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PubMed1981

Subcutaneous, isogeneic transplantation of duct-ligated pancreas in streptozotocin-diabetic mice. Relationships between carbohydrate tolerance and hormone content in transplant or host pancreas.

Mice with subcutaneous, isogeneic transplants of duct-ligated pancreas from either two or five donors displayed impaired glucose tolerance to gastric or intravenous glucose, or to an overnight fast followed by a 15-min meal of mouse food. Compared with peripheral insulin responses in normal controls, those of transplant recipients were less after gastric or intravenous glucose, but no different after the meal. Despite normalization of peak insulin levels in the peripheral circulation of isografted mice, glucose clearance was still impaired and this probably resulted, in part, from a relative insufficiency of insulin in the portal circulation. Results of glucose tolerance tests, after transplant recipients consumed a relatively small amount of food, suggested that near-normal glucose homeostasis may be present for these mice under normal feeding conditions. In mice that received incremental doses of streptozotocin, impaired glucose tolerance to a meal was observed if pancreatic insulin content fell below 26% of normal. We failed to show a similar relationship between glucose tolerance and pancreatic insulin content in transplant recipients since all showed impaired glucose tolerance. Despite the initial transplantation of different amounts of islet tissue, at termination insulin content (transplant plus endogenous pancreas) was essentially the same for all recipients, totaling 19-22% of that found in a normal mouse pancreas.

Animals↗

PubMed1992

Characterization of the regulon controlled by the leucine-responsive regulatory protein in Escherichia coli.

The leucine-responsive regulatory protein (Lrp) has been shown to regulate, either positively or negatively, the transcription of several Escherichia coli genes in response to leucine. We have used two-dimensional gel electrophoresis to analyze the patterns of polypeptide expression in isogenic lrp+ and lrp mutant strains in the presence or absence of leucine. The absence of a functional Lrp protein alters the expression of at least 30 polypeptides. The expression of the majority of these polypeptides is not affected by the presence or absence of 10 mM exogenous leucine. Outer membrane porins OmpC and OmpF, glutamine synthetase (GlnA), the small subunit of glutamate synthase (GltD), lysyl-tRNA synthetase form II (LysU), a high-affinity periplasmic binding protein specific for branched-chain amino acids (LivJ), W protein, and the enzymes of the pathway converting threonine to glycine, namely, threonine dehydrogenase (Tdh) and 2-amino-3-ketobutyrate coenzyme A ligase (Kbl), were identified as members of the Lrp regulon by electrophoretic analysis. We have shown that Lrp is a positive regulator of glutamate synthase and glutamine synthetase and that exogenous leucine has little or no effect on the expression of these proteins. In strains carrying a glnL deletion and in strains carrying the glnL2302 allele, which directs the synthesis of a GlnL protein that is constitutively active, expression of glutamine synthetase is no longer regulated by Lrp, demonstrating that the effect of Lrp on glutamine synthetase levels is indirect and requires an intact glnL gene. lrp::Tn10 strains grow poorly when arginine or ornithine is present as the sole nitrogen source in the medium. On the bases of present studies and previous research, we propose that Lrp is involved in the adaptation of E. coli cells to major shifts in environment, such as those which occur when E. coli leaves the intestinal tract of its animal host. Several genes required for amino acid and peptide transport and catabolism are negatively regulated by Lrp, and other genes required for amino acid biosynthesis and ammonia assimilation in a nitrogen-poor environment are positively regulated by Lrp.

Amino Acids↗

PubMed1982

Morphogenetic potential of rat growth cartilages as isogeneic transplants in the interparietal suture area.

The proximal end of the tibia or the spheno-occipital synchondrosis with some adjoining bone were isogeneically transplanted across the interparietal suture of 10-day-old rats. As a sham procedure, a piece of calvarium traversed by a part of the interparietal suture was interchanged between pairs of animals. Untreated rats served as controls. The animals, injected with Alizarin red S, were killed 25-75 days after the operation, 10 in each group. The transverse dimensions of the neurocranium were larger in the rats with the cartilage transplants than in the controls, particularly at 25 days after the operation; the differences persisted longer in the animals with the synchondroseal transplant. The orientation of the bone interdigitation at the anterior lambdoidal suture changed temporarily in response to the excessive lateral displacement of the parietal bones. The observations indicate that basicranial synchondroses, like epiphyseal cartilage, are endowed with a tissue-separating property that may persist for a prolonged period under transplant conditions. As such cartilaginous structures may also be affected by environmental influences, there must be a two-way interaction between the synchondroses and their immediate environment.

Animals↗

PubMed2001

The Saccharomyces cerevisiae RAD9 cell cycle checkpoint gene is required for optimal repair of UV-induced pyrimidine dimers in both G(1) and G(2)/M phases of the cell cycle.

Cells respond to DNA damage by activating both cellular growth arrest and DNA repair processes. In Saccharomyces cerevesiae the RAD9 gene controls DNA damage-mediated cell cycle arrest that is known to allow efficient repair. To ascertain whether RAD9 plays a role in DNA repair per se, the removal of UV-induced photolesions was assessed in synchronized isogenic normal and rad9 cells using the high resolution primer extension technique. The results show that RAD9 is indeed involved in the removal of photolesions from both the transcribed and the non-transcribed strands of the reporter GAL10 gene, in G(1)- as well as G(2)/M-arrested cells. Interestingly, these data also reveal that in both normal and rad9 mutant, the repair strand bias towards the transcribed stand is more pronounced in G(2)/M- than in G(1)-arrested cells. These data indicate that RAD9 coordinate the cellular response to DNA damage by activating both cell cycle checkpoint and excision repair.

Cell Cycle↗

PubMed2006

Characterization of a Kluyveromyces lactis mutant with altered regulation of mitochondrial alcohol dehydrogenases.

KlADH3 and KlADH4 are Kluyveromyces lactis genes encoding the two mitochondrial alcohol dehydrogenase activities located within mitochondria. In this yeast, ethanol induces the transcription of KlADH4 and, conversely, represses that of KlADH3. In this study, we describe the effects of the aar900 mutation on such regulation. This mutation, firstly isolated in a strain devoid of alcohol dehydrogenase genes except KlADH4, conferred to cells resistance to allyl alcohol because of the absence of the KlAdh4p activity. When the mutation was transferred by crosses to an isogenic strain containing all the alcohol dehydrogenase genes, we found that the KlADH3 gene was highly expressed even in the presence of ethanol. In addition, we observed that the absence of KlAdh4p resulted from a post-transcriptional control in that KlADH4 was transcriptionally induced by ethanol. We also found that KlPDC1, another ethanol-repressible gene, was not transcribed in the mutant in the presence of this carbon source, indicating that the escape of KlADH3 from ethanol repression was a peculiar feature of this gene. Genetic analysis showed a Mendelian segregation of the mutation that was mapped in a region of chromosome III close to the ade1 locus. Interestingly, the aar900 mutants had a pleiotropic phenotype and showed increased resistance to monovalent cations and benomyl, suggesting that the mutation could also affect genes other than the alcohol dehydrogenase ones. Strains carrying the aar900 mutation could represent useful tools to unravel the peculiar regulation of KlADH3 and KlADH4 by ethanol.

Alcohol Dehydrogenase↗

PubMed1982

B cell differentiation antigens as probes for functional B cell subsets.

In this review I have discussed the serological, biochemical and functional characterization of two differentiation antigens, Lyb3 and Ia. W39, which have the same time distribution; namely, they are selectively expressed on a late maturing subset of B cells (Lyb3 and Ia. W39) and antigen presenting macrophages (Ia. W39, Lyb3?) Antisera against both determinants were raised in xid defective F1 male mice, which were immunized with spleen cells from the normal parent. Lyb3 is an isogenic specificity expressed without allelic forms in all mouse strains, whereas Ia.W39 is a private specificity, encoded by a gene(s) within the I-Ab subregion of the H-2 complex. Interestingly, the xid gene does not control the synthesis of these differentiation antigens, but affects their membrane expression (shown for Ia. W39.) Lyb3 is a polypeptide of 68,000d MW which has a similar IE point in all mouse strains. The molecule bearing Ia. W39 has an identical 2-chain structure (a and beta) and 2-D gel profile as the molecule expressing all the conventional Ia specificities encoded by the I-Ab subregion. However, from the difference in the ontological appearance and the turnover rate and from sequential immunoprecipitation studies we concluded that there are two kinds of glycoproteins containing Aa and Abeta chains; both would express the conventional specificities, and one would, in addition, bear Ia. W39. Functionally, we have defined Lyb3 as a receptor for triggering signals and Ia. W39 as a specific Ir gene epitope.

Animals↗

PubMedDate not supplied

[Effects of H2 blockers on the spreading of macrophages in vitro].

PURPOSE: To verify if cimetidine, ranitidine, and famotidine, when inoculated by ip route in mice, do enhance macrophage activation and whether or not such activation is altered with prior use of sodium thioglycolate. KIND OF STUDY: Experimental. ANIMALS: isogenic, Balb/c mice, 19-21 g. INTERVENTION: The authors used 8 groups of 10 animals. The mice were treated by ip route with cimetidine (100 mg/kg), ranitidine (62.5 mg/kg), and famotidine (50 mg/kg), and were compared to a control group (saline). The method of spreading of macrophages was applied twenty-four hours after the inoculation. The same procedure was repeated on a later stage in groups submitted to a prior treatment with sodium thioglycolate (15 mg/kg). EVALUATION: Kruskal-Wallis and Mann-Whitney's tests were used. RESULTS: Inoculation of anti-H2 drugs in the peritoneal cavity of mice significantly enhances the spreading of macrophages independently of the presence of a peritoneal irritant.

Animals↗

PubMed1991

Heat shock factor-independent heat control of transcription of the CTT1 gene encoding the cytosolic catalase T of Saccharomyces cerevisiae.

Transcription of the Saccharomyces cerevisiae CTT1 gene encoding the cytosolic catalase T has been previously shown to be derepressed by nutrient stress. To investigate whether expression of this gene is also affected by other types of stress, the influence of heat shock on CTT1 expression was studied. The results obtained show that expression of the gene is low at 23 degrees C and is induced rapidly at 37 degrees C. By deletion analysis, a promoter element necessary for high level induction by heat shock was located between base pairs -340 and -364 upstream of the translation start codon. This region was demonstrated to be sufficient for heat shock control by placing it upstream of a S. cerevisiae LEU2-lacZ fusion gene. Mutagenesis of the region showed that the response to heat shock is not mediated by a sequence similar to canonical heat shock elements, but by DNA elements also involved in nutrient control of transcription. Catalase T appears to have a function in protecting yeast cells against oxidative damage under stress conditions. Catalase T-containing strains are less sensitive to exposure to 50 degrees C ("lethal heat shock") than isogenic catalase T-deficient mutants, and catalase T-containing strains pretreated by incubation at 37 degrees C are less sensitive to H2O2 than pretreated catalase-deficient mutants.

Base Sequence↗

PubMed1994

Ecological Similarity and Coexistence of Epiphytic Ice-Nucleating (Ice) Pseudomonas syringae Strains and a Non-Ice-Nucleating (Ice) Biological Control Agent.

De Wit replacement series were used to study competitive interactions between epiphytic IcePseudomonas syringae strains and the biological frost control agents IceP. syringae TLP2del1 and Pseudomonas fluorescens A506. Mixtures containing two strains in different proportions but at a constant total population size were inoculated onto potato leaves. The population sizes of each strain and the total population size were determined when the community had reached equilibrium. A near-isogenic P. syringae strain pair exhibited an interaction similar to that expected for strains competing equally for limiting environmental resources. Replacement series with nonisogenic Ice and IceP. syringae strain pairs suggested that these strains competed for limiting resources according to their relative competitive abilities. There was no evidence of any niche differentiation between the IceP. syringae strains and the IceP. syringae strain. The growth responses of epiphytes following addition of nutrients to the phyllosphere indicated that the epiphytic P. syringae populations were nutrient limited and that, under growth chamber conditions, the populations were more limited by the availability of carbon than by the availability of nitrogen. Determination of in vitro carbon source utilization profiles provided further evidence for the lack of niche differentiation between the Ice and the IceP. syringae strains. Niche overlap indices calculated for the IceP. syringae strains with respect to IceP. syringae TLP2del1 were uniformly high, indicating ecological similarity, and were consistent with the observed low level of coexistence. The biological frost control agent P. fluorescens A506 replaced P. syringae. This was correlated with a high degree of niche overlap between these species.

Journal Article↗

PubMed1999

Repair of DNA double-strand breaks and radiosensitivity to killing in an isogenic group of p53 mutant cell lines.

PURPOSE: Accumulation of the p53 protein can result in G1 arrest that may facilitate DNA repair, or alternatively, it may lead to apoptosis. Mutations that alter p53's ability to mediate these responses are expected to alter cell radiosensitivity to killing. However, the relationship between p53 status and cell radiosensitivity has proven to be complex. Several studies have suggested that p53 mutations are associated with increased radioresistance to killing, while others have shown no such correlation. These differences may be derived from the fact that different mutations of p53 exert different effects on cell radiosensitivity. METHODS AND MATERIALS: To address this question, we examined a group of isogenic cell lines that express different "hot spot" mutant forms of p53. These cells were generated from human osteosarcoma (SAOS) cells, a p53 null cell line, by transfection with vectors expressing different p53 mutants. Vectors with the following p53 mutations were utilized: 143Ala, 175His, 248Try, 273His, and 281Gly. As controls, we used the original SAOS cells and cells transfected with the vector alone. Results were compared to those obtained with a cell line expressing wild-type p53 (wt p53). Radiosensitivity to killing was determined in the exponential phase of growth by measuring loss of colony-forming ability. Induction and repair of DNA double-strand breaks (dsb) was measured in irradiated cells using pulsed-field gel electrophoresis. Apoptosis was assessed using morphologic evaluation of DAPI-stained cells after treatment either with radiation or paclitaxel. RESULTS: Transfected SAOS-2 cell lines expressed a mutant form of p53 that could not be induced by radiation, and which was transcriptionally inactive. Among the 7 cell lines studied, we observed no difference in cellular radiosensitivity to killing (p = NS). When examining DNA repair, no difference in either the induction or repair of DNA dsb was noted in any of the cell lines studied (p = NS). Also, induction of apoptosis, either after exposure to radiation or paclitaxel, was low, and similar in all cell lines (p = NS). Non-isogenic cells expressing wt p53 were more radioresistant to killing by radiation, but showed similar kinetics of dsb rejoining. CONCLUSION: The results suggest that expression of different p53 mutants does not alter the yields of radiation-induced dsb, or the ability of cells to repair this type of lesion. In addition, the same p53 mutants do not affect cellular radiosensitivity to killing, or the induction of apoptosis after exposure to radiation or paclitaxel.

Apoptosis↗

PubMed1996

Trehalose-6-phosphate synthase A affects citrate accumulation by Aspergillus niger under conditions of high glycolytic flux.

Accumulation of citric acid by Aspergillus niger depends on a high flux through glycolysis. We have investigated the possibility of control of this flux by trehalose 6-phosphate, an inhibitor of hexokinase of Saccharomyces cerevisiae and other eukaryotes (Blasquez et al., FEBS Lett. (1993) 329, 51-54). Hexokinase of A. niger was shown in vitro to be only weakly inhibited by trehalose 6-phosphate (KI 1.5-2 mM). To investigate the in vivo relevance of this inhibition, we used isogenic strains of A. niger, carrying either a disruption or an amplification of the trehalose-6-phosphate synthase A (T6PSA)-encoding gene (ggsA) and exhibiting corresponding differences in T6PSA activity. These strains produced citric acid at comparable rates and with similar yields on 1 or 2.5% (w/v) sucrose. At 5-14% (w/v) sucrose, the ggsA disrupted strain initiated citric acid accumulation earlier, whereas the multicopy strain showed the reverse effect. When sucrose was replaced by lactose, which enabled only low rates of catabolism irrespective of its concentration (1-8%), no differences in the initiation or rate of citric acid accumulation were observed between the three strains. The possible mechanisms by which ggsA controls glycolytic flux in A. niger in the presence of high sugar concentrations are discussed.

Aspergillus niger↗

PubMed1998

Genetic localization of a bruchid resistance gene and its relationship to insecticidal cyclopeptide alkaloids, the vignatic acids, in mungbean (Vigna radiata L. Wilczek).

Bruchid resistance, controlled by a single dominant gene (Br) in a wild mungbean accession (TC1966), has been incorporated into cultivated mungbean (Vigna radiata). The resistance gene simultaneously confers inhibitory activity against the bean bug, Riptortus clavatus Thunberg (Hemiptera: Alydidae). The resultant isogenic line (BC20 generation) was characterized by the presence of a group of novel cyclopeptide alkaloids, called vignatic acids. A linkage map was constructed for Br and the vignatic acid gene (Va) using restriction fragment length polymorphism (RFLP) markers and a segregating BC20F2 population. By screening resistant and susceptible parental lines with 479 primers, eight randomly amplified polymorphic DNA (RAPD) markers linked to Br were identified and cloned for use as RFLP probes. All eight RAPD-based markers, one mungbean, and four common bean genomic clones were effectively integrated around Br within a 3.7-cM interval. Br was mapped to a 0.7-cM segment between a cluster consisting of six markers and a common bean RFLP marker, Bng110. The six markers are closest to the bruchid resistance gene, approximately 0.2 cM away. The vignatic acid gene, Va, cosegregated with bruchid resistance. However, one individual was identified in the BC20F2 population that retained vignatic acids in spite of its bruchid susceptibility. Consequently, Va was mapped to a single locus at the same position as the cluster of markers and 0.2 cM away from Br. These results suggest that the vignatic acids are not the principal factors responsible for bruchid resistance in V. radiata but will facilitate the use of map-based cloning strategies to isolate the Br gene.

Alkaloids↗

PubMed2005

Inhibition of nuclear factor kappaB by IkappaB superrepressor gene transfer ameliorates ischemia-reperfusion injury after experimental lung transplantation.

OBJECTIVES: Ischemia-reperfusion injury after lung transplantation is associated with significant morbidity and mortality. The activation of the transcription factor nuclear factor kappaB is central to the 2 important pathways that characterize ischemia-reperfusion injury, namely the inflammatory response and apoptosis. The purpose of this study was to determine the effects of nuclear factor kappaB inhibition on experimental lung transplant ischemia-reperfusion injury with gene transfer of the nuclear factor kappaB inhibitor IkappaB in a superrepressor form (IkappaBSR). METHODS: An orthotopic left lung transplant model in isogeneic rats was used, with 18 hours of prolonged cold storage of donor lung grafts used to create severe ischemia-reperfusion injury. Donor rats underwent endobronchial gene transfection with saline alone or adenovirus encoding either beta-galactosidase control or IkappaBSR 48 hours before harvest. The function of transplanted lung grafts was assessed on the basis of isolated graft oxygenation, wet/dry lung weight ratio, and myeloperoxidase activity. Nuclear factor kappaB activation was assessed by means of enzyme-linked immunosorbent assay. Apoptotic cell death was assessed by evaluating the levels of histone-associated DNA fragments and caspase-3 activity. RESULTS: Treatment of donor lung grafts with IkappaBSR resulted in significantly improved oxygenation compared with that seen in control tissue 24 hours after transplantation. IkappaBSR-treated lungs also demonstrated less pulmonary edema and reduced neutrophil infiltration 24 hours after reperfusion. Nuclear factor kappaB activation and apoptotic cell death induction 2 hours after transplantation was significantly reduced in IkappaBSR-treated lungs compared with in control lungs. CONCLUSIONS: Inhibition of nuclear factor kappaB activation by IkappaBSR gene transfer improves transplanted lung graft oxygenation, decreases pulmonary edema and neutrophil sequestration, and reduces apoptotic cell death after experimental lung transplantation.

Adenoviridae↗

PubMedDate not supplied

Electrophysiological characterization of the Cyclophilin D-deleted mitochondrial permeability transition pore.

Mitochondria isolated from engineered mice lacking Cyclophilin D (CypD), a component of the Permeability Transition Pore (PTP) complex, can still undergo a Ca2+ -dependent but Cyclosporin A-insensitive permeabilization of the inner membrane. Higher Ca2+ concentrations are required than for wild-type controls. The characteristics of the pore formed in this system were not known, and it has been proposed that they might differ substantially from those of the normal PTP. To test this hypothesis, we have characterized the PTP of isogenic wild-type and CypD- mouse liver mitochondria in patch clamp experiments, which allow biophysical characterization. The pores observed in the two cases, very similar to those of rat liver mitochondria, are indistinguishable according to a number of criteria. The only clear difference is in their sensitivity to Cyclosporin A. CypD is thus shown to be an auxiliary, modulatory component of the "standard" PTP, which forms and has essentially the same properties even in its absence. The observations suggest that Ca2+, CypD, and presumably other inducers and inhibitors act at the level of an activation or assembly process. Activation is separate and upstream of the gating observable on a short or medium-term time scale. Once the pore is activated, its molecular dynamics and biophysical properties may thus be predicted not to depend on the details of the induction process.

Animals↗

PubMed1999

Influence of in vitro susceptibility phenotype against thrombin-induced platelet microbicidal protein on treatment and prophylaxis outcomes of experimental Staphylococcus aureus endocarditis.

Thrombin-induced platelet microbicidal protein-1 (tPMP-1) is a small, cationic staphylocidal peptide from rabbit platelets. In the current study, the outcomes of vancomycin treatment and prophylaxis were compared in experimental infective endocarditis (IE) caused by an isogenic Staphylococcus aureus strain pair differing in tPMP-1 susceptibility (tPMPS) or resistance (tPMPR) in vitro (ISP479C and ISP479R, respectively). Vancomycin therapy (selected for its intrinsically slow bactericidal activity) reduced ISP479C (but not ISP479R) densities in vegetations compared with controls (P<.01). In contrast, prophylactic administration of vancomycin yielded no differences in efficacies for the 2 challenge strains. These data suggest that the tPMPR phenotype in vitro has a negative effect on the antimicrobial therapy (but not the prophylaxis) of experimental S. aureus IE. These disparate results may be explained in part by the requirement for microbicidal effects in the treatment of established IE, whereas prophylactic efficacy depends more on growth inhibitory and antiadhesion effects.

Animals↗

PubMed1986

Immune reactivity in congenic mice after allogeneic or isogeneic transfusion.

Pretransplant donor-specific blood transfusion has a beneficial effect on subsequent skin graft survival in B10 congenic mice when donor and recipient differ at both the K and I regions of the H-2 complex, but has no effect on graft survival in donor-recipient combinations differing at the K region alone. Immune reactivity was studied in B10.A recipients after a single allogeneic transfusion from B10 donors differing either at both K and I regions or at K alone, or after a transfusion of isogeneic B10.A blood. Spleen cells were assayed for the ability to respond in mixed lymphocyte culture (MLC) and cell mediated lymphocytotoxicity (CML) assays against blood donor and third-party at 2, 9, 16, and 30 days after transfusion. MLC and CML responses were nonspecifically suppressed in recipients transfused with allogeneic blood from K or K and I region disparate donors. The responses of mice receiving K region disparate blood were partially recovered by 9 days posttransfusion and had returned to normal 16 days posttransfusion. Responses of mice receiving K and I region disparate blood remained suppressed 30 days post transfusion. Isogeneic transfusion induced a suppression of the MLC response. Two days posttransfusion, the MLC response was suppressed to a variety of stimulators representing disparity at K alone; K and I; or K, I, and D. The response to K region disparate stimulators had recovered 9 days after isogeneic transfusion, but the response to K and I or K, I, and D region disparate stimulators remained suppressed 30 days after transfusion. The suppression observed after allogeneic or isogeneic transfusion was not due to a shift in the day of peak response in the transfused mice. Kinetic studies showed that the day of peak MLC and CML responses in the transfused mice corresponded to that of normal controls, but the level of response was significantly lower in the transfused mice.

Animals↗

PubMed1997

Complement activation as a cause for primary graft failure in an isogeneic rat model of hypothermic lung preservation and transplantation.

Although agents that inhibit complement activation may be beneficial in discordant xenotransplantation, it is not known whether local complement activation occurs and is deleterious after isogeneic lung transplantation. Lungs were harvested from Lewis rats subjected to 4 degrees C 6-hr preservation followed by transplantation into strain-, gender-, and weight-matched recipients. Transplanted lungs demonstrated increased immunostaining for C5b-9 compared with nontransplanted controls, confirming local complement activation in this isograft model. To investigate the physiologic relevance of complement activation in the transplanted lung, the native pulmonary artery was ligated after transplantation, and pulmonary vascular resistance (mmHg/ml/min), arterial oxygenation (mmHg), graft neutrophil infiltration (myeloperoxidase activity, deltaAbs 460 nm/min), and recipient survival were measured at 30 min. Animals received either saline (control; n=22) or soluble complement receptor type-1 (sCR1, 15 mg/kg; n=19) 2 min before reperfusion. Animals treated with sCR1 showed a marked reduction in serum complement hemolytic activity (CH50; 90% lower than that of control animals, P<0.001). Compared with controls, sCR1-treated animals showed reduced pulmonary vascular resistance (2.9+/-1.1 vs. 8.5+/-1.5 mmHg/ml/min, P<0.05), improved arterial oxygenation (194+/-34 vs. 91+/-17 mmHg, P<0.05), decreased neutrophil infiltration (35% decrease, P<0.005), and improved recipient survival (74% vs. 23%, P<0.005). In parallel with the reduction in complement hemolytic activity in sCR1-treated animals, immunohistology of the transplanted lung revealed decreased C5b-9 deposition compared with controls. Taken together, these data indicate that complement activation occurs after lung preservation and transplantation in an isograft model, and that inhibiting complement activation improves outcome after transplantation.

Animals↗

PubMed1996

Role of the Bordetella pertussis P.69/pertactin protein and the P.69/pertactin RGD motif in the adherence to and invasion of mammalian cells.

The role of the Bordetella pertussis P.69/pertactin protein in mammalian cell adhesion and invasion was investigated. Salmonella strains expressing surface-associated P.69/pertactin from a chromosomally located prn gene were significantly more invasive than isogenic parental strains. This effect was most pronounced in the poorly invasive, semi-rough S. typhimurium strain LB5010. Escherichia coli K-12 strain HB101 harbouring the plasmid p41869D, which encodes the full-length prn gene under the control of the tac promoter on the broad-host-range plasmid pMMB66EH, was significantly more adhesive to HEp-2 and Chinese Hamster Ovary (CHO) cells growing in culture than E. coli HB101(pMMB66EH). However, the ability of E. coli to invade mammalian cells was not affected by P.69/pertactin expression. P.69/pertactin-mediated adhesiveness of HB101 to HEp-2 and CHO cells was not influenced by the viability of the bacterial cells. However, adherence was markedly reduced when assays were performed for less than 3 h, at 4 degrees C or in the presence of cycloheximide, suggesting the active participation of the eukaryotic cell in bacterial adhesion. Site-directed mutagenesis was used to mutate Asp to Glu in an Arg-Gly-Asp (RGD-->RGE) sequence present in mature P.69/pertactin and the mutated gene was cloned in the same broad-host-range vector (plasmid p41869E). This mutation had no detectable influence on the ability of P.69/pertactin to mediate adhesion of HB101 to HEp-2 or CHO cells. Plasmids p41869D and p41869E were introduced into the bvg-negative B. pertussis strain BP347. Expression of P.69RGD or P.69RGE did not enhance the adhesiveness of BP347 for epithelial (HEp-2 and CHO) cells.

Animals↗