Modulation of plumbagin production in Plumbago zeylanica using a single-chain variable fragment antibody against plumbagin

A single-chain variable fragment antibody (scFv) against plumbagin (PL) accumulated the PL production in the hairy roots of Plumbago zeylanica. Recombinant Agrobacterium rhizogenes (ATCC 15834) containing an scFv gene against PL (PL-scFv) were obtained through triparental mating and transformed into P. zeylanica to induce PL-scFv protein in the hairy roots. Up to 40 μg recombinant PL-scFv were expressed per milligram of soluble protein in transgenic P. zeylanica hairy root cultures. The mean PL content obtained from transgenic hairy roots (12.24 μg/100 mg dry weight) exhibited 2.2 times higher than those obtained from wild-type (5.48 μg/100 mg dry weight). The high correlation between the PL-scFv expression level and PL content of the recombinant plants suggested that the PL biosynthesis pathway had been modulated by the expression of PL-scFv protein in the hairy roots of P. zeylanica.     

Angiotensin-II and MARCKS: A HYDROGEN PEROXIDE- AND RAC1-DEPENDENT SIGNALING PATHWAY IN VASCULAR ENDOTHELIUM

MARCKS is an actin-binding protein that modulates vascular endothelial cell migration and cytoskeleton signaling (Kalwa, H., and Michel, T. (2011) J. Biol. Chem. 286, 2320-2330). Angiotensin-II is a vasoactive peptide implicated in vascular physiology as well as pathophysiology; the pathways connecting angiotensin-II and cytoskeletal remodeling are incompletely understood. Here we show that MARCKS is expressed in intact arterial preparations, with prominent staining of the endothelium. In endothelial cells, angiotensin-II-promoted MARCKS phosphorylation is abrogated by PEG-catalase, implicating endogenous H(2)O(2) in the angiotensin-II response. Studies using the H(2)O(2) biosensor HyPer2 reveal that angiotensin-II promotes increases in intracellular H(2)O(2). We used a Rac1 FRET biosensor to show that angiotensin-II promotes Rac1 activation that is attenuated by PEG-catalase. siRNA-mediated Rac1 knockdown blocks angiotensin-II-stimulated MARCKS phosphorylation. Cell imaging studies using a phosphoinositide 4,5-bisphosphate (PIP(2)) biosensor revealed that angiotensin-II PIP(2) regulation depends on MARCKS and H(2)O(2). siRNA-mediated knockdown of MARCKS or Rac1 attenuates receptor-mediated activation of the tyrosine kinase c-Abl and disrupts actin fiber formation. These studies establish a critical role for H(2)O(2) in angiotensin-II signaling to the endothelial cytoskeleton in a novel pathway that is critically dependent on MARCKS, Rac1, and c-Abl.     

Characterization of cancer stem cell properties of CD24 and CD26-positive human malignant mesothelioma cells

Malignant mesothelioma (MM) is an asbestos-related malignancy characterized by rapid growth and poor prognosis. In our previous study, we have demonstrated that several cancer stem cell (CSC) markers correlated with CSC properties in MM cells. Among these markers, we focused on two: CD24, the common CSC marker, and CD26, the additional CSC marker. We further analyzed the CSC properties of CD24 and CD26-positve MM cells. We established RNAi-knockdown cells and found that these markers were significantly correlated with chemoresistance, proliferation, and invasion potentials in vitro. Interestingly, while Meso-1 cells expressed both CD24 and CD26, the presence of each of these two markers was correlated with different CSC property. In addition, downstream signaling of these markers was explored by microarray analysis, which revealed that their expressions were correlated with several cancer-related genes. Furthermore, phosphorylation of ERK by EGF stimulation was significantly affected by the expression of CD26, but not CD24. These results suggest that CD24 and CD26 differentially regulate the CSC potentials of MM and could be promising targets for CSC-oriented therapy.     

Involvement of GABAergic and glutamatergic systems in the anticonvulsant activity of 3-alkynyl selenophene in 21?day-old rats

In this study, we investigated the role of GABAergic and glutamatergic systems in the anticonvulsant action of 3-alkynyl selenophene (3-ASP) in a pilocarpine (PC) model of seizures. To this purpose, 21 day-old rats were administered with an anticonvulsant dose of 3-ASP (50 mg/kg, per oral, p.o.), and [(3)H]γ-aminobutyric acid (GABA) and [(3)H]glutamate uptakes were carried out in slices of cerebral cortex and hippocampus. [(3)H]GABA uptake was decreased in cerebral cortex (64%) and hippocampus (58%) slices of 21 day-old rats treated with 3-ASP. In contrast, no alteration was observed in [(3)H]glutamate uptake in cerebral cortex and hippocampus slices of 21 day-old rats that received 3-ASP. Considering the drugs that increase synaptic GABA levels, by inhibiting its uptake or catabolism, are effective anticonvulsants, we further investigated the possible interaction between sub-effective doses of 3-ASP and GABA uptake or GABA transaminase (GABA-T) inhibitors in PC-induced seizures in 21 day-old rats. For this end, sub-effective doses of 3-ASP (10 mg/kg, p.o.) and DL-2,4-diamino-n-butyric acid hydrochloride (DABA, an inhibitor of GABA uptake--2 mg/kg, intraperitoneally; i.p.) or aminooxyacetic acid hemihydrochloride (AOAA; a GABA-T inhibitor--10 mg/kg, i.p.) were co-administrated to 21 day-old rats before PC (400 mg/kg; i.p.) treatment, and the appearance of seizures was recorded. Results demonstrated that treatment with AOAA and 3-ASP or DABA and 3-ASP significantly abolished the number of convulsing animals induced by PC. The present study indicates that 3-ASP reduced [(3)H]GABA uptake, suggesting that its anticonvulsant action is related to an increase in inhibitory tonus.     

Synthesis of organosoluble chitosan derivatives with polyphenolic side chains

A one-pot synthesis was used to produce chitosan derivatives with polyphenolic side chains via a regioselective phenolic coupling reaction. Under Mannich reaction conditions, treatment of chitosan with formaldehyde and methyl 2,4-dihydroxybenzoate gave N-(2,6-dihydroxy-3-methoxycarbonylphenyl)methylated chitosan in good yield (87%). Formation of a CC bond occurred regioselectively at the C(3) position of methyl 2,4-dihydroxybenzoate. Chitosan derivatives having various phenolic compounds as a side chain were easily synthesized in a similar manner. The chitosan derivatives showed good biodegradability and improved their solubility in methanol (9.8mgmL(-1)) and 2-methoxyethanol (> 10mgmL(-1)). The UV protection provided by the derivatives with phenolic benzophenone side chain was evaluated using UV spectra of polyethylene terephthalate and poly(vinyl butyral-co-vinyl alcohol-co-vinyl acetate) films coated with the derivatives and the derivatives absorbed effectively in the UV-A region (<60%). Self-aggregation of the chitosan derivatives with the phenolic side chain was observed by using a fluorescent probe in aqueous solution.     

Cooperative effect of p150Glued and microtubule stabilization to suppress excitotoxicity-induced axon degeneration

Glutamate excitotoxicity is implicated in chronic neurological disorders and acute CNS insults and causes neuronal degeneration including axons. The molecular mechanism underlying excitotoxicity-induced axon degeneration is poorly understood. Recently, we found that components of the dynein-dynactin complex that governs microtubule-dependent retrograde transport play important roles in modulating the process of excitotoxicity-induced neurodegeneration. Here we used hippocampal cultures and searched for pathways that function in concert with the components of the dynein-dynactin complex and identified microtubule stabilization as a cooperative pathway to suppress axon degeneration. We find that overexpression of p150Glued, a major component of the dynactin complex, and microtubule stabilization cooperatively suppress axon degeneration. The protective effect of p150Glued is dependent on the C-terminal region as excitotoxicity-induced C-terminal truncated form of p150Glued was unable to interact with APP cargo and altered the localization of APP in neurites when overexpressed. C-terminal truncation of p150Glued is not rescued by microtubule stabilization suggesting that the downstream effects of p150Glued and microtubule stabilization to protect axon degeneration are mutually exclusive.     

Variation in liana abundance and biomass along an elevational gradient in the tropical Atlantic Forest (Brazil)

Lianas play a key role in forest structure, species diversity, as well as functional aspects of tropical forests. Although the study of lianas in the tropics has increased dramatically in recent years, basic information on liana communities for the Brazilian Atlantic Forest is still scarce. To understand general patterns of liana abundance and biomass along an elevational gradient (0–1,100 m asl) of coastal Atlantic Forest, we carried out a standard census for lianas ≥1 cm in five 1-ha plots distributed across different forest sites. On average, we found a twofold variation in liana abundance and biomass between lowland and other forest types. Large lianas (≥10 cm) accounted for 26–35% of total liana biomass at lower elevations, but they were not recorded in montane forests. Although the abundance of lianas displayed strong spatial structure at short distances, the present local forest structure played a minor role structuring liana communities at the scale of 0.01 ha. Compared to similar moist and wet Neotropical forests, lianas are slightly less abundant in the Atlantic Forest, but the total biomass is similar. Our study highlights two important points: (1) despite some studies have shown the importance of small-scale canopy disturbance and support availability, the spatial scale of the relationships between lianas and forest structure can vary greatly among tropical forests; (2) our results add to the evidence that past canopy disturbance levels and minimum temperature variation exert influence on the structure of liana communities in tropical moist forests, particularly along short and steep elevational gradients.     

Temperature stress-induced changes in the proteomic profiles of Ecklonia cava (Laminariales, Phaeophyceae)

Proteomic profiling on Ecklonia cava Kjellman grown under various seawater temperatures was conducted to search for biomarkers that were useful to evaluate the health of the colonies and formulate actions for the maintenance of marine forests. In the cultivated strains, protein expression was not significantly changed when the cultivation temperature was lowered from 15°C (control) to 10°C. On the contrary, it was markedly changed, i.e., photosynthesis-related proteins were up-regulated and metabolic enzymes were down-regulated, when the temperature was heightened to 20°C. With the cultivation at 30°C, 25 spots within 27 spots expressed at this temperature peculiarly could be identified and classified into ten proteins. Of the distinctive 27 spots at 30°C, 20 spots were detected in the wild strains cultured at the same temperature for a brief time. It is presumed that the proteins including vanadium-dependent bromoperoxidase are heat stress-induced proteins.     

Baupain, a plant cysteine proteinase that hinders thrombin-induced human platelet aggregation

Bauninia forficata is trivially known as cow paw, and popularly used in Brazil for treatment of diabetes mellitus. Denominated baupain a cysteine proteinase was purified from B. forficata leaves. In this study, we investigated the baupain effect on aggregation of isolated human platelets in vitro and the results show that baupain hinders thrombin - but not ADP- and collagen- induced platelet aggregation. With synthetic quenched-fluorescent peptides, the kinetics of the cleavage site of human proteinase-activated receptor 1 / 2 / 3 and 4 [PAR-1 / 2 / 3 and 4] by baupain was determined. In conclusion, similar to bromelain and papain, baupain hinders human platelets aggregation, probably through an unspecific cleavage in the Phe-Leu bond of PAR1.