International journal of biochemistry and molecular biology最新文献

Bacillus cereus can cause endophthalmitis through secretion of virulence factors, including hemolysin BL (Hbl) and nonhemolytic entertoxin (Nhe). Carvacrol is an extract from oregano oil, with potential for curtailing B. cereus endophthalmitis, due to antimicrobial and anti-inflammatory qualities. However, sublethal levels of carvacrol increases B. cereus virulence. The goal of this study was to investigate the increase in B. cereus virulence potential in response stress induced by a subinhibitory concentration (SIC) of carvacrol. Enterotoxin production and tissue damage were examined during ocular infections in vitro and in vivo. We hypothesized that the SIC of carvacrol would significantly increase toxin production in B. cereus without progressing systemically. RT-PCR determined SIC carvacrol-treated B. cereus had significantly higher hblC and nheA mRNA expression levels than controls in vitro. ELISA and RPLA analysis revealed a 46.8% and 50% increase in NheA and HblC toxin levels, respectively, in SIC-treated cultures. Caenorhabditis elegans-fed SIC carvacrol-treated B. cereus had a significantly higher mean mortality rate than nematodes fed untreated B. cereus. Significantly higher TNF-α levels were observed in SIC carvacrol-treated B. cereus mice compared to other treatment groups except for mice infected with B. cereus alone. Significantly higher IL-6 levels were also found in SIC-B. cereus mice. Histological analysis using Rose-Bengal and DAPI determined that the eyes of mice infected with SIC carvacrol-treated B. cereus had significantly more damage than eyes treated with B. cereus alone. The SIC of carvacrol increased B. cereus virulence in vitro and in vivo, with a mild systemic infection noted.

Genome editing is a powerful tool to modify a specific gene and to correct a disease-causing mutation. Recently developed new techniques, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9), significantly facilitate the progression in this field. However, mutations associated with the double strand DNA breaks (DSBs) introduced by these systems hampered their direct usage in clinic. In order to prevent the mutations caused by DSBs, we have designed a novel mean to induce homology-directed recombination (HDR) without DSBs, i.e., the fusion protein of RecA with cell-penetrating peptide (CPP). The involvement of RecA in these fusion proteins will play important roles in formation of the nucleoprotein filament with single strand DNA (ssDNA) in vitro and promoting HDR in vivo; whereas the involvement of CPP in these fusion proteins will mainly play a role in facilitating cellular intake/uptake of the nucleoprotein filaments. Our results indicated that certain amount of the fusion proteins expressed in bacteria is in soluble fraction, whereas majority of the fusion proteins expressed in baby hamster kidney (BHK) cells is in soluble fraction. Interestingly, expression of these fusion proteins in bacteria completely blocked cell growth, whereas expression of them in BHK cells significantly inhibited cell growth, implying that these fusion proteins may bind to ssDNA regions, such as ssDNA regions in DNA replication forks, and inhibit cell growth. These results suggest that we have functional RecA.CPP fusion proteins ready to test our novel idea of inducing HDR without DSB.

Environmental exposure to paraquat has been reported to be associated with Parkinson's disease (PD). In experimental animal models paraquat reproduces features of PD, however, the exact mechanism of PD-induced neurotoxicity has not been fully established. This study was designed to investigate paraquat-mediated interference with mitochondrial function and autophagy, and determine the impact of the modulation of autophagy flux on paraquat-induced cell toxicity. Rat adrenal pheochromocytoma PC12 cells were treated with paraquat for 24 h to establish a cellular mode of PD induced neurotoxicity. Pre-incubation of PC12 cells with an antioxidant N-acetyl-L-cysteine (NAC) or autophagy modulators rapamycin and chloroquine was conducted to determine the effect of modulation of oxidative status and autophagy flux on paraquat-elicited cytotoxicity. Mitochondrial functions and dynamics were analyzed by measuring oxygen consumption in a high-resolution oxygraph and imaging with a fluorescent mitochondrial dye (MitoTracker). Reactive oxygen species was determined by flow cytometry using fluorescent probe DCFH-DA. Autophagic flux was determined by Western blot analysis of autophagy marker LC3-II as well as p62 expression. It was found that treatment of cells with paraquat caused a concentration-dependent loss of cell viability that was accompanied by a decrease in cell respiration and reduction of polarized mitochondria, which was prevented by pretreatment of cells with NAC. Analysis of autophagy showed that NAC inhibited basic autophagy flux of PC12 cells, as evidenced by a decrease in LC3-II level and an increase in p62 expression. However, this modulation of autophagy by NAC may not be implicated into its cellular protective mechanism over paraquat cytotoxicity as inhibition of autophagy by chloroquine significantly enhanced paraquat induced cytotoxicity. Furthermore, the autophagy inducer rapamycin dramatically decreased paraquat induced cellular toxicity in PC12 cells. The present study demonstrates that basal autophagy plays a protective role in paraquat-induced cell toxicity. Antioxidant NAC confers protective role in paraquat toxicity mainly through maintaining mitochondrial dynamics and function, other than a modulation of autophagy flux.

The acquisition of resistance to anticancer drugs is widely viewed as a key obstacle to successful cancer therapy. However, detailed knowledge of the initial molecular events in the response of cancer cells to these chemotherapeutic and stress responses, and how these lead to the development of chemoresistance, remains incompletely understood. Using microRNA array and washout and rechallenge experiments, we found that short term treatment of leukemia cells with etoposide led a few days later to transient resistance that was associated with a corresponding transient increase in expression of ABCB1 mRNA, as well as microRNA (miR)-135b and miR-196b. This phenomenon was associated with short-term exposure to genotoxic agents, such as etoposide, topotecan, doxorubicin and ionizing radiation, but not agents that do not directly damage DNA. Further, this appeared to be histiotype-specific, and was seen in leukemic cells, but not in cell lines derived from solid tumors. Treatment of leukemic cells with either 5-aza-deoxycytidine or tricostatin A produced similar increased expression of ABCB1, miR-135b, and miR-196b, suggesting a role for epigenetic regulation of this phenomenon. Bioinformatics analyses revealed that CACNA1E, ARHGEF2, PTK2, SIAH1, ARHGAP6, and NME4 may be involved in the initial events in the development of drug resistance following the upregulation of ABCB1, miR-135b and miR-196b. In summary, we report herein that short-term exposure of cells to DNA damaging agents leads to transient drug resistance, which is associated with elevations in ABCB1, miR-135b and miR-196b, and suggests novel components that may be involved in the development of anticancer drug resistance.

Human organic anion transporter 4 (hOAT4) belongs to a family of organic anion transporters which play critical roles in the body disposition of clinically important drugs. hOAT4 is expressed in the kidney and placenta. In the current study, we examined the inhibitory effects of 101 anticancer drugs from a clinical drug library on hOAT4 transport activity. The studies were carried out in hOAT4-expressing human kidney HEK-293 cells and human placenta BeWo cells. Among these drugs, only chlorambucil and cabazitaxel demonstrated more than 50% cis-inhibitory effect on hOAT4-mediated uptake of (3)H-labeled estrone sulfate, a prototypical substrate for the transporter. The IC50 values for chlorambucil and cabazitaxel were 44.28 and 3.5 µM respectively. Dixon plot analysis revealed that inhibition by chlorambucil was competitive with a Ki = 55.73 µM whereas inhibition by cabazitaxel was non-competitive with a Ki = 1.78 µM. Our results demonstrated that chlorambucil and cabazitaxel were inhibitors of hOAT4. Furthermore, by comparing our data with clinically relevant exposures of these drugs, we conclude that the propensity for chlorambucil and cabazitaxel to cause drug-drug interaction through inhibition of hOAT4 is low.

Human organic anion transporter 1 (hOAT1) belongs to a family of organic anion transporters that play critical roles in the body disposition of clinically important drugs, including anti-viral therapeutics, anti-cancer drugs, antibiotics, antihypertensives, and anti-inflammatories. hOAT1 is abundantly expressed in the kidney and brain. In the current study, we examined the regulation of hOAT1 by serum- and glucocorticoid-inducible kinase 2 (sgk2) in the kidney COS-7 cells. We showed that sgk2 stimulated hOAT1 transport activity. Such stimulation mainly resulted from an increased cell surface expression of the transporter, kinetically revealed as an increased maximal transport velocity V max without significant change in substrate-binding affinity K m. We further showed that stimulation of hOAT1 activity by sgk2 was achieved by preventing hOAT1 degradation. Our co-immunoprecipitation experiment revealed that the effect of sgk2 on hOAT1 was through a direct interaction between these two proteins. In conclusion, our study demonstrated that sgk2 stimulates hOAT1 transport activity by enhancing the stability of the transporter. This study provides the insights into sgk2 regulation of hOAT1-mediated transport in normal physiology and disease.

Aspergillus niger is the most commonly used fungus for commercial amylase production, the increase of amylase activity will be beneficial to the amylase industry. Herein we report a high α-amylase producing (HAP) A. niger WLB42 mutated from A. niger A4 by ethyl methanesulfonate treatment. The fermentation conditions for the amylase production were optimized. The results showed that both the amylase activity and total protein content reached highest after 48-h incubation in liquid medium using starch as the sole carbon source. The enzyme production reached maximum at temperature of 30°C, pH 7, with 40 g/L starch in the medium inoculated with 1.4% v/v spore. When 0.3% w/v urea was added to the liquid medium as a nitrogen source, the amylase activity was elevated by 20%. Nine monosaccharides and derivatives were tested for α-amylase induction, glucose was the best inducer. Furthermore, the enzymology characterization of amylase was conducted. The molecular weight of amylase was determined to be 50 kD by SDS-PAGE. The amylase had maximum activity at 45°C and pH 7. The activity could be dramatically triggered by adding 1 mM Co(2+), increased to 250%. The activity was inhibited by detergents SDS and Triton X-100. Six different brands of starch were tested for amylase activity, the results demonstrated that the more soluble of the starch, the higher hydrolyzability of the substrate by amylase.

Interleukin-34 (IL-34) is a cytokine consisting of a 39kD homodimer, shown to be a ligand for both the Macrophage Colony Stimulating Factor (M-CSF/CSF-1) receptor and the Receptor-like protein tyrosine phosphatase-zeta (RPTP-ƺ). IL-34 has been shown to promote monocyte viability and proliferation as well as the differentiation of bone marrow cells into macrophage progenitors. Published work on IL-34 involves its effects on normal hematopoietic and osteoclast progenitors. However, it is not known whether IL-34 has biologic effects in cancer, including leukemia. Here we report that the biological effects of IL-34 include induction of differential expression of Interleukins-1α and -1β as well as induction of differentiation of U937, HL-60 and THP-1 leukemia cell lines demonstrating monocyte-like characteristics. The ability of IL-34 to induce monocytic-like differentiation is supported by strong morphological and functional evidence. Cell surface markers of myeloid lineage, CD64 and CD86, remain constant while the levels of CD11b and CD71 decline with IL-34 treatment. IL-34 also induced increases in CD14 and CD68 expression, further supporting maturation toward monocytic character. IL-34-induced differentiated U937 and THP-1 cell lines exhibited biological functions such as endocytosis and respiratory burst activities. Collectively, we conclude that while IL-34 does not induce cell growth or proliferation, it is able to induce differentiation of leukemia cell lines from monoblastic precursor cells towards monocyte- and macrophage-like cells, mediated through the JAK/STAT and PI3K/Akt pathways. To our knowledge, this is the first report that IL-34 induces differentiation in human leukemic cells, let alone any cancer model.

We have shown that the receptor tyrosine kinase ErbB4 signals neuregulin1-stimulated proliferation of human cells. Some isoforms of ErbB4 are cleaved to release the soluble intracellular domain p80; however, the function of p80 in cell proliferation remained unclear. Here we propose the possibility for p80 as a negative feedback modulator of ErbB4-mediated cell proliferation. Cells exposed to lower doses of neuregulin1 displayed a stimulated proliferation and contained ErbB4 but barely p80. By contrast, cells exposed to its higher doses displayed a suppressed proliferation and contained p80 but barely ErbB4. Analyses with cells overexpressing the p80 wild type and mutants indicated that nuclear p80 inhibits cell proliferation independently of the tyrosine kinase activity. A screen for a novel protein that interacts with p80 identified α-enolase, which is reported as a transcriptional inhibitor for the proliferation-associated c-myc gene. The c-myc mRNA expression was induced by lower doses of neuregulin1 but was suppressed by its higher doses. Subcellular fractionation demonstrated the localization of not only p80 and α-enolase but also the decrease of the functional c-myc amount in the nuclei of cells exposed to higher doses of neuregulin1. These results suggested that p80, which is generated from ErbB4 and translocates to the nuclei, interacts with α-enolase and inhibits neuregulin1-dependent ErbB4-mediated cell proliferation by impairing the c-myc gene transcription.

Ectopic expression of multi-transgenic copies can result in reduced expression of the transgene and can induce silence of endogenous gene; this process is called as co-suppression. Using a transgene-mediated co-suppression technique, we demonstrated the biological function of DNA topoisomerase-1 (top-1) in C. elegans development. Introduction of full-length top-1 transgene sufficiently induced the co-suppression of endogenous top-1 gene, causing embryonic lethality and abnormal germline development. We also found that the co-suppression of top-1 gene affected morphogenesis, lifespan and larval growth that were not observed in top-1 (RNAi) animals. Strikingly, co-suppression effects were significantly reduced by the elimination of top-1 introns, suggesting that efficient co-suppression may require intron(s) in C. elegans. Sequence analysis revealed that the introns 1 and 2 of top-1 gene possess consensus binding sites for several transcription factors, including MAB-3, LIN-14, TTX-3/CEH-10, CEH-1, and CEH-22. Among them, we examined a genetic link between ceh-22 and top-1. The ceh-22 is partially required for the specification of distal tip cells (DTC), which functions as a stem cell niche in the C. elegans gonad. Intriguingly, top-1 (RNAi) significantly enhanced DTC loss in ceh-22 mutant gonads, indicating that top-1 may play an important role in CEH-22-mediated DTC fate specification. Therefore, our findings suggest that transgene-mediated co-suppression facilitates the silencing of the specific genes and the study of gene function in vivo.