Hyperforin changes the zinc-storage capacities of brain cells.

In vitro and in vivo experiments were carried out to investigate the consequences on brain cells of a chronic treatment with hyperforin, a plant extract known to dissipate the mitochondrial membrane potential and to release Zn(2+) and Ca(2+) from these organelles. Dissociated cortical neurons were grown in a culture medium supplemented with 1 µM hyperforin. Live-cell imaging experiments with the fluorescent probes FluoZin-3 and Fluo-4 show that a 3 day-hyperforin treatment diminishes the size of the hyperforin-sensitive pools of Ca(2+) and Zn(2+) whereas it increases the size of the DTDP-sensitive pool of Zn(2+) without affecting the ionomycin-sensitive pool of Ca(2+). When assayed by quantitative PCR the levels of mRNA coding for metallothioneins (MTs) I, II and III were increased in cortical neurons after a 3 day-hyperforin treatment. This was prevented by the zinc chelator TPEN, indicating that the plant extract controls the expression of MTs in a zinc-dependent manner. Brains of adult mice who received a daily injection (i.p.) of hyperforin (4 mg/kg/day) for 4 weeks had a higher sulphur content than control animals. They also exhibited an enhanced expression of the genes coding for MTs. However, the long-term treatment did not affect the brain levels of calcium and zinc. Based on these results showing that hyperforin influences the size of the internal pools of Zn(2+), the expression of MTs and the brain cellular sulphur content, it is proposed that hyperforin changes the Zn-storage capacity of brain cells and interferes with their thiol status.

Functional characterization of NRAMP3 and NRAMP4 from the metal hyperaccumulator Thlaspi caerulescens.

The ability of metal hyperaccumulating plants to tolerate and accumulate heavy metals results from adaptations of metal homeostasis. NRAMP metal transporters were found to be highly expressed in some hyperaccumulating plant species. Here, we identified TcNRAMP3 and TcNRAMP4, the closest homologues to AtNRAMP3 and AtNRAMP4 in Thlaspi caerulescens and characterized them by expression analysis, confocal imaging and heterologous expression in yeast and Arabidopsis thaliana. TcNRAMP3 and TcNRAMP4 are expressed at higher levels than their A. thaliana homologues. When expressed in yeast TcNRAMP3 and TcNRAMP4 transport the same metals as their respective A. thaliana orthologues: iron (Fe), manganese (Mn) and cadmium (Cd) but not zinc (Zn) for NRAMP3; Fe, Mn, Cd and Zn for NRAMP4. They also localize at the vacuolar membrane in A. thaliana protoplasts. Inactivation of AtNRAMP3 and AtNRAMP4 in A. thaliana results in strong Cd and Zn hypersensitivity, which is fully rescued by TcNRAMP3 or TcNRAMP4 expression. However, metal tolerance conferred by TcNRAMP expression in nramp3nramp4 mutant does not exceed that of wild-type A. thaliana. Our data indicate that the difference between TcNRAMP3 and TcNRAMP4 and their A. thaliana orthologues does not lie in a different protein function, but probably resides in a different expression level or expression pattern.

Results of the phase II EORTC 22971 trial evaluating combined accelerated external radiation and chemotherapy with 5FU and cisplatin in patients with muscle invasive transitional cell carcinoma of the bladder.

INTRODUCTION: We prospectively evaluated concomitant radiotherapy and chemotherapy for advanced bladder cancer in a phase II EORTC trial to test whether it could be further studied as a potential treatment of bladder cancer. PATIENTS AND METHODS: Patients up to 75 years of age with invasive transitional-cell carcinoma of the bladder up to 5 cm, stage pT2 to pT3b, N0M0, without residual macroscopical tumour after transurethral excision were eligible. Radiotherapy consisted of 2 fractions of 1.2 Gy daily up to 60 Gy delivered in a period of 5 weeks. During the first and the last week, cisplatin 20 mg/m(2)/day and 5 FU 375 mg/m(2)/day were given concomitantly. RESULTS: The study was interrupted early due to poor recruitment. Nine patients of the originally 43 planned were treated. Mean age was 63 years. Five patients had tumour stage pT2, 1 stage pT3a and 3 stage pT3b. All patients completed radiotherapy and chemotherapy as scheduled. Only one grade 3 and no grade 4 toxicity was seen. All patients were evaluated 3 months after treatment: eight patients had no detectable tumour and one had para-aortic lymph nodes. During further follow-up, a second patient got lymph node metastases and two patients developed distant metastases (lung in the patient with enlarged lymph nodes at the first evaluation and abdominal in one other). Those three patients died at respectively 19, 14, and 18 months after registration. Late toxicity was limited and often temporary. After 26 to 57 months of follow-up, no local recurrences were seen. Six patients remained alive without disease. DISCUSSION: Despite the small cohort, this combination of concomitant chemotherapy and accelerated hyperfractionated radiotherapy for invasive bladder cancer seemed to be well tolerated and to result in satisfactory local control with limited early and late toxicity. It could therefore be considered for study in further clinical trials.