Distinct regulation of salinity and genotoxic stress responses by Arabidopsis MAP kinase phosphatase 1

The Arabidopsis genome contains 20 genes encoding mitogen-activated protein kinases (MAPKs), which drastically outnumbers genes for their negative regulators, MAP kinase phosphatases (MKPs) (five at most). This contrasts sharply with genomes of other eukaryotes where the number of MAPKs and MKPs is approximately equal. MKPs may therefore play an important role in signal integration in plants, through concerted regulation of several MAPKs. Our previous studies identified Arabidopsis MKP1 and showed that its deficiency in the mkp1 mutant results in plant hypersensitivity to genotoxic stress. Here, we identify a set of MAPKs that interact with MKP1, and show that the activity level of one of these, MPK6, is regulated by MKP1 in vivo. Moreover, using expression profiling, we identified a specific group of genes that probably represent targets of MKP1 regulation. Surprisingly, the identity of these genes and interacting MAPKs suggested involvement of MKP1 in salt stress responses. Indeed, mkp1 plants have increased resistance to salinity. Thus MKP1 apparently plays a pivotal role in the integration and fine-tuning of plant responses to various environmental challenges.     

In vitro activity of oxazaphosphorines in childhood acute leukemia: preliminary report

Glufosfamide (beta-D-glucosyl-ifosfamide mustard) is a new agent for cancer chemotherapy. Its pharmacology is similar to commonly used oxazaphosphorines, but it does not require activation by hepatic cytochrome P-450 and preclinically demonstrates lower nephrotoxicity and myelosuppression than ifosfamide. The aim of the study was a comparison of the drug resistance profiles of glufosfamide and other oxazaphosphorines in childhood acute leukemias. Leukemic cells, taken from children with ALL on diagnosis (n = 41), ALL on relapse (n = 12) and AML on diagnosis (n = 13) were analyzed by means of the MTT assay. The following drugs were tested: glufosfamide (GLU), 4-HOO-ifosfamide (IFO), 4-HOO-cyclophosphamide (CYC) and mafosfamide cyclohexylamine salt (MAF). In the group of initial ALL samples median cytotoxicity values for GLU, IFO, CYC and MAF were 15.5, 33.8, 15.7 and 7.8 microM, respectively. In comparison with initial ALL samples, the relative resistance for GLU and IFO in relapsed ALL samples was 1.9 (p = 0.049) and 1.3 (ns), and in initial AML samples 31 (p < 0.001) and 5 (p = 0.001), respectively. All oxazaphosphorines presented highly significant cross-resistance. Glufosfamide presented high activity against lymphoblasts both on diagnosis and on relapse.     

Allograft rejection by primed/memory CD8+ T cells is CD154 blockade resistant: therapeutic implications for sensitized transplant recipients

We have shown that CD8(+) CTLs are the key mediators of accelerated rejection, and that CD8(+) T cells represent the prime targets of CD154 blockade in sensitized mouse recipients of cardiac allografts. However, the current protocols require CD154 blockade at the time of sensitization, whereas delayed treatment fails to affect graft rejection in sensitized recipients. To elucidate the mechanisms of costimulation blockade-resistant rejection and to improve the efficacy of CD154-targeted therapy, we found that alloreactive CD8(+) T cells were activated despite the CD154 blockade in sensitized hosts. Comparative CD8 T cell activation study in naive vs primed hosts has shown that although both naive and primed/memory CD8(+) T cells relied on the CD28 costimulation for their activation, only naive, not primed/memory, CD8(+) T cells depend on CD154 signaling to differentiate into CTL effector cells. Adjunctive therapy was designed accordingly to deplete primed/memory CD8(+) T cells before the CD154 blockade. Indeed, unlike anti-CD154 monotherapy, transient depletion of CD8(+) T cells around the time of cardiac engraftment significantly improved the efficacy of delayed CD154 blockade in sensitized hosts. Hence, this report provides evidence for 1) differential requirement of CD154 costimulation signals for naive vs primed/memory CD8(+) T cells, and 2) successful treatment of clinically relevant sensitized recipients to achieve stable long term graft acceptance.     

Cyanogenesis in glucosinolate-producing plants: Carica papaya and Carica quercifolia

(R)-2-(beta-D-Glucopyranosyloxy)-2-phenylacetonitrile (prunasin) was isolated from Carica papaya L. and C. quercifolia (A. St.-Hil.) Hieron. (syn. C. hastata Brign.). Earlier reported presence of cyclopentanoid cyanohydrin glycosides in C. papaya could not be confirmed, and no cyclopentanoid amino acids could be detected in extracts of C. papaya and C. quercifolia. Conversion of [2,3,4,5,6-3H]phenylalanine into tritiated prunasin was demonstrated in both species. On the other hand, when the plants were administered [2-14C]-2-(2'cyclopentenyl)glycine, extracted, and the extracts hydrolyzed with beta-glucosidase (Helix pomatia), formation of labelled cyanide was not observed. The absence of cyclopentanoids, which are typical for the Passifloraceae, and the inability of Carica species to utilize 2-(2'-cyclopentenyl)glycine as a precursor of cyanogenic glycosides are in agreement with the relative phylogenetic position of the Caricaceae and the Passifloraceae. Carica species are thus rare examples of taxa in which glucosinolates and cyanogenic glycosides co-occur, both types of natural products being derived from the same amino acid, phenylalanine.     

Stimulation of oxygen evolution in photosystem II by copper(II) ions

We have found that copper(II) ions at about equimolar Cu2+/photosystem II (PS II) reaction center proportions stimulate oxygen evolution nearly twofold. This high affinity Cu-binding site is different from the binding sites of Mn and Ca ions. The analysis of the Cu2+ content in PS II preparations isolated from wild-type tobacco and a tobacco mutant deficient in light-harvesting complex suggests that Cu2+ may be a native component of PS II and may take part in the oxygen evolution process. At higher concentrations, Cu2+ ions inhibit oxygen evolution and quench fluorescence.     

Calcium transients in the model of rapidly induced anoxic tolerance in rat cortical slices: involvement of NMDA receptors

In this study, we investigated the effects of NMDA receptor antagonists on calcium transients induced by a single 2-min preconditioning anoxia (PA) in rat olfactory cortical slices, and on the ability of PA to prevent pathological calcium overload induced by subsequent 10-min test anoxia (TA). Relative changes in the intracellular Ca(2+) concentration (Ca(i)) and in the level of Ca(2+) bound to intracellular hydrophobic domains (Ca(b)) were monitored using fura-2 and chlortetracycline, respectively. Our results confirmed that TA induces prominent long-lasting increases in Ca(i) and Ca(b), reflecting cellular calcium overload. It was found that PA produces moderate increases in both Ca(2+) pools and prevents Ca(2+) overload induced by TA carried out 90 min later. Calcium transients and the protective effects of PA were significantly suppressed in slices treated with NMDA receptor antagonists during and 30 min after PA. These results indicate that moderate activation of the NMDA receptors participates in the mechanism of the PA-induced anoxic tolerance of cortical neurons.     

Ex vivo apoptotic potential of peripheral blood mononuclear cells of the elderly human subject

Studies of immunosenescence have led to a detailed knowledge of immune system dysfunctions in the ageing human being. Apoptosis seems to be one of the process regulating an immune response after the antigenic stimulation. We examined whether commonly used methods of assessing apoptosis in the elderly human subject produce comparable results to young subjects. PBMC of young and elderly volunteers were isolated from the venous blood and cultured for 6 or 24 h with antigens of anti-influenza vaccine or PMA. The intensity of apoptosis was measured using the annexinV test, flow cytometric evaluation of DNA content (sub-G1 peak in DNA histograms), 'ladder' by DNA gel electrophoresis, and fluorescence microscope. Apoptosis in 6 h-lasting cultures of the elderly was more intense in annexinV test, while it was decreased assessing subG1 peak. Additionally, in the aged group, those changes were associated with cell cycle arrest. Our results suggest that the apoptosis after the stimulation with the vaccine antigens seems to be some kind of activation-induced cell death (AICD). Different patterns of apoptosis after stimulation may be associated with the cell cycle arrest of the PBMC in the elderly.     

Deletion of deoxyribonucleic acid binding domain of the vitamin D receptor abrogates genomic and nongenomic functions of vitamin D

The vitamin D hormone 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], the biologically active form of vitamin D, is essential for an intact mineral metabolism. Using gene targeting, we sought to generate vitamin D receptor (VDR) null mutant mice carrying the reporter gene lacZ driven by the endogenous VDR promoter. Here we show that our gene-targeted mutant mice express a VDR with an intact hormone binding domain, but lacking the first zinc finger necessary for DNA binding. Expression of the lacZ reporter gene was widely distributed during embryogenesis and postnatally. Strong lacZ expression was found in bones, cartilage, intestine, kidney, skin, brain, heart, and parathyroid glands. Homozygous mice are a phenocopy of mice totally lacking the VDR protein and showed growth retardation, rickets, secondary hyperparathyroidism, and alopecia. Feeding of a diet high in calcium, phosphorus, and lactose normalized blood calcium and serum PTH levels, but revealed a profound renal calcium leak in normocalcemic homozygous mutants. When mice were treated with pharmacological doses of vitamin D metabolites, responses in skin, bone, intestine, parathyroid glands, and kidney were absent in homozygous mice, indicating that the mutant receptor is nonfunctioning and that vitamin D signaling pathways other than those mediated through the classical nuclear receptor are of minor physiological importance. Furthermore, rapid, nongenomic responses to 1,25-(OH)(2)D(3) in osteoblasts were abrogated in homozygous mice, supporting the conclusion that the classical VDR mediates the nongenomic actions of 1,25-(OH)(2)D(3).