Induction of Tumor Cell Death through Targeting Tubulin and Evoking Dysregulation of Cell Cycle Regulatory Proteins by Multifunctional Cinnamaldehydes

Multifunctional trans-cinnamaldehyde (CA) and its analogs display anti-cancer properties, with 2-benzoyloxycinnamaldehyde (BCA) and 5-fluoro-2-hydroxycinnamaldehyde (FHCA) being identified as the ortho-substituted analogs that possess potent anti-tumor activities. In this study, BCA, FHCA and a novel analog 5-fluoro-2-benzoyloxycinnamaldehyde (FBCA), were demonstrated to decrease growth and colony formation of human colon-derived HCT 116 and mammary-derived MCF-7 carcinoma cells under non-adhesive conditions. The 2-benzoyloxy and 5-fluoro substituents rendered FBCA more potent than BCA and equipotent to FHCA. The cellular events by which these cinnamaldehydes caused G₂/M phase arrest and halted proliferation of HCT 116 cells were thereby investigated. Lack of significant accumulation of mitosis marker phospho-histone H3 in cinnamaldehyde-treated cells indicated that the analogs arrested cells in G₂ phase. G₂ arrest was brought about partly by cinnamaldehyde-mediated depletion of cell cycle proteins involved in regulating G₂ to M transition and spindle assembly, namely cdk1, cdc25C, mad2, cdc20 and survivin. Cyclin B1 levels were found to be increased, which in the absence of active cdk1, would fail to drive cells into M phase. Concentrations of cinnamaldehydes that brought about dysregulation of levels of cell cycle proteins also caused tubulin aggregation, as evident from immunodetection of dose-dependent tubulin accumulation in the insoluble cell lysate fractions. In a cell-free system, reduced biotin-conjugated iodoacetamide (BIAM) labeling of tubulin protein pretreated with cinnamaldehydes was indicative of drug interaction with the sulfhydryl groups in tubulin. In conclusion, cinnamaldehydes treatment at proapoptotic concentrations caused tubulin aggregation and dysegulation of cell cycle regulatory proteins cdk1 and cdc25C that contributed at least in part to arresting cells at G₂ phase, resulting in apoptotic cell death characterized by emergence of cleaved forms of caspase 3 and poly (ADP-ribose) polymerase (PARP). Results presented in this study have thus provided further insights into the intricate network of cellular events by which cinnamaldehydes induce tumor cell death.     

Characterization of Tannase Protein Sequences of Bacteria and Fungi: An In Silico Study

The tannase protein sequences of 149 bacteria and 36 fungi were retrieved from NCBI database. Among them only 77 bacterial and 31 fungal tannase sequences were taken which have different amino acid compositions. These sequences were analysed for different physical and chemical properties, superfamily search, multiple sequence alignment, phylogenetic tree construction and motif finding to find out the functional motif and the evolutionary relationship among them. The superfamily search for these tannase exposed the occurrence of proline iminopeptidase-like, biotin biosynthesis protein BioH, O-acetyltransferase, carboxylesterase/thioesterase 1, carbon–carbon bond hydrolase, haloperoxidase, prolyl oligopeptidase, C-terminal domain and mycobacterial antigens families and alpha/beta hydrolase superfamily. Some bacterial and fungal sequence showed similarity with different families individually. The multiple sequence alignment of these tannase protein sequences showed conserved regions at different stretches with maximum homology from amino acid residues 389–469 and 482–523 which could be used for designing degenerate primers or probes specific for tannase producing bacterial and fungal species. Phylogenetic tree showed two different clusters; one has only bacteria and another have both fungi and bacteria showing some relationship between these different genera. Although in second cluster near about all fungal species were found together in a corner which indicates the sequence level similarity among fungal genera. The distributions of fourteen motifs analysis revealed Motif 1 with a signature amino acid sequence of 29 amino acids, i.e. GCSTGGREALKQAQRWPHDYDGIIANNPA, was uniformly observed in 83.3 % of studied tannase sequences representing its participation with the structure and enzymatic function.     

Polymorphic toxin systems: comprehensive characterization of trafficking modes, processing, mechanisms of action, immunity and ecology using comparative genomics

ABSTRACT: BACKGROUND: Proteinaceous toxins are observed across all levels of inter-organismal and intra-genomic conflicts. These include recently discovered prokaryotic polymorphic toxin systems implicated in intra-specific conflict. They are characterized by a remarkable diversity of C-terminal toxin domains generated by recombination with standalone toxin-coding cassettes. Prior analysis revealed a striking diversity of nuclease and deaminase domains among the toxin modules. We systematically investigated polymorphic toxin systems using comparative genomics, sequence and structure analysis. RESULTS: Polymorphic toxin systems are distributed across all major bacterial lineages and are delivered by at least eight distinct secretory systems. In addition to type-II, these include type-V, VI, VII (ESX), and the poorly characterized "Photorhabdus virulence cassettes (PVC)", PrsW-dependent and MuF phage-capsid-like systems. We present evidence that trafficking of these toxins is often accompanied by autoproteolytic processing catalyzed by HINT, ZU5, PrsW, caspase-like, papain-like, and a novel metallopeptidase associated with the PVC system. We identified over 150 distinct toxin domains in these systems. These span an extraordinary catalytic spectrum to include 23 distinct clades of peptidases, numerous previously unrecognized versions of nucleases and deaminases, ADP-ribosyltransferases, ADP ribosyl cyclases, RelA/SpoT-like nucleotidyltransferases, glycosyltranferases and other enzymes predicted to modify lipids and carbohydrates, and a pore-forming toxin domain. Several of these toxin domains are shared with host-directed effectors of pathogenic bacteria. Over 90 families of immunity proteins might neutralize anywhere between a single to at least 27 distinct types of toxin domains. In some organisms multiple tandem immunity genes or immunity protein domains are organized into polyimmunity loci or polyimmunity proteins. Gene-neighborhood-analysis of polymorphic toxin systems predicts the presence of novel trafficking-related components, and also the organizational logic that allows toxin diversification through recombination. Domain architecture and protein-length analysis revealed that these toxins might be deployed as secreted factors, through directed injection, or via inter-cellular contact facilitated by filamentous structures formed by RHS/YD, filamentous hemagglutinin and other repeats. Phyletic pattern and life-style analysis indicate that polymorphic toxins and polyimmunity loci participate in cooperative behavior and facultative 'cheating' in several ecosystems such as the human oral cavity and soil. Multiple domains from these systems have also been repeatedly transferred to eukaryotes and their viruses, such as the nucleo-cytoplasmic large DNA viruses. CONCLUSIONS: Along with a comprehensive inventory of toxins and immunity proteins, we present several testable predictions regarding active sites and catalytic mechanisms of toxins, their processing and trafficking and their role in intra-specific and inter-specific interactions between bacteria. These systems provide insights regarding the emergence of key systems at different points in eukaryotic evolution, such as ADP ribosylation, interaction of myosin VI with cargo proteins, mediation of apoptosis, hyphal heteroincompatibility, hedgehog signaling, arthropod toxins, cell-cell interaction molecules like teneurins and different signaling messengers.     

BRED: A Simple and Powerful Tool for Constructing Mutant and Recombinant Bacteriophage Genomes

Advances in DNA sequencing technology have facilitated the determination of hundreds of complete genome sequences both for bacteria and their bacteriophages. Some of these bacteria have well-developed and facile genetic systems for constructing mutants to determine gene function, and recombineering is a particularly effective tool. However, generally applicable methods for constructing defined mutants of bacteriophages are poorly developed, in part because of the inability to use selectable markers such as drug resistance genes during viral lytic growth. Here we describe a method for simple and effective directed mutagenesis of bacteriophage genomes using Bacteriophage Recombineering of Electroporated DNA (BRED), in which a highly efficient recombineering system is utilized directly on electroporated phage DNA; no selection is required and mutants can be readily detected by PCR. We describe the use of BRED to construct unmarked gene deletions, in-frame internal deletions, base substitutions, precise gene replacements, and the addition of gene tags.     

Heterologous expression of lipase in Escherichia coli is limited by folding and disulfide bond formation

Functional expression of lipase B from Pseudozyma antarctica (PalB) in the cytoplasm of Escherichia coli BL21(DE3) and its mutant derivative Origami B(DE3) was explored. Coexpression of DsbA was found to be effective in enhancing PalB expression. The improvement was particularly pronounced with Origami B(DE3) as a host, suggesting that both folding and disulfide bond formation may be major factors limiting PalB expression. Fusion tag technique was also explored by constructing several PalB fusions for the evaluation of their expression performance. While the solubility was enhanced for most PalB fusions, only the DsbA tag was effective in boosting PalB activity, possibly by both enhanced solubility and correct disulfide bond formation. Our results suggest that PalB activity is closely associated with correct disulfide bond formation, and increased solubilization by PalB fusions does not necessarily result in activity enhancement.     

Enhancing functional expression of heterologous lipase in the periplasm of <Emphasis Type="Italic">Escherichia</Emphasis><Emphasis Type="Bold"> </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Functional expression of heterologous Pseudozyma antarctica lipase B (PalB) in the periplasm of Escherichia coli was explored using four fusion tags, i.e. DsbC, DsbA, maltose-binding protein (MBP), and FLAG in the sequence of increasing expression efficacy. Amongst these fusion tags, FLAG and MBP appear to be the most effective ones in terms of boosting enzyme activity and enhancing solubility of PalB, respectively. Overexpression of these PalB fusions often resulted in concomitant formation of insoluble inclusion bodies. Coexpression of a selection of periplasmic folding factors, including DegP (and its mutant variant of DegP_S210A), FkpA, DsbA, DsbC, and a cocktail of SurA, FkpA, DsbA, and DsbC, could improve the expression performance. Coexpression of DsbA appeared to be the most effective in reducing the formation of inclusion bodies for all the four PalB fusions, implying that functional expression of PalB could be limited by initial bridging of disulfide bonds. Culture performance was optimized by overexpressing FLAG-PalB with DsbA coexpression, resulting in a high volumetric PalB activity of 360 U/L.     

Synthesis, SAR, and Evaluation of 4-[2,4-Difluoro-5-(cyclopropylcarbamoyl)phenylamino]pyrrolo[2,1-f][1,2,4]triazine-based VEGFR-2 kinase inhibitors

Introduction of the 2,4-difluoro-5-(cyclopropylcarbamoyl)phenylamino group at the C-4 position of the pyrrolo[2,1-f][1,2,4] triazine scaffold led to the discovery of a novel sub-series of inhibitors of VEGFR-2 kinase activity. Subsequent SAR studies on the 1,3,5-oxadiazole ring appended to the C-6 position of this new sub-family of pyrrolotriazines resulted in the identification of low nanomolar inhibitors of VEGFR-2. Antitumor efficacy was observed with compound 37 against L2987 human lung carcinoma xenografts in athymic mice.     

Elevation of Defense Network in Chilli Against <Emphasis Type="Italic">Colletotrichum capsici</Emphasis> by Phyllospheric <Emphasis Type="Italic">Trichoderma</Emphasis> Strain

Biocontrol strategies have been mainly focused on proposing the use of biocontrol agents (BCAs) isolated from the rhizospheric region of the plant for protection against phytopathogens. The present study evaluates the effectiveness of phyllospheric Trichoderma isolates in elevating the defense responses in chilli against Colletotrichum capsici infection and comparing its efficiency to the conventionally recommended rhizospheric Trichoderma strains. The elicitation of the defense network in the plants was analyzed using biochemical assays for important enzymes, that is, PAL, PO, PPO, TPC, SOD along with the total protein level in challenged plants over untreated and unchallenged control plants. The results recorded 2.1, 5.18, 3, 0.67, and 0.5-fold increases in TPC, PAL, PO, PPO, and total protein content in BHUF4 (phyllopsheric Trichoderma isolate)-treated plants when compared to control plants under C. capsici challenge. This was at par with the increment recorded in T16A (rhizospheric Trichoderma isolate)-treated chilli plants. The increment in growth parameters was also recorded after treatment with the isolated Trichoderma strains. Interestingly, the phyllospheric isolate (BHUF4) treatment recorded comparable growth promotion in chilli plants recording 36, 62, and 60 % increases in one of the major parameters of plant growth, that is, root length, no. of leaves, and dry weight, respectively. This study proposes the use of combined application of both rhizospheric as well as phyllospheric Trichoderma isolates for better and all around protection of plants against foliar as well as soil phytopathogens. This would be a novel approach in biological control strategy for better management of anthracnose disease of chilli.