Lipid peroxidation in cataract of the human

Lipid peroxidation was investigated as one of the possible mechanisms of cataractogenesis in the human. Malondialdehyde (MDA), a major breakdown product of lipid peroxides, was significantly higher in cataractous lenses as compared to that in normal lenses. 2-Thiobarbituric acid-reactive material, isolated from cortical cataracts and purified by Sephadex G-10 column chromatography, was identified as MDA. In cataractous lenses the enzymic defenses against reactive species of O2 were impaired as evidenced by the significant decrease in activities of superoxide dismutase, catalase and glutathione peroxidase. Hydrogen peroxide in aqueous humor and vitreous humor of human eyes associated with cataract was increased 2-3 fold. It is possible that carbonyl groups of MDA could interact with primary amino groups of proteins and phospholipids of lenticular plasmalemmae by a cross-linking reaction forming Schiff-base conjugates and these mechanisms might be involved in the pathogenesis of cataract.     

Stopped-flow NMR measurement of hydrogen exchange rates in reduced horse cytochrome c under strongly destabilizing conditions

A procedure to measure exchange rates of fast exchanging protein amide hydrogens by time-resolved NMR spectroscopy following in situ initiation of the reaction by diluting a native protein solution into an exchanging deuterated buffer is described. The method has been used to measure exchange rates of a small set of amide hydrogens of reduced cytochrome c, maintained in a strictly anaerobic atmosphere, in the presence of an otherwise inaccessible range of guanidinium deuterochloride concentrations. The results for the measured protons indicate that hydrogen exchange in the unfolding transition region of cytochrome c reach the EX2 limit, but emphasize the difficulty in interpretation of the exchange mechanism in protein hydrogen exchange studies. Comparison of free energies of structure opening for the measured hydrogens with the global unfolding free energy monitored by far-UV CD measurements has indicated the presence of at least one partially unfolded equilibrium species of reduced cytochrome c. The results provide the first report of measurement of free energy of opening of structure to exchange in the 0–2-kcal/mol range. Proteins 32:241–247, 1998. © 1998 Wiley-Liss, Inc.     

Exploration and validation of diphosphate‐based Plasmodium LytB inhibitors using computational approaches

LytB or IspH is an indispensable enzyme and a suitable drug target of Plasmodium falciparum that participate in isoprenoid biosynthesis of nonmevalonate pathway (MEP). Recently, we have investigated the structural dynamics of Plasmodium LytB and proposed some novel diphosphate‐based inhibitors using molecular modeling and docking studies. Here, we have tried to characterize those previously screened molecules by quantitative structure activity relationships and pharmacophore‐based analyses, as well as validated the dynamics of their interactions with LytB protein. Five total compounds having PubChem CID 516 , 125696 , 165275 , 448012 , and 9921431 were predicted with significant inhibitory activity by quantitative structure activity relationships and pharmacophore models. Again, the molecular dynamics simulation results showed that these five compounds are able to form stable complexes with the receptor through many direct and water mediated interactions. The binding free energies calculated by Poisson‐Boltzmann surface area method resulted within the range between ?99.77 and ? 43.74 kcal/mol, which favoured their profound inhibitory affinity. Residues of LytB like His41, His74, Ser222, Ser223, and Asn224 in LytB were the main protagonists in contributing the majority of interaction energies to the ligands. Finally, the ADMET, toxicity, and drug‐likeness scores also affirm these compounds to be considered for further development of new antimalarial inhibitor in the future.     

Isolation of two antifungal compounds fromNeolitsea cuipala and their activity against some plant pathogenic fungi

Zeylanidine and zeylanicine isolated fromNeolitsea cuipala fruit peels are found to be potential antifungal agents againstHelminthosporium oryzae, Fusarium oxysporum, Alternaria solani, Curvularia varuciformis andColletotrichum gleosporioides.     

Light meromyosin paracrystal formation

Studies of paracrystal formation by column purified light meromyosin (LMM) prepared in a variety of ways led to the following conclusions: (a) different portions of the myosin rod may be coded for different stagger relationships. This was concluded from observations that paracrystals with different axial repeat periodicities could be obtained either with LMM framents of different lengths prepared with the same enzyme, or with LMM fragments of identical lengths but prepared with different enzymes. (b) Paracrystals with a 14-nm axial repeat periodicity are most likely formed by the aggregation of sheets with a 44-nm axial repeat within the sheets which are staggered by 14 nm. All of the axial repeat patterns expected from one sheet or aggregates of more than one sheet, on this basis, were observed in the same electron micrograph. (c) C-protein binding probably occurs preferentially to LMM molecules related in some specific way. This was concluded from the observation that the same axial repeat pattern was obtained in paracrystals formed from different LMM preparations in the presence of C-protein, regardless of differences in the axial repeat obtained in the absence of C-protein. (d) Nucleic acid is responsible for the 43-nm axial repeat patterns observed in paracrystals formed by the ethanol-resistant fraction of LMM. In the absence of nuclei acid, paracrystals with a 14nm axial repeat are obtained. (e) The 43-nm axial repeat pattern observed with the ethanol-resistant fraction of LMM is different for LMM preparations obtained by trypsin and papain digestions.     

Cytotoxicity, mutations and DNA damage produced in Chinese hamster cells treated with streptozotocin, its analogs, and N-methyl-N'-nitro-N-nitrosoguanidine.

The activities of streptozotocin (SZ), three structural analogs of SZ, and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in producing cytotoxicity, mutations to 8-azaguanine (8-AzG) resistance, and DNA damage (single-strand breaks) in V79 Chinese hamster cells have been examined. These three biological processes appear to be associated. MNNG was about 10(3) times more active on a molar basis than SZ, and the activities of the analogs fell within these extremes.     

Small interfering RNAs mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor 3

Small interfering (si) and short hairpin (sh) RNAs induce robust degradation of homologous mRNAs, making them a potent tool to achieve gene silencing in mammalian cells. Silencing by siRNAs is used widely because it is considered highly specific for the targeted gene, although a recent report suggests that siRNA also induce signaling through the type I IFN system. When human embryonic kidney 293 (HEK293) or keratinocyte (HaCaT) cell lines or human primary dendritic cells or macrophages were transfected with siRNA or shRNAs, suppression of nontargeted mRNA expression was detected. Additionally, siRNA and shRNA, independent of their sequences, initiated immune activation, including IFN-alpha and TNF-alpha production and increased HLA-DR expression, in transfected macrophages and dendritic cells. The siRNAs induced low, but significant, levels of IFN-beta in HEK293 and HaCaT cells. Secretion of these cytokines increased tremendously when HEK293 cells overexpressed Toll-like receptor 3 (TLR3), and the increased secretion of IFN-beta was inhibited by coexpression of an inhibitor of TIR domain-containing adapter-inducing IFN-beta, the TLR3 adaptor protein linked to IFN regulatory factor 3 signaling. Although siRNA and shRNA knockdown of genes represents a new and powerful tool, it is not without nonspecific effects, which we demonstrate are mediated in part by signaling through TLR3.