Mobilizations of pBC16 and pAND006, containing the replicon of the Bacillus thuringiensis subsp. israelensis plasmid pTX14-3, between strains of B. thuringiensis subsp. israelensis were examined. Transconjugants appeared after a few minutes and reached a maximum frequency after approximately 2 h. Plasmid pBC16 was mobilized at a frequency approximately 200 times that of pAND006. However, pAND006 was consistently transferred, suggesting that the replicon of pTX14-3 is sufficient to sustain mobilization in B. thuringiensis subsp. israelensis. A specific protease-sensitive coaggregation between strains of B. thuringiensis subsp. israelensis was found to be unambiguously correlated with plasmid transfer. Two aggregation phenotypes, Agr+ and Agr-, were identified in this subspecies. Aggregation disappeared when the optical density of the mating mixture at 600 nm exceeded approximately 1, and it did not reappear upon dilution. Aggregation was shown to involve interactions of cells with opposite aggregation phenotypes, and evidence of a proteinaceous molecule on the surface of the Agr-that is cells involved in aggregation formation is presented. Matings and selection for the presence of two antibiotic resistance plasmids followed by identification of the host cell revealed that mobilization was unidirectional, from the Agr+ cell to the Agr-cell. The aggregation phenotype was found to be transferred with high frequency ("100%) in broth matings, and the appearance of Agr-isolates from Agr+ strains suggested that the loci involved in aggregation formation are located on a plasmid. No excreted aggregation-inducing signals were detected in the supernatant or culture filtrate of either the donor, the recipient, or the mating mixture.Conjugation, the direct transfer of genes from one bacterial cell to another, is often mediated by large conjugative plasmids. These large plasmids are transferred at high frequency, and occasionally chromosomal genes and smaller nonconjugative plasmids are cotransferred by the transfer apparatus of the conjugative plasmids. In gram-negative bacteria many small nonconjugative plasmids can be transferred (mobilized) if an additional self-transmissible plasmid is present in the host (13, 46, 47). Recent reports have described similar mobilizations of small plasmids in gram-positive bacteria (2,5,26,34,40). The first studied and best understood of the conjugative plasmids is the F plasmid, which can replicate in Escherichia coli and certain closely related enteric bacteria. The mechanism of F plasmid-mediated conjugation involves physical contact of the cells and transfer of plasmid DNA brought about by the sex pilus (23). Studies of the gram-positive bacterium Enterococcus (formerly Streptococcus) faecalis suggest a mechanism quite different from that in the better-studied gram-negative systems. Pili do not play any role in conjugation between strains of this organism, but pheromone-induced aggregation between cells has been shown to be a major characteristic (11,15).
The aggregation phenotypes Agr؉ and Agr ؊ of Bacillus thuringiensis subsp. israelensis are correlated with a conjugation-like plasmid transfer and characterized by the formation of aggregates when the bacteria are socialized during exponential growth. We present evidence for the association of the Agr ؉ phenotype with the presence of the large (135-MDa) self-transmissible plasmid pXO16.
BackgroundIn humans and animal models, excessive intake of dietary fat, fructose and cholesterol has been linked to the development of non-alcoholic fatty liver disease (NAFLD). However, the individual roles of the dietary components remain unclear. To investigate this further, we compared the effects of a high-fat diet, a high-fructose diet and a combination diet with added cholesterol on the development of NAFLD in rats.MethodsForty male Sprague–Dawley rats were randomized into four groups receiving either a control-diet (Control: 10% fat); a high-fat diet (HFD: 60% fat, 20% carbohydrate), a high-fructose diet [HFr: 10% fat, 70% carbohydrate (mainly fructose)] or a high-fat/high-fructose/high-cholesterol-diet (NASH: 40% fat, 40% carbohydrate (mainly fructose), 2% cholesterol) for 16 weeks.ResultsAfter 16 weeks, liver histology revealed extensive steatosis and inflammation in both NASH- and HFD-fed rats, while hepatic changes in HFr-rats were much more subtle. These findings were corroborated by significantly elevated hepatic triglyceride content in both NASH- (p < 0.01) and HFD-fed rats (p < 0.0001), elevated hepatic cholesterol levels in NASH-fed rats (p < 0.0001), but no changes in HFr-fed rats, compared to Control. On the contrary, only HFr-fed rats developed dyslipidemia as characterized by higher levels of plasma triglycerides compared to all other groups (p < 0.0001). Hepatic dysfunction and inflammation was confirmed in HFD-fed rats by elevated levels of hepatic MCP-1 (p < 0.0001), TNF-alpha (p < 0.001) and plasma β-hydroxybutyrate (p < 0.0001), and in NASH-fed rats by elevated levels of hepatic MCP-1 (p < 0.01), increased hepatic macrophage infiltration (p < 0.001), and higher plasma levels of alanine aminotransferase (p < 0.0001) aspartate aminotransferase (p < 0.05), haptoglobin (p < 0.001) and TIMP-1 (p < 0.01) compared to Control.ConclusionThese findings show that dietary fat and cholesterol are the primary drivers of NAFLD development and progression in rats, while fructose mostly exerts its effect on the circulating lipid pool.
Obesity and type 2 diabetes are associated with an increased risk for development of certain forms of cancer, including colon cancer. The publication of highly controversial epidemiological studies in 2009 raised the possibility that use of the insulin analog glargine increases this risk further. However, it is not clear how mitogenic effects of insulin and insulin analogs measured in vitro correlate with tumor growth-promoting effects in vivo. The aim of this study was to examine possible growth-promoting effects of native human insulin, insulin X10 and IGF-1, which are considered positive controls in vitro, in a short-term animal model of an obesity- and diabetes-relevant cancer. We characterized insulin and IGF-1 receptor expression and the response to treatment with insulin, X10 and IGF-1 in the murine colon cancer cell line (MC38 cells) in vitro and in vivo. Furthermore, we examined pharmacokinetics and pharmacodynamics and monitored growth of MC38 cell allografts in mice with diet-induced obesity treated with human insulin, X10 and IGF-1. Treatment with X10 and IGF-1 significantly increased growth of MC38 cell allografts in mice with diet-induced obesity and we can therefore conclude that supra-pharmacological doses of the insulin analog X10, which is super-mitogenic in vitro and increased the incidence of mammary tumors in female rats in a 12-month toxicity study, also increase growth of tumor allografts in a short-term animal model.
Using the HF/HS-STZ rat as an animal model for type 2 diabetes, we find that insulin therapy modulates fat distribution. Specifically, our data show that insulin has a relatively positive effect on CVD-associated parameters, including abdominal fat distribution, lean body mass, adiponectin, TGs and HDL in HF/HS-STZ rats, despite a modest gain in weight.
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