Discovery and Validation of Nitroxoline as a Novel STAT3 Inhibitor in Drug-resistant Urothelial Bladder Cancer

Repeated cycles of first-line chemotherapy drugs such as doxorubicin (DOX) and cisplatin (CIS) trigger frequent chemoresistance in recurrent urothelial bladder cancer (UBC). Nitroxoline (NTX), an antibiotic to treat urinary tract infections, has been recently repurposed for cancer treatment. Here we aimed to investigate whether NTX suppresses drug-resistant UBC and its molecular mechanism. The drug-resistant cell lines T24/DOX and T24/CIS were established by continual exposure of parental cell line T24 to DOX and CIS, respectively. T24/DOX and T24/CIS cells were resistant to DOX and CIS, respectively, but they were sensitive to NTX time- and dose-dependently. Overexpressions of STAT3 and P-glycoprotein (P-gp) were identified in T24/DOX and T24/CIS, which could be reversed by NTX. Western blot revealed that NTX downregulated p-STAT3, c-Myc, Cyclin D1, CDK4, CDK6, Bcl-xL, Mcl-1, and Survivin, which were further confirmed by Stattic, a selective STAT3 inhibitor. In vivo, NTX exhibited the significant anti-tumor effect in T24/DOX and T24/CIS tumor-bearing mice. These results suggested that NTX-induced P-gp reversal, G0/G1 arrest, and apoptosis in drug-resistant UBC were mediated by inhibition of STAT3 signaling. Our findings repurpose NTX as a novel STAT3 inhibitor to induce P-gp reversal, G0/G1 arrest, and apoptosis in drug-resistant UBC.     

Prevention of hypercholesterolemia and atherosclerosis in the hyperlipidemia- and atherosclerosis-prone Japanese (LAP) quail by taurine supplementation

The effects of taurine supplementation on the serum cholesterol levels and the progression of atherosclerosis were investigated in the hyperlipidemia- and atherosclerosis-prone Japanese (LAP) quail. The ingestion of a high-cholesterol diet containing 1% cholesterol by LAP quails for 60 days resulted in a marked elevation in serum non-HDL cholesterol and triglyceride, as well as severe aortic lesions with lipid droplets. An immunohistochemical study showed that the lesion consisted of mainly lipid-rich macrophages and T cells. Sixty-day taurine supplementation (1% in drinking tap water) to LAP quails fed high-cholesterol diet containing 1% cholesterol significantly reduced serum non-HDL cholesterol from 4,549 to 2,350 mg/dl. The serum triglyceride level also decreased after taurine supplementation from 703 to 392 mg/dl. Although the HDL cholesterol level significantly decreased due to the high-cholesterol diet, it recovered to the control level fed a regular diet in response to taurine. Bile acid production was stimulated and hepatic cholesterol was reduced by taurine supplementation. A quantitative analysis using aortic cross-sections showed that areas of oil-red O positive lipid accumulation significantly decreased by 74% after taurine supplementation. These results demonstrated the lipid-lowering and anti-atherosclerotic effects of taurine in a diet-induced hyperlipidemic LAP quail model. The prevention of atherosclerosis by taurine is mainly attributed to an improvement in the serum cholesterol and triglyceride levels, which may be related to changes in the hepatic cholesterol metabolism.     

Rapid enumeration of respiratory active mycobacteria with fluorescent double staining

A new method for rapid enumeration of physiologically active mycobacteria was developed by acid-fast bacilli staining (Auramine O) following formazan reduction. Results can be obtained within 90 min by the optimized procedure, while more than one week is required for the widely used culture-dependent approach.     

Aberrant interchain disulfide bridge of tissue-nonspecific alkaline phosphatase with an Arg433-->Cys substitution associated with severe hypophosphatasia

Various mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene are responsible for hypophosphatasia characterized by defective bone and tooth mineralization; however, the underlying molecular mechanisms remain largely to be elucidated. Substitution of an arginine at position 433 with a histidine [TNSALP(R433H)] or a cysteine [TNSALP(R433C)] was reported in patients diagnosed with the mild or severe form of hypophosphatasia, respectively. To define the molecular phenotype of the two TNSALP mutants, we sought to examine them in transient (COS-1) and conditional (CHO-K1 Tet-On) heterologous expression systems. In contrast to an 80 kDa mature form of the wild-type and TNSALP(R433H), a unique disulfide-bonded 160 kDa molecular species appeared on the cell surface of the cells expressing TNSALP(R433C). Sucrose density gradient centrifugation demonstrated that TNSALP(R433C) forms a disulfide-bonded dimer, instead of being noncovalently assembled like the wild-type. Of the five cysteine residues per subunit of the wild-type, only Cys102 is thought to be present in a free form. Replacement of Cys102 with serine did not affect the dimerization state of TNSALP(R433C), implying that TNSALP(R433C) forms a disulfide bridge between the cysteine residues at position 433 on each subunit. Although the cross-linking did not significantly interfere with the intracellular transport and cell surface expression of TNSALP(R433C), it strongly inhibited its alkaline phosphatase activity. This is in contrast to TNSALP(R433H), which shows enzyme activity comparable to that of the wild-type. Importantly, addition of dithiothreitol to the culture medium was found to partially reduce the amount of the cross-linked form in the cells expressing TNSALP(R433C), concomitantly with a significant increase in enzyme activity, suggesting that the cross-link between two subunits distorts the overall structure of the enzyme such that it no longer efficiently carries out its catalytic function. Increased susceptibility to proteases confirmed a gross conformational change of TNSALP(R433C) compared with the wild-type. Thus, loss of function resulting from the interchain disulfide bridge is the molecular basis for the lethal hypophosphatasia associated with TNSALP(R433C).     

Detection of bacteria carrying the stx2 gene by in situ loop-mediated isothermal amplification

A new in situ DNA amplification technique for microscopic detection of bacteria carrying a specific gene is described. Loop-mediated isothermal amplification (LAMP) was used to detect stxA(2) in Escherichia coli O157:H7 cells. The mild permeabilization conditions and low isothermal temperature used in the in situ LAMP method caused less cell damage than in situ PCR. It allowed use of fluorescent antibody labeling in the bacterial mixture after the DNA amplification for identification of E. coli O157:H7 cells with an stxA(2) gene. Higher-contrast images were obtained with this method than with in situ PCR.     

Reaction of substituted phenols with thermostable laccase bound to Bacillus subtilis spores

A strain of B. subtilis produced 1.8 times more laccase on sporulation medium than on non-sporulation medium. Spores oxidized mono- and di-methoxyphenols (0.1 mM) at 50 °C. The half-life of laccase bound to spores was about 2 d and the substrate was repeatedly removed by spores recovered from the reaction mixture.     

Development of an adhesive sheet for direct counting of bacteria on solid surfaces

An adhesive sheet was developed for direct counting of microorganisms on solid surfaces. The sheet consists of a polyurethane film base and water insoluble adhesive. SYBR Green II (for total direct counting) or 6-carboxyfluorescein diacetate (6CFDA) (for fluorescent vital staining) was used for fluorescent microscopy of bacteria collected on the adhesive face of the sheet. Adhesive sheet sampling showed a higher recovery rate for microbial enumeration than conventional swab method or stamp agar. This method is simple, rapid, inexpensive and reproducible.     

Daily spawning and development of sensitivity to gonadotropin and maturation-inducing steroid in the oocytes of the bambooleaf wrasse, Pseudolabrus japonicus

The cycle of oocyte development of the bambooleaf wrasse, Pseudolabrus japonicus, was studied to elucidate the endocrinological mechanism of oocyte maturation in a marine teleost. A single female reared with two males spawned every day for 17 days in captivity, indicating that this species is a daily spawner. Ovarian histology revealed that germinal vesicle migration of the largest oocytes progressed from 12:00 to 3:00 h, and germinal vesicle breakdown (GVBD) was completed at 6:00 h. Ovulation and spawning occurred between 6:00 and 9:00 h. The effectiveness of human chorionic gonadotropin (HCG) and 17,20-dihydroxy-4-pregnen-3-one (17,20-P), which is one of the most potent steroidal inducers of GVBD in bambooleaf wrasse oocytes, in inducing final oocyte maturation was examined at eight different times of the day. The responsiveness of the oocyte to HCG and steroid differed at different times of the day. The GVBD could be induced by HCG but not 17,20-P at 9:00 h. Between 12:00 and 18:00 h, not only HCG but also 17,20-P induced GVBD. Both GVBD and ovulation spontaneously occurred between 0:00 and 6:00 h without any hormonal treatment. These results clearly showed that the oocyte of the bambooleaf wrasse possessed a diurnal maturation cycle. Responsiveness of oocytes to HCG appeared earlier than responsiveness to 17,20-P. This suggests that sensitivity to 17,20 -P is induced by gonadotropic hormone (GTH).     

Activated sludge process performance using a multistage tower aeration tank

This study's objective was to clarify both experimentally and theoretically whether a vertical multistage tower aeration tank system is advantageous as compared with a completely mixed system, particularly with respect to purification efficiency, sludge settleability, and excess sludge production. In comparing the two systems: (1) purification efficiency in the multistage tower aeration system with partial fluid mixing with a large Peclet number was higher than in a corresponding completely mixed system for all applied organic loadings; (2) the multistage tower aeration system had some definite advantages with respect to sludge settleability and excess sludge production; (3) the activated sludge system's higher performance with partial fluid mixing was shown quantitatively with the axial dispersion model in conjunction with growth kinetics which involved rapid uptake such as biosorption and subsequent oxidative biodegradation processes of organic substances. (c) 1993 John Wiley & Sons, Inc.