In Vitro Production of Echioidinin, 7-O-Methywogonin from Callus Cultures of Andrographis lineata and Their Cytotoxicity on Cancer Cells

Andrographis lineata is an herbal medicinal plant used in traditional medicine as a substitute for Andrographis paniculata. Here, using mature leaf explants of A. lineata we demonstrate for the first time the callus induction established on MS medium containing 1.0 mg l^–1 IAA. Dried callus was subjected to solvent extraction with acetone. Further the acetone residue was separated by silica gel column chromatography, crystallized and characterized on the basis of nuclear magnetic resonance (proton and c13) and liquid chromatographic mass spectroscopy. This analysis revealed the occurrence of two known flavones namely, 7-O-methylwogonin (MW) and Echioidinin (ED). Furthermore, these compounds were tested for their cytotoxicity against leukemic cell line, CEM. We identify that ED and MW induced cytotoxicity in a time- and concentration-dependent manner. Further increase in the LDH release upon treatment with ED and MW further confirmed our cytotoxicity results against leukemic cell line. Strikingly, MW was more potent than ED when compared by trypan blue and MTT assays. Our results recapitulate the utility of callus cultures for the production of plant specific bioactive secondary metabolites instead of using wild plants. Together, our in vitro studies provide new insights of A. lineata callus cultures serving as a source for cancer chemotherapeutic agents.     

Forest cover and fruit crop size differentially influence frugivory of select rainforest tree species in Western Ghats,India

Forest fragmentation and habitat loss are major disruptors of plant–frugivore interactions, affecting seed dispersal and altering recruitment patterns of the dependent tree species. In a heterogeneous production landscape (primarily tea and coffee plantations) in the southern Western Ghats, India, we examined effects of surrounding forest cover and fruit crop size on frugivory of four rainforest bird-dispersed tree species (N = 131 trees, ≥30 trees per species, observed for 623 hr). Frugivore composition differed among the four tree species with the large-seeded Canarium strictum and Myristica dactyloides being exclusively dependent on large-bodied avian frugivores, whereas medium-seeded Persea macrantha and Heynea trijuga were predominantly visited by small-bodied and large-bodied avian frugivores, respectively. Using the seed-dispersal-effectiveness framework, we identified effective frugivores and examined their responses to forest cover and fruit crop size. Results were idiosyncratic and were governed by plant and frugivore traits. Visitations to medium-seeded Persea had a positive relationship with forest cover but the relationship was negative for the large-seeded Myristica. In addition, two of the three effective frugivores for Persea responded to the interactive effect of forest cover and fruit crop size. Frugivore visitations to Heynea were not related to forest cover or fruit crop, and there were too few visitations to Canarium to discern any trends. These results highlight the context-specific responses of plant–frugivore interactions to forest cover and fruit crop size influenced by plant and frugivore traits.     

Early generation selection for agronomic characters in a potato breeding programme

Summary A random sample of seedlings representing high, medium and poor vigour was studied for tuber colour, tuber shape, eye depth, tuber cracking, tuber yield per plant, average tuber weight and number of tubers per plant in four successive generations (F₁, F₁, F₁C₂, and F₁C₃). Based on the performance of vigour groups in various generations and inter-generation correlation coefficients, we propose a procedure for the elimination of unproductive genotypes early in the breeding programme. The data indicates that seedlings of poor vigour can be discarded at the seedling stage prior to transplantation in the field. The rejection of clones on the basis of tuber colour, tuber shape, eye depth and tuber cracking can also be initiated at the seedling stage. For tuber yield and average tuber weight a negative selection (rejection of poor phenotypes) is suggested from the first clonal generation and for number of tubers, from second clonal generation, until statistically sound replicated trials can be conducted for carrying out positive selection.     

Point of Care Tuberculosis Sero-Diagnosis Kit for Wild Animals: Combination of Proteins for Improving the Diagnostic Sensitivity and Specificity

Tuberculosis is a significant problem globally for domestic animals as well as captive and free ranging wild life. Rapid point of care (POC) serology kits are well suited for the diagnosis of TB in wild animals. However, wild animals are invariably exposed to environmental non-pathogenic mycobacterium species with the development of cross reacting antibodies. In the present study, POC TB diagnosis kit was developed using a combination of pathogenic Mycobacteria specific recombinant antigens and purified protein derivatives of pathogenic and non-pathogenic Mycobacteria. To benchmark the TB antibody detection kit, particularly in respect to specificity which could not be determined in wildlife due to the lack of samples from confirmed uninfected animals, we first tested well-characterized sera from 100 M. bovis infected and 100 uninfected cattle. Then we investigated the kit’s performance using sera samples from wildlife, namely Sloth Bears (n = 74), Elephants (n = 9), Cervidae (n = 14), Felidae (n = 21), Cape buffalo (n = 2), Wild bear (n = 1) and Wild dog (n = 1).In cattle, a sensitivity of 81% and a specificity of 90% were obtained. The diagnostic sensitivity of the kit was 94% when the kit was tested using known TB positive sloth bear sera samples. 47.4% of the in-contact sloth bears turned seropositive using the rapid POC TB diagnostic kit. Seropositivity in other wild animals was 25% when the sera samples were tested using the kit. A point of care TB sero-diagnostic kit with the combination of proteins was developed and the kit was validated using the sera samples of wild animals.     

Structural differences between the resting eggs of Brachionus plicatilis and Brachionus rotundiformis (Rotifers, Brachionidae): an electron microscopic study

Surface topography and layering of resting eggs of Brachionus plicatilis and B. rotundiformis are described, based on scanning and transmission electron microscope studies. In B. plicatilis, the resting eggs are spherical with smooth wavy ridges on the surface, whereas these ridges are small and condensed in B. rotundiformis. The distribution of pores on the egg surface clearly distinguishes the two species.Ultrastructure of the egg membranes of both species varies greatly with regard to size, shape and sculpturing of each membrane. The alveolar and dense sublayers, which constitute the outer egg membrane (S1) are very thick (10–12 µm) in B. rotundiformis compared to B. plicatilis (4–5 µm). Thus, each species has a characteristic surface and membrane architecture. The functional roles of these membranes, during the resting phase of rotifers, are discussed.     

Physiological causes of cotton fruit abscission under conditions of high temperature and enhanced ultraviolet-B radiation

An experiment was conducted in sunlit controlled environment growth chambers to determine the physiological mechanisms of fruit abscission of cotton ( Gossypium hirsutum L. cv. NuCOTN 33B) grown in high temperature and enhanced ultraviolet (UV)-B radiation. Six treatments included two levels of optimum (30/22°C) and high (36/28°C) day/night temperatures and three levels of biologically effective UV-B radiation (0, 7, and 14 kJ m⁻² per day). Both the temperature and UV-B treatments were imposed from seedling emergence through 79 days after emergence (DAE). High temperature did not negatively affect either leaf net photosynthetic rates (Pn) or abscission of cotton squares (floral buds with bracts) but significantly decreased boll retention. Plants exposed to 7 kJ UV-B radiation retained 56% less bolls than the 0 kJ UV-B control plants at 79 DAE, despite no significant differences in leaf Pn measured at squaring and flowering. At 53 DAE, leaf Pn of plants grown in high UV-B radiation (14 kJ m⁻² per day) decreased by 11%, whereas total non-structural carbohydrate (TNC) concentrations in the leaves, floral buds, and young bolls decreased by 34, 32, and 20%, respectively, compared with the control plants. The high UV-B radiation significantly increased square abscission. Square abscission was not related to leaf TNC concentration but closely correlated with TNC in floral buds ( r  = −0.68, P  < 0.001). Young boll abscission was highly correlated with TNC concentrations in both the leaves ( r  = −0.40, P  < 0.01) and the bolls ( r  = −0.80, P  < 0.001). Our results indicate that non-structural carbohydrate limitation in reproductive parts was a major factor associated with fruit abscission of cotton grown under high temperature and enhanced UV-B radiation conditions.     

Variation of mitochondrial size during the cell cycle: A multiparameter flow cytometric and microscopic study.

BACKGROUND: Changes in mitochondrial structure and size are observed in response to alterations in cell physiology. Flow cytometry provides a useful tool to study these changes in intact cells. We have used flow cytometry and digital fluorescence microscopy to analyze the variations in mitochondrial size in relation to specific phases of the cell cycle. METHODS: Supravital staining of rat fibroblasts was done with Hoechst 33342 and rhodamine 123, and cells were analyzed in a dual-laser flow cytometer. Synchronized cells at various stages of the cell cycle were analyzed for changes in mitochondrial size. These cells were also examined by electron microscopy, digital fluorescence microscopy and computerized image analysis to compare the lengths of the mitochondria. RESULTS: By using fluorescence pulse width analysis, we observed two populations of mitochondria in intact cells. The percentage of cells with small and large mitochondria at specific stages of the cell cycle indicated that mitochondrial size increases during the cell cycle; early G1 phase cells had the smallest mitochondria and the mitotic phase cells had the largest mitochondria. These results were confirmed by microscopic analysis of cells. CONCLUSIONS: Flow cytometry can distinguish the relative mitochondrial size in intact cells, and in combination with digital microscopy it can be used to study mitochondrial variation during the cell cycle.     

A Structural Study of CESA1 Catalytic Domain of Arabidopsis Cellulose Synthesis Complex: Evidence for CESA Trimers

A cellulose synthesis complex with a “rosette” shape is responsible for synthesis of cellulose chains and their assembly into microfibrils within the cell walls of land plants and their charophyte algal progenitors. The number of cellulose synthase proteins in this large multisubunit transmembrane protein complex and the number of cellulose chains in a microfibril have been debated for many years. This work reports a low resolution structure of the catalytic domain of CESA1 from Arabidopsis (Arabidopsis thaliana; AtCESA1CatD) determined by small-angle scattering techniques and provides the first experimental evidence for the self-assembly of CESA into a stable trimer in solution. The catalytic domain was overexpressed in Escherichia coli, and using a two-step procedure, it was possible to isolate monomeric and trimeric forms of AtCESA1CatD. The conformation of monomeric and trimeric AtCESA1CatD proteins were studied using small-angle neutron scattering and small-angle x-ray scattering. A series of AtCESA1CatD trimer computational models were compared with the small-angle x-ray scattering trimer profile to explore the possible arrangement of the monomers in the trimers. Several candidate trimers were identified with monomers oriented such that the newly synthesized cellulose chains project toward the cell membrane. In these models, the class-specific region is found at the periphery of the complex, and the plant-conserved region forms the base of the trimer. This study strongly supports the “hexamer of trimers” model for the rosette cellulose synthesis complex that synthesizes an 18-chain cellulose microfibril as its fundamental product.Cellulose, the most abundant biopolymer on Earth, is composed of linear chains of β-1,4 linked d-Glc monomers with repeating structural units of the disaccharide cellobiose. Numerous cellulose polymers cocrystallize to form microfibrils, which provide mechanical strength and rigidity to plants. Its natural abundance makes it an attractive target for many industrial applications, including paper and pulping, construction, and textile manufacture. More recently, cellulose has been used for production of biofuels, such as ethanol (Ragauskas et al., 2006; Langan et al., 2014), and in the form of nanocellulose as a component in advanced composite materials (Reddy et al., 2013; Habibi, 2014). Cellulose microfibrils are synthesized by a large membrane-bound protein complex. In the land plants and charophycean algae, the cellulose synthesis complex (CSC) has a “rosette” shape (Mueller et al., 1976; Mueller and Brown, 1980b; Kimura et al., 1999), and the entire CSC has reported diameters between 24 to 30 nm (Lerouxel et al., 2006). This structural information was revealed by freeze-fracture transmission electron microscopy, showing six lobes in a hexagonal arrangement at the point where the transmembrane helices of multiple cellulose synthase proteins (CESAs) cross the plasma membrane. Recently KORRIGAN, a protein with cellulase activity, has also been implicated as an integral component of the CSC (Vain et al., 2014).Vascular plants produce several different CESA isoforms. For example, Arabidopsis (Arabidopsis thaliana) has 10 different isoforms with 64% to 98% sequence identity (Holland et al., 2000; Richmond, 2000; McFarlane et al., 2014). The different CESA isoforms play specific roles in cellulose synthesis during plant development. In Arabidopsis, CESA1, CESA3, and CESA6 are required for primary cell wall synthesis, while CESA4, CESA7, and CESA8 are required for secondary cell wall synthesis (Gardiner et al., 2003; Taylor et al., 2003; Persson et al., 2007). CESA2, CESA5, and CESA9 play roles in tissue-specific processes and are partially redundant with CESA6, whereas CESA10 is closely related to AtCESA1 but evidently has a minor role in plant development (Somerville, 2006). The absolute number of CESA proteins present in a CSC remains a subject of much speculation, largely because the stoichiometry of the cellulose microfibril remains unresolved (Cosgrove, 2014). The traditional representation of the microfibril has 36 cellulose chains, and based on this, one would expect that each lobe of the rosette CSC contains six CESA proteins responsible for the synthesis of six glucan chains for a total of 36 CESA proteins per rosette CSC (Herth, 1983; Perrin, 2001; Doblin et al., 2002). However, recent studies using different analytical techniques combined with computation report 18 to 24 cellulose chains per microfibril (Fernandes et al., 2011; Thomas et al., 2013; Oehme et al., 2015). A study of cellulose from mung bean (Vigna radiata) primary cell walls, using x-ray diffraction, solid-state NMR, and computational analysis, supports an 18-chain model for a cellulose microfibril (Newman et al., 2013). This implies that the CSC is composed of fewer than 36 CESA proteins or that not all of the proteins in a CSC are simultaneously active. Further, it has been recently reported that the stoichiometry of CESAs 1, 3, and 6 and CESAs 4, 7, and 8 in the primary and secondary cell walls, respectively, is 1:1:1 (Gonneau et al., 2014; Hill et al., 2014). Together, these reports suggest a rosette CSC composed of 18 CESA proteins with three CESAs per lobe as the most likely composition of a rosette CSC to account for an 18-chain cellulose microfibril (Newman et al., 2013; Gonneau et al., 2014; Hill et al., 2014). In addition, it should also be noted that 24 CESA proteins in a rosette CSC with four proteins per lobe is incompatible with a 1:1:1 CESA stoichiometry.Numerous efforts to isolate active CESA proteins directly from plants or by recombinant expression have not been successful, preventing a detailed structural analysis of CESA proteins or the mechanism of plant cellulose synthesis. In contrast, the recently reported crystal structure of cellulose synthase from Rhodoobacter sphaeroides (Morgan et al., 2013) clearly showed that only a single cellulose synthase polypeptide is required for glucan polymerization and also identified the conserved sequence motifs responsible for catalysis. In addition, based on the presence of an 18-residue glucan chain in the protein tunnel, a mechanism for cellulose synthesis and translocation across a cytoplasmic membrane was proposed that also addressed how the alternate d-Glc molecules are inverted during polymer synthesis (Morgan et al., 2013; Omadjela et al., 2013). However, this structure cannot provide insight into the formation of microfibrils from the cellulose chains synthesized by single polypeptides of CESA.The CESA proteins of land plants and their charophycean algal relatives are multidomain single polypeptide chains of approximately 1000 amino acids. They are predicted to have eight transmembrane helices and to have their N- and C-terminal regions facing the cytoplasm (Pear et al., 1996). Although they share sequence similarity with the bacterial counterpart, they also have unique structural features not found in the bacterial enzymes. The N-terminal domain contains a Zn-binding site that may play a role in oligomerization of CESA proteins (Kurek et al., 2002). The putative cytosolic domain, which is flanked by a two-helix N-terminal transmembrane domain and a six-helix C-terminal transmembrane domain (McFarlane et al., 2014; Slabaugh et al., 2014), has D, D, D, QxxRW motifs that are conserved substrate binding and catalytic residues in the glycosyltransferase-2 superfamily (Nagahashi et al., 1995; Pear et al., 1996; Saxena and Brown, 1997; Yoshida et al., 2000). This domain also has a plant-conserved region (P-CR) and a class-specific region (CSR) that are only found in CESAs that form rosette CSCs. Although the roles of these regions are unknown, they are proposed to be involved in regulatory functions, such as interactions with other proteins and oligomerization to form the rosette shape. In the Arabidopsis CESAs, the sequence identity within the P-CR regions is greater than 80%, while in CSR regions, it is only about 40%. A recent computational model of the cytosolic domain of cotton (Gossypium hirsutum) CESA1 provides the first detailed structural model of the catalytic domain of CESA (Sethaphong et al., 2013). This model structure aligns well with the crystal structure of the bacterial cellulose synthase, indicating that a common mechanism exists for cellulose synthesis in bacteria and plants and that CESAs within rosette CSCs contain a single active synthetic site. In addition, this model made it possible to test possible configurations for the assembly of CESA monomers into a functional rosette CSC (Newman et al., 2013; Sethaphong et al., 2013).Our understanding of the mechanism of cellulose biosynthesis in plants at the molecular level is hampered by the lack of an atomic level CESA model. To gain deeper insight into the structure and role of the catalytic domain of CESA in rosette formation, we carried out a structural characterization of the cytosolic domain of Arabidopsis CESA1, a protein that is essential for cellulose synthesis in the primary cell wall (Arioli et al., 1998). The recombinant protein was purified from Escherichia coli in a two-step process that allowed us to obtain low-resolution structural information about the monomeric and trimeric forms of the recombinant protein using small-angle scattering (SAS) techniques. This study provides the first experimental evidence to support the self-assembly of CESAs into a stable trimer complex, revealing the possible role of the catalytic domain in the formation of the rosette CSC. Comparison of the size of the catalytic domain trimer with dimensions of rosette CSCs obtained from TEM studies strongly supports the “hexamer of trimers” model for rosette CSCs. Computational analysis of the scattering data suggested configurations for how the monomers, including the plant-specific P-CR and CSR domains, may be arranged in the trimeric lobes of the rosette CSC. Knowledge of how CESA proteins assemble in the CSC will enable approaches for rational genetic manipulation of plant cell wall synthesis, which offers enormous opportunities to improve feedstocks for the production of sustainable fuels and chemicals.