A single membrane-embedded negative charge is critical for recognizing positively charged drugs by the Escherichia coli multidrug resistance protein MdfA.
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The nature of the broad substrate specificity phenomenon, as manifested by multidrug resistance proteins, is not yet understood. In the Escherichia coli multidrug transporter, MdfA, the hydrophobicity profile and PhoA fusion analysis have so far identified only one membrane-embedded charged amino acid residue (E26). In order to determine whether this negatively charged residue may play a role in multidrug recognition, we evaluated the expression and function of MdfA constructs mutated at this position. Replacing E26 with the positively charged residue lysine abolished the multidrug resistance activity against positively charged drugs, but retained chloramphenicol efflux and resistance. In contrast, when the negative charge was preserved in a mutant with aspartate instead of E26, chloramphenicol recognition and transport were drastically inhibited; however, the mutant exhibited almost wild-type multidrug resistance activity against lipophilic cations. These results suggest that although the negative charge at position 26 is not essential for active transport, it dictates the multidrug resistance character of MdfA. We show that such a negative charge is also found in other drug resistance transporters, and its possible significance regarding multidrug resistance is discussed. (+info)
H5 Histone and DNA-relaxing enzyme of chicken erythrocytes. Interaction with superhelical DNA.
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The interaction of closed circular duplex DNA with the lysine-rich H5 histone fraction of avian erythrocytes has been studied. H5, like H1 histone, interacts preferentially with superhelical DNA. The extent of interaction increases with increasing negative or positive superhelicity. Salt-extracted lysine-rich histones show the same specificity for interaction with superhelices as do acid-extracted preparations. Chicken erythrocyte nuclei contain DNA-relaxing enzyme. This enzyme is extracted from the nuclei at lower salt concentrations than those required to extract H1 and H5 histones and is, therefore, probably a function of a protein distinct from H1 and H5 histones. (+info)
All 16 centromere DNAs from Saccharomyces cerevisiae show DNA curvature.
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All 16 centromere DNA regions of Saccharomyces cerevisiae including 90 bp framing sequences on either side were cloned. These 300 bp long centromere regions were analysed by native polyacrylamide gel electrophoresis and found to display a reduced mobility indicative of DNA curvature. The degree of curvature is centromere dependent. The experimental data were confirmed by computer analysis of the 3-dimensional structure of the CEN DNAs. Altogether these data provide further evidence for a model for budding yeast centromeres in which CEN DNA structure could be important for the assembly, activity and/or regulation of the centromere protein-DNA complex. (+info)
Conformational state of DNA in chromatin subunits. Circular dichroism, melting, and ethidium bromide binding analysis.
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This study compares some physical properties of DNA in native chromatin and mono-, di-, trinucleosomes obtained after mild micrococcal nuclease digestion. Melting curves and derivatives are shown to be very similar from one sample to another although a shift from 79 to 82 degrees C is observed between the mainly monophasic peak of multimers and chromatin. Careful analysis of the positive band of the circular dichroism spectra shows the appearance of a shoulder at 275nm, the intensity of which increases from the mono- to the di- and trinucleosome. This shoulder is maximum for native chromatin. At the same time binding isotherms of ethidium - bromide are characterized by two highly fluorescent binding sites for all the samples but the product KN of the apparent binding constant of the higher affinity binding sites by the apparent number of those sites increases from the mono- to the di- and trinucleosome. There again the valus is maximum for native chromatin. Such results strongly suggest that the native state of chromatin requires something more than the indefinite repeat of an elementary subunit. (+info)
The effect of mannitol versus dimethyl thiourea at attenuating ischemia/reperfusion-induced injury to skeletal muscle.
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OBJECTIVE: Mannitol is used as a treatment for skeletal muscle ischemia/reperfusion (I/R) injury in humans, despite the fact that its effectiveness in vivo is still disputed. The purpose of this study was to determine the efficacy of mannitol in attenuating I/R injury at the microcirculatory level. METHODS: The study was designed as an experimental study with male Wistar rats. The main outcome measures were intravital microscopy, which was used to measure capillary perfusion, capillary and venular red blood cell velocity (VRBC), and leukocyte-endothelial interactions in the extensor digitorum longus muscle of the rat hind limb before and after ischemia. In addition, tissue injury was assessed during reperfusion with the fluorescent vital dyes bisbenzimide and ethidium bromide. Dimethyl thiourea (DMTU), a highly effective therapeutic agent of experimental I/R injury, was used as a positive control. RESULTS: No-flow ischemia (2 hour) resulted in a 40% drop in capillary perfusion, a decline in capillary and venular VRBC, and increased leukocyte venular adherence and tissue infiltration. Tissue injury increased to a constant level during reperfusion. Mannitol attenuated capillary malperfusion during the first 60 minutes of reperfusion and prevented a decline in capillary VRBC. However, mannitol did not reduce tissue injury or leukocyte adherence and infiltration during reperfusion. By comparison, DMTU not only prevented the perfusion deficits and the increases in leukocyte venular adherence and tissue infiltration but significantly reduced the magnitude of tissue injury. CONCLUSION: Our findings suggest that mannitol may be of limited value for the prevention of early reperfusion-induced injury after no-flow ischemia in skeletal muscle. By comparison, DMTU was highly efficacious by not only reducing microvascular perfusion deficits but by also reducing leukocyte-endothelial cell interactions and the incidence of cellular injury. (+info)
No loss of sst receptors gene expression in advanced stages of colorectal cancer.
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As demonstrated by several studies, the pan-inhibitory peptide somatostatin (SS) is implicated in a large variety of physiological processes in the gastrointestinal tractus. SS inhibits hormonal and gastric acid secretions, and decreases gastric and intestinal motility, mesenteric blood flow and intestinal absorption. In vitro and in vivo studies showed also that the antiproliferative potency of SS analogs may be a target to improve the prognosis of colorectal cancer. Here we report the expression profile of the five SS receptor subtypes (hsst1-5) mRNAs in a large set of tumoral and normal colon. Using reverse transcription-PCR, we showed that hsst5, hsst1 and hsst2 mRNA subtypes were the most frequently expressed hsst mRNA subtypes in normal and pathological colon. Interestingly, we found that the frequency of hsst5 mRNA expression in the left colon was significantly higher in tumors than in normal samples: 81. 2% (13/16) and 36.4% (4/11) respectively (0.025>P>0.01, chi2 test with Yates' correction). We did not find any influence of Dukes' stage on hsst mRNAs expression. Of interest, no loss of hsst2 and hsst5 mRNA expression in advanced stages was noted. Some differences in the frequency of expression of hsst mRNAs according to the origin of the tissue (left or right colon) were evident. The expression of hsst5 and hsst2 mRNA in advanced colorectal carcinoma associated with the development of new SS analogs boost the relevance of colorectal cancer treatment by somatostatin analogs. (+info)
Reactive oxygen species-induced apoptosis and necrosis in bovine corneal endothelial cells.
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PURPOSE: The loss of corneal endothelial cells associated with aging and possibly other causes has been speculated to be related to exposure to reactive oxygen species (ROS). The current study was conducted to investigate, by use of photosensitizers, the underlying mechanisms involved in the death of bovine corneal endothelial cells (BCENs) caused by ROS. METHODS: BCEN cells in primary culture were treated with a photosensitizer (riboflavin or rose bengal) with light exposure. The patterns of cell damage and death were assessed using an acridine orange-ethidium bromide differential staining method, TdT-mediated dUTP nick-end labeling (TUNEL) assay, and transmission electron microscopy. The cytotoxicity was assayed by mitochondrial function using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) testing. Antioxidants, including catalase, L-histidine, salicylic acid, and superoxide dismutase, were used to determine the types of ROS involved. Activation of nuclear factor (NF)-kappaB was examined by fluorescent immunocytochemistry with anti-p65 antibody. RESULTS: Light-irradiated riboflavin or rose bengal resulted in a significant decrease in viability of BCEN cells. Chromosomal condensation and fragmentation were observed in apoptotic cells, and membrane lysis and damage of cell ultrastructures were observed in necrotic cells. Riboflavin induced apoptosis at 30 minutes and thereafter and induced necrosis after 2 hours. Rose bengal was shown to cause similar effects within half the time required for the effects of riboflavin. Catalase and salicylic acid were found to provide protection for BCENs from cytotoxic effects of riboflavin, and L-histidine was found to protect BCENs from cytotoxicity induced by rose bengal. Kinetic studies using immunocytochemistry showed that NF-kappaB was translocated into the nucleus within 15 minutes and 30 minutes after treatment with rose bengal and riboflavin, respectively. CONCLUSIONS: The cytotoxic effects of photo-irradiated riboflavin and rose bengal are shown to be mediated by two distinct but parallel pathways, one leading to apoptosis and the other to necrosis. Possible involvement of NF-kappaB in cell death is suggested. These findings provide potential leads for future investigation into the molecular mechanisms of loss of corneal endothelial cells related to aging, oxidative stress, and possibly other similar causes. (+info)
Differential chemosensitivity of breast cancer cells to ganciclovir treatment following adenovirus-mediated herpes simplex virus thymidine kinase gene transfer.
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The development of resistance to radiation and chemotherapeutic agents that cause DNA damage is a major problem for the treatment of breast and other cancers. The p53 tumor suppressor gene plays a direct role in the signaling of cell cycle arrest and apoptosis in response to DNA damage, and p53 gene mutations have been correlated with increased resistance to DNA-damaging agents. Herpes simplex virus thymidine kinase (HSV-tk) gene transfer followed by ganciclovir (GCV) treatment is a novel tumor ablation strategy that has shown good success in a variety of experimental tumor models. However, GCV cytotoxicity is believed to be mediated by DNA damage-induced apoptosis, and the relationship between p53 gene status, p53-mediated apoptosis, and the sensitivity of human tumors to HSV-tk/GCV treatment has not been firmly established. To address this issue, we compared the therapeutic efficacy of adenovirus-mediated HSV-tk gene transfer and GCV treatment in two human breast cancer cell lines: MCF-7 cells, which express wild-type p53, and MDA-MB-468 cells, which express high levels of a mutant p53 (273 Arg-His). Treating MCF-7 cells with AdHSV-tk/GCV led to the predicted increase in endogenous p53 and p21WAF1/CIP1 protein levels, and apoptosis was observed in a significant proportion of the target cell population. However, treating MDA-MB-468 cells under the same conditions resulted in a much stronger apoptotic response in the absence of induction in p21WAF1/CIP1 protein levels. This latter result suggested that HSV-tk/GCV treatment can activate a strong p53-independent apoptotic response in tumor cells that lack functional p53. To confirm this observation, four additional human breast cancer cell lines expressing mutant p53 were examined. Although a significant degree of variability in GCV chemosensitivity was observed in these cell lines, all displayed a greater reduction in cell viability than MCF-7 or normal mammary cells treated under the same conditions. These results suggest that endogenous p53 status does not correlate with chemosensitivity to HSV-tk/GCV treatment. Furthermore, evidence for a p53-independent apoptotic response serves to extend the potential of this therapeutic strategy to tumors that express mutant p53 and that may have developed resistance to conventional genotoxic agents. (+info)