Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic cell death of human myeloid leukemia HL-60 cells by a dietary compound withaferin A with concomitant protection by <Emphasis Type="Italic">N</Emphasis>-acetyl cysteine

Induction of apoptosis in cancer cells has become the major focus of anti-cancer therapeutics development. WithaferinA, a major chemical constituent of Withania somnifera, reportedly shows cytotoxicity in a variety of tumor cell lines while its molecular mechanisms of action are not fully understood. We observed that withaferinA primarily induces oxidative stress in human leukemia HL-60 cells and in several other cancer cell lines. The withanolide induced early ROS generation and mitochondrial membrane potential (Δψ_mt) loss, which preceded release of cytochrome c, translocation of Bax to mitochondria and apoptosis inducing factor to cell nuclei. These events paralleled activation of caspases −9, −3 and PARP cleavage. WA also activated extrinsic pathway significantly as evidenced by time dependent increase in caspase-8 activity vis-à-vis TNFR-1 over expression. WA mediated decreased expression of Bid may be an important event for cross talk between intrinsic and extrinsic signaling. Furthermore, withaferinA inhibited DNA binding of NF-κB and caused nuclear cleavage of p65/Rel by activated caspase-3. N-acetyl-cysteine rescued all these events suggesting thereby a pro-oxidant effect of withaferinA. The results of our studies demonstrate that withaferinA induced early ROS generation and mitochondrial dysfunction in cancer cells trigger events responsible for mitochondrial -dependent and -independent apoptosis pathways.     

A triterpenediol from <Emphasis Type="Italic">Boswellia serrata</Emphasis> induces apoptosis through both the intrinsic and extrinsic apoptotic pathways in human leukemia HL-60 cells

A triterpenediol (TPD) comprising of isomeric mixture of 3α, 24-dihydroxyurs-12-ene and 3α, 24-dihydroxyolean-12-ene from Boswellia serrata induces apoptosis in cancer cells. An attempt was made in this study to investigate the mechanism of cell death by TPD in human leukemia HL-60 cells. It inhibited cell proliferation with IC₅₀ ∼ 12 μg/ml and produced apoptosis as measured by various biological end points e.g. increased sub-G0 DNA fraction, DNA ladder formation, enhanced AnnexinV-FITC binding of the cells. Further, initial events involved massive reactive oxygen species (ROS) and nitric oxide (NO) formation, which were significantly inhibited by their respective inhibitors. Persistent high levels of NO and ROS caused Bcl-2 cleavage and translocation of Bax to mitochondria, which lead to loss of mitochondrial membrane potential (Δψ_m) and release of cytochrome c, AIF, Smac/DIABLO to the cytosol. These events were associated with decreased expression of survivin and ICAD with attendant activation of caspases leading to PARP cleavage. Furthermore, TPD up regulated the expression of cell death receptors DR4 and TNF-R1 level, leading to caspase-8 activation. These studies thus demonstrate that TPD produces oxidative stress in cancer cells that triggers self-demise by ROS and NO regulated activation of both the intrinsic and extrinsic signaling cascades.     

The closed structure of presequence protease PreP forms a unique 10,000 Angstroms3 chamber for proteolysis

Presequence protease PreP is a novel protease that degrades targeting peptides as well as other unstructured peptides in both mitochondria and chloroplasts. The first structure of PreP from Arabidopsis thaliana refined at 2.1 Angstroms resolution shows how the 995-residue polypeptide forms a unique proteolytic chamber of more than 10,000 Angstroms(3) in which the active site resides. Although there is no visible opening to the chamber, a peptide is bound to the active site. The closed conformation places previously unidentified residues from the C-terminal domain at the active site, separated by almost 800 residues in sequence to active site residues located in the N-terminal domain. Based on the structure, a novel mechanism for proteolysis is proposed involving hinge-bending motions that cause the protease to open and close in response to substrate binding. In support of this model, cysteine double mutants designed to keep the chamber covalently locked show no activity under oxidizing conditions. The manner in which substrates are processed inside the chamber is reminiscent of the proteasome; therefore, we refer to this protein as a peptidasome.     

Chromatographic separation of enantiomers of non-protein alpha-amino acids after derivatization with Marfey's reagent and its four variants

Some non-protein α-amino acids were derivatized with 1-fluoro-2,4-dinitrophenyl-5-l-alaninamide (Marfey’s reagent, MR, FDNP-l-Ala-NH₂,) and four of its structural variants FDNP-l-Phe-NH₂, FDNP-l-Val-NH₂, FDNP-l-Leu-NH₂ and FDNP-l-Pro-NH₂. The resultant diastereomers were separated by normal and reversed phase thin layer chromatography (TLC) and reversed phase HPLC. In normal phase TLC, best resolution was obtained with solvent combination of phenol-water (3:1) while in reversed phase TLC mixtures of acetonitrile with triethylammonium phosphate buffer were found successful for resolution of diastereomers. The separation behavior of diastereomers prepared with different reagents was compared. The diastereomers of most of the amino acids prepared with FDNP-l-Leu-NH₂ were best separated while those prepared with FDNP-l-Pro-NH₂ failed to separate in most of the cases. The diastereomers were also separated on a reversed phase C₈ column with gradient elution using mixture of aqueous-trifluoroacetic acid (TFA) and acetonitrile and with detection at 340 nm. The effects of TFA concentration, flow rate and run time on HPLC separation were studied.     

Application of (S)-N-(4-Nitrophenoxycarbonyl) phenylalanine methoxyethyl ester as a chiral derivatizing reagent for reversed-phase high-performance liquid chromatographic separation of diastereomers of amino alcohols, non-protein amino acids, and PenA

Indirect enantioresolution of 15 primary and secondary amino group containing compounds (amino alcohols, non-protein amino acids, PenA) was done using the reagent (S)-N-(4-Nitrophenoxycarbonyl) phenylalanine methoxyethyl ester [(S)-NIFE] by reversed-phase high-performance liquid chromatography. The diastereomeric derivatives were analyzed under reversed-phase conditions using linear gradient. The detection was at 205 nm and sharp peaks were obtained. The reagent used is comparatively economic than the other derivatizing reagents. Method validation was also done.     

Selective isolation,evaluation and characterization of antagonistic actinomycetes against <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The baiting bag method was found to be useful for isolating antagonistic actinomycetes from terrestrial habitat. Out of total 110 actinomycetes isolated from rhizospheric and non-rhizospheric soil of Indo Gangetic Plains (IGP) of India, 9 isolates exhibited aggressive antagonism against Rhizoctonia solani, screened through dual culture, well diffusion and sealed plate technique. Maximum growth inhibition was recorded up to 50% under well diffusion (S. toxytricini vh22) and 52.6% in a direct confrontation (Actinomycetales bacterium vh41). Whereas maximum disease suppression (53.33%) under green house condition was achieved on seedling treated with S. tricolor vh85. Scanning electron microscopy of antagonists and pathogen interaction exhibited pore formation and hyphal degradation of test pathogen. Physiological and molecular characterization of selected isolates showed wide diversity and uncommon species has been encountered through the selective isolation technique.     

Mode of Binding of the Tuberculosis Prodrug Isoniazid to Heme Peroxidases: BINDING STUDIES AND CRYSTAL STRUCTURE OF BOVINE LACTOPEROXIDASE WITH ISONIAZID AT 2.7 ? RESOLUTION*

Isoniazid (INH) is an anti-tuberculosis prodrug that is activated by mammalian lactoperoxidase and Mycobacterium tuberculosis catalase peroxidase (MtCP). We report here binding studies, an enzyme assay involving INH, and the crystal structure of the complex of bovine lactoperoxidase (LPO) with INH to illuminate binding properties and INH activation as well as the mode of diffusion and interactions together with a detailed structural and functional comparison with MtCP. The structure determination shows that isoniazid binds to LPO at the substrate binding site on the distal heme side. The substrate binding site is connected to the protein surface through a long hydrophobic channel. The acyl hydrazide moiety of isoniazid interacts with Phe⁴²² O, Gln⁴²³ O^ϵ1, and Phe²⁵⁴ O. In this arrangement, pyridinyl nitrogen forms a hydrogen bond with a water molecule, W-1, which in turn forms three hydrogen bonds with Fe³⁺, His¹⁰⁹ N^ϵ2, and Gln¹⁰⁵ N^ϵ2. The remaining two sides of isoniazid form hydrophobic interactions with the atoms of heme pyrrole ring A, C^β and C^γ atoms of Glu²⁵⁸, and C^γ and C^δ atoms of Arg²⁵⁵. The binding studies indicate that INH binds to LPO with a value of 0.9 × 10⁻⁶ m for the dissociation constant. The nitro blue tetrazolium reduction assay shows that INH is activated by the reaction of LPO-H₂O₂ with INH. This suggests that LPO can be used for INH activation. It also indicates that the conversion of INH into isonicotinoyl radical by LPO may be the cause of INH toxicity.     

Two novel mitochondrial and chloroplastic targeting-peptide-degrading peptidasomes in A. thaliana, AtPreP1 and AtPreP2

Two novel metalloendopeptidases in Arabidopsis thaliana, AtPreP1 and AtPreP2, are responsible for the degradation of targeting peptides in mitochondria and chloroplasts. Both AtPreP1 and AtPreP2 contain ambiguous targeting peptides and are dually targeted to both organelles. The proteases also have the capacity to degrade unstructured peptides of up to 65 amino acid residues, but not small proteins. The catalysis occurs in a huge catalytic chamber revealed by the crystal structure of AtPreP1 at 2.1 A. The enzymes show a preference for basic and small uncharged amino acids or serines at the cleavage sites. Despite similarities in cleavage specificities, cleavage-site recognition differs for both proteases and is context- and structure-dependent. The AtPreP1 and AtPreP2 genes are differentially expressed in Arabidopsis.