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.     

Redox regulation of the VEGF signaling path and tissue vascularization: Hydrogen peroxide, the common link between physical exercise and cutaneous wound healing

Vascularization, under physiological or pathophysiological conditions, typically takes place by one or more of the following processes: angiogenesis, vasculogenesis, arteriogenesis, and lymphangiogenesis. Although all of these mechanisms of vascularization have sufficient contrasting features to warrant consideration under separate cover, one common feature shared by all is their sensitivity to the VEGF signaling pathway. Conditions such as wound healing and physical exercise result in increased production of reactive oxygen species such as H(2)O(2), and both are associated with increased tissue vascularization. Understanding these two scenarios of adult tissue vascularization in tandem offers the potential to unlock the significance of redox regulation of the VEGF signaling pathway. Does H(2)O(2) support tissue vascularization? H(2)O(2) induces the expression of the most angiogenic form of VEGF, VEGF-A, by a HIF-independent and Sp1-dependent mechanism. Ligation of VEGF-A to VEGFR2 results in signal transduction leading to tissue vascularization. Such ligation generates H(2)O(2) via an NADPH oxidase-dependent mechanism. Disruption of VEGF-VEGFR2 ligation-dependent H(2)O(2) production or decomposition of such H(2)O(2) stalls VEGFR2 signaling. Numerous antioxidants exhibit antiangiogenic properties. Current evidence lends firm credence to the hypothesis that low-level endogenous H(2)O(2) supports vascular growth.     

Purification and characterization of a trypsin inhibitor from Putranjiva roxburghii seeds

A highly stable and potent trypsin inhibitor was purified to homogeneity from the seeds of Putranjiva roxburghii belonging to Euphorbiaceae family by acid precipitation, cation-exchange and anion-exchange chromatography. SDS-PAGE analysis, under reducing condition, showed that protein consists of a single polypeptide chain with molecular mass of approximately 34 kDa. The purified inhibitor inhibited bovine trypsin in 1:1 molar ratio. Kinetic studies showed that the protein is a competitive inhibitor with an equilibrium dissociation constant of 1.4x10(-11) M. The inhibitor retained the inhibitory activity over a broad range of pH (pH 2-12), temperature (20-80 degrees C) and in DTT (up to100 mM). The complete loss of inhibitory activity was observed above 90 degrees C. CD studies, at increasing temperatures, demonstrated the structural stability of inhibitor at high temperatures. The polypeptide backbone folding was retained up to 80 degrees C. The CD spectra of inhibitor at room temperature exhibited an alpha, beta pattern. N-terminal amino acid sequence of 10 residues did not show any similarities to known serine proteinase inhibitors, however, two peptides obtained by internal partial sequencing showed significant resemblance to Kunitz-type inhibitors.     

Identification of a Peptide-like Compound with Antimicrobial and Trypsin Inhibitory Activity from Seeds of Bottle Gourd (Lagenaria siceraria)

A low molecular mass peptide like compound with antimicrobial and trypsin inhibitory activity was isolated from the seeds of Lagenaria siceraria. It was purified by ion-exchange and reverse-phase chromatography. The molecular weight of the compound was 678.9 Dalton as determined by MALDI-MS. The infra-red absorbance at 1639 cm^?1, characteristic of an amide bond, by FTIR spectroscopic studies, and absorption at 214 nm on spectrophotometer indicates the peptidic nature of the compound. The compound exhibited antimicrobial activity when tested against Escherichia coli with minimum inhibitory concentration of 20 μM, and trypsin inhibitory activity inhibiting trypsin at a molar ratio of 1:2.     

Microwave-assisted sample preparation for rapid and sensitive analysis of H. pylori lipid A applicable to a single colony

The lipid A of Gram-negative bacteria plays a major role in the pathogenesis of bacterial infections. Lipid A diversity is observed both in the number and length of fatty-acid side chains and in the presence of terminal phosphate residues and associated modifications. In this report, we describe a new sample preparation method based on microwave-assisted enzymatic digestion and detergent-free mild hydrolysis, in conjunction with a MALDI-time-of-flight (TOF)/TOF analysis, to determine the structures of lipid A from Helicobacter pylori. The total time for sample preparation and mass spectrometric analysis is within 2 h and applicable to profiling the lipid A structures from dried bacterial cells on as little as 1 μg. The reliability of the technique was further demonstrated through the analysis of the lipid A from bacterial cells of different H. pylori strains. The phosphorylation and acylation patterns of lipid A could be elucidated using material from a single colony. Furthermore, we found unusual heptaacyl lipid A species present in H. pylori mutant that have not been previously reported, although the abundance was relatively low. The present study provides the first characterization of the lipid A component from a single bacterial colony sample by mass spectrometry.     

Synonymous codon usage pattern among the S,M, and L segments in Crimean-congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever (CCHF) virus is one among the major zoonosis viral diseases that use the Hyalomma ticks as their transmission vector to cause viral infection to the human and mammalian community. The fatality of infectious is high across the world especially in Africa, Asia, Middle East, and Europe. This study regarding codon usage bias of S, M, and L segments of the CCHF virus pertaining to the host Homo sapiens, reveals in-depth information about the evolutionary characteristics of CCHFV. Relative Synonymous Codon Usage (RSCU), Effective number of codons (ENC) were calculated, to determine the codon usage pattern in each segment. Correlation analysis between Codon adaptation index (CAI), GRAVY (Hydrophobicity), AROMO (Aromaticity), and nucleotide composition revealed bias in the codon usage pattern. There was no strong codon bias found among any segments of the CCHF virus, indicating both the factors i.e., natural selection and mutational pressure shapes the codon usage bias.