Sustained release varnish containing chlorhexidine for prevention of Streptococcus mutans biofilm formation on voice prosthesis surface: an in vitro study

International Microbiology - In this study, we aimed to develop a novel, sustained release varnish (SRV) for voice prostheses (VP) releasing chlorhexidine (CHX), for the prevention of biofilm...     

A role for the polyproline domain of p53 in its regulation by Mdm2

The p53 protein plays a key role in the cellular response to stress by inducing cell growth arrest or apoptosis. The polyproline region of p53 has been shown to be important for its growth suppression activity. p53 protein lacking the polyproline region has impaired apoptotic activity and altered specificity for certain apoptotic target genes. Here we describe the role of this region in the regulation of p53 by its inhibitor Mdm2. p53 lacking the polyproline region was identified to be more susceptible to inhibition by Mdm2. Furthermore, the absence of this region renders p53 more accessible to ubiquitination, nuclear export, and Mdm2-mediated degradation. This increased sensitivity to Mdm2 results from an enhanced affinity of Mdm2 toward p53 lacking the polyproline region. Our results provide a new explanation for the impaired growth suppression activity of p53 lacking this region. The polyproline region is proposed to be important in the modulation of the inhibitory effects of Mdm2 on p53 activities and stability.     

The promyelocytic leukemia protein protects p53 from Mdm2-mediated inhibition and degradation

The p53 protein is kept labile under normal conditions. This regulation is governed largely by its major negative regulator, Mdm2. In response to stress however, p53 accumulates and becomes activated. For this to occur, the inhibitory effects of Mdm2 have to be neutralized. Here we investigated the role of the promyelocytic leukemia protein (PML) in the activation of p53 in response to stress. We found that PML is critical for the accumulation of p53 in response to DNA damage under physiological conditions. PML protects p53 from Mdm2-mediated ubiquitination and degradation, and from inhibition of apoptosis. PML neutralizes the inhibitory effects of Mdm2 by prolonging the stress-induced phosphorylation of p53 on serine 20, a site of the checkpoint kinase 2 (Chk2). PML recruits Chk2 and p53 into the PML nuclear bodies and enhances p53/Chk2 interaction. Our results provide a novel mechanistic explanation for the cooperation between PML and p53 in response to DNA damage.     

Polyene and cytokine treatment of experimental aspergillosis

The aim of the present study was to assess the efficacy of amphotericin B (AMB), AMB-intralipid admixture (AMB-IL) or nystatin-IL formulation (Ny-IL) in combination with granulocyte colony stimulating factor (G-CSF) in treatment of experimental murine aspergillosis. ICR mice were immunosuppressed by cyclophosphamide and 3 days later inoculated intravenously (i.v.) with Aspergillus fumigatus. Treatment was initiated 2 h later and administered for 5 consecutive days (polyenes, i.v.; G-CSF, intraperitoneally). Combination therapy, particularly G-CSF with AMB or AMB-IL, significantly increased the survival rate (up to 87.3%) and prolonged the mean survival time (MST) (up to 28.8 days) in comparison to untreated controls (0% survival, MST 6.7 days) and to treatment with polyenes alone (up to 51.5% survival, MST 18.4 days). These data indicate that combination therapy could be beneficial for management of disseminated aspergillosis in humans.     

Cryptococcus neoformans Overcomes Stress of Azole Drugs by Formation of Disomy in Specific Multiple Chromosomes

Cryptococcus neoformans is a haploid environmental organism and the major cause of fungal meningoencephalitis in AIDS patients. Fluconazole (FLC), a triazole, is widely used for the maintenance therapy of cryptococcosis. Heteroresistance to FLC, an adaptive mode of azole resistance, was associated with FLC therapy failure cases but the mechanism underlying the resistance was unknown. We used comparative genome hybridization and quantitative real-time PCR in order to show that C. neoformans adapts to high concentrations of FLC by duplication of multiple chromosomes. Formation of disomic chromosomes in response to FLC stress was observed in both serotype A and D strains. Strains that adapted to FLC concentrations higher than their minimal inhibitory concentration (MIC) contained disomies of chromosome 1 and stepwise exposure to even higher drug concentrations induced additional duplications of several other specific chromosomes. The number of disomic chromosomes in each resistant strain directly correlated with the concentration of FLC tolerated by each strain. Upon removal of the drug pressure, strains that had adapted to high concentrations of FLC returned to their original level of susceptibility by initially losing the extra copy of chromosome 1 followed by loss of the extra copies of the remaining disomic chromosomes. The duplication of chromosome 1 was closely associated with two of its resident genes: ERG11, the target of FLC and AFR1, the major transporter of azoles in C. neoformans. This adaptive mechanism in C. neoformans may play an important role in FLC therapy failure of cryptococcosis leading to relapse during azole maintenance therapy.     

The unique killing of embryo-derived teratocarcinoma cells by nonactivated murine macrophages is not due to a lack of H-2 antigen expression.

It is well documented that activated macrophages, but not nonactivated ones, kill tumor cells in vitro without damaging normal cells. We, however, have previously shown that embryo-derived teratocarcinoma cells (F9, P19, PCC4) are efficiently killed by nonactivated macrophages as well as by activated ones. Whereas other tumor cells are killed extracellularly by macrophages, we found that F9 teratocarcinoma cells are phagocytosed alive by macrophages and subsequently killed intracellularly by a process dependent on intact lysosomal function. Neither the H-2 antigens nor the mRNAs for the alpha-chain and beta 2-microglobulin are detectable in embryo-derived teratocarcinoma cells. An obvious explanation for this unique killing is that the nonactivated macrophages recognize and kill these cells due to their lack of class I MHC antigen expression, assuming that class I MHC gene products on the target cells switch off the cytolytic machinery of nonactivated macrophages. Our present findings demonstrate that there is no correlation between H-2 antigen expression on tumor cells and their susceptibility to killing by macrophages. Retinoic acid-differentiated F9 cells and P19 cells expressing H-2 antigen after exposure to MAF (IFN-gamma) were sensitive to the killing by nonactivated macrophages. Hybrids that arose from fusion of P19 teratocarcinoma cells with embryonal normal fibroblasts (C57BL/6), which displayed the morphology of embryonal carcinoma stem cells and expressed H-2 antigens, were also sensitive to the killing by nonactivated macrophages. On the other hand, the H-2-negative testicular 402AX teratocarcinoma cells and K1735P melanoma cells were both resistant to the killing by nonactivated macrophages. We concluded that the unique killing of embryo-derived teratocarcinoma cells by nonactivated murine macrophages is not related to a lack of H-2 antigen expression.     

c-Abl regulates p53 levels under normal and stress conditions by preventing its nuclear export and ubiquitination

The p53 protein is subject to Mdm2-mediated degradation by the ubiquitin-proteasome pathway. This degradation requires interaction between p53 and Mdm2 and the subsequent ubiquitination and nuclear export of p53. Exposure of cells to DNA damage results in the stabilization of the p53 protein in the nucleus. However, the underlying mechanism of this effect is poorly defined. Here we demonstrate a key role for c-Abl in the nuclear accumulation of endogenous p53 in cells exposed to DNA damage. This effect of c-Abl is achieved by preventing the ubiquitination and nuclear export of p53 by Mdm2, or by human papillomavirus E6. c-Abl null cells fail to accumulate p53 efficiently following DNA damage. Reconstitution of these cells with physiological levels of c-Abl is sufficient to promote the normal response of p53 to DNA damage via nuclear retention. Our results help to explain how p53 is accumulated in the nucleus in response to DNA damage.     

Differential regulation of FBXW7 isoforms by various stress stimuli

Fbxw7 is a tumor suppressor mutated in a wide range of human cancers. It serves as the substrate recognition component of SCF E3 ubiquitin ligases, and intensive effort was made to identify its substrates. Some of the substrates are central regulators of the cell cycle, cell fate determination, and cellular survival. Unlike the many efforts aimed at identifying novel targets, little is known about the regulation of Fbw7 isoform expression. In this study, we examined the mRNA expression of different FBXW7 isoforms during the cell cycle and after exposure to various stress stimuli. We observed that Fbw7β is induced by all the stress stimuli tested, mostly, but not exclusively, in a p53-dependent manner. In fact, FBXW7β was found to be the most potently induced p53 target gene in HCT-116 cells. Expression of FBXWα and γ is p53-independent and their responsiveness to most stress stimuli is limited. Furthermore, their pattern of stress responsiveness is very different from that of the β isoform. Under certain conditions, the same genotoxic agent stimulates induction of β and repression of α. Analysis of FACS-sorted cells in specific phases of the cell cycle by using fluorescent ubiquitination-based cell cycle indicator (FUCCI), showed a significant repression of the γ isoform during the S phase of normal cycling HCT-116 cells. Altogether, this study suggests differential regulation of the 3 Fbw7 isoforms.