Effects of a brassinosteroid on growth and on H+-extrusion in isolated radish cotyledons: comparison with the effects of benzyladenine

We investigated the effect of 24-epibrassinolide (BR) on the cytokinin-bioassay based on growth of isolated radish ( Raphanus sativus L. cv. Tondo Rosso Quarantino) cotyledons. BR stimulated growth of the cotyledons by about 50% when applied at saturating concentrations (3 μ M ). This effect was much lower than that of saturating concentrations of benzyladenine (BA) which was about 150% at 10 μ M BA. The effects of saturating concentrations of BR and BA were additive. BR, but not BA, stimulated H⁺-secretion by the cotyledons (measured as acidification of the incubation medium) slightly but reproducibly. These results indicate that the modes of action of the two phytoregulators are at least partially different.
BR-induced stimulation of H⁺-secretion was of similar amplitude to that induced by a concentration of fusicoccin (30 n M ) such as to stimulate growth to the same extent as BR. These results suggest that BR-induced stimulation of radish cotyledon growth might depend, at least in part, on BR-induced acidification of the wall space, similar to that observed on Azuki bean epicotyls and maize roots.     

Anoxybacillus thermarum sp. nov., a novel thermophilic bacterium isolated from thermal mud in Euganean hot springs, Abano Terme, Italy

A novel aerobe thermophilic endospore-forming bacterium designated strain AF/04^T was isolated from thermal mud located in Euganean hot springs, Abano Terme, Padova, Italy. Strain AF/04^T was Gram-positive, motile, rod-shaped, occurring in pairs, or filamentous. The isolate grew between 55 and 67°C (optimum 65°C) and at pH 6.0–7.5 (optimum pH 7.2). The strain was aerobic and grew on maltose, trehalose, and sodium acetate as sole carbon sources. The G + C content of DNA was 53.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain AF/04^T falls within the genus Anoxybacillus. Levels of 16S rRNA gene sequence similarity between strain AF/04^T and the type strains of recognized Anoxybacillus species ranged from 95 to 99%. Chemotaxonomic data (major isoprenoid quinone–menaquinone-7; major fatty acid iso-C15:0 and anteiso-C17:0) supported the affiliation of strain AF/04^T to the genus Anoxybacillus. Based on phenotypic and chemotaxonomic characteristics, 16S rRNA gene sequence analysis and DNA–DNA hybridization data, it was proposed that strain AF/04^T (=DSM 17141^T = ATCC BAA 1156^T) should be placed in the genus Anoxybacillus as the type strain of a novel species, Anoxybacillus thermarum sp. nov.     

Methotrexate chronotherapy is effective against rheumatoid arthritis

Methotrexate (MTX) is the most important drug for treating rheumatoid arthritis (RA). It has been stated that cytokines play an important role in the pathogenesis of RA, and that cytokine levels increase and show 24-h rhythms in RA patients. Previously, we found that arthritis was relieved after the administration of MTX at specific times in synchronization with the 24-h rhythm of tumor necrosis factor (TNF)-α in collagen-induced arthritis (CIA) animals. Based on our findings in an earlier study of the dosing time-dependent effects of MTX in MRL/lpr mice, which develop autoimmune disorders that share similarities with human RA, we examined here the utility of MTX chronotherapy in Japanese RA patients. In an initial animal modeling study, we collected blood from MRL/lpr mice at different times (2, 6, 10, 14, 18, or 22 hours after the light was turned on [HALO]), and we measured TNF-α mRNA expression in leukocytes. MTX was administered to the mice at two different dosing times (6 or 18 HALO), and various blood parameters were measured to estimate arthritis activity. TNF-α mRNA levels showed a clear 24-h rhythm with a peak at 22 HALO and a trough at 18 HALO after RA had developed. In these MRL/lpr mice, inflammation and TNF-α were markedly reduced when the MTX dosing time was matched to the time (18 HALO) when the TNF-α level began to increase. We then applied these findings to Japanese RA patients by switching them from the standard MTX three times/wk (day 1: after breakfast and supper; day 2: after breakfast schedule), to chronotherapy, in which the dose and number of doses/wk were not changed but MTX was administered once-a-day at bedtime. Disease Activity Score (DAS)28, modified health assessment questionnaire (MHAQ), and adverse effects were assessed. With MTX chronotherapy, DAS28, which is commonly used to quantitatively assess RA symptoms, was significantly improved at all follow-up clinical visit times compared with the baseline (vs. 1 mo: p?=?.0197, 2 mos: p?=?.0107, 3 mos: p?=?.0087). Significant symptom recovery was observed in 41.2% of patients, and 23.5% of patients achieved clinical remission during the 3 mos of follow-up. Functional capacity of RA patients, as indicated by the MHAQ, was markedly improved by chronotherapy. There were no severe adverse effects. Thus, we demonstrated (i) inflammation and plasma TNF-α concentrations were significantly reduced in MRL/lpr mice treated with MTX at 18 HALO, the time when TNF-α mRNA level began to increase; and (ii) MTX bedtime chronotherapy was safe, markedly reduced disease activity, and improved the functional capacity of RA patients. The findings on RA patients show that bedtime MTX chronotherapy can improve RA symptoms compared to the current standard dosing methods.     

Peroxisome reintroduction in Hansenula polymorpha requires Pex25 and Rho1

We identified two proteins, Pex25 and Rho1, which are involved in reintroduction of peroxisomes in peroxisome-deficient yeast cells. These are, together with Pex3, the first proteins identified as essential for this process. Of the three members of the Hansenula polymorpha Pex11 protein family-Pex11, Pex25, and Pex11C-only Pex25 was required for reintroduction of peroxisomes into a peroxisome-deficient mutant strain. In peroxisome-deficient pex3 cells, Pex25 localized to structures adjacent to the ER, whereas in wild-type cells it localized to peroxisomes. Pex25 cells were not themselves peroxisome deficient but instead contained a slightly increased number of peroxisomes. Interestingly, pex11 pex25 double deletion cells, in which both peroxisome fission (due to the deletion of PEX11) and reintroduction (due to deletion of PEX25) was blocked, did display a peroxisome-deficient phenotype. Peroxisomes reappeared in pex11 pex25 cells upon synthesis of Pex25, but not of Pex11. Reintroduction in the presence of Pex25 required the function of the GTPase Rho1. These data therefore provide new and detailed insight into factors important for de novo peroxisome formation in yeast.     

Polyclonal T-cell activation protocol stimulates preferential expansion of EBV-specific T-cell clones in vitro

Purpose Allogeneic bone marrow transplantation (AlloBMT) can be curative for patients with leukemia. The most important anti-leukemic effect may be mediated by the T-cells contained within the graft; however, the allogeneic T-cells may also give rise to graft-vs-host disease (GVHD). One way to control GVHD might be to transduce the donor T-cells with a drug-inducible suicide gene. If a retrovirus vector is to be used for this transduction, activation of the T-cells is required for integration of the transgene to occur. The activation protocol should ensure expansion of a broad repertoire of donor T-cells. Notably, T-cells specific for herpes virus family antigens are important for adoptive immunoprotection.Methods To define optimal activation conditions for retrovirus-mediated suicide gene transduction of donor T-cells, we examined the repertoire of CD8+ T-cells in general, and Epstein-Barr virus (EBV) specific T-cells in particular, following two different activation and expansion procedures.Results We found that repeated CD3/CD28 stimulation resulted in a high level of activation-induced T-cell death, affecting in vivo expanded clones, some of which were specific for EBV, in particular. In contrast, initial CD3/CD28 activation followed by proliferation in interleukin-2 lead to expansion of EBV-specific clones over and above the expansion observed for CD8+ T-cells in general.Conclusion These results should impact on protocols for ex vivo activation of T-cells prior to suicide gene transduction.