Sensitivity to in vitro lipid peroxidation in liver and brain of aged rats.

Lipid peroxidation in rat liver and brain has been studied to see if it increases with old age. No significant differences in the level of endogenous, nonstimulated lipid peroxidation (TBA-RS) is found between 9 month-old (mature adults) and 28 month-old animals in liver or cerebral cortex. Liver homogenates subjected in vitro to an oxidative stress (ascorbate-Fe++), show a clearly slower peroxidation rate in old than in young animals. On the other hand, the in vitro peroxidation rate of cerebral homogenates was similar in young and old animals. The in vitro peroxidation rate was much higher in brain than in liver tissue. These results do not support the view that old rats liver and brain are more susceptible to free radical oxidative damage than those of young ones.     

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.     

The rate of action of tetrodotoxin on sodium conductance in the squid giant axon.

When tetrodotoxin is applied to or washed away from the squid giant axon, the rates at which the sodium conductatnce is blocked and unblocked are an order of magnitude smaller than those reported for the isolated node of Ranvier. This slowing is to be expected if in squid the tetrodotoxin binding sites act as a saturable sink in series with the barrier to free diffusion imposed by the presence of the Schwann cell. A comparison has been made between the rates observed experimentally and those calculated for a computer model of the system, in order to estimate the apparent density in the membrane of both specific and non-specific tetrodotoxin binding sites. The figure thus obtained for the number of sodium channels in the squid giant axon, several hundred per square micrometre, agrees well with those derived from other lines of argument.