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PubMedDate not supplied

Warning: the use of heterogeneous mice may seriously damage your research.

Genetically heterogeneous (GH) mice and rats continue to be widely used in research even though the case for using isogenic strains has been argued repeatedly. The paper by Miller et al. in this issue appears to be the only one in the last 22 to attempt a scientific justification for the continued use of (a limited subset of) GH stocks. However, although they are to be commended for bravery, they fail to make their case. GH stocks represent poor material for controlled studies because genetic heterogeneity normally leads to phenotypic variability and a decline in experimental sensitivity. To counter this argument, Miller et al. claim that phenotypic variability may actually be smaller in GH animals than in their isogenic parents. Were this so (e.g., all mice being short lived, small, and aggressive), it is difficult to see how the use of such a stock could increase the generality of research results based on it, as claimed by Miller et al. Isogenic strains are a vital, proven, and powerful resource for biomedical research, and should be used in preference to GH stocks by all scientists who use laboratory rodents.

Animals↗
PubMed2004

atxA controls Bacillus anthracis capsule synthesis via acpA and a newly discovered regulator, acpB.

Two regulatory genes, acpA and atxA, have been reported to control expression of the Bacillus anthracis capsule biosynthesis operon capBCAD. The atxA gene is located on the virulence plasmid pXO1, while pXO2 carries acpA and the cap genes. acpA has been viewed as the major regulator of the cap operon because it is essential for capsule gene expression in a pXO1(-) pXO2(+) strain. atxA is essential for toxin gene transcription but has also been implicated in control of the cap genes. The molecular functions of the regulatory proteins are unknown. We examined cap gene expression in a genetically complete pXO1(+) pXO2(+) strain. Our results indicate that another pXO2 gene, acpB (previously called pXO2-53; accession no. NC002146.1:49418-50866), has a role in cap expression. The predicted amino acid sequence of AcpB is 62% similar to that of AcpA and 50% similar to that of AtxA. Assessment of cap gene transcription revealed that cap expression was not affected in a pXO1(+) pXO2(+) acpB-null mutant and was slightly reduced in an isogenic acpA mutant. However, cap gene expression was abolished in an acpA acpB double mutant. Microscopic examination of capsule synthesis by the mutants corroborated these findings. acpA and acpB expression is controlled by atxA; capsule synthesis and transcription of acpA and acpB were markedly reduced in an atxA mutant. The data suggest that, in a strain containing both virulence plasmids, atxA is the major regulator of capsule synthesis and controls capBCAD expression indirectly, via positive regulation of acpA and acpB.

Amino Acid Sequence↗
PubMed1999

Transcription factor GCN4 for control of amino acid biosynthesis also regulates the expression of the gene for lipoamide dehydrogenase.

The yeast LPD1 gene encoding lipoamide dehydrogenase is subject to the general control of amino acid biosynthesis mediated by the GCN4 transcription factor. This is striking in that it demonstrates that GCN4-mediated regulation extends much farther upstream than simply to the direct pathways for amino acid and purine biosynthesis. In yeast, lipoamide dehydrogenase functions in at least three multienzyme complexes: pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase (which function in the entry of pyruvate into, and metabolism via, the citric acid cycle) and glycine decarboxylase. When wild-type cells were shifted from growth on amino acid-rich to amino acid-deficient medium, the expression of lipoamide dehydrogenase was induced approx. 2-fold. In a similar experiment no such induction was observed in isogenic gcn4 mutant cells. Northern analysis indicated that amino acid starvation affected levels of the LPD1 transcript. In the upstream region of LPD1 are three matches to the consensus for control mediated by GCN4. Directed mutagenesis of each site, and of all combinations of sites, suggests that only one site might be important for the general control response under the conditions tested. Gel-retardation analysis with GCN4 protein synthesized in vitro has indicated that GCN4 can bind in vitro to at least two of the consensus motifs.

Amino Acid Sequence↗
PubMed1973

Structure and development of neuronal connections in isogenic organisms: variations and similarities in the optic system of Daphnia magna.

It is readily apparent upon examination and comparison of organisms of the same and related species that to a large extent genes control morphological features. One means to ascertain the role and degree of genetic control is to study in detail the anatomy and development of a particular structure, say an identifiable neuron, in a population with a fixed genome, and at different stages of morphogenesis. The small crustacean, Daphnia, is well suited for this type of study, since it reproduces parthenogenically, its development is easily staged, and its nervous system is reasonably simple. In this paper comparisons of detailed morphology of neurons in the visual system of adult Daphnia magna are considered. Results indicate that gross features of the system are reproduced within the clone, but some of the finer details are not reproduced.

Animals↗
PubMed1997

A Method for Selection and Characterization of Rhizosphere-Competent Bacteria of Chickpea

A greenhouse assay was developed to evaluate the root-colonizing capability of the native chickpea rhizospheric bacterial population. In this assay system, screening time was reduced on two counts. First, spontaneous chromosomal rifampicin-resistant (Rifr) strains were directly inoculated to seeds without any check for the stability of the mutation, and second, no attempts were made to taxonomically identify all the strains being screened for chickpea rhizosphere competence. Only two chickpea rhizosphere-competent Rifr strains from the group of six good chickpea rhizosphere colonizers forming 10(7) to 10(8) colony-forming units (cfu)/g root were taxonomically identified as Pseudomonas fluorescens NB13R and Pseudomonas spp. NB49R, after screening 49 bacteria. Both the strains showed no difference from their corresponding wild-type strains P. fluorescens NB13 and Pseudomonas spp. NB49 in terms of chickpea rhizosphere competence. Isogenic or equally rhizospheric competitive second non-isogenic bacterial isolate, when present in tenfold higher amount, pre-empted the colonization of the soil by the bacterium, which was present in smaller ratio. These findings indicate that the isogenic or equally rhizospheric competitive second non-isogenic Rifr strains should be compared for their survival and competition with that of the isogenic parent and with each other for specific ecological niche, before using a mixture of isolates, for stable and consistent biological seed treatment to control soilborn pathogens or pests or to promote plant growth.

Journal Article↗
PubMed1996

Interleukin-1 alpha and interleukin-6 in the urine, kidney, and bladder of mice inoculated with Escherichia coli.

After bladder inoculation of mice using the pyelonephritogenic Escherichia coli strain DS17, urinary interleukin-1 alpha (IL-1 alpha) peaked at 0.5 h post infection (mean 233 pg/ml), interleukin-6 (IL-6) at 2 h (mean 572 pg/ml), and leukocyturia at 4 h, all three persisting for more than 24 h. In the kidneys IL-1 alpha peaked at 2 h, persisted over 24 h (mean 900-1,000 pg/ml), and decreased over 2-6 days post inoculation to a mean value of 208 pg/ml. Control kidneys showed low IL-1 alpha values. IL-6 in the kidneys peaked at 5 h (mean 9,999 pg/ml) but normalized, i.e., similar to control kidneys, by 48 h (mean 105 pg/ml). A similar cytokine response, but with tenfold lower levels, was found in the bladder tissue. The isogenic P-negative E. coli mutant DS178 elicited lower IL-6 in the kidneys at 5 h, but persisted in higher numbers in the kidneys at 6 days. Bacterial counts and cytokine levels correlated both in the kidneys and in bladder tissue, (r = 0.50-0.76, P < 0.001). Characterization of the immune response gives a better understanding of the relative importance of different bacterial characteristics for the local inflammatory process and is needed for studies of its pharmacological down-regulation.

Animals↗
PubMed1998

Endothelin-A and -B antagonists protect myocardial and endothelial function after ischemia/reperfusion in a rat heart transplantation model.

OBJECTIVE: Previous studies suggested that endothelin-1 (ET-1) may play a pathophysiological role in myocardial ischemia/reperfusion injury. This study was designed to investigate the effects of the selective ET-A receptor antagonist BQ123 and the selective ET-B receptor antagonist BQ788 on myocardial and endothelial function after reversible deep hypothermic ischemia in a heterotopic rat heart transplantation model. METHODS: Isogenic intraabdominal heterotopic transplantation was performed in Lewis rats. After 1 h of cold ischemic preservation reperfusion was started either after application of placebo (control), BQ123 (3 mumol/kg/min). BQ788 (3 mumol/kg/min), ET-1 (8 pmol/kg/min) or simultaneous infusion of BQ123 or BQ788 and ET-1, respectively (n = 12 each). An implanted balloon was used to obtain pressure-volume relations of the transplanted heart. Myocardial blood flow (MBF) was assessed by the hydrogen-clearance method. Measurements were taken after 1 and 24 h of reperfusion. Endothelium-dependent vasodilation to acetylcholine (ACH) and endothelium-independent vasodilation to sodium nitroprusside were also determined. RESULTS: Both BQ123 and BQ788 significantly improved myocardial and endothelial functional recovery during early reperfusion, whereas ET-1 significantly impaired myocardial and endothelial function. Simultaneous infusion of ET-1 diminished the effects of BQ123 and BQ788. Although myocardial function and baseline MBF were similar in all groups after 24 h of reperfusion, endothelium dependent vasodilation to ACH was still significantly higher in the BQ123 and BQ788 groups and lower in the ET-1 groups (p < 0.05). CONCLUSIONS: These results suggest that endogenous ET release is involved in the pathogenesis of reperfusion injury after heart transplantation. ET-A and ET-B receptor antagonists may be useful to reduce ischemia/reperfusion injury.

Acetylcholine↗
PubMed1998

The role of autolysins during vegetative growth of Bacillus subtilis 168.

A set of isogenic mutants of Bacillus subtilis 168, insertionally inactivated in the genes encoding a number of lytic enzymes and a sigma factor (sigma D, which controls the expression of a number of autolysins) was constructed. Phenotypic analysis of the mutants determined the individual and combined roles of the autolysins in vegetative growth. The major vegetative autolysins of B. subtilis, LytC (50 kDa amidase) and LytD (90 kDa glucosaminidase), were shown to have roles in cell separation, cell wall turnover, antibiotic-induced lysis and motility. LytC was also shown to have a role in general cell lysis induced by sodium azide. Renaturing SDS-PAGE of cell-wall-binding protein extracts of the mutant strains revealed the presence of a novel autolysin that was previously masked by LytC. This 49 kDa enzyme was shown to be sigma D-controlled and was identified as a candidate cell separation and cell wall turnover enzyme. A multiple mutant strain, lacking LytC, LytD and the 49 kDa enzyme, retained at least ten bands of autolytic activity. These may correspond to individual or proteolytically processed novel autolysins, the functions of which are unknown. The multiple mutant strains facilitate the study of these, and other lytic enzymes, to determine their cellular functions.

Anti-Bacterial Agents↗
PubMed1976

In vivo transcription of R-plasmid deoxyribonucleic acid in Escherichia coli strains with altered antibiotic resistance levels and/or conjugal proficiency.

The amounts of plasmid deoxyribonucleic acid (DNA) and the levels of the in vivo transcription of the Escherichia coli plasmids R538-1 (repressed for conjugal transfer) and R538-1drd (derepressed for transfer) were determined by DNA-DNA hybridization and DNA-ribonucleic acid hybridization, respectively. The results demonstrate that the level of plasmid transcription is increased by two-fold in the strain carrying the derepressed plasmid, compared to an isogenic strain carrying the repressed plasmid, whereas the amount of plasmid DNA is approximately the same, suggesting that the transfer genes are under transcriptional control. Levels of plasmid DNA, plasmid DNA transcription, and chloramphenicol acetyltransferase activity were also compared in a mutant strain that carried the R538-1drd plasmid and was resistant to high levels of antibiotics. This strain produces about 13 copies of plasmid DNA per chromosome compared to five copies for the parent strain. The level of transcription of plasmid DNA was found to be twofold higher in the high-level resistant strain, whereas the level of chloramphenition, acetyltransferase activity was increased by 10-fold. In addition the levels of plasmid DNA transcription and chloramphenicol acetyltransferase activity in the high-level resistant strain were found to be further increased by the presence of high levels of chloramphenicol in the growth medium. The amount of plasmid DNA remained constant under these conditions, indicating that high levels of chloramphenicol can stimulate the expression of plasmid genes at the level of transcription in this strain.

Acetyltransferases↗
PubMed2005

Development and validation of molecular markers linked to an Aegilops umbellulata-derived leaf-rust-resistance gene, Lr9, for marker-assisted selection in bread wheat.

An Aegilops umbellulata-derived leaf-rust-resistance gene, Lr9, was tagged with 3 random amplified polymorphic DNA (RAPD) markers, which mapped within 1.8 cM of gene Lr9 located on chromosome 6BL of wheat. The markers were identified in an F2 population segregating for leaf-rust resistance, which was generated from a cross between 2 near-isogenic lines that differed in the alien gene Lr9 in a widely adopted agronomic background of cultivar 'HD 2329'. Disease phenotyping was done in controlled environmental conditions by inoculating the population with the most virulent pathotype, 121 R63-1 of Puccinia triticina. One RAPD marker, S5550, located at a distance of 0.8+/-0.008 cM from the Lr9 locus, was converted to sequence-characterized amplified region (SCAR) marker SCS5550. The SCAR marker was validated for its specificity to gene Lr9 against 44 of the 50 known Lr genes and 10 wheat cultivars possessing the gene Lr9. Marker SCS5550 was used with another SCAR marker, SCS73719, previously identified as being linked to gene Lr24 on a segregating F2 population to select for genes Lr9 and Lr24, respectively, demonstrating the utility of the 2 markers in marker-assisted gene pyramiding for leaf-rust resistance in wheat.

DNA, Plant↗
PubMed2004

Intravenous administration of mesenchymal stem cells improves cardiac function in rats with acute myocardial infarction through angiogenesis and myogenesis.

Mesenchymal stem cells (MSCs) are pluripotent cells that differentiate into a variety of cells, including cardiomyocytes and endothelial cells. However, little information is available regarding the therapeutic potency of systemically delivered MSCs for myocardial infarction. Accordingly, we investigated whether intravenously transplanted MSCs induce angiogenesis and myogenesis and improve cardiac function in rats with acute myocardial infarction. MSCs were isolated from bone marrow aspirates of isogenic adult rats and expanded ex vivo. At 3 h after coronary ligation, 5 x 10(6) MSCs (MSC group, n=12) or vehicle (control group, n=12) was intravenously administered to Lewis rats. Transplanted MSCs were preferentially attracted to the infarcted, but not the noninfarcted, myocardium. The engrafted MSCs were positive for cardiac markers: desmin, cardiac troponin T, and connexin43. On the other hand, some of the transplanted MSCs were positive for von Willebrand factor and formed vascular structures. Capillary density was markedly increased after MSC transplantation. Cardiac infarct size was significantly smaller in the MSC than in the control group (24 +/- 2 vs. 33 +/- 2%, P <0.05). MSC transplantation decreased left ventricular end-diastolic pressure and increased left ventricular maximum dP/dt (both P <0.05 vs. control). These results suggest that intravenous administration of MSCs improves cardiac function after acute myocardial infarction through enhancement of angiogenesis and myogenesis in the ischemic myocardium.

Animals↗
PubMed2005

Gene transfer of a broad spectrum CC-chemokine inhibitor reduces vein graft atherosclerosis in apolipoprotein E-knockout mice.

BACKGROUND: Accelerated atherosclerosis is a major cause of vein graft failure after bypass surgery. Several CC-chemokines (CC-CKs) mediate monocyte/macrophage recruitment in native atherosclerotic plaques; we hypothesized that CC-CKs may be critical in the development of accelerated atherosclerosis in vein grafts. METHODS AND RESULTS: Using in vivo gene transfer, we administered a soluble CC-CK binding protein ("35K") to apolipoprotein E-knockout (ApoE-/-) mice that underwent interposition bypass grafting of the vena cava from isogenic donor mice to the common carotid artery. Two days before operation, a recombinant adenovirus encoding either 35K (Ad35K) or green fluorescent protein (AdGFP; control) was injected into recipient mice via the tail vein. 35K greatly reduced CC-CK activity in mouse plasma. After 14 days, vein graft atherosclerotic lesion area, smooth muscle alpha-actin-positive neointimal area, and total vessel wall thickness were strikingly reduced by Ad35K gene transfer compared with AdGFP controls. Furthermore, 35K gene transfer dramatically reduced macrophage content by approximately 90% and cell proliferation by 95%. After 28 days, lesion area and vessel wall thickness remained significantly less in Ad35K mice. CONCLUSIONS: A single intravenous injection of the CC-CK inhibitor 35K significantly reduced atherosclerosis in carotid-caval vein grafts in ApoE-/- mice. This study highlights the importance of the CC-CK class in accelerated atherosclerosis, and its role as a potential target for improving vein graft patency.

Adenoviridae↗
PubMed2006

Dynamic reprogramming of DNA methylation at an epigenetically sensitive allele in mice.

There is increasing evidence in both plants and animals that epigenetic marks are not always cleared between generations. Incomplete erasure at genes associated with a measurable phenotype results in unusual patterns of inheritance from one generation to the next, termed transgenerational epigenetic inheritance. The Agouti viable yellow (A(vy)) allele is the best-studied example of this phenomenon in mice. The A(vy) allele is the result of a retrotransposon insertion upstream of the Agouti gene. Expression at this locus is controlled by the long terminal repeat (LTR) of the retrotransposon, and expression results in a yellow coat and correlates with hypomethylation of the LTR. Isogenic mice display variable expressivity, resulting in mice with a range of coat colours, from yellow through to agouti. Agouti mice have a methylated LTR. The locus displays epigenetic inheritance following maternal but not paternal transmission; yellow mothers produce more yellow offspring than agouti mothers. We have analysed the DNA methylation in mature gametes, zygotes, and blastocysts and found that the paternally and maternally inherited alleles are treated differently. The paternally inherited allele is demethylated rapidly, and the maternal allele is demethylated more slowly, in a manner similar to that of nonimprinted single-copy genes. Interestingly, following maternal transmission of the allele, there is no DNA methylation in the blastocyst, suggesting that DNA methylation is not the inherited mark. We have independent support for this conclusion from studies that do not involve direct analysis of DNA methylation. Haplo-insufficiency for Mel18, a polycomb group protein, introduces epigenetic inheritance at a paternally derived A(vy) allele, and the pedigrees reveal that this occurs after zygotic genome activation and, therefore, despite the rapid demethylation of the locus.

Alleles↗
PubMedDate not supplied

[Influence of tumor necrosis factor on the growth of vegetative and nonculturable forms of Salmonella].

The growth rate of the vegetative forms and the recultivation rate of the uncultivable forms of Salmonella isogenous strains, one of these strains carrying mutation in gene pqi, were studied. The multiplication rate of the vegetative and uncultivable forms of Salmonella control strain in the spleen of infected animals at the initial stages of the infectious process was shown (in vivo) to be considerably accelerated after the preliminary incubation of the culture with cytokine (tumor necrosis factor). The multiplication rate, in vivo and in vitro, of Salmonella vegetative and uncultivable forms with mutation in gene pqi did not change after the incubation of the cells with cytokine, which is indicative of an important role played by the product of this gene in the process of the interaction of bacteria with cytokines. The full nucleotide sequence Salmonella gene pqi was determined.

Amino Acid Sequence↗
PubMed1991

T-dependent destruction of thyroid isografts exposed to IFN-gamma.

Several autoimmune diseases are accompanied by tissue-specific expression of class II molecules of the MHC, and it has been suggested that this elicits a T cell response against tissue-specific Ag to which the individual is not tolerant. However, recent transgenic studies have indicated that non-lymphoid expression of class II genes in the pancreas, liver, and kidney is either innocuous or induces peripheral tolerance. To test this hypothesis in another organ-specific autoimmune disease, we attempted to induce autoimmune thyroiditis in normal mice with class II+ thyroid tissue. Normal thyroid lobes were cultured with and without IFN-gamma and then transplanted to adult isogeneic recipients. The thyroid that had been induced to express class II genes by IFN-gamma was destroyed in normal mice, whereas the control cultured thyroid and the native cervical gland survived. Both types of transplants remained intact and functional in congenic nu/nu recipients, indicating that neither exposure to IFN-gamma nor expression of class II genes compromised the thyroid. Thus, in some tissues, exposure to IFN-gamma and/or the induction of class II expression can lead to T-dependent autoimmune disease.

Animals↗
PubMed1977

Intrahepatic transplantation of pancreatic islets in the rat.

Islets of Langerhans from isogeneic donor rats were transplanted directly into the hepatic parenchyma of recipients which had been made severely diabetic by streptozotocin (glycaemia ranging between 400 and 1090 mg%). Complete control lasting up to 13 months was achieved in 65% of recipients by using 600-800 islets. Following intravenous glucose administration, each rat responded similarly to normal rats with a rapid but reduced release of insulin. Cytoimmunofluorescence and electron microscopic studies demonstrated the presence of both functional insulin and glucagon cells, within the transplanted islets. It is suggested that for various reasons direct intrahepatic transplantation might become the preferred method for islets.

Animals↗
PubMed1998

Pleiotropic mutations alter the kinetics of calcium transport, competence regulation, autolysis and experimental virulence in Streptococcus pneumoniae.

Streptococcus pneumoniae is a pathogen in which the extracellular calcium concentration plays a major physiological role, in growth as well as in the induction of competence for genetic transformation and activation of autolysis. Both responses are under the control of a protein activator exported in the medium. We have checked the impact of mutations which alter the regulation of competence and autolysis on experimental virulence. Isogenic encapsulated derivatives carrying the relevant mutations were serotype 3 smooth clones, obtained by transformation of the relevant rough strains with DNA from a serotype 3 smooth isolate. Survival kinetics and bacterial clearance from the blood were followed after intraperitoneal infection of Swiss mice with the different bacterial cultures. In this model, mutants showing an attenuation of virulence relative to the wild type fell into two classes. In the first, represented by the lytA::ery mutant V1095 defective for calcium-induced autolysis, attenuated virulence could be correlated with rapid bacterial clearance from the blood. In the second, represented by the dmb mutants V2200 and V3300, attenuation was associated with delayed bacterial clearance from the blood, and correlated with altered kinetics of calcium transport and of regulation of competence and autolysis. It appeared unlikely that attenuation of virulence for strains V2200 and V3300 was a direct consequence of their competence phenotype, since the com::ery mutants V1008 and V1019, defective for the production of the competence activator, were as virulent as the wild-type strain. Autolysis involving an N-acetyl-muramyl-alanine amidase encoded by lytA was also regulated by calcium. The inserted allele lytA0::ery further reduced virulence in the dmb1 background (V2200). This additive effect of lytA- to dmb1 points to different routes of virulence regulation by LYT and DMB1 and suggests that the kinetics of calcium traffic controls several pathways involved in the virulence of pneumococcus.

Animals↗
PubMed1978

Syngeneic transplantation of fetal rat pancreas. II. Effect of insulin treatment on the growth and differentiation of pancreatic implants fifteen days after transplantation.

Eight 18-days-postcoitum fetal pancreases were transplanted to isogenic alloxan-diabetic male rats. Some recipients were treated with insulin for seven days immediately after transplantation. Eight animals in both the insulin-treated group and control group were killed 15days after transplantation for morphologic and hormonal studies of the transplanted tissue. Using the morphometric technique of linear scanning, the insulin, glucagon, and somatostatin immunocytochemically positive, cell masses of the fetal pancreatic implants were quantitated. The beta cell mass of the implants from the control animals increased roughly eightfold from the time of transplant; insulin treatment resulted in a further two- to threefold increase. The insulin content of the implants increased more than did the beta cell mass, resulting in the fivefold increase in insulin per beta cell. The alpha cell and delta cell masses did not change during the transplant site, the mass of functional beta cells, and the cell-to-cell content of the implanted tissue. These results are discussed in relation to previous quantitative studies of pancreatic islet cell growth. The relationships of the transplant site, the mass of functional beta cell, and the cell-to-cell interaction within the islet to the maintenance of glucose homeostasis are also discussed.

Animals↗