Electrophoretic Mobility In Agarose Gels Research Articles

Non-intertwined strands of plasmid DNA contradict the Watson and Crick model of DNA structure

According to Watson and Crick (W/C) model of DNA structure, a DNA molecule consists of two antiparallel polynucleotide chains, intertwined with each other. Although W/C model is accepted widely, some researchers have raised questions against it and proposed alternative structures for DNA. In the present study, we examined W/C model using plasmid DNA. It was hypothesized that two strands of plasmid will remain intertwined (and not separate from each other) under denaturing conditions if it follows W/C model. To test this, plasmid DNA was denatured using sodium hydroxide (NaOH) and analyzed by agarose gel electrophoresis. It was observed that addition of 0.5 N NaOH to pUC19 or pBR322 plasmids resulted in a new form of DNA having higher electrophoretic mobility in agarose gel. Higher electrophoretic mobility DNA (HmDNA) in NaOH-denatured pUC19 was digestible with S1 nuclease, but not with HindIII and ‘exonuclease I + alkaline phosphatase’. These results demonstrated that HmDNA is single-stranded circular DNA, formed due to separation of two strands of NaOH-denatured plasmid. Single-stranded and circular nature of HmDNA was corroborated by its comparable electrophoretic mobility with purified top and bottom strands of plasmid DNA. Next, we examined whether HmDNA can re-anneal into the native plasmid. Interestingly, when subjected to renaturing conditions, HmDNA from NaOH-denatured pUC19 re-annealed to form native pUC19 plasmid, which was digestible with HindIII and induced ampicillin resistance in Escherichia coli. These findings demonstrated the reversible separation of two strands of plasmid DNA and contradicted the W/C model of DNA structure.

Non-intertwined strands of plasmid DNA contradicts the Watson and Crick model of DNA structure

According to Watson and Crick (W/C) model of DNA structure, a DNA molecule consists of two antiparallel polynucleotide chains, intertwined with each other. Although W/C model is accepted widely, some researchers have raised questions against it and proposed alternative structures for DNA. In the present study, we examined W/C model using plasmid DNA. It was hypothesized that two strands of plasmid will remain intertwined (and not separate from each other) under denaturing conditions if it follows W/C model. To test this, plasmid DNA was denatured using sodium hydroxide (NaOH) and analyzed by agarose gel electrophoresis. It was observed that addition of NaOH to pUC19 and pBR322 plasmids resulted in a new form of DNA having higher electrophoretic mobility in agarose gel. Higher electrophoretic mobility DNA (HmDNA) in NaOH-denatured pUC19 was digestible with S1 nuclease, but not with HindIII and ‘exonuclease I + alkaline phosphatase’. These results demonstrated that HmDNA is single-stranded circular DNA, formed due to separation of two strands of NaOH-denatured plasmid. Single-stranded and circular nature of HmDNA was corroborated by its comparable electrophoretic mobility with purified top and bottom strands of plasmid DNA. Next, we examined whether HmDNA can re-anneal into the native plasmid. Interestingly, when HmDNA from NaOH-denatured pUC19 was subjected to renaturing conditions, it formed native pUC19 plasmid, which was digestible with HindIII and induced ampicillin resistance in Escherichia coli cells. These findings demonstrated the reversible separation of two strands of plasmid DNA and contradicted the W/C model of DNA structure.

Non-intertwined strands of plasmid DNA contradicts the Watson and Crick model of DNA structure

According to Watson and Crick (W/C) model, a DNA molecule consists of two antiparallel polynucleotide chains, intertwined with each other. Although the W/C model is accepted widely, a number of researchers have raised questions against it and proposed alternative structures for DNA. In the present study, the W/C model was examined using plasmid DNA. It was hypothesized that two strands of plasmid DNA will remain intertwined and not separate from each other under denaturing conditions, if it follows the W/C model. To test this, plasmid DNA was denatured using sodium hydroxide (NaOH) and analyzed by gel electrophoresis. It was observed that addition of NaOH to pUC19 and pBR322 plasmids resulted in new form of DNA with higher electrophoretic mobility in agarose gel. DNA corresponding to higher electrophoretic mobility band of pUC19 (hmP19) was single-stranded and circular, indicating the separation of two strands of pUC19 plasmid. Next, we examined whether hmP19 DNA can re-anneal to form native pUC19 plasmid. It was observed that neutralization of NaOH resulted in the appearance of native pUC19 plasmid in denatured DNA. Native pUC19 was also formed by hmP19 DNA extracted from agarose gel and was found to be digestible with Hind III. Ability to confer ampicillin resistance in transformed Escherichia coli demonstrated the functionality of pUC19 plasmid formed by extracted hmP19 DNA. Reversible separation of two strands of plasmid into single-stranded circular DNA shows that DNA strands are not intertwined with each other and contradicts the W/C model of DNA structure.

Interaction between TATA-Binding Protein (TBP) and Multiprotein Bridging Factor-1 (MBF1) from the Filamentous Insect Pathogenic Fungus Beauveria bassiana.

TATA-binding protein (TBP) is a ubiquitous component of eukaryotic transcription factors that acts to nucleate assembly and position pre-initiation complexes. Multiprotein bridging factor 1 (MBF1) is thought to interconnect TBP with gene specific transcriptional activators, modulating transcriptional networks in response to specific signal and developmental programs. The insect pathogen, Beauveria bassiana, is a cosmopolitan fungus found in most ecosystems where it acts as an important regulator of insect populations and can form intimate associations with certain plants. In order to gain a better understanding of the function of MBF1 in filamentous fungi, its interaction with TBP was demonstrated. The MBF1 and TBP homologs in B. bassiana were cloned and purified from a heterologous E. coli expression system. Whereas purified BbTBP was shown to be able to bind oligonucleotide sequences containing the TATA-motif (Kd ≈ 1.3 nM) including sequences derived from the promoters of the B. bassiana chitinase and protease genes. In contrast, BbMBF1 was unable to bind to these same target sequences. However, the formation of a ternary complex between BbMBF1, BbTBP, and a TATA-containing target DNA sequence was seen in agarose gel electrophoretic mobility shift assays (EMSA). These data indicate that BbMBF1 forms direct interactions with BbTBP, and that the complex is capable of binding to DNA sequences containing TATA-motifs, confirming that BbTBP can link BbMBF1 to target sequences as part of the RNA transcriptional machinery in fungi.

The identification of proteoglycans and glycosaminoglycans in archaeological human bones and teeth.

Bone tissue is mineralized dense connective tissue consisting mainly of a mineral component (hydroxyapatite) and an organic matrix comprised of collagens, non-collagenous proteins and proteoglycans (PGs). Extracellular matrix proteins and PGs bind tightly to hydroxyapatite which would protect these molecules from the destructive effects of temperature and chemical agents after death. DNA and proteins have been successfully extracted from archaeological skeletons from which valuable information has been obtained; however, to date neither PGs nor glycosaminoglycan (GAG) chains have been studied in archaeological skeletons. PGs and GAGs play a major role in bone morphogenesis, homeostasis and degenerative bone disease. The ability to isolate and characterize PG and GAG content from archaeological skeletons would unveil valuable paleontological information. We therefore optimized methods for the extraction of both PGs and GAGs from archaeological human skeletons. PGs and GAGs were successfully extracted from both archaeological human bones and teeth, and characterized by their electrophoretic mobility in agarose gel, degradation by specific enzymes and HPLC. The GAG populations isolated were chondroitin sulfate (CS) and hyaluronic acid (HA). In addition, a CSPG was detected. The localization of CS, HA, three small leucine rich PGs (biglycan, decorin and fibromodulin) and glypican was analyzed in archaeological human bone slices. Staining patterns were different for juvenile and adult bones, whilst adolescent bones had a similar staining pattern to adult bones. The finding that significant quantities of PGs and GAGs persist in archaeological bones and teeth opens novel venues for the field of Paleontology.

A plasmid containing the human metallothionein II gene can function as an antibody-assisted electrophoretic biosensor for heavy metals

Different forms of heavy metals affect biochemical systems in characteristic ways that cannot be detected with typical metal analysis methods like atomic absorption spectrometry. Further, using living systems to analyze interaction of heavy metals with biochemical systems can be laborious and unreliable. To generate a reliable easy-to-use biologically-based biosensor system, the entire human metallothionein-II (MT-II) gene was incorporated into a plasmid (pUC57-MT) easily replicated in Escherichia coli. In this system, a commercial polyclonal antibody raised against human metal-responsive transcription factor-1 protein (MTF-1 protein) could modify the electrophoretic migration patterns (i.e. cause specific decreases in agarose gel electrophoretic mobility) of the plasmid in the presence or absence of heavy metals other than zinc (Zn). In the study here, heavy metals, MTF-1 protein, and polyclonal anti-MTF-1 antibody were used to assess pUC57-MT plasmid antibody-assisted electrophoretic mobility. Anti-MTF-1 antibody bound both MTF-1 protein and pUC57-MT plasmid in a non-competitive fashion such that it could be used to differentiate specific heavy metal binding. The results showed that antibody-inhibited plasmid migration was heavy metal level-dependent. Zinc caused a unique mobility shift pattern opposite to that of other metals tested, i.e. Zn blocked the antibody ability to inhibit plasmid migration, despite a greatly increased affinity for DNA by the antibody when Zn was present. The Zn effect was reversed/modified by adding MTF-1 protein. Additionally, antibody inhibition of plasmid mobility was resistant to heat pre-treatment and trypsinization, indicating absence of residual DNA extraction-resistant bacterial DNA binding proteins. DNA binding by anti-DNA antibodies may be commonly enhanced by xenobiotic heavy metals and elevated levels of Zn, thus making them potentially effective tools for assessment of heavy metal bioavailability in aqueous solutions and fluid obtained from metal implant sites.

Co-delivery of plasmid DNA and antisense oligodeoxyribonucleotide into human carcinoma cells by cationic liposomes.

This study aimed to evaluate the co-delivery of cationic liposome/plasmid DNA complexes and cationic liposome/antisense oligodeoxyribonucleotide (AS ODN) complexes in HeLa human cervical carcinoma cells. Dimethyldioctadecyl ammonium bromide (DDAB): dioleoyl phosphatidylethanolamine (DOPE) liposome/plasmid DNA complexes, and DDAB:DOPE liposome/AS ODN complexes were formulated and characterized in terms of agarose gel electrophoretic mobility, particle size and zeta potential. The complexes were evaluated for delivery of pEGFP plasmid DNA and AS ODN in HeLa cells. Cell growth inhibition was evaluated using p53 plasmid DNA and bcl-2 AS ODN, by codelivery of DDAB:DOPE liposome/p53 plasmid DNA and DDAB:DOPE liposome/bcl-2 AS ODN complexes. The particle size of DDAB:DOPE liposome/plasmid DNA complexes, and DDAB:DOPE liposome/AS ODN complexes were 180.6±2.0 to 372.3±2.4 nm, and zeta potentials were -26.7±1.2 to +6.8±0.4 mV, respectively. The AS ODN uptake and green fluorescent protein (GFP) expression upon their co-delivery by DDAB:DOPE liposomes were both high. Treatment of the cells with the co-delivery of DDAB:DOPE liposome/p53 plasmid DNA complexes and DDAB:DOPE liposome/ bcl-2 AS ODN complexes inhibited cell growth to a greater degree than that with either DDAB:DOPE liposome/p53 plasmid DNA complexes or DDAB:DOPE liposome/bcl-2 AS ODN complexes alone. These data suggest that co-delivery of cationic liposome/p53 plasmid DNA and cationic liposome/bcl-2 AS ODN complexes is an effective strategy to achieve enhanced therapeutic activities.

Abstract 408: Macrophage-mediated Low Density Lipoprotein Modification Reduces Foam Cell Formation

Macrophage metabolism of LDL-derived cholesterol is thought to be a key process regulating the composition and progression of atherosclerotic lesions, including the generation of cholesterol-loaded macrophage foam cells and the lipid-rich necrotic core. Many mechanisms contribute to macrophage endocytosis of LDL, and we recently reported that selective cholesteryl ester (CE) uptake contributes to LDL-induced macrophage cholesterol accumulation. Selective uptake is expected to result in the net removal of cholesteryl ester from LDL; however, the effect of this and other LDL modifications that may occur during incubation with macrophages is not understood. In the current investigation, we pre-incubated LDL for 1-2 days with macrophages in order to promote macrophage-mediated LDL modification. Surprisingly, when macrophage-conditioned LDL was transferred to fresh macrophages, much less cholesterol accumulation resulted compared to cells incubated with unconditioned LDL, as indicated by cholesterol quantification and by neutral lipid staining with Oil Red-O. This difference was not accounted for by changes in uptake of the modified 125I-LDLs, but instead was associated with a large decrease in selective [3H]CE uptake. In contrast, uptake of the non-hydrolyzable CE analog [3H]cholesteryl ether was not reduced, suggesting that reduced uptake in macrophage-conditioned LDL may be due to changes in the hydrolysis of LDL-derived CE or its subsequent metabolism. Cell-mediated LDL oxidation was considered as a possible mechanism for changes in selective CE uptake. However, agarose gel electrophoretic mobility of LDL and analysis of LDL cholesteryl esters indicated that reduced selective uptake and foam cell formation did not depend on extensive oxidative modifications of LDL. These results indicate the importance of selective CE and suggest that macrophage-mediated LDL modification may reduce foam cell formation in some circumstances.

Anion exchange chromatographic distribution of human monoclonal immunoglobulin G is determined by heavy chain subclass and level of sialic acid expression

Anion exchange chromatography is widely accepted method for purification of immunoglobulins. In this work, we used human monoclonal immunoglobulin G (IgG) with structure and solubility of normal human IgG as a model for studying chromatographic behavior of particular molecular forms of IgG. Human sera with monoclonal IgG were fractionated on strong anion exchanger, Q Sepharose Fast Flow. With 20 mM Tris pH 7.5 as a start buffer, 42% of human monoclonal IgG passed through column, and 58% of them remained adsorbed. Bound monoclonal IgG were eluted from the exchanger by linear increasing of concentration of NaCl from 0 to 0.5 M. Chromatographic distribution of human monoclonal IgG correlated with their electrophoretic mobility in agarose gels, and it was dependent on ? heavy chain isotype. Light chain type, as well as serum concentration of monoclonal IgG did not influence their chromatographic behavior. The level of heavy chain sialic acid expression, but not of galactose and N-acetylglucosamine, significantly determined chromatographic distribution of serum monoclonal IgG. Additionally to the information on the chromatographic behavior of human monoclonal IgG, we believe that presented data could provide useful information about the possible use of Q Sepharose Fast Flow matrix for the isolation of specific molecular forms of human IgG.

Electrophoretic mobility in agarose gel as an isolating technique and correlation with synergestic cytotoxicity enhancement

Electrophoretic mobility in agarose gel as an isolating technique and correlation with synergestic cytotoxicity enhancement

Oxidized Low-Density Lipoprotein and Atherosclerosis

Chance and serendipity play major roles in the history of science. Too often, though, their contributions do not show up in formal publications. We all tend to shape history according to the styles of the times and according to our own biases. Acknowledged or unacknowledged, there is a tendency to want the scientific “story” to be neat and more or less linear. The genesis of the oxidized low-density lipoprotein (OxLDL) hypothesis owed a great deal to happenstance, but that may not be readily apparent from the formal articles. We take this opportunity to tell the whole story, at least as we remember it. Several apparently unrelated events occurring at about the same time in Oslo, Norway; in Cleveland, Ohio; in La Jolla, California; in New York, New York; and in Dallas, Texas converged to lay the groundwork for the hypothesis that oxidative modification of LDL might be important in atherogenesis. Later developments regarding the relationship between OxLDL and the immune system in atherogenesis, again, were often smiled on by chance and serendipity. In 1979, a young Norwegian researcher, Tore Henriksen, got in touch with our laboratory asking whether he could come to La Jolla to learn more about lipoproteins. He and his colleagues in Oslo had observed that under certain conditions, LDL was highly toxic for endothelial cells in culture, leading to cell death in just 24 hours.1 Almost simultaneously and independently, Chisolm and coworkers in Cleveland made very similar observations.2 Both groups noted that addition of whole serum or purified high-density lipoprotein to the medium completely prevented the toxic effects of the LDL, but the mechanism(s) involved remained unclear. Henriksen arrived in La Jolla in the summer of 1980 and demonstrated to us how drastically LDL affected the cultured cells. We agreed that the phenomenon was worth …

Apolipoprotein M expression increases the size of nascent preβ HDL formed by ATP binding cassette transporter A1

Apolipoprotein M (apoM) is a novel apolipoprotein that is reportedly necessary for pre beta HDL formation; however, its detailed function remains unknown. We investigated the biogenesis and properties of apoM and its effects on the initial steps of nascent pre beta HDL assembly by ABCA1 in HEK293 cells. Transiently transfected apoM was localized primarily in the endomembrane compartment. Pulse-chase analyses demonstrated that apoM is inefficiently secreted, relative to human serum albumin, and that approximately 50% remains membrane-associated after extraction with sodium carbonate, pH 11.5. To investigate the role of apoM in nascent pre beta HDL formation, ABCA1-expressing or control cells, transfected with empty vector, apoM, or C-terminal epitope-tagged apoM (apoM-C-FLAG), were incubated with (125)I-apoA-I for 24 h. Conditioned media were harvested and fractionated by fast-protein liquid chromatography (FPLC) to monitor HDL particle size. Pre beta HDL particles were formed effectively in the absence of apoM expression; however, increased apoM expression stimulated the formation of larger-sized nascent pre beta HDLs. Immunoprecipitation with anti-apoA-I antibody followed by apoM Western blot analysis revealed that little secreted apoM was physically associated with pre beta HDL. Our results suggest that apoM is an atypical secretory protein that is not necessary for ABCA1-dependent pre beta HDL formation but does stimulate the formation of larger-sized pre beta HDL. We propose that apoM may function catalytically at an intracellular site to transfer lipid onto pre beta HDL during or after their formation by ABCA1.

Synthesis, Characterization and Biological Activity of trans‐Platinum(II) and trans‐Platinum(IV) Complexes with 4‐Hydroxymethylpyridine

The synthesis and chemical characterization of two new trans platinum complexes, trans-[PtCl(2)NH(3)(4-hydroxymethylpyridine)] (1) and trans-[PtCl(4)NH(3)(4-hydroxymethylpyridine)] (2) are described. Their ability to interact with 5'-GMP by themselves and in the presence of reducing agents in the case of trans-[PtCl(4)NH(3)(4-hydroxymethylpyridine)] were tested. Circular dichroism, electrophoretic mobility in agarose gel, and atomic force microscopy studies showed that the interaction of complex 1 with DNA is stronger than that of complex 2. Cytotoxicity tests against HL-60 tumor cells also showed higher activity for trans-[PtCl(2)NH(3)(4-hydroxymethylpyridine)] than for trans-[PtCl(4)NH(3)(4-hydroxymethylpyridine)]. Complex 1 presents similar behavior to cisplatin, but with a lower IC(50) at 24 h. Complex 1 also showed high apoptosis induction.

Both HDL3 and HDL2 Exert a Powerful Anti-Oxidative and Protective Effect against Acceleration of Oxidative Modification of LDL by Ascorbic Acid

The objective of the present study was to establish whether high-density lipoprotein 3 (HDL3) or high-density lipoprotein 2 (HDL2) might show an anti-oxidative effect on the acceleration of the oxidative modification of low-density lipoprotein (LDL) by ascorbic acid from measurement of the agarose gel electrophoretic mobility of LDL. LDL was incubated without adding transitional-metal ions for 48 or 96 h in phosphate-buffered saline (PBS) alone, with ascorbic acid (20 microg/mL), or with both ascorbic acid (20 microg/mL) and HDL3 (200 microg protein/mL). The LDL autoxidation occurred in PBS alone. Although ascorbic acid significantly suppressed oxidative modification of LDL after incubation for 48 h, the opposite was true after 96 h. However, since the anti-oxidative ability of HDL2 shows a weaker tendency than that of HDL3, both HDL3 and HDL2 significantly inhibited this acceleration of oxidative modification of LDL by ascorbic acid as assessed by electrophoretic mobility. If there is an augmented oxidative modification of LDL due to ascorbic acid in vivo, HDL3 or HDL2 may thus have an important role in inhibiting this ascorbic acid-accelerated oxidation of LDL.

Characterization of Genetic Variation in Intron 4 of The Surfactant Protein B Gene.

Expression of the surfactant protein B gene is required for function of the pulmonary surfactant. The 9.5 kb surfactant protein B gene includes 10 translated and exons and 1 untranslated exon. Genetic variants in intron 4 characterized by insertions or deletions of 11 distinct motifs have been associated with respiratory distress in some populations of infants, adult respiratory distress syndrome, and risk of squamous cell carcinoma of the lung. Due to polymerase enzyme stutter, characterization of allelic variation by direct sequencing has been difficult. To examine genetic variation in intron 4 in a cohort of Missouri infants (n=240), we identified a polymerase enzyme with high fidelity for intron 4 amplification and analyzed product length by agarose gel electrophoretic mobility. In 480 alleles, we found 14.4% (69/480) variant alleles, 9.4% with insertions and 5.0% with deletions. Allelic diversity was significantly greater among African-Americans (n=204, 19.1% insertion alleles, 2.9% deletion alleles) than Caucasians (n=244, 2.0% insertion alleles, 7.4% deletion alleles) (p<.001). Insertion variants were strongly associated with African Americans (28/31), and deletions were associated with Caucasians (15/20) (p<.001). We then cloned fragments from a subset of 42 infants (22 African-American, 19 Caucasian, 1 Hispanic). Automated sequencing of the 32 variant alleles revealed 6 previously unreported variants (all insertions) that accounted for an unexpectedly high proportion (18/32) of variant alleles sequenced. Two different insertion variants shared agarose gel electrophoretic mobility (Mr~570 bps), as did 2 other insertion variants (Mr~600 bps). When analyzed by automated sequencing, these variants differed in motif insertion or deletion or in length of dinucleotide (CA) repeat. The C genotype at genomic position 1580, a C/T nonsynonymous, single nucleotide polymorphism in exon 4, was strongly associated with insertion (28/31 = T) (p<.001). We conclude that genotype at genomic position 1580 and race are associated with intron 4 variation. Characterization of intron 4 by agarose gel electrophoretic mobility alone may fail to detect important differences in genetic variation in intron 4 that contribute to risk for respiratory disease.

Activation of adherent vascular neutrophils in the lung during acute endotoxemia.

BackgroundNeutrophils constitute the first line of defense against invading microorganisms. Whereas these cells readily undergo apoptosis under homeostatic conditions, their survival is prolonged during inflammatory reactions and they become biochemically and functionally activated. In the present study, we analyzed the effects of acute endotoxemia on the response of a unique subpopulation of neutrophils tightly adhered to the lung vasculature.MethodsRats were treated with 5 mg/kg lipopolysaccharide (i.v.) to induce acute endotoxemia. Adherent neutrophils were isolated from the lung vasculature by collagenase digestion and sequential filtering. Agarose gel electrophoresis, RT-PCR, western blotting and electrophoretic mobility shift assays were used to evaluate neutrophil activity.ResultsAdherent vascular neutrophils isolated from endotoxemic animals exhibited decreased apoptosis when compared to cells from control animals. This was associated with a marked increase in expression of the anti-apoptotic protein, Mcl-1. Cells isolated 0.5–2 hours after endotoxin administration were more chemotactic than cells from control animals and expressed increased tumor necrosis factor-alpha and cyclooxygenase-2 mRNA and protein, demonstrating that they are functionally activated. Endotoxin treatment of the animals also induced p38 and p44/42 mitogen activated protein kinases in the adherent lung neutrophils, as well as nuclear binding activity of the transcription factors, NF-κB and cAMP response element binding protein.ConclusionThese data demonstrate that adherent vascular lung neutrophils are highly responsive to endotoxin and that pathways regulating apoptosis and cellular activation are upregulated in these cells.

Effects of Catechins and Ground Green Tea Drinking on the Susceptibility of Plasma and LDL to the Oxidation In Vitro and Ex Vivo

We investigated the effects of catechins and ground green tea drinking on the susceptibilities of human low density lipoprotein (LDL) and plasma to the oxidation by CuSO4 in vitro and ex vivo. The effects of (-)epicatechin (EC) and (-)epigallocatechin gallate (EGCg) on LDL oxidation in vitro were estimated by measuring the lag time of conjugated diene formation, lipid hydroperoxide and agarose gel electrophoretic mobility. EC and EGCg had antioxidant activities for LDL oxidation, and the antioxidant activity of EGCg to LDL oxidation was stronger than that of EC in vitro. In an ex vivo study, we investigated the effects of ground green tea drinking on LDL and plasma oxidation after the consumption of a cup of green tea (5g). Plasma total catechin contents were increased at 1 and 3h after the oral ingestion, and we detected (-)epigallocatechin (EGC), EC, EGCg and (+)catechin (CA) in plasma. The lag time of conjugated diene formation of plasma was increased in all subjects after consumption of green tea (p<0.028), but the susceptibility of LDL oxidation ex vivo was different among individual subjects. Although the β-carotene, α-tocopherol and uric acid contents in plasma did not change after green tea consumption, ascorbic acid contents in plasma increased at 3h after green tea ingestion. These findings suggest that catechins suppressed the susceptibilities of human LDL to oxidation by CuSO4 in vitro and plasma oxidation ex vivo after ground green tea ingestion.

Changes of NF-kB, p53, Bcl-2 and caspase in apoptosis induced by JTE-522 in human gastric adenocarcinoma cell line AGS cells: role of reactive oxygen species.

To identify whether JTE-522 can induce apoptosis in AGS cells and ROS also involved in the process, and to investigate the changes in NF-kB, p53, bcl-2 and caspase in the apoptosis process. Cell culture, MTT, Electromicroscopy, agarose gel electrophoresis, lucigenin, Western blot and electrophoretic mobility shift assay (EMSA) analysis were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanisms. JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Lucigenin assay showed the generation of ROS in cells under incubation with JTE-522. The increased ROS generation might contribute to the induction of AGS cells to apoptosis. EMSA and Western blot revealed that NF-kB activity was almost completely inhibited by preventing the degradation of IkBalpha. Additionally, by using Western blot we confirmed that the level of bcl-2 was decreased, whereas p53 showed a great increase following JTE-522 treatment. Their changes were in a dose-dependent manner. These findings suggest that reactive oxygen species, NF-kB, p53, bcl-2 and caspase-3 may play an important role in the induction of apoptosis in AGS cells after treatment with JTE-522.

Effects of native and oxidized low-density lipoproteins on macrophage chemiluminescence

Effects of low-density lipoproteins (LDL) obtained from healthy donors and patients with hypercholesterolemia on spontaneous luminol-dependent and zymosan-induced chemiluminescence of rat macrophages were studied. Unlike LDL from healthy donors, native LDL from patients with hypercholesterolemia inhibited spontaneous chemiluminescence of macrophages. Simultaneous incubation with endotheliocytes from the umbilical vein led to the appearance of inhibitory effect of LDL from healthy donors (incubation for 24 h) and potentiated this effect of LDL from patients with hypercholesterolemia (incubation for 6 and 24 h). The inhibitory effect was more pronounced in LDL incubated with umbilical endotheliocytes under ischemic conditions then after aerobic incubations. This corresponded to higher oxidation of LDL confirmed by accumulation of thiobarbituric acid-reactive substances, increased fluorescence, and high electrophoretic mobility in agarose gel. These data suggest that the model system of spontaneous and zymosan-induced chemiluminescence of macrophages can be used for evaluating the degree of oxidation and potential atherogenicity of LDL.

Palladium(II) 4,5-Diphenylimidazole Cyclometalated Complexes: DNA Interaction

In this paper, we show the synthesis of palladium(II) 4,5-phenylimidazole cyclometalated complexes. They have been characterized by IR, 1 H- and 13 C-NMR spectroscopy. The cyclometalated dimer compound 2 [Pd(C 15 H 11 N 2 1)(μ-OAc)] 2 and the cyclometalated monomer compound 5 [PdBr(SEt 2 )(C 15 H 11 N 2 )], having OAc and Br as leaving groups, interact with DNA, modifying its secondary structure (as measured by T m and CD), without modifying its tertiary structure (as determined by measurement of the electrophoretic mobility in agarose gels). The monomeric compound 5 seems to be the one that induces the highest alterations in DNA secondary structure since it strongly modifies the CD spectrum of the DNA. Melting data of drug-DNA complexes suggest that, at low drug concentration, the 4,5-Imd ligand intercalates between the base pairs in the DNA molecule, increasing the T m , while at high drug concentrations the palladium(II) centers destabilize the double helix, producing a lowering in T m values. These results indicate that complexes containing planar structures might selectively bind to DNA that is not supercoiled, and that therefore it only has a secondary structure.