Cells possessing only A-band LPS demonstrated the highest surface hydrophobicity, followed by the strain lacking both A- and B-band LPS. The presence of B-band LPS resulted in a more hydrophilic surface. Strains lacking B-band LPS (A+B- and A-B-) had more electronegative surfaces than those possessing B-band LPS (A+B+ and A-B+), with cells lacking both A- and B-band LPS showing the highest surface electronegativity. These data suggest that the main surface-charge-determining groups reside in the core region of the LPS molecule. Cells with the lowest surface hydrophobicity and lowest surface charge (A+B+, A-B+) adhered to glass the most efficiently, implying a role for electrostatic interaction, whereas adhesion to polystyrene mirrored the relative hydrophobicities of the strains (A+B- > A-B- > A+B+ > A-B+). It is postulated that phenotypic variation in the relative expression of A- and B-band LPS may be a mechanism by which P. aeruginosa can alter its overall surface characteristics in such a way as to influence adhesion and favour survival.

Bacterial Adhesion↗
PubMedDate not supplied

Effect of cortical perforations of both graft and host bed on onlay incorporation to the rat skull.

The mechanisms involved in the integration of autogeneic bone grafts still attract much interest due to their clinical importance. The purpose of this study was to obtain data on the effects of a combination of cortical bone perforations both at the recipient site and the inner layer of a bicortical graft. 12 adult rats obtained femoral or tibial bicortical bone grafts from isogeneic donors to the tibia, one on each leg. On the experimental side, both the recipient bed and the inner cortical graft layer received multiple perforations (0.25 mm in diameter), while on the control side, only perforations of the recipient cortical bed were made. The findings were assessed by routine histology and immunohistochemical analysis for some bone and cartilage matrix proteins after 4 and 20 weeks. The combined cortical perforations of the graft and the host bed induced a locally improved bony incorporation of the graft and a corticalization of the graft marrow, implying improved mechanical stability. The graft height persistence was similar between groups. Intense labelling of the bone matrix proteins was apparent in all bone tissue and by diversified intensity at its various components, demonstrating ongoing remodeling activities. PRELP and fibromodulin mainly outlined the soft tissues surrounding the graft and compact bone sealing off the graft marrow. Immunolabelling contributed a more delicate picture of the mechanisms involved in graft incorporation.

Animals↗
PubMed1998

Endogenous superoxide dismutase levels regulate iron-dependent hydroxyl radical formation in Escherichia coli exposed to hydrogen peroxide.

Aerobic organisms contain antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, to protect them from both direct and indirect effects of reactive oxygen species, such as O2.- and H2O2. Previous work by others has shown that Escherichia coli mutants lacking SOD not only are more susceptible to DNA damage and killing by H2O2 but also contain larger pools of intracellular free iron. The present study investigated if SOD-deficient E. coli cells are exposed to increased levels of hydroxyl radical (.OH) as a consequence of the reaction of H2O2 with this increased iron pool. When the parental E. coli strain AB1157 was exposed to H2O2 in the presence of an alpha-(4-pyridyl-1-oxide)-N-tert-butyl-nitrone (4-POBN)-ethanol spin-trapping system, the 4-POBN-.CH(CH3)OH spin adduct was detectable by electron paramagnetic resonance (EPR) spectroscopy, indicating .OH production. When the isogenic E. coli mutant JI132, lacking both Fe- and Mn-containing SODs, was exposed to H2O2 in a similar manner, the magnitude of .OH spin trapped was significantly greater than with the control strain. Preincubation of the bacteria with the iron chelator deferoxamine markedly inhibited the magnitude of .OH spin trapped. Exogenous SOD failed to inhibit .OH formation, indicating the need for intracellular SOD. Redox-active iron, defined as EPR-detectable ascorbyl radical, was greater in the SOD-deficient strain than in the control strain. These studies (i) extend recent data from others demonstrating increased levels of iron in E. coli SOD mutants and (ii) support the hypothesis that a resulting increase in .OH formation generated by Fenton chemistry is responsible for the observed enhancement of DNA damage and the increased susceptibility to H2O2-mediated killing seen in these mutants lacking SOD.

Escherichia coli↗
PubMed2003

Acid adaptation and starvation effects on Shiga toxin production by Escherichia coli O157:H7.

Reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay (ELISA), and gene fusion were used to determine differences in the expression of stx-II mRNA and the production of mature Stx protein following acid adaptation or starvation of Escherichia coli O157:H7 (ATCC 43895) and an isogenic rpoS mutant (FRIK 816-3) under static conditions and with shaking. The expression of stx-II mRNA in acid-adapted and starved cells was more extensive than that in nonstressed control cells. This effect was more pronounced for the rpoS mutant. Oxygenation (incubation with shaking) increased stx-II mRNA expression for both strains relative to the level of expression obtained with static conditions. ELISA results indicated that Stx production was enhanced more in the rpoS mutant than in its wild-type parent strain and that oxygenation enhanced Stx production for both strains but there were no detectable differences between stressed and nonstressed cells of either strain. The monitoring of the gene product of Stx-II alone with the use of stx-IIAB::lacZ gene fusions confirmed the induction of aeration and the absence of a stress effect for both the wild type and the rpoS mutant. These results indicate that oxygen enhances stx-II mRNA expression and Stx production in E. coli O157:H7. Stress conditions such as acid adaptation and starvation enhance stx-II toxin mRNA levels but do not enhance subsequent Stx toxin production.

Adaptation, Physiological↗
PubMed1986

Relationship of success in classical immunotherapy to the relative immunorejective strength of the tumor.

Six tumors of varying immunorejective strengths were used to compare the response of their isogenic hosts to standardized regimens of immunoprophylaxis and immunotherapy. The tumors were generated spontaneously or induced chemically [with 9,10-dimethyl-1,2-benzanthracene (CAS: 57-97-6)], virally (with murine leukemia virus), or radiogenically (with strontium-90). The hosts were C57BL/6J or BALB/cByJ mice. Immunoprophylaxis and immunotherapy were performed with isogenic irradiated tumor cells, with Corynebacterium parvum, or with both. The results of challenge experiments were quantified as the doses of viable tumor cells that produced 50% tumor deaths for immunized and for control mice. The results for these quantitative "classical" immunoprophylaxis and immunotherapy experiments were consistent with two theses: that only immunorejective tumors give positive results with classical immunotherapy and that classical immunoprophylaxis is more effective than classical immunotherapy when identical materials are used for immunizations. These results have important consequences for the clinical use of classical immunotherapy.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1985

Numbers of metrial gland cells in the placental labyrinth of congenic resistant and inbred strains of mice, with observations on the size of the labyrinth.

The size of the placental labyrinth and the number of metrial gland cells in it were investigated, from the eleventh to the sixteenth day of gestation, in C57BL/10Sn, B10.A/SgSn, SWR/J and DBA/2J isogenic matings and C57BL/10Sn X B10.A/SgSn and B10.A/SgSn X C57BL/10Sn congenic matings. It was found that both H-2 disparity between mother and conceptus and some other strain-specific trait under genetic control influence the size of the placental labyrinth, while the number of labyrinthine metrial gland cells is affected only by the strain-specific trait.

Animal Husbandry↗
PubMed1993

Growth of costochondral fragments transplanted from mature to young isogeneic rats.

Experiments were carried out to determine whether growth of costochondral grafts is associated with age-related humoral factors. Rib fragments containing either short (without germinative zone) or long (with proliferative and germinative zones) cartilaginous ends were excised from adult rats, and the sections transplanted across the interparietal suture in 10-day-old isogeneic rats. The animals with short cartilage transplants were killed 15 or 25 days later; those with long cartilage transplants 25 days postoperatively. Measurements on dry skulls with short transplants showed neurocranial width to be greater in the experimental rats than in the unoperated controls at 25 days, whereas 10 days later the difference was no longer significant. By the latter time, however, the neurocranium was significantly wider in the long cartilage group than in the other groups. It is suggested that the declining growth potential of the costal cartilages from the adult rats was reactivated when transplanted to younger animals, i.e., the growth of costochondral transplants is related to humoral factors.

Age Factors↗
PubMed1993

The mlo resistance alleles to powdery mildew infection in barley trigger a developmentally controlled defence mimic phenotype.

Recessive mlo resistance alleles of the Mlo locus in barley control a non race-specific resistance response to infection by the obligate biotrophic fungus Erysiphe graminis f.sp. hordei. All the mlo alleles analysed stop fungal growth at the same developmental stage within a subcellularly restricted, highly localized cell wall apposition directly beneath the site of abortive fungal penetration. We report that near-isogenic lines carrying the alleles mlo1, mlo3 or mlo5 undergo dramatic spontaneous formation of cell wall appositions, not only in the absence of the fungal pathogen but also in sterile grown plants. A comparative study of spontaneous and infection-triggered cell wall appositions reveals a high degree of similarity with respect to structure, chemical composition and distinct localization within plant tissue. We show that the rate of spontaneous apposition formation is dependent on the genetic background of the plant and that its onset is under developmental control. Furthermore, spontaneous formation of wall appositions is specifically triggered by mlo alleles, since it is unaffected in the presence of the race-specific resistance allele Mlg. We propose a model for the function of the Mlo locus that suggests that both Mlo and mlo alleles control qualitatively the same apposition-based resistance mechanism, which, in the presence of the wild-type Mlo allele, is merely less efficient to provide protection against the currently common races of E. graminis f.sp. hordei.

Alleles↗
PubMed2005

Global analysis of cellular factors and responses involved in Pseudomonas aeruginosa resistance to arsenite.

The impact of arsenite [As(III)] on several levels of cellular metabolism and gene regulation was examined in Pseudomonas aeruginosa. P. aeruginosa isogenic mutants devoid of antioxidant enzymes or defective in various metabolic pathways, DNA repair systems, metal storage proteins, global regulators, or quorum sensing circuitry were examined for their sensitivity to As(III). Mutants lacking the As(III) translocator (ArsB), superoxide dismutase (SOD), catabolite repression control protein (Crc), or glutathione reductase (Gor) were more sensitive to As(III) than wild-type bacteria. The MICs of As(III) under aerobic conditions were 0.2, 0.3, 0.8, and 1.9 mM for arsB, sodA sodB, crc, and gor mutants, respectively, and were 1.5- to 13-fold less than the MIC for the wild-type strain. A two-dimensional gel/matrix-assisted laser desorption ionization-time of flight analysis of As(III)-treated wild-type bacteria showed significantly (>40-fold) increased levels of a heat shock protein (IbpA) and a putative allo-threonine aldolase (GlyI). Smaller increases (up to 3.1-fold) in expression were observed for acetyl-coenzyme A acetyltransferase (AtoB), a probable aldehyde dehydrogenase (KauB), ribosomal protein L25 (RplY), and the probable DNA-binding stress protein (PA0962). In contrast, decreased levels of a heme oxygenase (HemO/PigA) were found upon As(III) treatment. Isogenic mutants were successfully constructed for six of the eight genes encoding the aforementioned proteins. When treated with sublethal concentrations of As(III), each mutant revealed a marginal to significant lag period prior to resumption of apparent normal growth compared to that observed in the wild-type strain. Our results suggest that As(III) exposure results in an oxidative stress-like response in P. aeruginosa, although activities of classic oxidative stress enzymes are not increased. Instead, relief from As(III)-based oxidative stress is accomplished from the collective activities of ArsB, glutathione reductase, and the global regulator Crc. SOD appears to be involved, but its function may be in the protection of superoxide-sensitive sulfhydryl groups.

Arsenites↗
PubMed1995

Glial cell transplants that are subsequently rejected can be used to influence regeneration of glial cell environments in the CNS.

Transplantation of glial cells into demyelinating lesions in CNS offers an experimental approach which allows investigation of the complex interactions that occur between CNS glia, Schwann cells, and axons during remyelination and repair. Earlier studies have shown that 1) transplanted astrocytes are able to prevent Schwann cells from participating in CNS remyelination, but that they are only able to do so with the cooperation of cells of the oligodendrocyte lineage, and 2) transplanted mouse oligodendrocytes can remyelinate rat axons provided their rejection is controlled by immunosuppression. On the basis of these observations, we have been able to prevent the Schwann cell remyelination that normally follows ethidium bromide demyelination in the rat spinal cord by co-transplanting isogeneic astrocytes with a potentially rejectable population of mouse oligodendrocyte lineage cells. Since male mouse cells were used it was possible to demonstrate their presence in immunosuppressed recipients using a mouse Y-chromosome probe by in situ hydridisation. When myelinating mouse cells were rejected by removal of immunosuppression, the demyelinated axons were remyelinated by host oligodendrocytes rather than Schwann cells, whose entry was prevented by the persistence of the transplanted isogeneic astrocytes. The oligodendrocyte remyelination was extensive and rapid, indicating that the inflammation associated with cell rejection did not impede repair. If this host oligodendrocyte remyelination was prevented by local X-irradiation, the lesion consisted of demyelinated axons surrounded by processes from the transplanted astrocytes. By this approach, it was possible to create an environment which resembled the chronic plaques of multiple sclerosis. Thus, these experiments demonstrate that in appropriate circumstances the temporary presence of a population of glial cells can alter the outcome of damage to the CNS.

Animals↗
PubMed2000

Development of a noninvasive reporter system for gene expression in Porphyromonas gingivalis.

Several reports have supported the association of Porphyromonas gingivalis with periodontal disease. Genetic studies are vital for understanding the relative importance of virulence factors in this organism. Thus, gene reporters may prove useful for the study of gene expression in this organism. We have investigated the use of the green fluorescent protein (GFP), bacterial luciferase, and bifunctional xylosidase/arabinosidase enzyme (XA) as reporters of gene expression in P. gingivalis. Fusion cassettes containing the promoterless tetracycline resistant gene [tetA(A)Q2] and the promoterless gfp, luxAB, or xa gene were placed under the control of the rgpA promoter in P. gingivalis W83 using recombinational allelic exchange. The rgpA gene encodes for an arginine-specific protease in P. gingivalis. No GFP activity was detected in P. gingivalis isogenic mutants carrying the rgpA::gfp-tetA(Q)2 fusion construct. Luciferase activity in P. gingivalis mutants carrying the rgpA::luxAB-tetA(Q)2 fusion was only detected in the presence of exogenous FMNH(2). xa gene expression in P. gingivalis with the rgpA::xa-tetA(Q)2 fusion construct was detected in crude extracts using rho-nitrophenol derivatives as substrate and on agar plates with methylumbelliferyl derivatives under long-wave ultraviolet light. This indicates that both luxAB and xa genes can be used as reporters of gene expression in P. gingivalis. However, only the xa gene can be used as a noninvasive reporter gene.

Artificial Gene Fusion↗
PubMed2002

CENP-B is not critical for meiotic chromosome segregation in male mice.

Centromere protein B (CENP-B) is a constitutive protein that binds to a highly conserved 17bp motif located at most mammalian centromeres. To determine whether disruption of this gene affects chromosome segregation in male germ cells, we evaluated the frequencies of disomic and diploid sperm in CENP-B heterozygous and homozygous null mice using the mouse epididymal sperm aneuploidy (m-ESA) assay, a multicolor FISH method with probes for chromosomes X, Y and 8. The specificity and sensitivity of the m-ESA assay was demonstrated using Robertsonian (2.8) translocation heterozygotes as positive controls for sperm aneuploidy. Our results show that the frequencies of disomic and diploid sperm did not differ significantly between CENP-B heterozygous and homozygous null mice (P> or = 0.5) or from 129/Swiss isogenic mice (P> or = 0.5) and B6C3F1 mice (P> or = 0.2). These findings indicate that CENP-B does not have an essential role during chromosome segregation in male meiosis.

Aneuploidy↗
PubMed2003

Attenuated virulence of Streptococcus agalactiae deficient in D-alanyl-lipoteichoic acid is due to an increased susceptibility to defensins and phagocytic cells.

D-alanylation of lipoteichoic acid (LTA), allows Gram-positive bacteria to modulate their surface charge, regulate ligand binding and control the electromechanical properties of the cell wall. In this study, the role of D-alanyl LTA in the virulence of the extracellular pathogen Streptococcus agalactiae was investigated. We demonstrate that a DltA- isogenic mutant displays an increased susceptibility to host defence peptides such as human defensins and animal-derived cationic peptides. Accordingly, the mutant strain is more susceptible to killing by mice bone marrow-derived macrophages and human neutrophils than the wild-type strain. In addition, the virulence of the DltA- mutant is severely impaired in mouse and neonatal rat models. This mutant was eliminated more rapidly than the wild-type strain from the lung of three-week-old mice inoculated intranasally and, consequently, is unable to induce a pneumonia. Finally, after intravenous injection of three-week-old mice, the survival of the DltA- mutant is markedly reduced in the blood in comparison to that of the wild-type strain. We hypothesize that the decreased virulence of the DltA- mutant is a consequence of its increased susceptibility to cationic antimicrobial peptides and to killing by phagocytes. These results demonstrate that the D-alanylation of LTA contributes to the virulence of S. agalactiae.

Animals↗
PubMed2005

The rb7 matrix attachment region increases the likelihood and magnitude of transgene expression in tobacco cells: a flow cytometric study.

Many studies in both plant and animal systems have shown that matrix attachment regions (MARs) can increase expression of transgenes in whole organisms or cells in culture. Because histochemical assays often indicate variegated transgene expression, a question arises: Do MARs increase transgene expression by increasing the percentage of cells expressing the transgene (likelihood), by increasing the level of expression in expressing cells (magnitude), or both? To address this question, we used flow cytometry to measure green fluorescent protein (GFP) expression in individual tobacco (Nicotiana tabacum) cells from lines transformed by Agrobacterium tumefaciens. We conclude that MAR-mediated overall increases in transgene expression involve both likelihood and magnitude. On average, cell lines transformed with the Rb7 MAR-containing vector expressed GFP at levels 2.0- to 3.7-fold higher than controls. MAR lines had fewer nonexpressing cells than control lines (10% versus 45%), and the magnitude of GFP expression in expressing cells was greater in MAR lines by 1.9- to 2.9-fold. We also show that flow cytometry measurements on cells from isogenic lines are consistent with those from populations of independently transformed cell lines. By obviating the need to establish isogenic lines, this use of flow cytometry could greatly simplify the evaluation of MARs or other sequence elements that affect transgene expression.

Agrobacterium tumefaciens↗
PubMed2001

[Implantation of skeletal muscle stem cells in inhibition of fibroatrophy of ischemic myocardium: an experimental study].

OBJECTIVE: To investigate the effect of stem cells derived from autogenous skeletal muscle, namely satellite cells, and implanted into ischemic myocardium on inhibition of myocardium fibroatrophy. METHODS: The left anterodecendant arteries (LAD) of 12 adult dogs were ligated so as to establish animal model of acute myocardiac infarction. Satellite cells isolated from the greatest gluteal muscle of dogs were labeled with 4' 6-dimidino-2-phenylindone (DAPI), and then infused into the ventricular myocardium of the isogenic dogs through LAD. Specimens of ischemic myocardium were taken 2, 4, and 8 weeks after myoblast implantation. Histologic sections are examined under common light microscope and fluorescent microscope. Twelve dogs were used as controls. RESULTS: In the ischemic myocardium where satellite cells had been implanted, fibroaastrophy was efeectively inhibited. The implanted satellite cells had differentiated into fully developed striated muscle cells. Vitreous degeneration and disorder of basic structure could be observed in the ischemic myocardium in the control group. CONCLUSION: The satellite cells from autologous skeletal muscle differentiate into cardiac muscle cell-like cells in the ischemic area and inhibit fibroatrophy of the ischemic myocardium after implantation, thus bringing a hope of a new cure for myocardial damage.

Animals↗
PubMed1994

Repair of osteochondral defects with grafts of cultured chondrocytes. Comparison of allografts and isografts.

Isogeneic and allogeneic chondrocytes cultured in collagen gel were transplanted into osteochondral defects in knee joints of inbred strains of rats. At 12 weeks, all eight isografted defects had healed successfully, compared with only four of eight allografted defects (p < 0.05). At 26 and 52 weeks, all defects except one had healed successfully and there was no significant difference in the success rate between the isografted and allografted groups. Control defects that had been implanted with only collagen gel did not heal successfully.

Animals↗