Metaphloem development in the Arabidopsis root tip.

The phloem transport network is a major evolutionary innovation that enabled plants to dominate terrestrial ecosystems. In the growth apices, the meristems, apical stem cells continuously produce early 'protophloem'. This is easily observed in Arabidopsis root meristems, in which the differentiation of individual protophloem sieve element precursors into interconnected conducting sieve tubes is laid out in a spatio-temporal gradient. The mature protophloem eventually collapses as the neighboring metaphloem takes over its function further distal from the stem cell niche. Compared with protophloem, metaphloem ontogenesis is poorly characterized, primarily because its visualization is challenging. Here, we describe the improved TetSee protocol to investigate metaphloem development in Arabidopsis root tips in combination with a set of molecular markers. We found that mature metaphloem sieve elements are only observed in the late post-meristematic root, although their specification is initiated as soon as protophloem sieve elements enucleate. Moreover, unlike protophloem sieve elements, metaphloem sieve elements only differentiate once they have fully elongated. Finally, our results suggest that metaphloem differentiation is not directly controlled by protophloem-derived cues but rather follows a distinct, robust developmental trajectory.

Improving the productivity of 19,20-epoxy-cytochalasin Q in Xylaria sp. sof11 with culture condition optimization.

19,20-Epoxy-cytochalasin Q (B5A) is a cytochalasin with a wide range of biological activities, which can be produced by Xylaria sp. sof11, a strain isolated from the seafloor of the northern South China Sea. Since the low titer of B5A has greatly limited its further studies, we have systematically conducted the fermentative optimization for B5A production in this article. The effects of major medium components, including the carbon and organic nitrogen sources, as well as of the concentration of sea salt, were respectively investigated through single-factor experiments. As a result, sucrose and fish meal were determined to be the key factors affecting the production of B5A. Then three important variables, sucrose, fish meal, and filling volume, were screened out by the Plackett-Burman (PB) design. The optimal level of these variables was further confirmed by response surface analysis. The final formulated medium was set as 35.2 g/L sucrose and 18.0 g/L fish meal, with filling volume of 34.6 mL, which could afford 440.3 mg/L production of B5A, approximately 4.4-fold higher than that in the original medium. The significantly improved productivity of B5A will facilitate the subsequent mechanistic and clinical studies of B5A.

Effect of Culture Conditions on Metabolite Production of Xylaria sp.

Seeking a strategy for triggering the cryptic natural product biosynthesis to yield novel compounds in the plant-associated fungus Xylaria sp., the effect of culture conditions on metabolite production was investigated. A shift in the production of five known cytochalasin-type analogues 1-5 to six new α-pyrone derivatives, xylapyrones A-F (compounds 6-11), from a solid to a liquid medium was observed. These compounds were identified by analysis of 1D and 2D NMR and HRMS data. Compounds 1-3 showed moderate cytotoxicity against HepG2 and Caski cancer cell lines with IC50 values ranging from 25 to 63 µM and compounds 4-11 were found to be inactive, with IC50 values>100 µM.

Bidirectional interactions between NOX2-type NADPH oxidase and the F-actin cytoskeleton in neuronal growth cones.

NADPH oxidases are important for neuronal function but detailed subcellular localization studies have not been performed. Here, we provide the first evidence for the presence of functional NADPH oxidase 2 (NOX2)-type complex in neuronal growth cones and its bidirectional relationship with the actin cytoskeleton. NADPH oxidase inhibition resulted in reduced F-actin content, retrograde F-actin flow, and neurite outgrowth. Stimulation of NADPH oxidase via protein kinase C activation increased levels of hydrogen peroxide in the growth cone periphery. The main enzymatic NADPH oxidase subunit NOX2/gp91(phox) localized to the growth cone plasma membrane and showed little overlap with the regulatory subunit p40(phox) . p40(phox) itself exhibited colocalization with filopodial actin bundles. Differential subcellular fractionation revealed preferential association of NOX2/gp91(phox) and p40(phox) with the membrane and the cytoskeletal fraction, respectively. When neurite growth was evoked with beads coated with the cell adhesion molecule apCAM, we observed a significant increase in colocalization of p40(phox) with NOX2/gp91(phox) at apCAM adhesion sites. Together, these findings suggest a bidirectional functional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones, which contributes to the control of neurite outgrowth. We have previously shown that reactive oxygen species (ROS) are critical for actin organization and dynamics in neuronal growth cones as well as neurite outgrowth. Here, we report that the cytosolic subunit p40(phox) of the NOX2-type NADPH oxidase complex is partially associated with F-actin in neuronal growth cones, while ROS produced by this complex regulates F-actin dynamics and neurite growth. These findings provide evidence for a bidirectional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones.

Nuclear receptor nur77 promotes cerebral cell apoptosis and induces early brain injury after experimental subarachnoid hemorrhage in rats.

Nur77 is a potent proapoptotic member of the nuclear receptor superfamily that is expressed predominantly in brain tissue. It has been demonstrated that Nur77 mediates apoptosis in multiple organs. Nur77-mediated early brain injury (EBI) involves a conformational change in BCL-2 and triggers cytochrome C (cytoC) release resulting in cellular apoptosis. This study investigates whether Nur77 can promote cerebral cell apoptosis after experimentally induced subarachnoid hemorrhage (SAH) in rats. Sprague Dawley rats were randomly assigned to three groups: 1) untreated group, 2) treatment control group, and 3) SAH group. The experimental SAH group was divided into four subgroups, corresponding to 12 hr, 24 hr, 48 hr, and 72 hr after experimentally induced SAH. It remains unclear whether Nur77 can play an important role during EBI after SAH as a proapoptotic protein in cerebral cells. Cytosporone B (Csn-B) was used to demonstrate that Nur77 could be enriched and used to aggravate EBI after SAH. Rats treated with Csn-B were given an intraperitoneal injection (13 mg/kg) 30 min after experimentally induced SAH. We found that Nur77 promotes cerebral cell apoptosis by mediating EBI and triggering a conformational change in BCL-2, resulting in cytoC release. Nur77 activity, along with cerebral cell apoptosis, peaked at 24 hr after SAH onset. After induction of SAH, an injection of Csn-B, an agonist for Nur77, enhanced the expression and function of Nur77. In summary, we have demonstrated the proapoptotic effect of Nur77 within cerebral cells, an effect that can be further exacerbated with Csn-B stimulation.

Cytochalasins as Modulators of Stem Cell Differentiation.

Regenerative medicine aims to identify new research strategies for the repair and restoration of tissues damaged by pathological or accidental events. Mesenchymal stem cells (MSCs) play a key role in regenerative medicine approaches due to their specific properties, such as the high rate of proliferation, the ability to differentiate into several cell lineages, the immunomodulatory potential, and their easy isolation with minimal ethical issues. One of the main goals of regenerative medicine is to modulate, both in vitro and in vivo, the differentiation potential of MSCs to improve their use in the repair of damaged tissues. Over the years, much evidence has been collected about the ability of cytochalasins, a large family of 60 metabolites isolated mainly from fungi, to modulate multiple properties of stem cells (SCs), such as proliferation, migration, and differentiation, by altering the organization of the cyto- and the nucleo-skeleton. In this review, we discussed the ability of two different cytochalasins, cytochalasins D and B, to influence specific SC differentiation programs modulated by several agents (chemical or physical) or intra- and extra-cellular factors, with particular attention to human MSCs (hMSCs).

Binding of phenochalasin A, an inhibitor of lipid droplet formation in mouse macrophages, on G-actin.

Phenochalasin A, a unique phenol-containing cytochalasin produced by the marine-derived fungus Phomopsis sp. FT-0211, was originally discovered in a cell morphological assay of observing the inhibition of lipid droplet formation in mouse peritoneal macrophages. To investigate the mode of action and binding proteins, phenochalasin A was radio-labeled by 125I. Iodinated phenochalasin A exhibited the same biological activity as phenochalasin A. [125I]Phenochalasin A was found to be associated with an approximately 40 kDa protein, which was identified as G-actin. Furthermore, detail analyses of F-actin formation in Chinese hamster ovary cells (CHO-K1 cells) indicated that phenochalasin A (2 µM) caused elimination of F-actin formation on the apical site of the cells, suggesting that actin-oriented specific function(s) in cytoskeletal processes are affected by phenochalasin A.

Spitzenkorper, exocyst, and polarisome components in Candida albicans hyphae show different patterns of localization and have distinct dynamic properties.

During the extreme polarized growth of fungal hyphae, secretory vesicles are thought to accumulate in a subapical region called the Spitzenkörper. The human fungal pathogen Candida albicans can grow in a budding yeast or hyphal form. When it grows as hyphae, Mlc1 accumulates in a subapical spot suggestive of a Spitzenkörper-like structure, while the polarisome components Spa2 and Bud6 localize to a surface crescent. Here we show that the vesicle-associated protein Sec4 also localizes to a spot, confirming that secretory vesicles accumulate in the putative C. albicans Spitzenkörper. In contrast, exocyst components localize to a surface crescent. Using a combination of fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) experiments and cytochalasin A to disrupt actin cables, we showed that Spitzenkörper-located proteins are highly dynamic. In contrast, exocyst and polarisome components are stably located at the cell surface. It is thought that in Saccharomyces cerevisiae exocyst components are transported to the cell surface on secretory vesicles along actin cables. If each vesicle carried its own complement of exocyst components, then it would be expected that exocyst components would be as dynamic as Sec4 and would have the same pattern of localization. This is not what we observe in C. albicans. We propose a model in which a stream of vesicles arrives at the tip and accumulates in the Spitzenkörper before onward delivery to the plasma membrane mediated by exocyst and polarisome components that are more stable residents of the cell surface.

An actin depolymerizing agent 19,20-epoxycytochalasin Q of Xylaria sp. BCC 1067 enhanced antifungal action of azole drugs through ROS-mediated cell death in yeast.

Multidrug resistance is a highly conserved phenomenon among all living organisms and a major veritable public health problem worldwide. Repetitive uses of antibiotics lead to antimicrobial drug resistance. Here, 19,20-epoxycytochalasin Q (ECQ) was isolated from endophytic fungus Xylaria sp. BCC 1067 and, its chemical structure was determined via chromatographic and spectral methods. ECQ displayed an antifungal activity with low MIC50 of 410 and 55 mg/l in the model yeast Saccharomyces cerevisiae wild-type and ScΔpdr5 strains, respectively. ECQ was a new inducer and potential substrate of key multi-drug efflux pumps S. cerevisiae ScPdr5 and Candida albicans CaCdr1. ECQ targeted actin filament, disrupting actin dynamics of yeast cells. ECQ also sensitized the ScΔsrv2 mutant, lacking suppressor of RasVal19. Overexpression of ScPDR5 or CaCDR1 genes prevented aggregation of actin and alleviated antifungal effect of ECQ. Additionally, ECQ induced high accumulation of reactive oxygen species, caused plasma membrane leakage and decreased yeast cell survival. Importantly, a discovery of ECQ implied a cellular connection between multi-drug resistance and actin stability, an important determinant of transporter mediated-drug resistance mechanism. Combination of ECQ and antifungal azoles displayed promising drug synergy against S. cerevisiae strains expressing multi-drug transporters, thereby providing potential solution for antifungal therapy and chemotherapeutic application.

Protoplast mutation and genome shuffling induce the endophytic fungus Tubercularia sp. TF5 to produce new compounds.

Tubercularia sp. TF5 is an endophytic fungal strain isolated from the medicinal plant Taxus mairei. Previously, taxol has been detected in the fermentation products of this strain. However, it lost the capability of producing taxol after long-term laboratory culture. Herein, we tried to reactivate the production of taxol by protoplast mutations and genome shuffling. The protoplasts of Tub. sp. TF5 were prepared from its mycelia, and mutated by UV and NTG. The mutant strains regenerated from the mutated protoplasts were selected and classified into four groups on the basis of their phenotypes, the profile of their metabolites analyzed by TLC, MS, and bioassay data. Then, genome shuffling was subsequently carried out with eight mutant strains, with two representatives from each protoplast mutant group, and genome shuffling mutant strains were obtained and screened using the same screening procedure. Although taxol has not been detected in any mutant, two important mutants, M-741 and G-444 were selected for metabolites isolation and determination due to their phenotypes, and differences in TLC analysis result from TF5 and other mutants. Three new sesquiterpenoids, namely tuberculariols A-C (1-3), and a known dihydroisocoumarin (4) were obtained from M-741. Eighteen novel compounds were isolated from G-444, including five new sesquiterpenoids (5-9), two new dihydroisocoumarins (10, 11), one new tetralone (12), together with 10 known compounds (13-20, 1, and 2). The compounds isolated from the M-741 and G-444 were different in structure types and substitutions from those of TF5 (15, 21-29). The results showed, for the first time, that protoplast mutations and genome shuffling are efficient approaches to mining natural products from endophytic fungi. Understanding the mechanisms of unlocking the biosynthesis of new metabolites will facilitate the manipulation of the secondary metabolism in fungi.

Genomics-Driven Discovery of Phytotoxic Cytochalasans Involved in the Virulence of the Wheat Pathogen Parastagonospora nodorum.

The etiology of fungal pathogenesis of grains is critical to global food security. The large number of orphan biosynthetic gene clusters uncovered in fungal plant pathogen genome sequencing projects suggests that we have a significant knowledge gap about the secondary metabolite repertoires of these pathogens and their roles in plant pathogenesis. Cytochalasans are a family of natural products of significant interest due to their ability to bind to actin and interfere with cellular processes that involved actin polymerization; however, our understanding of their biosynthesis and biological roles remains incomplete. Here, we identified a putative polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) gene cluster (phm) that was upregulated in the pathogen Parastagonospora nodorum during its infection on wheat. Overexpression of the transcription factor gene phmR encoded in the phm gene cluster resulted in the production of two leucine-derived cytochalasans, phomacins D and E (1 and 2, respectively), and an acetonyl adduct phomacin F. Heterologous expression of the PKS-NRPS gene phmA and the trans-enoyl reductase (ER) gene phmE in Aspergillus nidulans resulted in the production of a novel 2-pyrrolidone precursor prephomacin. Reverse genetics and wheat seedling infection assays showed that ΔphmA mutants exhibited significantly reduced virulence compared to the wild type. We further demonstrated that both 1 and 2 showed potent actin polymerization-inhibitory activities and exhibited potentially monocot-specific antigerminative activities. The findings from this study have advanced our knowledge based on the biosynthesis and biological roles of cytochalasans, the latter of which could have significant implications for our understanding of the molecular mechanisms of fungus-plant interactions.

Cytochalasin inhibits the rate of elongation of actin filament fragments.

Submicromolar concentrations of cytochalasin inhibit the rate of assembly of highly purified dictyostelium discoideum actin, using a cytochalasin concentration range in which the final extent of assembly is minimally affected. Cytochalasin D is a more effective inhibitor than cytochalasin B, which is in keeping with the effects that have been reported on cell motility and with binding to a class of high-affinity binding sites from human erythrocyte membranes (Lin and Lin. 1978. J. Biol. CHem. 253:1415; Lin and Lin. 1979. Proc. Natl. Acad. Sci. U.S.A. 76:2345); 5x10(-7) M cytochalasin B lowers it to 70 percent of the control value, whereas 10(-7) M cytochalasin B lowers the rate to 25 percent. Fragments of F-actin were used to increase the rate of assembly fivefold by providing more filament ends on to which monomers could add. Under these conditions, cytochalasin has an even more dramatic effect on the assembly rate; the concentrations of cytochalasin B and cytochalasin D required for half-maximal inhibition are 2x10(-7) M and 10(-8) M, respectively. The assembly rate is most sensitive to cytochalasin when actin assembly is carried out in the absence of ATP (with 3 mM ADP present to stabilize the actin). In this case, the concentrations of cytochalasin B and cytochalasin D required for half-maximal inhibition are 4x10(-8) M and 1x10(-9) M, respectively. A scatchard plot has been obtained using [(3)H]cytochalasin B binding to F-actin in the absence of ATP. The K(d) from this plot (approximately 4x10(-8) M) agrees well with the concentration of cytochalasin B required for half-maximal inhibition of the rate of assembly under these conditions. The number of cytochalasin binding sites is roughly one per F-actin filament, suggesting that cytochalasin has a specific action on actin filament ends.

Action of cytochalasin D on cells of established lines. II. Cortex and microfilaments.

Cells in culture exposed to cytochalasin D (CD) rapidly undergo a long-sustained tonic contraction. Coincident with this contracture the thin microfilaments of the cortex become compacted into feltlike masses. The ravelled filaments of these masses remain actinlike and bind heavy meromyosin; they are not disrupted or disaggregated, but rather, appear to represent a contracted state of the microfilament apparatus of the cell cortex. On continued exposure to CD, 'myoid' bundles, containing thick, dense filaments, and larger fusiform or ribbonlike, putatively myosinoid, aggregates may appear. These appearances are interpreted as consequences of a state of hypercontraction without relaxation induced by CD. They do not occur in CD-treated cells prevented from contracting by inhibitors of energy metabolism, and are readily reversible on withdrawal of CD. Extensive ordered arrays of thin microfilaments develop in cells which are reextending during early recovery.

Induction of chondrogenesis in limb mesenchymal cultures by disruption of the actin cytoskeleton.

Cell shape is known to influence the chondrogenic differentiation of cultured limb bud mesenchyme cells (Solursh, M., T. F. Linsenmayer, and K. L. Jensen, 1982, Dev. Biol., 94: 259-264). To test whether specific cytoskeletal components mediate this influence of cell shape, we examined different cytoskeleton disrupting agents for their ability to affect chondrogenesis. Limb bud cells cultured at subconfluent densities on plastic substrata normally become flattened, contain numerous cytoplasmic microtubules and actin bundles, and do not undergo spontaneous chondrogenesis. If such cultures are treated with 2 micrograms/ml cytochalasin D during the initial 3-24 h in culture, the cells round up, lose their actin cables, and undergo chondrogenesis, as indicated by the production of immunologically detectable type II collagen and a pericellular Alcian blue staining matrix. Cytochalasin D also induces cartilage formation by high-density cultures of proximal limb bud cells, which normally become blocked in a protodifferentiated state. In addition, cytochalasin D was found to reverse the normal inhibition by fibronectin of chondrogenesis by proximal limb bud cells cultured in hydrated collagen gels. Agents that disrupt microtubules have no apparent effect on the shape or chondrogenic differentiation of limb bud mesenchymal cells. These results suggest an involvement of the actin cytoskeleton in controlling cell shape and chondrogenic differentiation of limb bud mesenchyme. Interactions of the actin cytoskeleton and extracellular matrix components may provide a regulatory mechanism for mesenchyme cell differentiation into cartilage or fibrous connective tissue in the developing limb.

Armochaetoglasins A-I: Cytochalasan alkaloids from fermentation broth of Chaetomium globosum TW1-1 by feeding L-tyrosine.

By feeding L-tyrosine into the culture medium, nine undescribed compounds, termed as armochaetoglasins A-I, together with three known analogues, namely armochaetoglobin E, chaetoglobosin V, and chaetoglobosin J, were isolated and identified from the medicinal terrestrial arthropod-derived fungus Chaetomium globosum TW1-1. Their structures were elucidated by means of NMR spectroscopy, single-crystal X-ray crystallography, and comparison of their electronic circular dichroism (ECD) spectra. Structurally, armochaetoglasin A represented the first tyrosine-derived cytochalasan alkaloid characterized by a 13-membered carbocyclic ring system; armochaetoglasins B and C possessed a rare 19,20-seco-chaetoglobosin skeleton. Armochaetoglasin B, chaetoglobosin V, and chaetoglobosin J showed weak cytotoxic activity with IC50 values ranging from 19.5 to 34.72 µM.

Multimycotoxin analysis of South African Aspergillus clavatus isolates.

Aspergillus clavatus poisoning is a neuromycotoxicosis of ruminants that occurs sporadically across the world after ingestion of infected feedstuffs. Although various toxic metabolites are synthesized by the fungus, it is not clear which specific or group of mycotoxins induces the syndrome. A. clavatus isolates were deposited in the culture collection of the Biosystematics Division, Plant Protection Research Institute, Agricultural Research Council during incidences of livestock poisoning (1988-2016). Six isolates were still viable and these plus three other South African isolates that were also previously deposited in the collection were positively identified as A. clavatus based on morphology and ß-tubulin sequence data. The cultures were screened for multiple mycotoxins using a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method. Twelve A. clavatus metabolites were detected. The concentrations of the tremorgenic mycotoxins (i.e., tryptoquivaline A and its related metabolites deoxytryptoquivaline A and deoxynortryptoquivaline) were higher than patulin and cytochalasin E. Livestock owners should not feed A. clavatus-infected material to ruminants as all the South African A. clavatus isolates synthesized the same compounds when cultured under similar conditions.

One new cytochalasin metabolite isolated from a mangrove-derived fungus Daldinia eschscholtzii HJ001.

One new cytochalasin metabolite [11]-cytochalasa-5(6),13-diene-1,21-dione-7,18-dihydroxy-16,18-dimethyl-10-phenyl-(7S*,13E,16S*,18R*) (1), together with three known compounds (2-4) were obtained from the EtOAc extract of the endophytic fungus Daldinia eschscholtzii HJ001 isolated from the mangrove Brguiera sexangula var. rhynchopetala collected in the South China Sea. Their structures were elucidated by the detailed analysis of comprehensive spectroscopic data. Compounds 1 and 2 were evaluated for their antibacterial and cytotoxic activities.

Uptake and intracellular fate of Francisella tularensis in human macrophages.

Francisella tularensis is an intracellular pathogen that survives and multiplies within host mononuclear phagocytes. We have shown that uptake of the bacterium by human macrophages occurs by a novel process, "looping phagocytosis," in which the bacterium is engulfed in a spacious, asymmetric, pseudopod loop. Whereas looping phagocytosis is resistant to treatment of the F. tularensis with formalin, proteases, or heat, the process is abolished by oxidation of the bacterial carbohydrates with periodate, suggesting a role for preformed surface carbohydrate molecules in triggering looping phagocytosis. Following uptake, F. tularensis initially resides in a spacious vacuole at the periphery of the cell, but this vacuole rapidly shrinks in size. The nascent F. tularensis vacuole transiently acquires early endosomal markers, but subsequently exhibits an arrested maturation, manifest by only limited amounts of lysosome-associated membrane glycoproteins (consistent with limited interaction with late endosomes), nonfusion with lysosomes, and minimal acidification. In ultrastructural studies, we have observed that the F. tularensis phagosome displays a novel feature in that many of the phagosomes acquire an electron dense fibrillar coat. This fibrillar coat forms blebs and vesicles, and with time, is seen to be fragmented and disrupted. With increasing time after infection, increasing numbers of the F. tularensis are found free in the macrophage cytoplasm, such that by 14 h after infection, less than 15% of the bacteria are surrounded by any discernible phagosomal membrane. Further research is needed to determine the mechanisms underlying looping phagocytosis, and the maturational arrest, fibrillar coat formation, and disruption of the phagosome.

Correlation of unstable multidrug cross resistance in Chinese hamster ovary cells with a homogeneously staining region on chromosome 1.

An enrichment selection method using repeated pulses of low drug concentration (1 microgram/ml) was used to isolate CHO (AK412) variants that are 20-fold more resistant to cytochalasin D (CD). CD-resistant (CydR) variants possess a unique unstable phenotype, including a longer doubling time in nonselective medium, a higher frequency of multinucleate cells in the population (probably due to a defect in cytokinesis), an altered morphology, and increased resistance or sensitivity to a number of unrelated drugs. In each of two variant lines examined cytologically, this multiple phenotype is associated with a small homogeneously staining region on chromosome 1. The homogeneously staining region is present in the CydR variants, but absent both in the CD-sensitive parent and in a CD-sensitive revertant subpopulation. Studies of CD-displaceable binding of [3H]cytochalasin B show a fourfold reduction in CD binding or uptake when whole cells of the variant line were examined. Lactoperoxidase-catalyzed iodination and metabolic labeling with [H3]fucose of cell surface proteins of the CydR variants showed multiple differences in electrophoretic band migration when compared with parental proteins.