Differential Actions of Chlorhexidine on the Cell Wall of Bacillus subtilis and Escherichia coli

Chlorhexidine is a chlorinated phenolic disinfectant used commonly in mouthwash for its action against bacteria. However, a comparative study of the action of chlorhexidine on the cell morphology of Gram-positive and Gram-negative bacteria is lacking. In this study, the actions of chlorhexidine on the cell morphology were identified with the aids of electron microscopy. After exposure to chlorhexidine, numerous spots of indentation on the cell wall were found in both Bacillus subtilis and Escherichia coli. The number of indentation spots increased with time of incubation and increasing chlorhexidine concentration. Interestingly, the dented spots found in B. subtilis appeared mainly at the hemispherical caps of the cells, while in E. coli the dented spots were found all over the cells. After being exposed to chlorhexidine for a prolonged period, leakage of cellular contents and subsequent ghost cells were observed, especially from B subtilis. By using 2-D gel/MS-MS analysis, five proteins related to purine nucleoside interconversion and metabolism were preferentially induced in the cell wall of E. coli, while three proteins related to stress response and four others in amino acid biosynthesis were up-regulated in the cell wall materials of B. subtilis. The localized morphological damages together with the biochemical and protein analysis of the chlorhexidine-treated cells suggest that chlorhexidine may act on the differentially distributed lipids in the cell membranes/wall of B. subtilis and E. coli

Biodegradation of phenoxyacetic acid in soil by Pseudomonas putida PP0301(pR0103), a constitutive degrader of 2, 4–dichlorophenoxyacetate

The efficacy of using genetically engineered microbes (GEMs) to degrade recalcitrant environmental toxicants was demonstrated by the application of Pseudomonas putida PP0301(pR0103) to an Oregon agricultural soil amended with 500 u.g/g of a model xenobiotic, phenoxyacetic acid (PAA). P. putida PP0301(pR0103) is a constitutive degrader of 2, 4–dichlorophenoxyacetate (2, 4–D) and is also active on the non–inducing substrate, PAA. PAA is the parental compound of 2, 4–dichlorophenoxyacetic acid (2, 4–D) and whilst the indigenous soil microbiota degraded 500 ng/g 2, 4–D to less than 10 J–g/g, PAA degradation was insignificant during a 40–day period. No significant degradation of PAA occurred in soil inoculated with the parental strain P. putida PP0301 or the inducible 2, 4–D degrader P. putida PP0301(pR0101). Moreover, co–amendment of soil with 2, 4–D and PAA induced the microbiota to degrade 2, 4–D; PAA was not degraded. P. putida PP0301–(pR0103) mineralized 500–Μg/g PAA to trace levels within 13 days and relieved phytotoxicity of PAA to Raphanus sativus (radish) seeds with 100% germination in the presence of the GEM and 7% germination in its absence. In unamended soil, survival of the plasmid–free parental strain P. putida PP0301 was similar to the survival of the GEM strain P. putida PP0301(pR0103). However, in PAA amended soil, survival of the parent strain was over 10 000–fold lower (< 3 colony forming units per gram of soil) than survival of the GEM strain after 39 days.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75471/1/j.1365-294X.1992.tb00160.x.pd

Conditionally immortalized brain capillary endothelial cell lines established from a transgenic mouse harboring temperature-sensitive simian virus 40 large T-antigen gene

Five immortalized brain capillary endothelial cell lines (TM-BBB1-5) were established from 3 transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene (Tg mouse). These cell lines expressed active large T-antigen and grew well at 33°C with a doubling time of about 20 to 30 hours. TM-BBBs also grew at 37°C but not at 39°C. However, growth was restored when the temperature of the culture was lowered to 33°C Although significant amounts of large T-antigen were shown to be present in the cell culture at 33°C, there was less of this complex at 37°C and 39°C. TM-BBBs expressed the typical endothelial marker, von Willberand factor and exhibited acetylated lowdensity lipoprotein uptake activity. The alkaline phosphatase and γ-glutamyltranspeptidase activity in TM-BBBs were −10% and 50% to 80% of brain capillary fraction of normal mice, respectively. D-Mannitol transport in the both apical-to-basal and basal-to-apical directions across the TM-BBB was 2-fold greater than for inulin. TM-BBBs were found to express GLUT-1 but not GLUT-3, and exhibited concentration-dependent 3-O-methyl-D-glucose (3-OMG) uptake activity with a Michaelis-Menten constant of 6.59±1.16 mmol/1. Moreover, P-glycoprotein (P-gp) with a molecular weight of −170 kDa was expressed in all TM-BBBs. Both mdr 1a and mdr 1b mRNA were detected in TM-BBB4 using reverse transcription-polymerase chain reaction (RT-PCR) analysis. [3H]-Cyclosporin A uptake by TM-BBB was significantly increased in the presence of 100 μmol/1 verapamil and vincristine, suggesting that TM-BBB exhibits efflux transport activity via P-gp In conclusion, conditional brain capillary endothelial cell lines were established from Tg mice. This cell line expresses endothelial markers and transporters at the BBB and is able to regulate cell growth, due to the amount of active large T-antigen in the cell, by changing the culture temperature