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An increased pretreatment neutrophil-to-lymphocyte ratio is an independent predictor of recurrence. The combination of T stage and the neutrophil-to-lymphocyte ratio can be used to stratify recurrence risk in patients with nonmetastatic renal cell carcinoma.

It is believed that intracellular carbonic anhydrases (CAs) are essential components of carbon concentrating mechanisms in microalgae. In this study, putative CA-encoding genes were identified in the genome sequences of the marine diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. Subsequently, the subcellular localizations of the encoded proteins were determined. Nine and thirteen CA sequences were found in the genomes of P. tricornutum and T. pseudonana, respectively. Two of the β-CA genes in P. tricornutum corresponded to ptca1 and ptca2 identified previously. Immunostaining transmission electron microscopy of a PtCA1:YFP fusion expressed in the cells of P. tricornutum clearly showed the localization of PtCA1 within the central part of the pyrenoid structure in the chloroplast. Besides these two β-CA genes, P. tricornutum likely contains five α- and two γ-CA genes, whereas T. pseudonana has three α-, five γ-, four δ-, and one ζ-CA genes. Semi-quantitative reverse transcription PCR performed on mRNA from the two diatoms grown in changing light and CO(2) conditions revealed that levels of six putative α- and γ-CA mRNAs in P. tricornutum did not change between cells grown in air-level CO(2) and 5% CO(2). However, mRNA levels of one putative α-CA gene, CA-VII in P. tricornutum, were reduced in the dark compared to that in the light. In T. pseudonana, mRNA accumulation levels of putative α-CA (CA-1), ζ-CA (CA-3) and δ-CA (CA-7) were analyzed and all levels found to be significantly reduced when cells were grown in 0.16% CO(2). Intercellular localizations of eight putative CAs were analyzed by expressing GFP fusion in P. tricornutum and T. pseudonana. In P. tricornutum, CA-I and II localized in the periplastidial compartment, CA-III, VI, VII were found in the chloroplast endoplasmic reticulum, and CA-VIII was localized in the mitochondria. On the other hand, T. pseudonana CA-1 localized in the stroma and CA-3 was found in the periplasm. These results suggest that CAs are constitutively present in the four chloroplastic membrane systems in P. tricornutum and that CO(2) responsive CAs occur in the pyrenoid of P. tricornutum, and in the stroma and periplasm of T. pseudonana.

Summary This study was undertaken to investigate the intracellular induction of reactive oxygen species (ROS) by cis-dichlorodiammineplatinum (CDDP) and the augmentation of their cytotoxicity in bladder cancer cells (KU7) by enhancement of ROS generation by the glutathione (GSH) depletors buthionine sulphoximine (BSO) and diethylmaleate (DEM). CDDP-induced cytotoxicity in KU7 cells and its modulation by GSH depletors were determined using spectrophotometric measurement with crystal violet staining. The effects of GSH depletors on intracellular GSH levels were confirmed using the GSH reductase-DTNB recycling method. Intracellular ROS generation induced by CDDP with or without GSH depletors was estimated from the amount of intracellular dichlorofluorescein (DCF), an oxidized product of dichlorofluorescein (DCFH), which was measured with an anchored cell analysis and sorting system. The cytotoxic effects of CDDP (IC50 15.0 ± 2.5 gIM) were significantly enhanced by BSO (IC50 9.3 ± 2.6 gM, P < 0.01) and DEM (IC50 10.3 ± 0.3 gM, P < 0.01). BSO and DEM produced a significant depletion in intracellular GSH levels (9.6 ± 0.4 nmol 10-6 cells, 17.9 ± 1.0 nmol 10-6 cells) compared with the controls (30.5 ± 0.6 nmol 10-6 cells). Intracellular DCF production in KU7 cells treated with CDDP (1.35 ± 0.33 g1M) was significantly enhanced by the addition of BSO (4.43 ± 0.33 gM) or DEM (3.12 ± 0.22 gM) at 150 min. These results suggest that ROS may play a substantial role in CDDPinduced cytotoxicity and that GSH depletors augment its cytotoxicity through an enhancement of ROS generation in bladder cancer cells.