Highly dynamic mitotic-spindle microtubules are among the most successful targets for anticancer therapy. Microtubule-targeted drugs, including paclitaxel and Vinca alkaloids, were previously considered to work primarily by increasing or decreasing the cellular microtubule mass. Although these effects might have a role in their chemotherapeutic actions, we now know that at lower concentrations, microtubule-targeted drugs can suppress microtubule dynamics without changing microtubule mass; this action leads to mitotic block and apoptosis. In addition to the expanding array of chemically diverse antimitotic agents, some microtubule-targeted drugs can act as vascular-targeting agents, rapidly depolymerizing microtubules of newly formed vasculature to shut down the blood supply to tumours.

Microtubules as a target for anticancer drugs.

Summary 1 Summary All plants in natural ecosystems appear to be symbiotic with fungal endophytes. This highly diverse group of fungi can have profound impacts on plant communities through increasing fitness by conferring abiotic and biotic stress tolerance, increasing biomass and decreasing water consumption, or decreasing fitness by altering resource allocation. Despite more than 100 yr of research resulting in thousands of journal articles, the ecological significance of these fungi remains poorly characterized. Historically, two endophytic groups (clavicipitaceous (C) and nonclavicipitaceous (NC)) have been discriminated based on phylogeny and life history traits. Here, we show that NC-endophytes represent three distinct functional groups based on host colonization and transmission, in planta biodiversity and fitness benefits conferred to hosts. Using this framework, we contrast the life histories, interactions with hosts and potential roles in plant ecophysiology of C- and NC-endophytes, and highlight several key questions for future work in endophyte biology.

https://courses.washington.edu/cfr523/documents/2009TansleyReview.pdf

Fungal endophytes: diversity and functional roles.

Pectins: structure, biosynthesis, and oligogalacturonide-related signaling.

Based on its generally accessible I/II redox couple and bioavailability, copper plays a wide variety of roles in nature that mostly involve electron transfer (ET), O2 binding, activation and reduction, NO2− and N2O reduction and substrate activation. Copper sites that perform ET are the mononuclear blue Cu site that has a highly covalent CuII-S(Cys) bond and the binuclear CuA site that has a Cu2S(Cys)2 core with a Cu-Cu bond that keeps the site delocalized (Cu(1.5)2) in its oxidized state. In contrast to inorganic Cu complexes, these metalloprotein sites transfer electrons rapidly often over long distances, as has been previously reviewed.1–4 Blue Cu and CuA sites will only be considered here in their relation to intramolecular ET in multi-center enzymes. The focus of this review is on the Cu enzymes (Figure 1). Many are involved in O2 activation and reduction, which has mostly been thought to involve at least two electrons to overcome spin forbiddenness and the low potential of the one electron reduction to superoxide (Figure 2).5,6 Since the Cu(III) redox state has not been observed in biology, this requires either more than one Cu center or one copper and an additional redox active organic cofactor. The latter is formed in a biogenesis reaction of a residue (Tyr) that is also Cu catalyzed in the first turnover of the protein. Recently, however, there have been a number of enzymes suggested to utilize one Cu to activate O2 by 1e− reduction to form a Cu(II)-O2•− intermediate (an innersphere redox process) and it is important to understand the active site requirements to drive this reaction. The oxidases that catalyze the 4e−reduction of O2 to H2O are unique in that they effectively perform this reaction in one step indicating that the free energy barrier for the second two-electron reduction of the peroxide product of the first two-electron step is very low. In nature this requires either a trinuclear Cu cluster (in the multicopper oxidases) or a Cu/Tyr/Heme Fe cluster (in the cytochrome oxidases). The former accomplishes this with almost no overpotential maximizing its ability to oxidize substrates and its utility in biofuel cells, while the latter class of enzymes uses the excess energy to pump protons for ATP synthesis. In bacterial denitrification, a mononuclear Cu center catalyzes the 1e- reduction of nitrite to NO while a unique µ4S2−Cu4 cluster catalyzes the reduction of N2O to N2 and H2O, a 2e− process yet requiring 4Cu’s. Finally there are now several classes of enzymes that utilize an oxidized Cu(II) center to activate a covalently bound substrate to react with O2.



Figure 1

Copper active sites in biology.





Figure 2

Latimer Diagram for Oxygen Reduction at pH = 7.0 Adapted from References 5 and 6.



This review presents in depth discussions of all these classes of Cu enzymes and the correlations within and among these classes. For each class we review our present understanding of the enzymology, kinetics, geometric structures, electronic structures and the reaction mechanisms these have elucidated. While the emphasis here is on the enzymology, model studies have significantly contributed to our understanding of O2 activation by a number of Cu enzymes and are included in appropriate subsections of this review. In general we will consider how the covalency of a Cu(II)–substrate bond can activate the substrate for its spin forbidden reaction with O2, how in binuclear Cu enzymes the exchange coupling between Cu’s overcomes the spin forbiddenness of O2 binding and controls electron transfer to O2 to direct catalysis either to perform two e− electrophilic aromatic substitution or 1e− H-atom abstraction, the type of oxygen intermediate that is required for H-atom abstraction from the strong C-H bond of methane (104 kcal/mol) and how the trinuclear Cu cluster and the Cu/Tyr/Heme Fe cluster achieve their very low barriers for the reductive cleavage of the O-O bond.

Much of the insight available into these mechanisms in Cu biochemistry has come from the application of a wide range of spectroscopies and the correlation of spectroscopic results to electronic structure calculations. Thus we start with a tutorial on the different spectroscopic methods utilized to study mononuclear and multinuclear Cu enzymes and their correlations to different levels of electronic structure calculations.

/pdf/copper-active-sites-in-biology-lhhabv47if.pdf

Copper Active Sites in Biology

Lead toxicity induces lipid peroxidation and alters the activities of antioxidant enzymes in growing rice plants

. Effects of paraquat were monitored by electron microscopic, cytochemical, and in vivo spectrophotometric procedures. Electron microscopy of paraquat-treated pea leaf discs indicates that the chloroplasts are affected primarily with both thylakoid dilation and apparent lamellar fusions, resulting in a ‘honeycomb’ of lamellae. No ultra-structural effects were noted when leaf discs were incubated in both DCMU and paraquat. Paraquat-induced peroxide was located cytochemically along the stroma lamellae, on the ends of the grana stack and in the dilated thylakoids. Less destruction was noted in those samples where peroxide was precipitated by cerium chloride, indicating that peroxide or one of its products is one of the major causes of paraquat toxicily. In C4 plants mesophyll plastids exhibited much more peroxide deposition than was detected in bundle sheath plastids. No plastid peroxide depositions were noted in a photobleaching mutant that is resistant to paraquat. In situ cytochrome f oxidation-reduction was followed during paraquat treatments and resulted in a hastening of the dark re-reduction of cytochrome f after only 1 h of treatment. This effect was prevented by DCMU or CeCI3. Electron transport, as measured by cytochrome f oxidation-reduction, was completely obliterated by a 24 h paraquat treatment. These in situ studies on paraquat confirm previous studies that the primary effect in the light is on the plastid and involves peroxide or further products generated by peroxide.

In situ localization of the sites of paraquat action

Cytological studies of dinitroaniline-resistant Eleusine

Two immunological approaches were used to determine if ribulose bisphosphate carboxylase oxygenase (RuBisCo) is present in guard cell chloroplasts. Immunocytochemistry on thin plastic sections using tissue samples that were processed using traditional glutaraldehyde/osmium fixation and then restored to antigenicity with metaperiodate treatment, resulted in labeling over wild-type mesophyll and guard cell plastids of several green and white variegated Pelargonium chimeras. The density of immunogold labeling in guard cell chloroplasts was only about one-seventh of that noted in mesophyll chloroplasts on a square micron basis. Because guard cell chloroplasts are much smaller than mesophyll chloroplasts, and occur at lower quantities/cell, the relative differences in RuBisCo concentration between the cell types indicate that guard cells have only 0.48% of the RuBisCo of mesophyll cells. No reaction was noted over 70S ribosomeless plastids of these chimeras even though adjacent green chloroplasts were heavily stained, indicating the high specificity of the reaction for RuBisCo. Spurr9s resin gave the most successful colloidal gold labeling in terms of low background staining and structural detail but L. R. White9s resin appeared to be superior for antigen retention. In the white leaf edges of the white and green Pelargonium chimeras, the only green, functional chloroplasts are in the guard cells. When either whole tissue or plastid enriched extracts from this white tissue were electrophoresed, blotted, and probed with anti-RuBisCo a large subunit band was detected, identical to that in the green tissue. These data indicate that a low, but detectable, level of RuBisCo is present in guard cell chloroplasts.

/pdf/two-immunological-approaches-to-the-detection-of-ribulose-1-3ybw2zw6w2.pdf

Two Immunological Approaches to the Detection of Ribulose-1,5-Bisphosphate Carboxylase in Guard Cell Chloroplasts

The developing spermatogenous cells of Ceratopteris richardii are ideal to investigate the plant cytoskeleton Spores sown on sterile medium produce gametophytes with antheridia at all developmental stages within a week of growth at 22⚬C Within an antheridium, however, all the cells are in synchrony Other unique aspects of this system for investigation of the cytoskeleton are the structurally discernible microtubule organizing centers (MTOCs) known as the blepharoplast and multilayered structure (MLS), the de novo production of centrioles, and the production of multiflagellated sperm cells Immunocytochemical localization studies reveal that the blepharoplast reacts with two monoclonal antibodies that recognize animal centrosomes The lamellar strip of the MLS reacts strongly with the centrosomal protein centrin These are the first reports of the specific localization of MTOC proteins/epitopes in structurally discernible land plant MTOCs Several of the stable microtubule arrays, the spline, basal body

Kevin C. Vaughn

Papers

In situ localization of the sites of paraquat action

Cytological studies of dinitroaniline-resistant Eleusine

Two Immunological Approaches to the Detection of Ribulose-1,5-Bisphosphate Carboxylase in Guard Cell Chloroplasts

Using the Developing Spermatogenous Cells of Ceratopteris to Unlock the Mysteries of the Plant Cytoskeleton

Structural and physiological studies of paraquat-resistant Conyza