Assessment of in vivo and in vitro cytotoxic activity of hydrolysable tannin extracted from Rhizophora apiculata barks

World Journal of Microbiology and Biotechnology - Rhizophora apiculata is a common mangrove tree in Malaysia. The bark of this tree has been reported to contain a chemical constituent such as...     

The role of gap phase processes in the biomass dynamics of tropical forests

The responses of tropical forests to global anthropogenic disturbances remain poorly understood. Above-ground woody biomass in some tropical forest plots has increased over the past several decades, potentially reflecting a widespread response to increased resource availability, for example, due to elevated atmospheric CO2 and/or nutrient deposition. However, previous studies of biomass dynamics have not accounted for natural patterns of disturbance and gap phase regeneration, making it difficult to quantify the importance of environmental changes. Using spatially explicit census data from large (50 ha) inventory plots, we investigated the influence of gap phase processes on the biomass dynamics of four 'old-growth' tropical forests (Barro Colorado Island (BCI), Panama; Pasoh and Lambir, Malaysia; and Huai Kha Khaeng (HKK), Thailand). We show that biomass increases were gradual and concentrated in earlier-phase forest patches, while biomass losses were generally of greater magnitude but concentrated in rarer later-phase patches. We then estimate the rate of biomass change at each site independent of gap phase dynamics using reduced major axis regressions and ANCOVA tests. Above-ground woody biomass increased significantly at Pasoh (+0.72% yr(-1)) and decreased at HKK (-0.56% yr(-1)) independent of changes in gap phase but remained stable at both BCI and Lambir. We conclude that gap phase processes play an important role in the biomass dynamics of tropical forests, and that quantifying the role of gap phase processes will help improve our understanding of the factors driving changes in forest biomass as well as their place in the global carbon budget.     

Genetic variation and possible origins of weedy rice found in California

Control of weeds in cultivated crops is a pivotal component in successful crop production allowing higher yield and higher quality. In rice‐growing regions worldwide, weedy rice (Oryza sativa f. spontanea Rosh.) is a weed related to cultivated rice which infests rice fields. With populations across the globe evolving a suite of phenotypic traits characteristic of weeds and of cultivated rice, varying hypotheses exist on the origin of weedy rice. Here, we investigated the genetic diversity and possible origin of weedy rice in California using 98 simple sequence repeat (SSR) markers and an Rc gene‐specific marker. By employing phylogenetic clustering analysis, we show that four to five genetically distinct biotypes of weedy rice exist in California. Analysis of population structure and genetic distance among individuals reveals diverse evolutionary origins of California weedy rice biotypes, with ancestry derived from indica, aus, and japonica cultivated rice as well as possible contributions from weedy rice from the southern United States and wild rice. Because this diverse parentage primarily consists of weedy, wild, and cultivated rice not found in California, most existing weedy rice biotypes likely originated outside California.     

The variation of tree beta diversity across a global network of forest plots

Aims With the aim of understanding why some of the world's forests exhibit higher tree beta diversity values than others, we asked: (1) what is the contribution of environmentally related variation versus pure spatial and local stochastic variation to tree beta diversity assessed at the forest plot scale; (2) at what resolution are these beta‐diversity components more apparent; and (3) what determines the variation in tree beta diversity observed across regions/continents? Location World‐wide. Methods We compiled an unprecedented data set of 10 large‐scale stem‐mapping forest plots differing in latitude, tree species richness and topographic variability. We assessed the tree beta diversity found within each forest plot separately. The non‐directional variation in tree species composition among cells of the plot was our measure of beta diversity. We compared the beta diversity of each plot with the value expected under a null model. We also apportioned the beta diversity into four components: pure topographic, spatially structured topographic, pure spatial and unexplained. We used linear mixed models to interpret the variation of beta diversity values across the plots. Results Total tree beta diversity within a forest plot decreased with increasing cell size, and increased with tree species richness and the amount of topographic variability of the plot. The topography‐related component of beta diversity was correlated with the amount of topographic variability but was unrelated to its species richness. The unexplained variation was correlated with the beta diversity expected under the null model and with species richness. Main conclusions Because different components of beta diversity have different determinants, comparisons of tree beta diversity across regions should quantify not only overall variation in species composition but also its components. Global‐scale patterns in tree beta diversity are largely coupled with changes in gamma richness due to the relationship between the latter and the variation generated by local stochastic assembly processes.     

Decreased hypertrophic differentiation accompanies enhanced matrix formation in co-cultures of outer meniscus cells with bone marrow mesenchymal stromal cells

Introduction

The main objective of this study was to determine whether meniscus cells from the outer (MCO) and inner (MCI) regions of the meniscus interact similarly to or differently with mesenchymal stromal stem cells (MSCs). Previous study had shown that co-culture of meniscus cells with bone marrow-derived MSCs result in enhanced matrix formation relative to mono-cultures of meniscus cells and MSCs. However, the study did not examine if cells from the different regions of the meniscus interacted similarly to or differently with MSCs.

Methods

Human menisci were harvested from four patients undergoing total knee replacements. Tissue from the outer and inner regions represented pieces taken from one third and two thirds of the radial distance of the meniscus, respectively. Meniscus cells were released from the menisci after collagenase treatment. Bone marrow MSCs were obtained from the iliac crest of two patients after plastic adherence and in vitro culture until passage 2. Primary meniscus cells from the outer (MCO) or inner (MCI) regions of the meniscus were co-cultured with MSCs in three-dimensional (3D) pellet cultures at 1:3 ratio, respectively, for 3 weeks in the presence of serum-free chondrogenic medium containing TGF-β1. Mono-cultures of MCO, MCI and MSCs served as experimental control groups. The tissue formed after 3 weeks was assessed biochemically, histochemically and by quantitative RT-PCR.

Results

Co-culture of inner (MCI) or outer (MCO) meniscus cells with MSCs resulted in neo-tissue with increased (up to 2.2-fold) proteoglycan (GAG) matrix content relative to tissues formed from mono-cultures of MSCs, MCI and MCO. Co-cultures of MCI or MCO with MSCs produced the same amount of matrix in the tissue formed. However, the expression level of aggrecan was highest in mono-cultures of MSCs but similar in the other four groups. The DNA content of the tissues from co-cultured cells was not statistically different from tissues formed from mono-cultures of MSCs, MCI and MCO. The expression of collagen I (COL1A2) mRNA increased in co-cultured cells relative to mono-cultures of MCO and MCI but not compared to MSC mono-cultures. Collagen II (COL2A1) mRNA expression increased significantly in co-cultures of both MCO and MCI with MSCs compared to their own controls (mono-cultures of MCO and MCI respectively) but only the co-cultures of MCO:MSCs were significantly increased compared to MSC control mono-cultures. Increased collagen II protein expression was visible by collagen II immuno-histochemistry. The mRNA expression level of Sox9 was similar in all pellet cultures. The expression of collagen × (COL10A1) mRNA was 2-fold higher in co-cultures of MCI:MSCs relative to co-cultures of MCO:MSCs. Additionally, other hypertrophic genes, MMP-13 and Indian Hedgehog (IHh), were highly expressed by 4-fold and 18-fold, respectively, in co-cultures of MCI:MSCs relative to co-cultures of MCO:MSCs.

Conclusions

Co-culture of primary MCI or MCO with MSCs resulted in enhanced matrix formation. MCI and MCO increased matrix formation similarly after co-culture with MSCs. However, MCO was more potent than MCI in suppressing hypertrophic differentiation of MSCs. These findings suggest that meniscus cells from the outer-vascular regions of the meniscus can be supplemented with MSCs in order to engineer functional grafts to reconstruct inner-avascular meniscus.     

Redox-regulation of Erk1/2-directed phosphatase by reactive oxygen species: role in signaling TPA-induced growth arrest in ML-1 cells

Extracellular signal-regulated kinase (Erk)1/2 activity signals myeloid cell differentiation induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Previously, we reported that Erk1/2 activation (phosphorylation) induced by TPA required reactive oxygen species (ROS) as a second messenger. Here, we hypothesized that ROS generated in response to TPA inhibit Erk1/2-directed phosphatase activity, which leads to an increase phosphorylation of Erk1/2 to signal p21(WAF1/Cip1)-mediated growth arrest in ML-1 cells. Incubation of ML-1 cells with TPA resulted in a marked accumulation of phosphorylated Erk1/2, and is subsequent to H2O2 generation. Interestingly, post-TPA-treatment with N-acetylcysteine (NAC) stimulated a marked and a rapid dephosphorylation of Erk1/2, suggesting a regeneration of Erk1/2-directed phospahatase activity by NAC. ROS generation in ML-1 cells induced by TPA was suggested to occur in the mitochondrial electron transport chain (METC) based on the following observations: (i) undifferentiated ML-1 cells not only lack p67-phox and but also express a low level of p47-phox key components required for NADPH oxidase enzymatic activity, (ii) pretreatment with DPI, an inhibitor of NADH- and NADPH-dependent enzymes, or rhein, an inhibitor of complex I, blocked the ROS generation, and (iii) examination of the microarray analysis data and Western blot analysis data revealed an induction of MnSOD expression at both mRNA and protein levels in response to TPA. MnSOD is a key member of the mitochondrial defense system against mitochondrial-derived superoxide. Together, this study suggested that TPA stimulated ROS generation as a second messenger to activate Erk1/2 via a redox-mediated inhibition of Erk1/2-directed phosphatase in ML-1 cells.     

MMP-9 Sheds the ��2 Integrin Subunit (CD18) from Macrophages

Activated macrophages are essential effectors of immunity and a rich source of matrix metalloproteinase-9 (MMP-9; gelatinase B). To search for cellular substrates of the enzyme, we subjected wild-type macrophages and macrophages expressing an autoactivating form of pro-MMP-9 (M9A macrophages) to proteomics analysis. Two-dimensional liquid chromatography together with tandem mass spectrometry identified 467 proteins in medium conditioned by M9A and/or wild-type macrophages. Subtractive proteomics identified 18 candidate MMP-9 substrates. Biochemical studies confirmed that two transmembrane proteins, β₂ integrin subunit (CD18) and amyloid protein precursor (APP), were enriched in the medium of M9A macrophages. To identify potential cleavage sites, we synthesized an overlapping library of peptides that spanned 60 residues of the ectodomain and transmembrane domain of β₂ integrin. Active MMP-9 cleaved a single peptide, ECVKGPNVAAIVGGT, at residues corresponding to Ala⁷⁰⁵ and Ile⁷⁰⁶ of the β₂ integrin. Peptides corresponding to this cleavage site were detected by tandem mass spectrometric analysis only in medium from M9A macrophages, strongly supporting the proposal that β₂ integrin is shed by autoactivating MMP-9. Our observations indicate that subtractive proteomics in concert with peptide substrate mapping is a powerful approach for identifying proteolytic substrates and suggest that MMP-9 plays previously unsuspected roles in the regulation and shedding of β₂ integrin.Matrix metalloproteinases (MMPs),¹ a subfamily of metazincins, are a structurally related group of zinc-dependent proteases (1). They are synthesized in latent form as pro-MMPs, and their prodomain must be removed or modified before they are proteolytically active. Some MMPs are secreted, whereas others are anchored to the cell surface, but their proteolytic activity is thought to be confined locally within the secretory pathway at the cell surface and nearby extracellular space (1–3). Individual MMPs have distinct substrate specificities and act on diverse extracellular and membrane proteins, such as chemokines, cell surface adhesion proteins, and extracellular matrix components. Proteolysis by MMPs plays an important role in a wide variety of normal and pathological processes, such as host defense, inflammation, and tumor progression (1–9).High levels of MMP-9 (gelatinase B) are expressed by activated macrophages (10), which are key effector cells of both innate and acquired immunity. In addition to having homeostatic functions, MMP-9 secreted by macrophages has been implicated in aneurysm formation, tumor progression, and disruption of atherosclerotic plaques (8, 9, 11, 12). Although the pathogenesis of those processes is generally thought to involve inappropriate degradation of extracellular matrix proteins, it has become increasingly clear that MMPs cleave a number of diverse substrates to mediate their varied functions (3, 13). Because MMP-9 can accumulate on the cell surface (14), it is likely to act on membrane proteins.To understand the specific roles of individual MMPs in inflammatory and immune responses, it is critical to identify their physiological substrates (3, 15–17). Most studies have focused on identifying substrates by their ability to be cleaved in defined in vitro reactions (18, 19), but this approach is biased in two ways. First, the candidate substrate must be selected a priori. Second, in vitro reactions fail to account for the complexity of the pericellular environment. Another method is to identify sequences in synthetic peptides that MMPs can cleave (20, 21). However, individual MMPs cleave different proteins at a variety of sites rather than at a consensus site. Moreover MMPs often interact with substrates through domains remote from the active site (exosites) (22), and exosites of MMP-2 have been used in a yeast two-hybrid system to trap candidate substrates (23). However, some substrates may bind weakly or not at all to exosites, limiting the utility of this approach for global substrate screening.An emerging strategy for finding MMP substrates is to conduct an unbiased, global search by coupling gel electrophoresis or liquid chromatography with MS-based protein identification. For example, two-dimensional (2D) gel electrophoresis (24) and derivatization of cysteine-containing peptides with an isotope affinity tag (25) have identified candidate substrates for membrane type-1 MMP (MT1-MMP) in plasma and cultured cells. Quantitative approaches using 2D difference gel electrophoresis have identified potential substrates of MMP-2 and MMP-9 in bronchoalveolar lavage fluid (26) and of MMP-9 and the related metalloproteinases ADAM-10 and ADAM-17 in cancer cells (27, 28). Lectin affinity chromatography detected glycosylated proteins that were selectively enriched in medium from a monocyte cell line expressing ADAM-17 and in phorbol ester-stimulated monocytes (16). Recently iTRAQ (isobaric tags for relative and absolute quantitation) labeling was used to identify substrates of MMP-2 (29). It is important to note, however, that proteases can affect protein abundance by pathways not involving proteolysis. Thus, an important limitation of many of these studies is that they fail to provide evidence that proteins with altered abundance in cells expressing a protease are direct substrates for proteolytic cleavage.In the current studies, we used subtractive proteomics to identify proteins enriched in the medium of a macrophage cell line. Subtractive proteomics compares two or more proteomes to identify proteins that are specifically enriched or depleted under certain conditions (30, 31). Our biochemical studies confirmed that two integral membrane proteins, amyloid precursor protein (APP) and the β₂ integrin subunit (CD18), were shed by macrophages expressing autoactivating MMP-9. We next used a peptide substrate mapping strategy to identify potential MMP-9 cleavage sites in β₂ integrin subunit. Targeted MS/MS analysis demonstrated that β₂ integrin subunit peptides with the same cleavage site were detected only in the medium of macrophages expressing autoactivating MMP-9, providing strong evidence that β₂ integrin is a direct substrate for proteolysis. Our observations indicate that subtractive proteomics in concert with peptide substrate mapping is a robust, high throughput technique for identifying cellular substrates that are proteolytically shed from macrophages.     

Potential for Plant-Based Remediation of Pesticide-Contaminated Soil and Water using Nontarget Plants such as Trees,Shrubs, and Grasses

Appropriate environmental management of pesticides includes their proper application, use of filter strips and riparian buffers to contain pesticides in runoff from fields, prompt cleanup of spills, and treatment processes for wastewater associated with manufacturing and equipment usage. Plants have beneficial effects in the management of pesticide-contaminated soil and water, including direct metabolism of many pesticides, stimulation of microbial activity in the root zone, extraction of contaminated water, and reduction of infiltrating contaminated water. In this work, we review the literature on nontarget plants that can grow in pesticide-contaminated soil and water, and the fate of pesticides in filter strips, riparian buffers, and vegetated remediation environments. Past research indicates that there are significant differences in the tolerance of plants to pesticides present in soil and water, and that some plants are more effective than others in the remediation of pesticide-contaminated soil and water. Thus, there is value in the identification of tolerant plants and favorable plant-based remediation technologies for management of pesticides and contaminated sites.     

The effect of inbreeding and larval feeding regime on immature development of Aedes albopictus

The fundamental approach to the biological control of Aedes albopictus requires the mass rearing of mosquitoes and the release of highly competitive adults in the field. As the fitness of adults is highly dependent on the development of immatures, we aimed to identify the minimum feeding regime required to produce viable and competitive adults by evaluating three response parameters: development duration, immature mortality, and adult wing length. Our study suggests at least 0.60 mg/larva/day of larval diet composed of dog food, dried beef liver, yeast, and milk powder in a weight ratio of 2:1:1:1 is required to maximize adult fitness. With standardized protocols in mass rearing, intensive studies can be readily conducted on mosquito colonies to facilitate comparisons across laboratories. This study also evaluated the differences in response of laboratory and field strains under different feeding regimes. We found that strain alone did not exert substantial effects on all response parameters. However, the field strain exhibited significantly lower immature mortality than the laboratory strain under the minimum feeding regime. Females and males of the laboratory strain had longer wing lengths under nutritional constraint due to the higher mortality that resulted in reduced interactions with the remaining larvae. Meanwhile, the field strain exhibited heterogeneous duration of immature development compared with the laboratory strain. The disparities demonstrated by the two strains in this study suggest the effect of inbreeding surfaced after a long term of laboratory colonization. Despite the trade‐offs resulting from laboratory colonization, the competitiveness of the laboratory strain of Ae. albopictus is comparable to the field strain, provided the larvae are fed optimally.