Enhancement of <Emphasis Type="Italic">Trichoderma</Emphasis> endochitinase secretion in tobacco cell cultures using an <Emphasis Type="Italic">α-</Emphasis>amylase signal peptide

An α-amylase signal peptide from rice was synthesized and fused with endochitinase (ech42) gene cloned from Trichoderma virens. The chimeric gene was designated as PSPα-amyech42, and this was transferred to a plant transformation vector, referred to as pMASGK. Leaf explants of tobacco cv White Burley were co-cultivated with Agrobacterium tumefaciens strain LBA4404 carrying ech42 with its own signal peptide(ech42SP) and PSPα-amyech42(pMASGK) separately. Putative transformants were selected on Murashige and Skoog (MS) medium, supplemented with 1 mg/l bezyladenine (BA), 0.5 mg/l naphthalene acetic acid(NAA), and containing 200 mg/l kanamycin and 200 mg/l cefotaxime. Transformation was further confirmed by PCR with specific primers and Southern blot hybridization. Endochitinase secretion was quantified in 1-week-old cell suspension cultures obtained from 3-week-old callus cultures of transformants carrying PSPα-amyech42, transformants with ech42 and of control (untransformed) plant. Callus cultures of PSPα-amyech42 showed higher endochitinase activity (9–12 times) than those carrying ech42SP (7–8 times) in both medium and cell extracts. Media collected (200 μg of total protein) from PSPα-amyech42 suspension cultures in Potato Dextrose Agar plates showed growth inhibition of 73 and 53% against Sclerotium rolfsii and Rhizoctonia bataticola, respectively, whereas media collected (200 μg of total protein) from ech42SP suspension culture showed inhibition of 14 and 24% against Sclerotium rolfsii and Rhizoctonia bataticola, respectively.     

Selection for salt tolerance in vitro using microspore-derived embryos ofBrassica napus cv. topas,and the characterization of putative tolerant plants

Microspore-derived embryos ofBrassica napus cv. Topas that survived salt stress, were obtained after selection against otherwise lethal doses (0.6 and 0.7%) of NaCl after mutagen treatment. A total of 10 salt-surviving embryos were obtained out of a possible 834 000 embryos that were mutagenized. One embryo out of a possible 845 000 obtained from nonmutagenized controls survived but failed to develop into a plant. Visual assessment after salt stress indicated that both the putative salt-tolerant plants and plants from control seeds behaved similarly. However, based on individual characteristics related to salt tolerance, one of the lines (PST-2) accumulated less sodium and retained more potassium, and hence was able to maintain a more favorable Na:K ratio as compared to the controls under salt stress. Also chlorophylla fluorescence induction and quenching signals indicated a high energetic state of the thylakoid membranes in PST-2 under salt stress. The other putative salt-tolerant line (PST-1) had a higher background level of proline that may have enabled it to survive salt stress during initial screening, although its later performance was no better than the control plants.     

Antagonistic molecular interactions of photosynthetic pigments with molecular disease targets: a new approach to treat AD and ALS

Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS) are progressive neurodegenerative diseases that affect the neurons in the brain and the spinal cord. Neuroinflamation and apoptosis are key players in the progressive damage of the neurons in AD and ALS. Currently, there is no drug to offer complete cure for both these diseases. Riluzole is the only available drug that can prolong the life time of the ALS patients for nearly 3 months. Molecules that offer good HIT to the molecular targets of ALS will help to treat AD and ALS patients. P53 kinase receptor (4AT3), EphA4 (3CKH) and histone deacetylase (3SFF) are the promising disease targets of AD and ALS. This paper discusses on a new approach to combat neurodegenerative diseases using photosynthetic pigments. The docking studies were performed with the Autodock Vina algorithm to predict the binding of the natural pigments such as β carotene, chlorophyll a, chlorophyll b, phycoerythrin and phycocyanin on these targets. The β carotene, phycoerythrin and phycocyanin had higher binding energies indicating the antagonistic activity to the disease targets. These pigments serve as a potential therapeutic molecule to treat neuroinflammation and apoptosis in the AD and ALS patients.     

Effects of oral dosing paradigms (gavage versus diet) on pharmacokinetics and pharmacodynamics

In cancer chemopreventive studies, test agents are typically administered via diet, while the preclinical safety studies normally employ oral gavage dosing. Correspondence in pharmacokinetic and pharmacodynamic profiles between the two dosing approaches cannot be assumed a priori. Sulindac, a non-steroidal anti-inflammatory agent with potential chemopreventive activity, was used to assess effects of the two oral dosing paradigms on its pharmacokinetics and pharmacodynamics. Time-dependent concentrations of sulindac and its sulfone metabolite were determined in plasma and potential target organ, mammary gland. Prostaglandin E(2) was used as a pharmacodynamic biomarker and measured in mammary gland. An inverse linear relationship was detected between pharmacodynamic and pharmacokinetic markers, area under the curve for prostaglandin E(2) levels and sulindac sulfone concentrations, respectively, in the mammary tissue. Marked differences in pharmacokinetics and pharmacodynamics were observed after administration of sulindac by the two oral dosing paradigms. In general, oral gavage resulted in higher peak and lower trough concentrations of sulindac in plasma and mammary tissue, higher area under concentration-time curve in plasma and mammary tissue, and greater effect on prostaglandin E(2) levels than the corresponding diet dosing. This study illustrates potential pitfalls and limitations in trying to generalize based on data obtained with different oral dosing schemes and their extrapolation to potential efficacy and health risks in humans.     

NF-kappaB and inhibitor of apoptosis proteins are required for apoptosis resistance of epithelial cells persistently infected with Chlamydophila pneumoniae

Infection with Chlamydophila pneumoniae (Cpn) renders host cells resistant to apoptosis induced by a variety of stimuli. While modulation of apoptosis has been extensively studied in cells acutely infected with Cpn, very little is known on how persistent chlamydial infection influences host cell survival. Here we show that epithelial cells persistently infected with Cpn resist apoptosis induced with TNFalpha or staurosporine. Cpn induced the activation of nuclear factor kappa B (NF-kappaB) and inhibition of NF-kappaB with a chemical inhibitor or by silencing expression of the p65 subunit sensitized infected cells for apoptosis induction by staurosporine or TNFalpha. Persistent infection resulted in the upregulation of the NF-kappaB regulated inhibitor of apoptosis protein 2 (cIAP-2) but not inhibitor of apoptosis protein 1 (cIAP-1). Interestingly, silencing of either cIAP-1 or cIAP-2 sensitized infected cells, suggesting that IAPs play an important role in the apoptosis resistance of persistently infected cells.     

Role of Melanoma Inhibitor of Apoptosis (ML-IAP) Protein, a Member of the Baculoviral IAP Repeat (BIR) Domain Family, in the Regulation of C-RAF Kinase and Cell Migration

Inhibitor of apoptosis (IAPs) proteins are characterized by the presence of evolutionarily conserved baculoviral inhibitor of apoptosis repeat (BIR) domains, predominantly known for their role in inhibiting caspases and, thereby, apoptosis. We have shown previously that multi-BIR domain-containing IAPs, cellular IAPs, and X-linked IAP can control tumor cell migration by directly regulating the protein stability of C-RAF kinase. Here, we extend our observations to a single BIR domain containing IAP family member melanoma-IAP (ML-IAP). We show that ML-IAP can directly bind to C-RAF and that ML-IAP depletion leads to an increase in C-RAF protein levels, MAPK activation, and cell migration in melanoma cells. Thus, our results unveil a thus far unknown role for ML-IAP in controlling C-RAF stability and cell migration.     

X-linked Inhibitor of Apoptosis Protein promotes the degradation of its antagonist, the pro-apoptotic ARTS protein

ARTS (Sept4_i2) is a mitochondrial pro-apoptotic tumor suppressor protein. In response to apoptotic signals, ARTS translocates to the cytosol where it promotes caspase activation through caspase de-repression and proteasome mediated degradation of X-linked Inhibitor of Apoptosis Protein (XIAP). Here we show that XIAP regulates the levels of ARTS by serving as its ubiquitin ligase, thereby providing a potential feedback mechanism to protect against unwanted apoptosis. Using both in vitro and in vivo ubiquitination assays we found that ARTS is directly ubiquitinated by XIAP. Moreover, we found that XIAP-induced ubiquitination and degradation is prevented by removal of the first four amino acids in the N-terminus of ARTS, which contains a single lysine residue at position 3. Thus, this lysine at position 3 is a likely target for ubiquitination by XIAP. Importantly, although the stabilized ARTS lacking its first 4 residues binds XIAP as well as the full length ARTS, it is more potent in promoting apoptosis than the full length ARTS. This suggests that increased stability of ARTS has a significant effect on its ability to induce apoptosis. Collectively, our data reveal a mutual regulatory mechanism by which ARTS and XIAP control each other's levels through the ubiquitin proteasome system.     

Interaction of silver and gold nanoparticles in mammalian cancer: as real topical bullet for wound healing— A comparative study

The present study evaluates in vitro cytotoxic effects and the mode of interaction of biologically synthesized Ag and Au nanoparticles (NPs) using Brassica oleracea L. var. capitata f. rubra (BOL) against HT-1080 cancer cells and bacterial cells as well as their wound healing efficacy using a mouse model. UV–visible spectroscopy, scanning electron microscopy, high-resolution transmission electron microscopy, and energy-dispersive X-ray analysis have ascertained the formation of nano-sized Ag and Au particles. Fourier transform infrared analysis has confirmed that polyphenol and amide groups in BOL act as capping as well as reducing agents. The free radical scavenging activity under in vitro conditions is found to be higher for the Ag NPs when compared to the Au NPs. Acridine orange–ethidium bromide dual staining and comet assay have indicated that the cytotoxic effects are mediated through nuclear morphological changes and DNA damage. The intracellular localization of Ag and Au NPs in HT-1080 cells and their subsequent effect on apoptosis and necrosis were analyzed by flow cytometry while the mode of interaction was established by scanning electron microscopy under field emission mode and by bio-transmission electron microscopy. These methods of analysis clearly revealed that the Ag and Au NPs have easily entered and accumulated into the cytosol and nucleus, resulting in activation of inflammatory and apoptosis pathways, which in turn cause damage in DNA. Further, mRNA and protein expression of caspase-3 and caspase-7, TNF-α, and NF-κB have provided sufficient clues for induction of intrinsic and extrinsic apoptosis and inflammatory pathways in Ag NP- and Au NP-treated cells. Evaluation of wound healing properties of Ag and Au NPs using a mouse model indicates rapid healing of wounds. In addition, no clear toxic effects and no nuclear abnormalities in peripheral blood cells are observed. Ag NPs appear to be a better anticancer therapeutic agent than Au NPs. Nonetheless, both Ag NPs and Au NPs show potential for promoting topical wound healing without any toxic effects.

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Graphical abstract Schematic representation of biological synthesis of Ag and Au NPs and its application on cancer and wound healing: