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PubMed1994

Pseudomonas aeruginosa cytotoxin as a pathogenicity factor in a systemic infection of leukopenic mice.

The effect of Pseudomonas aeruginosa cytotoxin was assessed in leukopenic outbred Swiss male mice (20 g) using a high cytotoxin-producing strain (PA158) and its cytotoxin-deficient isogenic mutant (PA114F5) generated by Tn7::Tn5 transposon mutagenesis of PA158. Leukopenia was induced by intraperitoneal (i.p.) administration of cyclophosphamide (150 micrograms/g). Anesthetized mice were infected via a 4 mm incision on the shaved back with 300 CFU/mouse (9 LD50; expected death rate 85%). Precleared mouse cytotoxin-specific heat inactivated rabbit polyclonal antibody (RPA) was administered i.p. (0.2 ml) 24 hr before challenge. Controls received i.p. normal rabbit serum, RPA, cyclophosphamide alone, or a sham procedure. Challenge with the high cytotoxin-producing strain PA158 caused earlier and a significantly greater mortality than that observed with a cytotoxin-deficient strain PA114F5 (P < 0.01). Cytotoxin-specific polyclonal antibody was protective. Pretreatment with antibody decreased the mortality rate following challenge with PA158 from 88.9% to 27.8% (P < 0.01). Pretreatment with antibody decreased the mortality rate following challenge with PA114F5 from 27.8% to 5.6% (P < 0.05). These results demonstrate that P. aeruginosa cytotoxin contributes to the pathogenicity of the organism and that cytotoxin antibody is protective in a systemic P. aeruginosa infection in leukopenic mice.

Analysis of Variance↗

PubMed2003

Transcoronary implantation of bone marrow stromal cells ameliorates cardiac function after myocardial infarction.

OBJECTIVES: Bone marrow stromal cells are capable of differentiating into cardiomyogenic cells. We tested the hypothesis that transcoronary implantation of bone marrow stromal cells may regenerate infarcted myocardium and reduce cardiac dysfunction. METHODS: Isolated bone marrow stromal cells from the isogenic donor rats were transfected with LacZ reporter gene for cell labeling. To induce cardiomyogenic differentiation, the bone marrow stromal cells were treated with 5-azacytidine before implantation. Two weeks after left coronary ligation, these cells (1 x 10(6) in 150 microL) were infused into the briefly distally occluded ascending aorta of the recipient rats (n = 15) to simulate direct coronary infusion clinically. Control animals were infused with cell-free medium (n = 14). Cardiac function was evaluated by echocardiography at preimplantation and 4 and 8 weeks postimplantation. The hearts were then immunohistochemically studied to identify phenotypic changes of implanted bone marrow stromal cells. RESULTS: Immediately after cell infusion, the bone marrow stromal cells were trapped within coronary vessels in both infarcted and noninfarcted areas. However, after 8 weeks, most of the cells were identified in the scar and periscar tissue, expressing sarcomeric myosin heavy chain and cardiomyocyte-specific protein troponin I-C. Some bone marrow stromal cells were found to be connected to the adjacent host cardiomyocytes with gap junction. Two-way repeated-measures analysis of variance revealed significant improvement in fractional shortening and end-diastolic and end-systolic diameter of the left ventricle (P =.0465,.002,.0004, respectively) in the bone marrow stromal cell group. CONCLUSIONS: Although bone marrow stromal cells had been reported to improve cardiac function when injected directly into the myocardial scar, this study demonstrated for the first time that bone marrow stromal cells can be delivered via the coronary artery, as they are capable of targeted migration and differentiation into cardiomyocytes in the scar tissue to improve cardiac function.

Animals↗

PubMed1979

Yeast killer toxin: purification and characterisation of the protein toxin from Saccharomyces cerevisiae.

Killer toxin from killer strains of Saccharomyces cerevisiae was isolated from concentrates of extracellular medium by precipitation in poly(ethylene glycol) and chromatography through glyceryl-controlled-pore glass. The toxin migrated as a single protein band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A molecular weight of 11470 was determined for the toxin protein from its electrophoretic mobility and amino acid composition. Gel filtration of the active toxin indicated that the 11,470-Mr monomer was the active unit. Electrophoretic comparison of extracellular concentrates from a killer strain and an isogenic non-killer showed the presence of the toxin protein only in the killer-derived material. The activity of the toxin was most stable between pH 4.2 and 4.6. At 30 degrees C toxin from a superkiller strain was more stable than that from a normal killer.

Amino Acids↗

PubMed1999

Accumulation of trehalose by overexpression of tps1, coding for trehalose-6-phosphate synthase, causes increased resistance to multiple stresses in the fission yeast schizosaccharomyces pombe

Recent studies have shown that heat shock proteins and trehalose synthesis are important factors in the thermotolerance of the fission yeast Schizosaccharomyces pombe. We examined the effects of trehalose-6-phosphate (trehalose-6P) synthase overexpression on resistance to several stresses in cells of S. pombe transformed with a plasmid bearing the tps1 gene, which codes for trehalose-6P synthase, under the control of the strong thiamine-repressible promoter. Upon induction of trehalose-6P synthase, the elevated levels of intracellular trehalose correlated not only with increased tolerance to heat shock but also with resistance to freezing and thawing, dehydration, osmostress, and toxic levels of ethanol, indicating that trehalose may be the stress metabolite underlying the overlap in induced tolerance to these stresses. Among the isogenic strains transformed with this construct, one in which the gene coding for the trehalose-hydrolyzing enzyme, neutral trehalase, was disrupted accumulated trehalose to a greater extent and was more resistant to the above stresses. Increased trehalose concentration is thus a major determinant of the general stress protection response in S. pombe.

Journal Article↗

PubMed1978

Surface membrane changes of T cells induced by syngeneic tumour cells. II. T-cell defects induced by small tumour cell inocula or tumour cell antigens.

Injection of a large number of tumour cells, like other strong immunogenic challenges, is followed within 6 h by the uptake of cytophilic Ig (probably complexes) by a subpopulation of T cells. This phenomenon, known as the "6-hour T-cell response" is abrogated when small tumour cell inocula (10(2)), or small amounts of a preparation from tumour cells, which contains tumour antigens, are injected prior to the immunogenic challenge Abrogation of the "6-hour T-cell response" resulted in a decrease in specific anti-tumour cell immunity as tested in vitro by measuring growth inhibition (cytostasis). It has also resulted in loss of the amplifying function on antibody formation against sheep erythrocytes, normally detected in a T-B cell co-operative system when T cells are used 6 h after priming with sheep erythrocytes. It is postulated that this T-cell defect may represent a mechanism by which tumour cells, in the early stages of their growth, interfere with inductive stages of the immune response for a sufficient period of time to allow the tumour to grow beyond immune control.

Animals↗

PubMed1998

Cytomegalovirus inhibits the engraftment of donor bone marrow cells by downregulation of hemopoietin gene expression in recipient stroma.

Cytomegalovirus (CMV) disease after bone marrow (BM) transplantation is often associated with BM graft failure. There are two possible reasons for such a correlation. First, a poor hematopoietic reconstitution of unrelated etiology could promote the progression of CMV infection by the lack of immune control. Alternatively, CMV infection could interfere with the engraftment of donor BM cells in recipient BM stroma. Evidence for a causative role of CMV in BM aplasia came from studies in long-term BM cultures and from the murine in vivo model of CMV-induced aplastic anemia. A deficiency in the expression of essential stromal hemopoietins, such as stem cell factor (SCF), has indicated a functional insufficiency of the stromal microenvironment. It remained open to question whether CMV mediates a negative regulation of hemopoietin gene expression (the downregulation model) or whether it causes the default of a positive regulator (the lack-of-induction model). Further, even though implicitly assumed, it has never been formally documented that CMV directly interferes with the engraftment of a BM cell transplant. We addressed these problems in a murine model of CMV infection after experimental male-into-female BM transplantation. The data indicate that the downregulation model applies. Quantitation of the male-sex-determining gene tdy demonstrated an impaired engraftment of donor BM cells in the BM stroma of the female recipients. This graft failure was reflected by a diminished population of SCF-receptor-expressing hematopoietic progenitor cells and correlated with a reduced level of stromal SCF gene expression. Interestingly, high doses of BM cells protected against stromal insufficiency by a mechanism unrelated to control of infection.

Animals↗

PubMed1981

Osteogenesis in bone grafts after short-term storage and topical antibiotic treatment. An experimental study in rats.

It has been shown in experimental animals that the living cells in a bone autograft can make an important contribution to osteogenesis. However, some common clinical techniques, such as the topical use of antibiotic powders on grafts or on the graft bed, are likely to damage or kill the cells. In this experimental study in rats, bone isografts dusted with chloramphenicol or methicillin powder or with Polybactrin spray before subcutaneous implantation produced little or no new bone over a period of two weeks whereas untreated, control grafts showed abundant osteogenesis, as did grafts pretreated with solutions of antibiotics. The effect of short-term storage of the grafts for 3 to 24 hours in air, saline or culture medium before implantation was also examined. Grafts stored in culture medium generally did as well as, or better than, fresh control grafts whereas immersion in saline inhibited osteogenesis. The importance of these results for clinical bone grafting is discussed.

Animals↗

PubMed1986

Development of pancreatic hyperplasia in female SHN mice receiving ectopic pituitary isografts.

The long-term effects on pancreas of ectopic pituitary grafting at various sites were studied in female SHN mice. Hyperprolactinemia induced by pituitary grafting resulted in an increase in the pancreatic weights, mainly due to hyperplastic proliferation of the pancreatic acinar glands. In addition, islet-cell hyperplasia and adenoma were found in the pituitary-grafted mice. Hyperplastic nodules resembling adenoma of the pancreatic acinar cells were also induced by pituitary grafting. The pancreata of mice with pituitary grafts frequently adhered to the uterus, ovary, and other organs; some of the pancreata further invaded these organs. Control mice bearing no pituitary grafts showed no pancreatic lesions. These results indicate the participation of prolactin in pancreatic tumorigenesis.

Animals↗

PubMed1996

Extracellular matrix proteins: bystanders or active participants in the allograft rejection cascade?

The role extracellular matrix (ECM) proteins may play in the host immune cascade triggered by organ transplantation is largely unknown. We have employed well-defined rat cardiac allograft model to test the hypothesis that the expression of ECM components, such as fibronectin (FN) or laminin (LN), represents an integral part of the host rejection response in transplant recipients. Indeed, treatment of rats with anti-LN Ab profoundly affected lymphocyte recirculation pathways, and selectively decreased deposition of alloreactive lymphocytes at the graft site and in host peripheral lymph nodes. Moreover, cardiac rejection was accompanied by markedly increased intragraft expression of both LN and FN. The infiltrating mononuclear cells, however, accumulated selectively in FN-rather than LN-rich cardiac areas. We also observed distinct temporal expression and spatial distribution patterns of FN variants in rat cardiac allografts and isografts. The local synthesis of FN, in both allografts and isografts was increased as early as at 3 h post-transplant. The expression of FN in cardiac allografts but not in isografts, remained high during later intervals to the point of ultimate rejection. The newly synthesized FNs derived from graft infiltrating macrophages and arterioles, and included EIIIA +, EIIIB +, and CS-I + splicing variants. The EIIIA was preferentially enriched in the earlier, whereas EIIIB and CS-I were selectively enhanced in the later, rejection phases. Finally, treatment of rat recipients with synthetic FN-CS-I peptides prevented the incidence of early (acute) and late (chronic) rejection, supporting the notion that distinct FNs may play a fundamental role in the host immune cascade triggered by organ transplantation. These data offer potential novel sites for intervention in the control of transplant rejection, and contribute to the development of refined therapeutic strategies based upon new concepts of host immunosuppression.

Animals↗

PubMed1992

Contractile proteins and sarcoplasmic reticulum calcium-ATPase gene expression in the hypertrophied and failing heart.

The physiology of myocardial contractility has been studied for over a century, but only recently has molecular biology provided new insights into the mechanisms responsible for the alterations of contraction and relaxation observed during cardiac hypertrophy and heart failure. Pressure and volume overload produce in the myocyte both qualitative changes characterized by protein isoform switches and quantitative changes characterized by modulation of single genes through a mechanogenic transduction the pathways of which are largely unknown. The qualitative changes involve differential expression of multigene families of contractile proteins, especially myosin heavy chain (MHC) and actin. All situations of pressure overload, or of combined pressure and volume overload activate the beta-MHC gene and deactivate the alpha-MHC one, which leads to a slower, more efficient contraction. In rat, pressure overload transitorily activates the alpha-skeletal actin gene, and both the timing and the distribution of the newly formed beta-MHC and alpha-skeletal actin mRNAs differ. We recently found that the isoactin pattern is the same in patients with end-stage heart failure as that of control human hearts. Moreover, both in rat and human, expression of isomyosins and isoactins are not coordinated, neither during ontogeny nor senescence. All this suggests the existence of several regulatory mechanisms activated during normal cardiac growth or by a mechanical trigger, and preliminary results indicate that it is possible to perform nuclear run-on assays in order to analyze the transcriptional step of these isogenes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Physiological↗

PubMed1997

Induction of oral tolerance in carp (Cyprinus carpio L.) after feeding protein antigens.

Induction of oral tolerance against ferritin, recombinant surface glycoprotein of viral haemorrhagic septicemia virus (KLG18) and ovalbumin (OVA) was studied in carp. Feeding of ferritin or KLG18 resulted in lower Ab titres compared to unprimed controls when animals were intramuscularly (i.m.) injected with protein 10 weeks later and sampled 21 days after this injection. After administration of OVA by different routes (oral, anal, i.m.) and i.m. injection with OVA + Freund's incomplete adjuvant 2 months later, only a few fish responded to OVA as measured by serum Ab titres. Responsiveness to OVA appeared to be carp strain dependent. When an isogenic carp strain was selected for an optimal response to i.m. injection with OVA, this carp strain did not develop oral tolerance after feeding. In contrast, 6 x feeding high doses of OVA on subsequent days, resulted in immunological memory formation. Oral tolerance can be induced in carp, but differences in tolerance induction may depend on the protein used. A possible role of genetic factors in the induction of oral tolerance in fish is discussed.

Administration, Oral↗

PubMed2002

Long-patch base excision repair of apurinic/apyrimidinic site DNA is decreased in mouse embryonic fibroblast cell lines treated with plumbagin: involvement of cyclin-dependent kinase inhibitor p21Waf-1/Cip-1.

Molecular interactions among cell cycle and DNA repair proteins have been described, but the impact of many of these interactions on cell cycle control and DNA repair remains unclear. The cyclin-dependent kinase inhibitor, p21, is known to be involved in DNA damage-induced cell cycle arrest and blocking DNA replication and repair. Participation of p21 has been implicated in nucleotide excision repair. However, the role of p21 in the base excision repair (BER) pathway has not been thoroughly studied. In the present investigation, we treated isogenic mouse embryonic fibroblast (MEF) cell lines containing wild-type (MEF-polbeta) or DNA polymerase beta (polbeta) gene-knockout (MEFpolbetaKO) with oxidative DNA-damaging agent, plumbagin, and examined its effect on p21 levels and BER activity. Plumbagin treatment caused a S-G(2)/M phase arrest and cell death of both MEF cell lines, induced p21 levels, and decreased p21-mediated long-patch (LP) BER by blocking DNA ligase activity in the polbeta-dependent pathway and by blocking both FEN1 and DNA ligase activity in polbeta-independent pathway. These findings suggest that plumbagin induced p21 levels play a regulatory role in cell cycle arrest, apoptosis, and polbeta-dependent and -independent LP-BER pathways in MEF cells.

Animals↗

PubMed2002

Role of CsrR, hyaluronic acid, and SpeB in the internalization of Streptococcus pyogenes M type 3 strain by epithelial cells.

Internalization of group A streptococcus by human epithelial cells has been extensively studied during the past 6 years. It is now clear that multiple mechanisms are involved in this process. We have previously demonstrated that the CsrR global regulator controls the internalization of an invasive M type 3 strain through regulation of the has (hyaluronic acid synthesis) operon, as well as another, unknown gene(s). Recently, it was reported that the CsrR-regulated cysteine protease (SpeB) is also involved in bacterial uptake. In this study we have examined the roles of CsrR, hyaluronic acid capsule, and SpeB in streptococcal internalization. We have constructed isogenic mutants of the M3 serotype deficient in the csrR, hasA, and speB genes and tested their ability to be internalized by HEp-2 epithelial cells. Inactivation of csrR abolished internalization, while inactivation of either hasA or speB increased the internalization efficiency. Mutation in csrR derepressed hasA transcription and lowered the activity of SpeB, while no effect on speB transcription was observed. The speB mutant expressed smaller amounts of capsule, while the hasA mutant transcribed more csrR and speB mRNAs. Thus, it seems that complex interactions between CsrR, SpeB, and capsule are involved in modulation of group A streptococcus internalization.

Amino Acid Sequence↗

PubMed2004

High-resolution fine mapping and fluorescence in situ hybridization analysis of sun, a locus controlling tomato fruit shape, reveals a region of the tomato genome prone to DNA rearrangements.

The locus sun on the short arm of tomato chromosome 7 controls morphology of the fruit. Alleles from wild relatives impart a round shape, while alleles from certain cultivated varieties impart an oval shape typical of roma-type tomatoes. We fine mapped the locus in two populations and investigated the genome organization of the region spanning and flanking sun. The first high-resolution genetic map of the sun locus was constructed using a nearly isogenic F(2) population derived from a cross between Lycopersicon pennellii introgression line IL7-4 and L. esculentum cv Sun1642. The mapping combined with results from pachytene FISH experiments demonstrated that the top of chromosome 7 is inverted in L. pennellii accession LA716. sun was located close to the chromosomal breakpoint and within the inversion, thereby precluding map-based cloning of the gene using this population. The fruit-shape locus was subsequently fine mapped in a population derived from a cross between L. esculentum Sun1642 and L. pimpinellifolium LA1589. Chromosome walking using clones identified from several large genomic insert libraries resulted in two noncontiguous contigs flanking sun. Fiber-FISH analysis showed that distance between the two contigs measured 68 kb in L. esculentum Sun1642 and 38 kb in L. pimpinellifolium LA1589, respectively. The sun locus mapped between the two contigs, suggesting that allelic variation at this locus may be due to an insertion/deletion event. The results demonstrate that sun is located in a highly dynamic region of the tomato genome.

Chromosome Mapping↗

PubMed1981

Subcutaneous, isogeneic transplantation of duct-ligated pancreas in streptozotocin diabetic mice II. Hormone storage as a function of time and of the recipient's initial glycemic state.

Between 7 and 18 wk after transplantation, the insulin contained in duct-ligated, pancreas transplants increased twofold whereas glucagon content showed no significant change. Insulin contents of the recipient's own endogenous pancreas, though severely depleted after streptozotocin injection, also showed a twofold increase. The combined insulin reserves in transplant in mice at 18 wk which totaled 30% of that in aged-matched, normal controls was still insufficient to promote normal tolerance and insulin response to intravenously injected glucose. In a second experiment reported herein, we found that the insulin and glucagon reserves in ten week-old transplants were not significantly different between recipients that were either diabetic (streptozotocin-treated) or nondiabetic at the time of transplantation. Thus, the initial glucose levels in the host environment, whether elevated or not, did not differentially effect the level of hormones eventually accumulated in transplants.

Animals↗

PubMed2002

Modulation of catecholamine responsiveness and beta-adrenergic receptor/adenylyl cyclase pathway during cardiac allograft rejection1 2.

BACKGROUND: This study investigated the changes of catecholamine responsiveness and beta-adrenergic receptor/adenylyl cyclase pathway during acute cardiac transplant rejection. METHODS: Isogeneic Lewis to Lewis and allogeneic Dark Agouti (DA) to Lewis rat cardiac transplants were studied 3 and 5 days after heterotopic intraabdominal transplantation (n=6/group). Myocardial blood flow (MBF), left ventricular systolic pressure (LVSP), maximum pressure development (+dP/dt), and end-diastolic pressure (LVEDP) were measured using an intraventricular balloon. Contractile response to dobutamine (5 microg/kg/min) was also assessed. In separate groups beta-adrenergic receptor density and adenylyl cyclase activity were measured in the grafts, in the recipients' native hearts and in native hearts of sham-operated controls. RESULTS: During mild to moderate rejection cardiac function indices remained unchanged, although MBF and contractile response to dobutamine decreased significantly (P<0.05) in the allogeneic group. The beta-adrenergic receptor density was significantly (P<0.05) increased in both isografts and allografts and in the native hearts of allografted recipients in comparison to native hearts of controls. Adenylyl cyclase activity showed a significant decrease (P<0.05) only in allografts. During severe rejection, LVSP and +dP/dt decreased and LVEDP increased in allografts in comparison to isografts (P<0.05). This was accompanied by a significant decrease in MBF, contractile response to dobutamine, beta-adrenergic receptor density, and adenylyl cyclase activity (P<0.05). CONCLUSIONS: Both microcirculatory disturbances and primary alteration in adenylyl cyclase activity may contribute to decreased contractile reserve in mild to moderate cardiac allograft rejection, whereas beta-adrenergic receptor density seems to be also influenced by cardiac denervation. Severe rejection leads to systolic and diastolic heart failure with complex dysregulation of the beta-adrenergic receptor/adenylyl cyclase pathway and impaired microcirculation.

Adenylyl Cyclases↗

PubMed2002

Genetic dissection of a genomic region for a quantitative trait locus, Hd3, into two loci, Hd3a and Hd3b, controlling heading date in rice.

The rice photoperiod sensitivity gene Hd3 was originally detected as a heading date-related quantitative trait locus localized on chromosome 6 of rice. High-resolution linkage mapping of Hd3 was performed using a large segregating population derived from advanced backcross progeny between a japonica variety, Nipponbare, and an indica variety, Kasalath. To determine the genotype of Hd3, we employed progeny testing under natural field and short-day conditions. As a result, two tightly linked loci, Hd3a and Hd3b, were identified in the Hd3 region. Nearly-isogenic lines for Hd3a and Hd3b were selected from progeny using marker-assisted selection. The inheritance mode of both Hd3a and Hd3b was found to be additive. Analysis of daylength response in nearly-isogenic lines of Hd3a and Hd3b showed that the Kasalath allele at Hd3a promotes heading under short-day conditions while that at Hd3b causes late heading under long-day and natural field conditions.

Journal Article↗

PubMed2005

Lack of consensus gene expression changes associated with radiation-induced chromosomal instability.

The relatively high frequency with which ionizing radiation induces genomic instability suggests that a gene mutation occurring after irradiation is an unlikely cause of the phenotype. To search for mechanism(s) of initiation and perpetuation of this instability phenotype, gene expression profiles of clones exhibiting delayed chromosomal instability were analyzed. Microarray analysis using two pools of isogenic radiation-induced chromosomally unstable clones compared to an irradiated but chromosomally stable clone uncovered a set of 68 differentially expressed genes using two methods of analysis. Unexpectedly, all 68 genes were under-expressed relative to the chromosomally stable reference clone. Further analysis of the candidates placed the differentially expressed genes into pathways implicating differential MAP kinase signaling, ubiquitin/proteasome function, DNA repair, cell cycle control, lipid signaling, nucleotide metabolism, and other potentially disrupted pathways. Validation studies using northern and western blotting, and functional assays concluded that although differences in some of these pathways exist, no single gene or molecular pathway was found to be differentially regulated in all of the chromosomally unstable clones tested. Inferred from these data is that there are multiple potential molecular pathways and/or events that maintain the unstable phenotype, and no single expression pattern is linked to instability in the unstable clones analyzed.

Animals↗