Partial intercalation with DNA of peptides containing two aromatic amino acids

The interactions with DNA of tetrapeptide amides containing lysine at the N-terminal position and aromatic amino acids at the second and fourth positions (Ala at position three), 1-6, have been investigated by nmr, CD, and viscometric methods. Tetrapeptides with N-terminal lysine and a single aromatic amino acid, 7-10, were investigated as controls. Significant decreases in DNA viscosity occurred on addition of 7, with the aromatic group at the second position, but not with any of the other single aromatic amino acid peptides. All of the tetrapeptides with two aromatic groups caused DNA viscosity decreases which were two to three times larger than with 7. Peptides with p-nitrophenylalanine (p-NO2Phe) as the aromatic group were synthesized for nmr studies because of its simpler aromatic nmr spectrum relative to Phe. Large upfield shifts of the aromatic proton signals were obtained when the amino acid in the second position was L-p-NO2Phe, and the fourth position contained either p-NO2Phe or Phe. Such peptides also caused the largest DNA viscosity decreases on complex formation. Smaller upfield shifts of the aromatic signals were obtained when the amino acid in the second position was L-Phe or a D isomer of Phe or p-NO2Phe. With all peptides, larger upfield nmr shifts were obtained with heat-denatured, recooled DNA than with native DNA under the same conditions. As with nmr, CD results are quite different for the peptides with L and D amino acids at the second position. All of the results can be interpreted in terms of a model in which lysine interacts stereospecifically with the backbone in a DNA double helix and the aromatic group at the second position stacks strongly with the base pairs when the amino acid is an L isomer. The aromatic group at the fourth position can also interact with the base pairs, but primarily through a sideways stacking of the aromatic group with base pairs for either L or D isomers. Because of covalent constraints on the separation distance for the two aromatic groups in the tetrapeptides, they must stack on opposite sides of the same base pair in violation of the neighbor exclusion principle observed with classical intercalators. This stacking at the same base pair no doubt accounts for the larger viscosity decreases in DNA with the peptides containing two aromatic groups relative to those with a single aromatic group.(ABSTRACT TRUNCATED AT 400 WORDS)     

Free radical scavenging action of Bio-catalyzer alpha.rho No.11 (Bio-normalyzer) and its by-product.

Bio-catalyzer alpha.rho No.11 (Bio-normalyzer) and its by-product are natural health products made by yeast fermentation of glucose, Carica papaya Linn., Pennisetum pupureum Schum., and Sechium edule Swartz. Their effects on free radicals were examined by electron spin resonance spectrometry using spin trapping agent 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). It was observed that both Bio-catalyzer and its by-product scavenged 95% of DMPO-OH spin adducts (89 x 10(15) spins/ml) generated by FeSO4-H2O2-diethylene triamine pentaacetic acid system at 45.45 mg/ml each. Five percent of DMPO-O2- spin adducts (27 x 10(15) spins/ml) generated by hypoxanthine-xanthine oxidase system and 11% of 1,1-diphenyl-2-picrylhydrazyl radicals (7 x 10(15) spins/ml) were quenched using 25 mg/ml of Bio-catalyzer while 5% of superoxide and nil DPPH radicals were scavenged by its by-product. Vivo tests showed that oral administration of 1-g/kg body weight of Bio-catalyzer significantly inhibited thiobarbituric acid reactive substances formation, which is an index of lipid peroxidation, in the FeCl3-induced epileptic focus of rats. These findings suggest that Bio-catalyzer or its by-product may be useful health foods against neural lipid peroxidation, traumatic epilepsy and aging.     

Hypothesis: the target cell of GM-CSF is a macrophage precursor capable to produce cells with the property to secrete a G-CSF like activity.

The induction of granulocyte and macrophage colony formation by the granulocyte-macrophage colony stimulating factor (GM-CSF) on bone marrow cells (BMC) was evaluated as a function of time in agar cultures. We found that while macrophage cell clusters were very abundant on the first two days of culture, granulocytic cell clusters did not appear until the third day. We also found that macrophage colonies were present from the fourth day of culture, while granulocyte colonies did not appear until the fifth day. When two day cell clusters were transferred to cultures with GM-CSF we observed that only macrophage-colonies developed. On the other hand, when four day clusters were transferred, both granulocyte and macrophage colony formation was obtained in a similar way as the one obtained when using GM-CSF with fresh BMC. Two day clusters did not respond to granulocyte colony stimulating factor (G-CSF) while fourth day clusters generated granulocytic colonies in a similar way as when G-CSF was used with fresh BMC. In order to test the hypothesis that granulocyte colony formation in these assays could be a result of the secretion of G-CSF by the macrophages previously induced by GM-CSF, lysates from macrophage colonies were used to induce colony formation on BMC. We observed that colonies, mainly granulocytic, were induced in a similar way as when G-CSF was used. Finally, the possibility that GM-CSF is just a macrophage inducer with the property to produce cells that secrete G-CSF is discussed.     

Vanadate,alcohol or both increase passive membrane permeability of neuro-2a cells; Lesser sensitivity of Hep-2 cells

Neuro-2a cells incubated for 1 hour with 0.1 mM vanadate showed an increase in cell membrane permeability. This effect is dose dependent, e.g. with 0.01 mM, 0.1 mM and 1 mM vanadate, there was {20, 30 and 40% increase. In contrast, no alteration in permeability was observed in HEp-2 cells under the same conditions.Ethanol (3%, 1 h incubation) also enhanced membrane permeability. The increase was also greater with Neuro-2a cells ({80%) than with HEp-2 cells (～30%). When the cells were incubated with ethanol plus vanadate (0.1 mM), there was a marked potentiation ({200%) in cell membrane permeability in Neuro-2a cells, and again a lesser increase in permeability ({50%) with HEp-2 cells.These results seem to be due to a preferential effect of vanadate on passive permeability of Neuro-2a cells because parallel measurements demonstrate equal inhibition of (Na⁺K) ATPase with both Neuro-2a and HEp-2 cells.     

Effect of physiological electron donors and acceptors on F1-ATPase

The hydrolytic activity of F1-ATPase isolated from rat liver was enhanced in the presence of NADH, FADH2, QH2 or reduced cyt c. The extent of this activation depended largely on substrate concentration. F1-ATPase sensitivity to bicarbonate or dinitrophenol activators decreased in the presence of any of those electron donors, which originated as well a slight sensitivity to oligomycin and a sensitivity increase to the inhibitory anion OCN-. In the presence of oxidized carriers the sensitivity to bicarbonate, dinitrophenol, or OCN- was not modified, and the enzyme remained oligomycin insensitive.     

Isolation of a mitochondrial factor from rat liver which potentiates the inactivation of glutamate dehydrogenase by lysosomes

Rat liver glutamate dehydrogenase (L-glutamate: NAD(P) oxidoreductase, deaminating) E.C. 1.4.1.3.) is inactivated by the mitochondrial matrix in combination with lysosomal preparations. Neither lysosomal or mitochondrial matrix extracts per se inactivate the enzyme appreciably under the conditions used. Fractionation of the matrix indicates that a low molecular weight factor is responsible for the potentiation of inactivation of glutamate dehydrogenase by lysosomes. Its absorption spectrum and chromatographic behaviour suggest that this factor is NADP.     

A comparison of the phenotypic and genetic stability of recombinant Trichoderma spp. generated by protoplast- and Agrobacterium-mediated transformation

Four different Trichoderma strains, T. harzianum CECT 2413, T. asperellum T53, T. atroviride T11 and T. longibrachiatum T52, which represent three of the four sections contained in this genus, were transformed by two different techniques: a protocol based on the isolation of protoplasts and a protocol based on Agrobacterium-mediated transformation. Both methods were set up using hygromycin B or phleomycin resistance as the selection markers. Using these techniques, we obtained phenotypically stable transformants of these four different strains. The highest transformation efficiencies were obtained with the T. longibrachiatum T52 strain: 65-70 transformants/microg DNA when transformed with the plasmid pAN7-1 (hygromycin B resistance) and 280 transformants/107 spores when the Agrobacterium-mediated transformation was performed with the plasmid pUR5750 (hygromycin B resistance). Overall, the genetic analysis of the transformants showed that some of the strains integrated and maintained the transforming DNA in their genome throughout the entire transformation and selection process. In other cases, the integrated DNA was lost.     

Metabolic engineering of isoprenoid biosynthesis in Arabidopsis for the production of taxadiene, the first committed precursor of Taxol

Paclitaxel (Taxol) is a widely used anticancer isoprenoid produced by the secondary metabolism of yew (Taxus sp.) trees. However, only limited amounts of Taxol or related metabolites (taxoids) can be obtained from the currently available sources. In this work we have taken the first step toward genetically engineering the biosynthesis of taxoids in angiosperms. The first committed step in Taxol biosynthesis is the production of taxadiene from geranylgeranyl diphosphate (GGPP), catalyzed by the plastid-localized enzyme taxadiene synthase (TXS). A recombinant T. baccata TXS lacking the putative plastid targeting peptide and fused to a C-terminal histidine (His) tag was shown to be enzymatically active in Escherichia coli. Constitutive production of the full-length His-tagged enzyme in Arabidopsis thaliana plants led to the accumulation of taxadiene and concomitant growth retardation and decreased levels of photosynthetic pigment in transgenic plants. Although these phenotypes may derive from a toxic effect of taxadiene, the lower accumulation of endogenous plastid isoprenoid products such as carotenoids and chlorophylls in transgenic plants also suggests that the constitutive production of an active TXS enzyme might alter the balance of the GGPP pool. Induction of transgene expression using a glucocorticoid-mediated system consistently resulted in a more efficient recruitment of GGPP for the production of taxadiene, which reached levels 30-fold higher than those in plants constitutively expressing the transgene. This accomplishment illustrates the possibility of engineering the production of taxoids and other GGPP-derived isoprenoids in crop plants despite the constraints associated with limited knowledge with regard to regulation of GGPP availability.     

Effect of Cattle on Salmonella Carriage,Diversity and Antimicrobial Resistance in Free-Ranging Wild Boar (Sus scrofa) in Northeastern Spain

Salmonella is distributed worldwide and is a pathogen of economic and public health importance. As a multi-host pathogen with a long environmental persistence, it is a suitable model for the study of wildlife-livestock interactions. In this work, we aim to explore the spill-over of Salmonella between free-ranging wild boar and livestock in a protected natural area in NE Spain and the presence of antimicrobial resistance. Salmonella prevalence, serotypes and diversity were compared between wild boars, sympatric cattle and wild boars from cattle-free areas. The effect of age, sex, cattle presence and cattle herd size on Salmonella probability of infection in wild boars was explored by means of Generalized Linear Models and a model selection based on the Akaike’s Information Criterion. Prevalence was higher in wild boars co-habiting with cattle (35.67%, CI 95% 28.19–43.70) than in wild boar from cattle-free areas (17.54%, CI 95% 8.74–29.91). Probability of a wild boar being a Salmonella carrier increased with cattle herd size but decreased with the host age. Serotypes Meleagridis, Anatum and Othmarschen were isolated concurrently from cattle and sympatric wild boars. Apart from serotypes shared with cattle, wild boars appear to have their own serotypes, which are also found in wild boars from cattle-free areas (Enteritidis, Mikawasima, 4:b:- and 35:r:z35). Serotype richness (diversity) was higher in wild boars co-habiting with cattle, but evenness was not altered by the introduction of serotypes from cattle. The finding of a S. Mbandaka strain resistant to sulfamethoxazole, streptomycin and chloramphenicol and a S. Enteritidis strain resistant to ciprofloxacin and nalidixic acid in wild boars is cause for public health concern.