MreB, a major component of the bacterial cytoskeleton, exhibits high structural homology to its eukaryotic counterpart actin. Live cell microscopy studies suggest that MreB molecules organize into large filamentous spirals that support the cell membrane and play a key shape-determining function. However, the basic properties of MreB filament assembly remain unknown. Here, we studied the assembly of Thermotoga maritima MreB triggered by ATP in vitro and compared it to the wellstudied assembly of actin. These studies show that MreB filament ultrastructure and polymerization depend crucially on temperature as well as the ions present on solution. At the optimal growth temperature of T. maritima, MreB assembly proceeded much faster than that of actin, without nucleation (or nucleation is highly favorable and fast) and with little or no contribution from filament end-to-end annealing. MreB exhibited rates of ATP hydrolysis and phosphate release similar to that of F-actin, however, with a critical concentration of ϳ3 nM, which is ϳ100-fold lower than that of actin. Furthermore, MreB assembled into filamentous bundles that have the ability to spontaneously form ring-like structures without auxiliary proteins. These findings suggest that despite high structural homology, MreB and actin display significantly different assembly properties.

The pH dependencies of the UV-vis and fluorescent spectra of new water-soluble dendritic porphyrins and tetrabenzoporphyrins were studied. Because of extended pi-conjugation and nonplanar distortion, the absorption and the emission bands of tetraaryltetrabenzoporphyrins (Ar(4)TBP) are red-shifted and do not overlap with those of regular tetraarylporphyrins (Ar(4)P). When encapsulated inside dendrimers with hydrophilic outer layers, Ar(4)Ps and Ar(4)TBPs become water soluble and can serve as pH indicators, with pK's adjustable by the peripheral charges on the dendrimers. Two new dendritic porphyrins, Gen 4 polyglutamic porphyrin dendrimer H(2)P-Glu(4)OH (1) with 64 peripheral carboxylates and Gen 1 poly(ester amide) Newkome-type tetrabenzoporphyrin dendrimer H(2)TBP-Nw(1)OH (2) with 36 peripheral carboxylates, were synthesized and characterized. The pK's of the encapsulated porphyrins (pK(H)()2(P)(-)(Glu)()4(OH) = 6.2 and pK(H)()2(TBP)(-)(Nw)()1(OH) = 6.3) were found to be strongly influenced by the dendrimers, revealing significant electrostatic shielding of the cores by the peripheral charges. The titration curves obtained by differential excitation using the mixtures of the dendrimers were shown to be identical to those determined for the dendrimers individually. Due to their peripheral carboxylates and nanometric molecular size, porphyrin dendrimers cannot penetrate through phospholipid membranes. Dendrimer 1 was captured inside phospholipid liposomes, which were suspended in a solution containing dendrimer 2. No response from 1 was detected upon pH changes in the bulk solution, while the response from 2 was predictably strong. When proton channels were created in the liposome walls, both compounds responded equally to the bulk pH changes. These results suggest that porphyrin dendrimers can be used as fluorescent pH indicators for proton gradient measurements.

SummaryThe isolation and characterization of cDNA and genomic clones encoding a proteinase inhibitor prorein (MPI) in maize is reported. Accumulation of the MPI mRNA is Induced in response to fungal infection in germinating maize embryos. The expression pattem of the MPI gene, in healthy and fungal infected maize tissues, was examined and compared with the expression pattern of a gene that codes for a pathogenesis-related protein (the PRms protein) from maize. These two genes are induced by fungal infection, however different signals trigger their activation. Accumulation of the proteinase inhibitor mRNA is more a consequence of the wound produced by the penetration and colonizetion of the host tissues by the pathogen, than the result of a direct molecular recognition of the pathogen by the plant, as is the case for the induction of the PRms gene. Wounding, or treatment with abscislc acid or methyl jasmonete, stimulate MPI mRNA accumulation, but not PRms mRNA accumulation. Local and systemic induction of the MPI gene expression in response to wounding occurs in maize plants. To the authors' knowledge, this is the first example of a gene from a monocotyledonous species that clearly shows a systemic wound response. The possible functional implications for the existence of different signal traneduction pathways that simultaneously activate a battery of defense mechanisms against potential pathogens are discussed.

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Oxidative stress is a common feature in neurodegenerative diseases associated with neuroinflammation, and therefore, has been proposed as a key target for novel therapies for these diseases. Recently, adipose-derived stem cell (ASC)-based cell therapy has emerged as a novel strategy for neuroprotection. In this study, we evaluate the therapeutic role of ASC-conditioned medium (ASC-CM) against HO-induced neurotoxicity in a new in vitro model of ec23/brain-derived neurotrophic factor (BDNF)-differentiated human SH-SY5Y neuron-like cells (SH-SY5Yd). In the presence of ASC-CM, stressed SH-SY5Yd cells recover normal axonal morphology (with an almost complete absence of HO-induced axonal beading), electrophysiological features, and cell viability. This beneficial effect of ASC-CM was associated with its antioxidant capacity and the presence of growth factors, namely, BDNF, glial cell line-derived neurotrophic factor, and transforming growth factor β1. Moreover, the neuroprotective effect of ASC-CM was very similar to that obtained from treatment with BDNF, an essential factor for SH-SY5Yd cell survival. Importantly, we also found that the addition of the antioxidant agent N-acetyl cysteine to ASC-CM abolished its restorative effect; this was associated with a strong reduction in reactive oxygen species (ROS), in contrast to the moderate decrease in ROS produced by ASC-CM alone. These results suggest that neuronal restorative effect of ASC-CM is associated with not only the release of essential neurotrophic factors, but also the maintenance of an appropriate redox state to preserve neuronal function.

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Using a genomic clone encoding a rice lipid transfer protein, LTP1, we analysed the activity of the 5' region of the Ltp1 gene in transgenic rice (Oryza sativa L.) during plant development and under pathogen attack. The -1176/+13, -556/+13 and -284/+13 regions of the promoter were fused upstream from the uidA reporter gene and nos 3' polyadenylation signal, resulting in the pdelta1176Gus, pdelta556Gus and pdelta284Gus constructs which were transferred to rice by microprojectile bombardment. Histochemical and fluorometric GUS assays and in situ detection of uidA transcripts in transgenic homozygous lines harbouring the pdelta1176Gus construct demonstrated that the Ltp1 promoter is preferentially active in aerial vegetative and reproductive organs and that both specificity and level of expression are regulated during organ development. In leaf sheath, GUS activity which is initially strictly localized in the epidermis of growing tissue, becomes restricted to the vascular system in mature tissues. In expanded leaf blade, expression of the uidA gene was restricted to the cutting level suggesting inducibility by wounding. Strong activity was detected in lemma and palea, sterile glumes, and immature anther walls and microspores but not in female reproductive organs. No GUS activity was detected during seed embryo maturation whereas the uidA gene was strongly expressed at early stages of somatic embryogenesis in scutellum tissue. The Ltp1 transcripts were found to strongly accumulate in response to inoculation with the fungal agent of the blast disease, Magnaporthe grisea, in two rice cultivars exhibiting compatible or incompatible host-pathogen interactions. Analysis of pdelta1176Gus leaf samples inoculated with the blast fungus demonstrated that the Ltp1 promoter is induced in all cell types of tissues surrounding the lesion and notably in stomata guard cells. In plants harbouring the Ltp1 promoter deletion construct pdelta556Gus, activity was solely detected in the vascular system of mature leaves whereas no uidA gene expression was observed in pdelta284Gus plants. These observations are consistent with the proposed role of LTP1 in strenghtening of structural barriers and organ protection against mechanical disruption and pathogen attack.

Plants respond to pathogen infection with the activation of the expression of pathogenesis-related genes, a response that involves Ca2+-regulated protein phosphorylation processes. We report here the isolation of a full-length complementary DNA encoding a calcium-dependent protein kinase (CPK) gene from maize. CPK genes occur in maize as members of a multigene family, but only one specific CPK gene, the ZmCPK10 gene here described, is transcriptionally activated in response to both fungal infection and treatment with fungal elicitors. Activation of the ZmCPK10 gene is extremely rapid. ZmCPK10 transcripts could be detected 5 min after elicitation and reached maximum levels at 30 min after treatment. Afterwards, there was a decline in the level of ZmCPK10 transcripts followed by a basal level of accumulation which is maintained over the time period of elicitor treatment. The activation of this kinase is accompanied by an increase in the level of PRms mRNA, the PRms being a pathogenesis-related protein from maize whose expression is induced in maize tissues in response to fungal infection and treatment with fungal elicitors. In situ mRNA hybridization analysis revealed a remarkable cell-type specific pattern of expression of ZmCPK10 during growth and development of the elicitor-treated or fungus-infected seedling. Moreover, the ZmCPK10 gene is expressed only in those specific cell types in which the PRms gene is also expressed. The involvement of ZmCPK10 in the elicitor-induced signal transduction pathway leading to the activation of PRms gene expression is discussed.