In vitro withanolide production by Withania somnifera L. cultures

In vitro multiple shoots, root, callus and cell suspension cultures of Withania somnifera exhibited the potentiality to produce pharmacologically active withanolides. Multiple shoots cultures exhibited an increase in withanolide A accumulation compared to shoots of the mother plant. In vitro generated root cultures as well as callus and suspension cultures also produced withanolides albeit at lower levels.     

Effects of calcium,alginate, and calcium-alginate immobilization on growth and tropane alkaloid levels of a stable suspension cell line of Datura innoxia Mill

Summary A stabilized two-year old suspension of a Datura innoxia cell line, producing small amounts of tropane alkaloids (scopolamine and hyoscyamine) was used in this study. Calcium alginate immobilization has been shown to be able to increase secondary metabolite (i. e. alkaloid) production. The effects of calcium and ungellified alginate were both beneficial for tropane alkaloid synthesis; a 10mM calcium chloride supply gave the best results, with a 10-fold yield increase.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - S standard cell culture medium - HPLC high performance liquid chromatography - FW fresh weight - FDA fluorescein diacetate - FW fresh weight     

Role of the host cell cycle in theAgrobacterium-mediated genetic transformation ofPetunia: Evidence of an S-phase control mechanism for T-DNA transfer

Chimeric -glucuronidase (GUS) gene expression in an efficientAgrobacterium-mediated transformation system utilising mesophyll cells ofPetunia hybrida synchronized with cell cycle phase-specific inhibitors (mimosine and colchicine) was used to show the absolute requirement of S-phase for transfer and/or integration of the transferred DNA (T-DNA). Flow-cytometric analysis of nuclear DNA content and immunohistological detection of bromodeoxyuridine (BrdUrd) incorporation showed that, prior to phytohormone treatment, most (98%) mesophyll cells were at GO-Gl-phase (quiescent phase) and no cell division was occurring. After 48 h and 72 h of phytohormone treatment, there was a rapid increase in S-G2-M-phase populations (> 75%) and a concomitant decrease (down to 24%) in G0–-G1-phase cells. Assays of GUS showed that maximum transformation (> 95% of explants) also occurred after this period. Our data showed that mimosine and colchicine blocked the mesophyll cells at late Gl-phase and M-phase, respectively. No transformation (= GUS expression) was observed in phytohormone-treated cells inhibited in late G1 by mimosine. However, after removal of mimosine, 82% of the explants were transformed, indicating the non-toxic and reversible effect of the inhibitor. On the other hand, a relatively high transformation frequency (65% of explants) was observed after blocking the cell cycle at M-phase with colchicine. However, only transient, but no stable, gene expression (= kanamycin-resistant callus formation) was observed in colchicine-treated M-phase-arrested cells. Similarly, endoreduplication of nuclear DNA, which occurred during the 48 h of phytohormone treatment in some mesophyll cells and cells located along the minor veins in the leaf explants, resulted in transient GUS expression only. These observations indicate a direct correlation between endoreduplication and transient GUS gene expression. Obviously, for stable GUS gene expression, cell division and proliferation are required, indicating that both DNA duplication (S-phase) and cell division (M-phase) are strongly related to stable transformation. We propose that the present system should facilitate further dissection of the process of T-DNA integration in the host genome and therefore should aid in developing new strategies for transformation of recalcitrant plants.Abbreviations BAP 6-benzylaminopurine - BM basal medium - BrdUrd bromodeoxyuridine - GUS -glucuronidase - Km^R kanamycin resistant - T-DNA transferred DNA     

Withaferin A induces apoptosis by activating p38 mitogen-activated protein kinase signaling cascade in leukemic cells of lymphoid and myeloid origin through mitochondrial death cascade

Withaferin A (WA) is present abundantly in Withania somnifera, a well-known Indian medicinal plant. Here we demonstrate how WA exhibits a strong growth-inhibitory effect on several human leukemic cell lines and on primary cells from patients with lymphoblastic and myeloid leukemia in a dose-dependent manner, showing no toxicity on normal human lymphocytes and primitive hematopoietic progenitor cells. WA-mediated decrease in cell viability was observed through apoptosis as demonstrated by externalization of phosphatidylserine, a time-dependent increase in Bax/Bcl-2 ratio; loss of mitochondrial transmembrane potential, cytochrome c release, caspases 9 and 3 activation; and accumulation of cells in sub-G0 region based on DNA fragmentation. A search for the downstream pathway further reveals that WA-induced apoptosis was mediated by an increase in phosphorylated p38MAPK expression, which further activated downstream signaling by phosphorylating ATF-2 and HSP27 in leukemic cells. The RNA interference of p38MAPK protected these cells from WA-induced apoptosis. The RNAi knockdown of p38MAPK inhibited active phosphorylation of p38MAPK, Bax expression, activation of caspase 3 and increase in Annexin V positivity. Altogether, these findings suggest that p38MAPK in leukemic cells promotes WA-induced apoptosis. WA caused increased levels of Bax in response to MAPK signaling, which resulted in the initiation of mitochondrial death cascade, and therefore it holds promise as a new, alternative, inexpensive chemotherapeutic agent for the treatment of patients with leukemia of both lymphoid and myeloid origin. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Piperine analogs as potent Staphylococcus aureus NorA efflux pump inhibitors

Based on our recent findings that piperine is a potent Staphylococcus aureus NorA efflux pump inhibitor (EPI), 38 piperine analogs were synthesized and bioevaluated for their EPI activity. Twenty-five of them were found active with potentiating activity equivalent or more than known EPIs like reserpine, carsonic acid and verapamil. The inhibitory mechanism of the compounds was confirmed by efflux inhibition assay using ethidium bromide as NorA substrate. The present communication describes the synthesis, bioevaluation and structure related activity of these efflux pump inhibitors.     

Role of vitronectin-like protein in Agrobacterium attachment and transformation of Arabidopsis cells

The role of plant vitronectin-like protein (Vn) in Agrobacterium-host plant interactions and receptor-specific bacterial attachment is unclear and still open to debate. Using a well-established Agrobacterium-mediated Arabidopsis transformation system, the marker gene beta-glucuronidase (GUS) of Escherichia coli, and biochemical and cytological methods, such as ELISA tests, immunoblots, immunolocalization, and functional in vitro binding assays, we have reassessed the role of Vn in receptor-specific bacterial attachment and transformation. We provide evidence that Vn is present in the host plant cells and anti-human vitronectin antibody cross-reacts with a 65-kDa protein from Arabidopsis cells. The specificity of the immunological cross-reactivity of anti-vitronectin antibodies was further demonstrated by ELISA competition experiments. Immunogold labeling showed that Vn is localized in the plant cell wall, and its level increased considerably after phytohormone treatment of the petiole explants. However, Agrobacterium attachment was unaffected, and no inhibition of petiole cell transformation was detected in the presence of human vitronectin and anti-vitronectin antibodies in the media. Additionally, no correlation between the occurrence of Vn, attachment of bacteria to the cells, and susceptibility to Agrobacterium-mediated transformation was observed. Taken together, our data do not support a functional role of plant Vn as the receptor for site-specific Agrobacterium attachment leading to the transformation of Arabidopsis cells.     

Cellulase production by six <Emphasis Type="Italic">Trichoderma</Emphasis> spp. fermented on medicinal plant processings

Capabilities of cellulase production, using delignified bioprocessings of medicinal and aromatic plants, viz. citronella (Cymbopogon winterianus) and Artemisia annua (known as marc of Artemisia) and garden waste (chiefly containing Cynodon dactylon), by the six species of Trichoderma were comparatively evaluated. Among the members of Trichoderma studied, T. citrinoviride was found to be the most efficient producer of cellulases along with a high level of β- glucosidase (produced 102.4 IU g⁻¹ on marc of Artemisia; 101.33 IU g⁻¹ on garden waste; 81.86 IU g⁻¹ on distillation waste of citronella and 94.77 IU g⁻¹ on pure cellulose). Although T. virens was noticed to be the minimal enzyme producer fungus, it interestingly could not produce complete cellulase enzyme complex on any test waste or pure cellulose, except on marc of Artemisia, where it produced all three enzymes of the complex. Immediate reduction in pH was also noticed during fermentation in the case of pure polymer (cellulose) by all tested fungi, while it was delayed with delignified agrowastes. The pH profile varied with the substrate used as well as with individual species of Trichoderma. On the other hand, no alteration in pH with any species of Trichoderma was noticed when grown on marc of A. annua, which might be due to the buffering capacity of this marc.     

Phylum Verrucomicrobia representatives share a compartmentalized cell plan with members of bacterial phylum Planctomycetes

Background  

The phylum Verrucomicrobia is a divergent phylum within domain Bacteria including members of the microbial communities of soil and fresh and marine waters; recently extremely acidophilic members from hot springs have been found to oxidize methane. At least one genus, Prosthecobacter, includes species with genes homologous to those encoding eukaryotic tubulins. A significant superphylum relationship of Verrucomicrobia with members of phylum Planctomycetes possessing a unique compartmentalized cell plan, and members of the phylum Chlamydiae including human pathogens with a complex intracellular life cycle, has been proposed. Based on the postulated superphylum relationship, we hypothesized that members of the two separate phyla Planctomycetes and Verrucomicrobia might share a similar ultrastructure plan differing from classical prokaryote organization.     

Properties of STAT1 and IRF1 enhancers and the influence of SNPs

Background

STAT1 and IRF1 collaborate to induce interferon-γ (IFNγ) stimulated genes (ISGs), but the extent to which they act alone or together is unclear. The effect of single nucleotide polymorphisms (SNPs) on in vivo binding is also largely unknown.

Results

We show that IRF1 binds at proximal or distant ISG sites twice as often as STAT1, increasing to sixfold at the MHC class I locus. STAT1 almost always bound with IRF1, while most IRF1 binding events were isolated. Dual binding sites at remote or proximal enhancers distinguished ISGs that were responsive to IFNγ versus cell-specific resistant ISGs, which showed fewer and mainly single binding events. Surprisingly, inducibility in one cell type predicted ISG-responsiveness in other cells. Several dbSNPs overlapped with STAT1 and IRF1 binding motifs, and we developed methodology to rapidly assess their effects. We show that in silico prediction of SNP effects accurately reflects altered binding both in vitro and in vivo.

Conclusions

These data reveal broad cooperation between STAT1 and IRF1, explain cell type specific differences in ISG-responsiveness, and identify genetic variants that may participate in the pathogenesis of immune disorders.