Sodium orthovanadate associated with pharmacological doses of ascorbate causes an increased generation of ROS in tumor cells that inhibits proliferation and triggers apoptosis

Pharmacological doses of ascorbate were evaluated for its ability to potentiate the toxicity of sodium orthovanadate (Na₃VO₄) in tumor cells. Cytotoxicity, inhibition of cell proliferation, generation of ROS and DNA fragmentation were assessed in T24 cells. Na₃VO₄ was cytotoxic against T24 cells (EC₅₀ = 5.8 μM at 24 h), but in the presence of ascorbate (100 μM) the EC₅₀ fell to 3.3 μM. Na₃VO₄ plus ascorbate caused a strong inhibition of cell proliferation (up to 20%) and increased the generation of ROS (4-fold). Na₃VO₄ did not directly cleave plasmid DNA, at this aspect no synergism was found occurring between Na₃VO₄ and ascorbate once the resulting action of the combination was no greater than that of both substances administered separately. Cells from Ehrlich ascites carcinoma-bearing mice were used to determine the activity of antioxidant enzymes, the extent of the oxidative damage and the type of cell death. Na₃VO₄ alone, or combined with ascorbate, increased catalase activity, but only Na₃VO₄ plus ascorbate increased superoxide dismutase activity (up to 4-fold). Oxidative damage on proteins and lipids was higher due to the treatment done with Na₃VO₄ plus ascorbate (2–3-fold). Ascorbate potentiated apoptosis in tumor cells from mice treated with Na₃VO₄. The results indicate that pharmacological doses of ascorbate enhance the generation of ROS induced by Na₃VO₄ in tumor cells causing inhibition of proliferation and apoptosis. Apoptosis induced by orthovanadate and ascorbate is closer related to inhibition on Bcl-xL and activation of Bax. Our data apparently rule out a mechanism of cell demise p53-dependent or related to Cdk2 impairment.     

Domain IV voltage-sensor movement is both sufficient and rate limiting for fast inactivation in sodium channels

Voltage-gated sodium channels are critical for the generation and propagation of electrical signals in most excitable cells. Activation of Na⁺ channels initiates an action potential, and fast inactivation facilitates repolarization of the membrane by the outward K⁺ current. Fast inactivation is also the main determinant of the refractory period between successive electrical impulses. Although the voltage sensor of domain IV (DIV) has been implicated in fast inactivation, it remains unclear whether the activation of DIV alone is sufficient for fast inactivation to occur. Here, we functionally neutralize each specific voltage sensor by mutating several critical arginines in the S4 segment to glutamines. We assess the individual role of each voltage-sensing domain in the voltage dependence and kinetics of fast inactivation upon its specific inhibition. We show that movement of the DIV voltage sensor is the rate-limiting step for both development and recovery from fast inactivation. Our data suggest that activation of the DIV voltage sensor alone is sufficient for fast inactivation to occur, and that activation of DIV before channel opening is the molecular mechanism for closed-state inactivation. We propose a kinetic model of sodium channel gating that can account for our major findings over a wide voltage range by postulating that DIV movement is both necessary and sufficient for fast inactivation.     

A quantitative method for assessing stages of the rat estrous cycle

The impact of gender and/or hormone variations on a wide variety of neural functions makes the choice between studying males or females (or both) of a given species difficult. Although female rats are widely used experimentally, few studies control for the stage of estrus. More detailed information about how to distinguish the various stages of the estrous cycle is needed. For the present study, vaginal smears were obtained once a day and stained using an adaptation of the Papanicolaou (PAP) procedure. Images are provided of unstained “wet” samples and the corresponding PAP stained smears illustrating the cellular profile for each stage of the cycle as well as post-ovariectomy. The different cell populations across the cycle were quantified and ratios determined to show trends between the predominant and other cell types in each stage of the estrous cycle. Both stained and unstained images and cell quantification data provide valuable guidelines for distinguishing the stages of the estrous cycle.     

Preparation of soybean seed samples for FT-IR microspectroscopy

Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4° C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample.     

Oxidation of bovine serum albumin initiated by the Fenton reaction—effect of EDTA,tert-butylhydroperoxide and tetrahydrofuran

Oxidation of bovine serum albumin (BSA) was investigated using different oxidants: The water-soluble azo-initiator 2,2′azo-bis-(2-amidinopropane) hydrochloride (AAPH), a combination of FeCl₃ and ascorbate or the Fenton oxidant consisting of FeCl₂, H₂O₂ and EDTA. In addition, the effects of exogenous compounds such as tert-butyl hydroperoxide (tBuOOH) or solvents such as tetrahydrofuran (THF), often used in model systems, was evaluated. The extent of protein damage was studied by measuring protein carbonyl groups and protein hydroperoxides. The interaction between Fenton oxidant and EDTA, THF or tBuOOH was further characterized using spin trapping electron spin resonance (ESR) spectroscopy. The results showed that the extent of protein oxidation depended on the oxidant used. The Fenton oxidant was the most reactive of the initiators tested. However, in the absence of EDTA, the Fenton system produced protein carbonyl groups on BSA equivalent to that obtained with the other oxidants, however, significantly more protein hydroperoxide was produced. Surprisingly, it was also found that addition of tBuOOH or THF to BSA reduced protein damage when the oxidation was initiated with the Fenton oxidant. ESR investigation showed that EDTA played a key role in the generation of free radicals. It was also revealed that in an EDTA containing system both tBuOOH and THF were able to react with radicals without inducing protein damage in effect protecting BSA from oxidative damage.     

A Rapid Two-Step Purification of Rat Liver Fetal Thymidine Kinase

The fetal isoenzyme of thymidine kinase was purified to apparent homogeneity from cytosols of rat fetuses liver. A two-step purification including anion exchange chromatography and affinity chromatography was developed. The purified enzyme appears as oligomeric with a relative molecular weight of 71 kDa. In denaturing media its molecular weight was 24 kDa, and its pHi 8.3.     

Transducing properties of a pre-structured α-helical DPT-peptide containing a short canine adenovirus type 2 E4orf4 PP2A1-binding sequence

Background

Induction of the death pathway resulting from the specific interaction of the PP2A₁ phosphatase with adenoviral E4orf4 protein is a promising approach for cancer therapy. With the aim of deregulating tumor pathways, and mimicking E4orf4 anti-cancer signal, we have previously proposed the DPT technology concept, based on design of specific PP1/PP2A interacting penetrating peptides.

Methods

Using biochemical, structural and cell survival experiments, we have characterized new DPT-peptides containing short PP2A binding sequences.

Results

We identified overlapping sequences, located within the N-terminal domain E4orf4_23-46 of canine adenoviral E4orf4 protein, that interact with the PP2A-Bα subunit of PP2A₁ holoenzyme. We characterized DPT-E4orf4₄ and TAT-E4orf4₄, two bi-partite cell penetrating peptides containing the 12 PP2A₁ binding residues of the canine type 2 E4orf4_27-38 sequence, respectively fused to the DPT-sh1 and TAT shuttle sequences. Surprisingly DPT-E4orf4₄, in contrast to inactive TAT-E4orf4₄, adopted a well defined α-helical structure and co-precipitated PP2A₁ from HeLa cell extracts. DPT-E4orf4₄ also internalized streptavidin-HRP and inhibited survival of HeLa cells more efficiently than TAT, TAT-E4orf4₄ or the previously published anti-tumor TAT-derived peptide shepherdin. DPT-E4orf4₄ also efficiently inhibited the survival of human adherent transformed cells, including wild type and p53 mutated colonic HCT116 cells, without affecting survival of human non-transformed fibroblasts.

Conclusions

We characterized the transducing properties of a new α-helical DPT-E4orf4₄ peptide containing a short PP2A-interacting sequence from canine Adenoviral E4orf4 protein.

General significance

Our results suggest that α-helical structured DPT peptides specifically interacting with PP2A could be a valuable anti-cancer drug design scaffold.     

Escherichia coli thymidylate kinase: molecular cloning, nucleotide sequence, and genetic organization of the corresponding tmk locus.

Thymidylate kinase (dTMP kinase; EC 2.7.4.9) catalyzes the phosphorylation of dTMP to form dTDP in both de novo and salvage pathways of dTTP synthesis. The nucleotide sequence of the tmk gene encoding this essential Escherichia coli enzyme is the last one among all the E. coli nucleoside and nucleotide kinase genes which has not yet been reported. By subcloning the 24.0-min region where the tmk gene has been previously mapped from the lambda phage 236 (E9G1) of the Kohara E. coli genomic library (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987), we precisely located tmk between acpP and holB genes. Here we report the nucleotide sequence of tmk, including the end portion of an upstream open reading frame (ORF 340) of unknown function that may be cotranscribed with the pabC gene. The tmk gene was located clockwise of and just upstream of the holB gene. Our sequencing data allowed the filling in of the unsequenced gap between the acpP and holB genes within the 24-min region of the E. coli chromosome. Identification of this region as the E. coli tmk gene was confirmed by functional complementation of a yeast dTMP kinase temperature-sensitive mutant and by in vitro enzyme assay of the thymidylate kinase activity in cell extracts of E. coli by use of tmk-overproducing plasmids. The deduced amino acid sequence of the E. coli tmk gene showed significant similarity to the sequences of the thymidylate kinases of vertebrates, yeasts, and viruses as well as two uncharacterized proteins of bacteria belonging to Bacillus and Haemophilus species.     

A nuclear gene for higher level phylogenetics: phosphoenolpyruvate carboxykinase tracks mesozoic-age divergences within Lepidoptera (Insecta)

The sequence of phosphoenolpyruvate carboxykinase (PEPCK) has been previously identified as a promising candidate for reconstructing Mesozoic-age divergences (Friedlander, Regier, and Mitter 1992, 1994). To test this hypothesis more rigorously, 597 nucleotides of aligned PEPCK coding sequence (approximately 30% of the coding region) were generated from 18 species representing Mesozoic-age lineages of moths (Insecta: Lepidoptera) and outgroup taxa. Relationships among basal Lepidoptera are well established by morphological analysis, providing a strong test for the utility of a gene which has not previously been used in systematics. Parsimony and other phylogenetic analyses were conducted on nucleotides by codon positions (nt1, nt2, nt3) separately and in combination, and on amino acids, for comparison to the test phylogeny. The highest concordance was achieved with nt1 + nt2, for which one of two most-parsimonious trees was identical to the test phylogeny, and with all nucleotides when nt3 was down-weighted sevenfold or higher, for which a single most-parsimonious tree identical to the test phylogeny resulted. Substitutions in nt3 approached saturation in many, but not all, pairwise comparisons and their exclusion or severe downweighting greatly increased the degree of concordance with the test phylogeny. Neighbor-joining analysis confirms this finding. The utility of PEPCK for phylogenetics is demonstrated over a time span for which few other suitable genes are currently available.