Surface roughness mediated adhesion forces between borosilicate glass and gram-positive bacteria.

It is well-known that a number of surface characteristics affect the extent of adhesion between two adjacent materials. One of such parameters is the surface roughness as surface asperities at the nanoscale level govern the overall adhesive forces. For example, the extent of bacterial adhesion is determined by the surface topography; also, once a bacteria colonizes a surface, proliferation of that species will take place and a biofilm may form, increasing the resistance of bacterial cells to removal. In this study, borosilicate glass was employed with varying surface roughness and coated with bovine serum albumin (BSA) in order to replicate the protein layer that covers orthopedic devices on implantation. As roughness is a scale-dependent process, relevant scan areas were analyzed using atomic force microscope (AFM) to determine Ra; furthermore, appropriate bacterial species were attached to the tip to measure the adhesion forces between cells and substrates. The bacterial species chosen (Staphylococci and Streptococci) are common pathogens associated with a number of implant related infections that are detrimental to the biomedical devices and patients. Correlation between adhesion forces and surface roughness (Ra) was generally better when the surface roughness was measured through scanned areas with size (2 × 2 µm) comparable to bacteria cells. Furthermore, the BSA coating altered the surface roughness without correlation with the initial values of such parameter; therefore, better correlations were found between adhesion forces and BSA-coated surfaces when actual surface roughness was used instead of the initial (nominal) values. It was also found that BSA induced a more hydrophilic and electron donor characteristic to the surfaces; in agreement with increasing adhesion forces of hydrophilic bacteria (as determined through microbial adhesion to solvents test) on BSA-coated substrates.

Escherichia coli uropathogenic-specific protein, Usp, is a bacteriocin-like genotoxin.

BACKGROUND: Bacterial genotoxins provoke DNA damage and carcinogenesis. The Escherichia coli uropathogenic-specific protein gene, usp, and its linked genes, imu1-3, are associated with strains from pyelonephritis, prostatitis, and bacteremia of urinary tract origin. While the Usp C-terminal domain exhibits similarity with DNase-like colicins and pyocins, its role and mechanisms of action, as well as those of the 3 associated proteins, is unknown. METHODS: We isolated Usp and Imu1-3 and examined their activity on plasmid DNA, human umbilical vein endothelial cells, and human embryonic kidney cells (cell line HEK293). The affect of Usp and Imu1-3 was assessed by MTT and Comet assays, infection assays, caspase 3/7 activity, fluorescently labeled actin staining, and Western blotting. RESULTS: Usp possesses DNase activity and, particularly when coapplied with Imu2, exhibits genotoxic activity in mammalian cells. Infection assays demonstrated that E. coli usp(+) imu1-3(+) affects the viability of mammalian cells, induces increased caspase 3/7 activity, and perturbs cell cytoskeleton structure. CONCLUSIONS: Usp is a novel E. coli genotoxin active against mammalian cells. Optimal in vivo activity of Usp requires Imu2. Infection with E. coli usp(+) imu1-3(+) induces a response characteristic of apoptosis.

DMSO modulates the pathway of apoptosis triggering.

We demonstrate here that distribution of caspase-9 influences the pathway of apoptosis triggering, since caspase-9 is activated efficiently only when it is distributed solely in the cytosol. Caspase-9 moves to the nuclei in a response to cell stress during isolation of primary hepatocytes; this is called preapoptotic cell stress response. The dimethyl sulfoxide (DMSO) treatment cannot prevent the migration of caspase-9 into the nuclei when it is added to primary hepatocytes immediately after isolation; however, it can trigger redistribution of caspase-9 from the nuclei into the cytosol when added 1 day post-isolation. This redistribution is temporary, since caspase-9 returns to the nuclei within 48 hours of DMSO treatment. Thereafter, some caspase-9 is retained in the nuclei of DMSO-treated hepatocytes for longer than in the nuclei of untreated hepatocytes. By measuring caspase activities, we demonstrate that the addition of DMSO to cell culture medium can temporarily normalize the susceptibility of hepatocytes for apoptosis triggering through the intrinsic pathway. DMSO contributes also to the prolonged pathway inactivation, i.e., by extending preapoptotic cell stress response. We propose that DMSO extends the survival of primary hepatocytes by modulating preapoptotic cell stress response, which could be exploited for extending the lifespan of other primary cell cultures.

Preapoptotic cell stress response of primary hepatocytes.

UNLABELLED: Primary hepatocytes are an important in vitro model for studying metabolism in man. Caspase-9 and Bcl-2-associated X protein (Bax) are regulators of the apoptotic pathway. Here we report on the translocation of procaspase-9 and Bax from cytoplasm to nuclei as well as on dispersion of mitochondria; these processes occur after isolation of primary hepatocytes. The observed changes appear similar to those at the beginning of apoptosis; however, the isolated hepatocytes are not apoptotic for the following reasons: (1) cells have a normal morphology and function; (2) the mitochondria are energized; (3) there is no apoptosis unless it is induced by, e.g., staurosporine or nodularin. Staurosporine does not trigger apoptosis through activation of caspase-9, as its activity is detected later than that of caspase-3. We propose that the translocation of procaspase-9 and Bax into the nuclei reduces the ability to trigger apoptosis through the intrinsic apoptotic pathway. The shifts of procaspase-9 and Bax are reversible in the absence of the apoptotic trigger; the spontaneous reversion was confirmed experimentally for procaspase-9, whereas Bax shifted from the nuclei to the cytosol and mitochondria after the initiation of apoptosis. To distinguish this process from apoptosis, we call it preapoptotic cell stress response. It shares some features with apoptosis; however, it is reversible and apoptosis has to be induced in addition to this process. CONCLUSION: Knowledge on preapoptotic cell stress response is important for assessing the quality of the cells used in cell therapies, in regenerative medicine, and of those used for modeling metabolic processes.

The riddle of mitochondrial caspase-3 from liver.

Caspase-3 is one of the main executors of apoptosis. Its zymogen procaspase-3 was localized to cytosol, mitochondria and nuclei. The subcellular location of procaspase-3 in liver was reported by several studies to be either cytosolic or cytosolic and mitochondrial. Our aim was to investigate these separate procaspase-3 pools to differentiate the pathways of their activation. By cell fractionation, immunocytochemistry, and confocal microscopy we report that there is a single procaspase-3 pool located to the cytosol in primary hepatocytes and in fractions of rat liver. In contrast, it depends on the isolation purity whether procaspase-3 is located in mitochondria of non-parenchymal liver cells, or not. All preparations with mitochondrial procaspase-3 fractions contain traces of haemoglobin, indicating the presence of some erythrocytes, which are the source of mitochondrial procaspase-3. Since erythrocytes migrate with mitochondria in subcellular fractionations, it is important to check for haemoglobin, before localizing the protein to mitochondria.