Aminoacid composition and hydrophobicity index of mitochondrial polypeptides

The aminoacid composition of protein stained bands in polyacrylamide gels, after electrophoresis of proteins from inner mitochondrial membranes, was investigated hydrolyzing directly the gel slices. The Hydrophobicity Index of 18 prominent polypeptide bands was calculated after their aminoacid analysis. The polypeptides less related to the membrane have low hydrophobicity as inferred from their Hydrophobicity Indexes.     

Mycoplasma pneumoniae infection and arthritis in man.

Seven patients developed arthritis after Mycoplasma pneumoniae infection. Their joint symptoms persisted for up to one year. One patient developed an articular erosion and in another rheumatoid factor was present in serum transiently. Mycoplasma infection often causes ill-defined arthralgias and myalgias, but the migratory polyarthropathy of middle-sized joints that occurred in these patients is much less common. The prognosis seems to be good.     

Escherichia coli ppGpp synthetase II activity requires spoT

Escherichia coli has two enzymes catalyzing the synthesis of guanosine tetraphosphate (ppGpp), designated ppGpp synthetase I (PSI = RelA) and II (PSII), whose activities are regulated differently. Until now, the gene for PSII had not been identified. Here, an E. coli relA1 strain that expresses lacZ from an rrnB P1 promoter was used to screen mutants with increased beta-galactosidase activity on 5-bromo-4-chloro-3-indoyl beta-D-galactoside indicator plates at 30 degrees C. About 15% of the mutants obtained in this manner had reduced levels of ppGpp at 30 degrees C and no detectable ppGpp at 43 degrees C. These mutants did not form colonies at 42 degrees C on minimal medium plates and had elevated ribosome concentrations and higher growth rates at 30 degrees C. Genetic mapping by phage P1 transduction and complementation analyses showed that the mutations were located in spoT and that they were recessive. Specific inhibition of SpoT-dependent ppGpp degradation activity with picolinic acid showed that two of the mutants tested were deficient in ppGpp synthesis activity. These results indicate that spoT is required for PSII activity, suggesting that spoT encodes both ppGpp degradation and synthesis activities and that these two functions can be affected independently by mutation.     

Grayanotoxin-I-modified eel electroplax sodium channels. Correlation with batrachotoxin and veratridine modifications.

To probe the structure-function relationships of voltage-dependent sodium channels, we have been examining the mechanisms of channel modification by batrachotoxin (BTX), veratridine (VTD), and grayanotoxin-I (GTX), investigating the unifying mechanisms that underlie the diverse modifications of this class of neurotoxins. In this paper, highly purified sodium channel polypeptides from the electric organ of the electric eel were incorporated into planar lipid bilayers in the presence of GTX for comparison with our previous studies of BTX (Recio-Pinto, E., D. S. Duch, S. R. Levinson, and B. W. Urban. 1987. J. Gen. Physiol. 90:375-395) and VTD (Duch, D. S., E. Recio-Pinto, C. Frenkel, S. R. Levinson, and B. W. Urban. 1989. J. Gen. Physiol. 94:813-831) modifications. GTX-modified channels had a single channel conductance of 16 pS. An additional large GTX-modified open state (40-55 pS) was found which occurred in bursts correlated with channel openings and closings. Two voltage-dependent processes controlling the open time of these modified channels were characterized: (a) a concentration-dependent removal of inactivation analogous to VTD-modified channels, and (b) activation gating similar to BTX-modified channels, but occurring at more hyperpolarized potentials. The voltage dependence of removal of inactivation correlated with parallel voltage-dependent changes in the estimated K1/2 of VTD and GTX modifications. Ranking either the single channel conductances or the depolarization required for 50% activation, the same sequence is obtained: unmodified > BTX > GTX > VTD. The efficacy of the toxins as activators follows the same ranking (Catterall, W. A. 1977. J. Biol. Chem. 252:8669-8676).     

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.     

Interaction of neurotensin with prostaglandin E2 and prostaglandin synthesis inhibitors: effects on colonic temperature in mice

Neurotensin (NT) administered intracisternally (i.c.) to adult mice produced a marked hypothermia while prostaglandin E₂, administered by the same route, produced hyperthermia. When administered concurrently the effects of the two substances were neutralized. The prostaglandin synthesis inhibitors, indomethacin and acetylsalicylic acid, were injected subcutaneously 30 min prior to i.c. administered NT and/or thyrotropin-releasing hormone (TRH). Both inhibitors failed to potentiate the hypothermia induced by NT or alter its antagonism by TRH in mice kept at 26°C. When mice were kept at 6°C, pretreatment with indomethacin, but not acetylsalicylic acid, potentiated NT-induced hypothermia and prevented its antagonism by TRH. Because indomethacin inhibits synthesis of prostaglandins within the central nervous system (CNS) as well as in peripheral organs while acetylsalicylic acid acts only in the periphery, it appears that NT-induced hypothermia in a cold environment is enhanced by a reduction of prostaglandins in the CNS.     

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.