Caspase-2 Short Isoform Interacts with Membrane-Associated Cytoskeleton Proteins to Inhibit Apoptosis

Caspase-2 (casp-2) is the most conserved caspase across species, and is one of the initiator caspases activated by various stimuli. The casp-2 gene produces several alternative splicing isoforms. It is believed that the long isoform, casp-2L, promotes apoptosis, whereas the short isoform, casp-2S, inhibits apoptosis. The actual effect of casp-2S on apoptosis is still controversial, however, and the underlying mechanism for casp-2S-mediated apoptosis inhibition is unclear. Here, we analyzed the effects of casp-2S on DNA damage induced apoptosis through “gain-of-function” and “loss-of-function” strategies in ovarian cancer cell lines. We clearly demonstrated that the over-expression of casp-2S inhibited, and the knockdown of casp-2S promoted, the cisplatin-induced apoptosis of ovarian cancer cells. To explore the mechanism by which casp-2S mediates apoptosis inhibition, we analyzed the proteins which interact with casp-2S in cells by using immunoprecipitation (IP) and mass spectrometry. We have identified two cytoskeleton proteins, Fodrin and α-Actinin 4, which interact with FLAG-tagged casp-2S in HeLa cells and confirmed this interaction through reciprocal IP. We further demonstrated that casp-2S (i) is responsible for inhibiting DNA damage-induced cytoplasmic Fodrin cleavage independent of cellular p53 status, and (ii) prevents cisplatin-induced membrane blebbing. Taken together, our data suggests that casp-2S affects cellular apoptosis through its interaction with membrane-associated cytoskeletal Fodrin protein.     

Site-specific gap-misrepair mutagenesis by O4-ethylthymine

The mutagenicity of the DNA base O-alkylation adduct, O4-ethylthymine, specifically incorporated into the plasmid vector pUC8 at the unique SalI and HincII recognition sites, was studied in vivo. Escherichia coli, Micrococcus luteus and AMV DNA polymerases catalyze the incorporation of O4-ethylTMP against template adenine and guanine residues, resulting in DNA sequence alteration during subsequent replication in the host E. coli K-12 strain JM83. The greatest mutation frequency was observed with error-prone AMV DNA polymerase. High levels of cognate restriction endonuclease-resistant mutant plasmid isolates were obtained by gap replication repair in the presence of O4-ethylTTP. The yields of mutant isolates were dependent upon the relative concentration of the competing pyrimidine deoxynucleoside triphosphates, TTP and dCTP, in the misreplication reaction. Repair of incorporated O4-ethylTMP of plasmid DNA by in vitro treatment with specific alkyltransferase, prior to transformation in the host, effectively increases the mutagenic efficiency of the adduct. The results obtained are consistent with the high miscoding potential O4-ethylthymine observed in in vitro studies and its ability to base-pair with noncomplementary guanine residues in DNA.     

Pterygodermatites variabilis n. sp. (Nematoda: Rictularioidea) from the Indian gerbils of Pakistan

Pterygodermatites variabilis n. sp. (Nematoda: Rictulalarioidea) from the Indian gerbils Tatera indica collected from Sind and Baluchistan Provinces of Pakistan is described and compared with closely related species of the genus recovered from small mammalian hosts. The new species is characterised by a combination of the following characters: it possesses a regular crown of 26 and 14–38 denticles in male and female specimens respectively, 40–47 pairs of pre-vulvar spines, and a variable position of the vulva in specimens collected from different localities: it also differs in the size of the spicules and gubernaculum.     

Effect of legume-based cropping systems on nitrogen mineralization potential of Vertisol

The quantity and patterns of net mineralization of soil nitrogen (N) were studied in Vertisols under different cropping systems in the semi-arid tropical areas. Eight cropping systems were selected; three contained pigeonpea (PP), one contained PP and cowpea (COP), and two contained chickpea (CP) as legume component crops, one included sequence cropping with nonlegumes during the rainy and postrainy seasons, and one system was kept fallow (F) during the rainy season and sown to sorghum (S) during the postrainy season. Cropping systems with PP as a component crop increased mineralizable N(N _o ) content two-fold in the soil compared with fallow + sorghum (F+S)–F+S system. The N mineralization rate constant (k) was not significantly affected by previous cropping history of the soil; however, a numerically higher rate constant was observed in the COP/PP intercrop, followed by sequential S+safflower (SF) system as compared to the other soils. Mineral N accumulation curves for six soils were more accurately described by the exponential model than the linear model. The active N fraction (N _o /N_tot %) varied between 8 and 16% for different systems and a direct relationship was observed between N _o /N_tot and total N for the soils under diverse cropping systems.ICRISAT JA (1638)     

Kinetics of formation of ferrioxamine B

Stopped-flow kinetic studies of the formation of ferrioxamine B were performed. Formation of the complex follows the rate law

where K_a is the acid dissociation constant of the iron(III) aquo species in 0.1 M formate buffer. At 25°C k₁ = 3.94 × 10²M^?1 sec^?1, k₂K_a = 1.18 × 10^?1 sec^?1, k₃ = 3.6 × 10^?1 sec^?1. Activation parameters for k₁ are ΔH^≠ = 11.7 kcal mole^?1 and ΔS^≠ = ?8 cal K^?1 mole^?1. An associative mechanism is proposed. Attachment of the first chelate ring is the slow step and favorably positions the second chelate ring for attachment. Coordination of two chelate rings favorably positions the third chelate ring for attachment. These results are compared to kinetics of formation of model complexes and to a previous study of the formation of ferrioxamine B in which attachment of the third chelate ring was proposed as the slow step     

Copper metabolism in the plaice, Pleuronectes platessa (L.)

Copper metabolism in a teleost, the plaice, Pleuronectes platessa (L.) from a natural environment has been studied. Distribution in the various tissues of the metal and of five key copper-dependent enzymes: ceruloplasmin EC 1.16.3.1; Superoxide dismutase EC 1.15.11; tryptophan oxygenase EC 1.13.1.12; cytochrome oxidase EC 1.9.3.1 and monoamine oxidase EC 1.4.3.4 have been determined. The copper distribution was found to be similar to that in mammals with the greatest concentrations in the brain and heart. Distribution of the copper enzymes is also similar to that found in mammals. A preliminary characterization of the copper enzymes showed that plaice cytochrome oxidase has a pH maximum 2 pH units more alkaline than the mammalian enzyme and that plaice tryptophan oxygenase is more sensitive to heat denaturation than the mammalian enzyme. The present data form a base-line against which studies on factors affecting the copper metabolism in a teleost can be assessed.     

A sequence specific endonuclease from Micrococcus radiodurans

A new sequence specific endonuclease, MraI has been purified from Micrococcus radiodurans. This enzyme cleaves bacteriophage λ DNA at three sites, adenovirus type 2 DNA at more than 12 sites and has a unique site on ΦX174 DNA. It has no sites on SV40, PM2 and pBR322 DNA. The three sites on phage λ DNA are different from those cleaved by SmaI, XmaI and XorII. The sites of cleavage are located at 0.424, 0.447 and 0.834 fractional lengths on the physical map of λ DNA. MraI is shown to be an isoschizomer of SacII and SstII recognizing the palindromic nucleotide sequence ′5-CCGC↓GG-3′. The enzyme shows an absolute requirement of Mg²⁺, but is active in the absence of added 2-mercaptoethanol. The enzyme shows activity at a broad range of temperature and pH with an optimum at 45°C and pH 7.0. MraI represents the first restriction enzyme from a bacterium whose DNA lacks modified methylated bases.     

Synergistic Interactions of Eugenol-tosylate and Its Congeners with Fluconazole against Candida albicans

We previously reported the antifungal properties of a monoterpene phenol “Eugenol” against different Candida strains and have observed that the addition of methyl group to eugenol drastically increased its antimicrobial potency. Based on the results and the importance of medicinal synthetic chemistry, we synthesized eugenol-tosylate and its congeners (E1-E6) and tested their antifungal activity against different clinical fluconazole (FLC)- susceptible and FLC- resistant C. albicans isolates alone and in combination with FLC by determining fractional inhibitory concentration indices (FICIs) and isobolograms calculated from microdilution assays. Minimum inhibitory concentration (MIC) results confirmed that all the tested C. albicans strains were variably susceptible to the semi-synthetic derivatives E1-E6, with MIC values ranging from 1–62 μg/ml. The test compounds in combination with FLC exhibited either synergy (36%), additive (41%) or indifferent (23%) interactions, however, no antagonistic interactions were observed. The MICs of FLC decreased 2–9 fold when used in combination with the test compounds. Like their precursor eugenol, all the derivatives showed significant impairment of ergosterol biosynthesis in all C. albicans strains coupled with down regulation of the important ergosterol biosynthesis pathway gene-ERG11. The results were further validated by docking studies, which revealed that the inhibitors snugly fitting the active site of the target enzyme, mimicking fluconazole, may well explain their excellent inhibitory activity. Our results suggest that these compounds have a great potential as antifungals, which can be used as chemosensitizing agents with the known antifungal drugs.