Design,synthesis and evaluation of a series of 5-methoxy-2,3-naphthalimide derivatives as AcrB inhibitors for the reversal of bacterial resistance

A series of novel 5-methoxy-2,3-naphthalimide derivatives were designed, synthesized and evaluated for their biological activities. In particular, the ability of the compounds to synergize with antimicrobials, to inhibit Nile Red efflux, and to target AcrB was assayed. The results showed that the most of the tested compounds more sensitized the Escherichia coli BW25113 to the antibiotics than the parent compounds 7c and 15, which were able to inhibit Nile Red efflux. Significantly, compound A5 possessed the most potent antibacterial synergizing activity in combination with levofloxacin by 4 times and 16 times at the concentration of 8 and 16?µg/mL, respectively, whilst A5 could effectively abolish Nile Red efflux at 100?μM. Additionally, target effect of A5 was confirmed in the outer- or inner membrane permeabilization assays. Therefore, A5 is an excellent lead compound for further structural optimization.     

Global Expanded Nutrient Supply (GENuS) Model: A New Method for Estimating the Global Dietary Supply of Nutrients

Insufficient data exist for accurate estimation of global nutrient supplies. Commonly used global datasets contain key weaknesses: 1) data with global coverage, such as the FAO food balance sheets, lack specific information about many individual foods and no information on micronutrient supplies nor heterogeneity among subnational populations, while 2) household surveys provide a closer approximation of consumption, but are often not nationally representative, do not commonly capture many foods consumed outside of the home, and only provide adequate information for a few select populations. Here, we attempt to improve upon these datasets by constructing a new model—the Global Expanded Nutrient Supply (GENuS) model—to estimate nutrient availabilities for 23 individual nutrients across 225 food categories for thirty-four age-sex groups in nearly all countries. Furthermore, the model provides historical trends in dietary nutritional supplies at the national level using data from 1961–2011. We determine supplies of edible food by expanding the food balance sheet data using FAO production and trade data to increase food supply estimates from 98 to 221 food groups, and then estimate the proportion of major cereals being processed to flours to increase to 225. Next, we estimate intake among twenty-six demographic groups (ages 20+, both sexes) in each country by using data taken from the Global Dietary Database, which uses nationally representative surveys to relate national averages of food consumption to individual age and sex-groups; for children and adolescents where GDD data does not yet exist, average calorie-adjusted amounts are assumed. Finally, we match food supplies with nutrient densities from regional food composition tables to estimate nutrient supplies, running Monte Carlo simulations to find the range of potential nutrient supplies provided by the diet. To validate our new method, we compare the GENuS estimates of nutrient supplies against independent estimates by the USDA for historical US nutrition and find very good agreement for 21 of 23 nutrients, though sodium and dietary fiber will require further improvement.     

UV-mediated regulation of the anti-senescence factor Tbx2

Several lines of evidence have implicated members of the developmentally important T-box gene family in cell cycle regulation and in cancer. Importantly, the highly related T-box factors Tbx2 and Tbx3 can suppress senescence through repressing the cyclin-dependent kinase inhibitors p19(ARF) and p21(WAF1/CIP1/SDII). Furthermore, Tbx2 is up-regulated in several cancers, including melanomas where it was shown to function as an anti-senescence factor, suggesting that this may be one of the mechanisms by which T-box proteins contribute to the oncogenic process. However, very little is known about whether Tbx2 is regulated by p21-mediated stress-induced senescence signaling pathways. In this study, using the MCF-7 breast cancer cell line known to overexpress Tbx2, we show that in response to stress induced by ultraviolet irradiation the Tbx2 protein is specifically phosphorylated by the p38 mitogen-activated protein kinase. Using site-directed mutagenesis and in vitro kinase assays, we have identified serine residues 336, 623, and 675 in the Tbx2 protein as the p38 target sites and show that these sites are phosphorylated in vivo. Importantly, we show by Western blotting, immunofluorescence, and reporter assays that this phosphorylation leads to increased Tbx2 protein levels, predominant nuclear localization of the protein, and an increase in the ability of Tbx2 to repress the p21(WAF1/CIP1/SDII) promoter. These results show for the first time that the ability of Tbx2 to repress the p21 gene is enhanced in response to a stress-induced senescence pathway, which leads to a better understanding of the regulation of the anti-senescence function of Tbx2.     

Expression of catalase mRNA and protein in adult rat brain: detection by nonradioactive in situ hybridization with signal amplification by catalyzed reporter deposition (ISH-CARD) and immunohistochemistry (IHC)/immunofluorescence (IF).

Catalase, the classical peroxisomal marker enzyme, decomposes hydrogen peroxide and is involved in the antioxidant defense mechanisms of mammalian cells. In addition, catalase can oxidize, by means of its peroxidatic activity, a variety of substrates such as methanol and ethanol, producing the corresponding aldehydes. The involvement of brain catalase in the oxidation of ethanol is well established, and severe afflictions of the CNS in hereditary peroxisomal diseases (e.g., Zellweger syndrome) are well known. Whereas the distribution of catalase in the CNS has been investigated by enzyme histochemistry and immunohistochemistry (IHC), very little is known about the exact localization of catalase mRNA in brain. Here we report the application of a tyramine/CARD (catalyzed reporter deposition)-enhanced nonradioactive in situ hybridization (ISH) protocol for detection of catalase mRNA in sections of perfusion-fixed, paraffin-embedded rat brain. Catalase mRNA could be demonstrated in a large number of neurons throughout the rat brain as a distinct cytoplasmic staining signal with excellent morphological resolution. Compared to our standard ISH protocol, the CARD-enhanced protocol for catalase mRNA detection in rat brain showed higher sensitivity and significantly better signal-to-noise ratio. In parallel IHC experiments, using an antigen retrieval method consisting of combined trypsin digestion and microwave treatment of paraffin sections, the catalase antigen was found as distinct cytoplasmic granules in most catalase mRNA-positive neurons. In addition, catalase-positive granules, presumably peroxisomes, were found by confocal laser scanning microscopy in glial cells, which were identified by double labeling immunofluorescence for GFAP and CNPase for astroglial cells and oligodentrocytes, respectively. The excellent preservation of morphology and sensitive detection of both mRNA and protein in our preparations warrant the application of the protocols described here for systematic studies of catalase and other peroxisomal proteins in diverse pathological conditions such as Alzheimer's disease and aging.     

Differential cellular and subcellular localization of heme-binding protein 23/peroxiredoxin I and heme oxygenase-1 in rat liver.

Heme-binding protein 23 (HBP23), also termed peroxiredoxin (Prx) I, and heme oxygenase-1 (HO-1) are distinct antioxidant stress proteins that are co-ordinately induced by oxidative stress. HBP23/Prx I has thioredoxin-dependent peroxidase activity with high binding affinity for the pro-oxidant heme, while HO-1 is the inducible isoform of the rate-limiting enzyme of heme degradation. We investigated the cellular and subcellular localization of both proteins in rat liver. Whereas by immunohistochemistry (IHC) a uniformly high level of HBP23/Prx I expression was observed in liver parenchymal and different sinusoidal cells, HO-1 expression was restricted to Kupffer cells. By immunoelectron microscopy using the protein A-gold technique, HBP23/Prx I immunoreactivity was detected in cytoplasm, nuclear matrix, mitochondria, and peroxisomes of parenchymal and non-parenchymal liver cell populations. In contrast, the secretory pathway, i.e., the endoplasmic reticulum and Golgi complex, was free of label. As determined by immunocytochemical (ICC) studies in liver cell cultures and by Western and Northern blotting analysis, HBP23/Prx I was highly expressed in cultures of isolated hepatocytes and Kupffer cells. In contrast, HO-1 was constitutively expressed only in Kupffer cell cultures but was also inducible in hepatocytes. These data suggest that HBP23/Prx I and HO-1 may have complementary antioxidant functions in different cell populations in rat liver.     

Degradation of excess peroxisomes in mammalian liver cells by autophagy and other mechanisms

Here we discuss the mechanisms for the degradation of excess peroxisomes in mammalian hepatocytes which include (a) autophagy, (b) the action of peroxisomal Lon protease and (c) the membrane disrupting effect of 15-lipoxygenase. A recent study using Atg7 conditional-knock-out mice revealed that 70–80% of excess peroxisomes are degraded by the autophagic process. The remaining 20–30% of excess peroxisomes is most probably degraded by the action of peroxisomal Lon protease. Finally, a selective disruption of the peroxisomal membrane has been shown to be mediated by 15-lipoxygenase activity which is followed by diffusion of matrix proteins into the cytoplasm and cytoplasmic proteolysis. Presented at the 50th Anniversary Symposium of the Society for Histochemistry, Interlaken, Switzerland, October 1–4, 2008.     

Identification of methionine oxidation in human recombinant erythropoietin by mass spectrometry: Comparative isoform distribution and biological activity analysis

Background: Oxidative degradation of human recombinant erythropoietin (hrEPO) may occur in manufacturing process or therapeutic applications. This unfavorable alteration may render EPO inefficient or inactive. We investigated the effect of methionine/54 oxidative changes on the amino acid sequences, glycoform distribution and biological activity of hrEPO. Methods: Mass spectrometry was applied to verify the sequence and determine the methionine oxidation level of hrEPO. Isoform distribution was studied by capillary zone electrophoresis method. In vivo normocythemic mice assay was used to assess the biological activity of three different batches (A, B, and C) of the proteins. Results: Nano-LC/ESI/MS/MS data analyses confirmed the amino acid sequences of all samples. The calculated area percent of three isoforms (2–4 of the 8 obtained isoforms) were decreased in samples of C, B, and A with 27.3, 16.7, and 6.8% of oxidation, respectively. Specific activities were estimated as 53671.54, 95826.47, and 112994.93?mg/mL for the samples of A, B, and C, respectively. Conclusion: The observed decrease in hrEPO biological activity, caused by increasing methionine oxidation levels, was rather independent of its amino acid structure and mainly associated with the higher contents of acidic isoforms.     

The peroxisome: still a mysterious organelle

More than half a century of research on peroxisomes has revealed unique features of this ubiquitous subcellular organelle, which have often been in disagreement with existing dogmas in cell biology. About 50 peroxisomal enzymes have so far been identified, which contribute to several crucial metabolic processes such as β-oxidation of fatty acids, biosynthesis of ether phospholipids and metabolism of reactive oxygen species, and render peroxisomes indispensable for human health and development. It became obvious that peroxisomes are highly dynamic organelles that rapidly assemble, multiply and degrade in response to metabolic needs. However, many aspects of peroxisome biology are still mysterious. This review addresses recent exciting discoveries on the biogenesis, formation and degradation of peroxisomes, on peroxisomal dynamics and division, as well as on the interaction and cross talk of peroxisomes with other subcellular compartments. Furthermore, recent advances on the role of peroxisomes in medicine and in the identification of novel peroxisomal proteins are discussed.     

Knocking-down the expression of nucleostemin significantly decreases rate of proliferation of rat bone marrow stromal stem cells in an apparently p53-independent manner

Abstract.  Objectives : Nucleostemin (NS) is a recently identified GTP-binding protein, predominantly expressed in embryonic and adult stem cells but not in terminally differentiated cells. NS is expressed in bone marrow-derived mesenchymal stem cells, and its expression ceases upon induction of neural differentiation. The major aim of this study was to determine whether down-regulation of NS expression acts as a promoter, or otherwise as a by-product of differentiation and senescence processes. Materials and methods : We used RNA interference protocols to specifically knock down NS in rat bone marrow-derived stromal stem cells. Changes in rate of proliferation and cell cycle profile after knocking-down of NS were measured. In addition, changes in expression of associated genes were studied by semiquantitative RT-PCR, Western blotting and immunocytochemistery. Results : Knocked-down expression of NS caused a significant decrease in the rate of cell proliferation with concomitant shutting off of expression of cyclin D1 and survivin, two other well-known regulators of cell proliferation. Interestingly, we noticed no obvious changes in expression level of p21, the main effector of p53 for its cell cycle repressing function. Conclusion : Our findings revealed a master role for NS in promoting proliferation of rat bone marrow-derived stromal stem cells. Moreover, we suggest that despite previous proposals, the cell cycle arrest/inhibitory role of NS is unlikely to be related to its proposed property of interaction with p53.