Modification of the gene 5 DNA unwinding protein with ruthenium

A chemical modification of the gene 5 DNA binding protein (G5BP) from bacteriophage fd was investigated using X-ray diffraction and difference Fourier analysis. The crystalline protein was reacted with pentaammineruthenium (III) trichloride, Ru(NH₃)₅Cl₃, a reagent believed specific for histidine residues and useful in NMR and chemical modification studies of proteins. The major ruthenium site was found by difference Fourier analysis to be 4 Å from histidine 64, the only histidine residue in the molecule. A second bipartite site, believed to be a ruthenium-anion pair, appeared to be salt-bridged to glutamic acid 40 and arginine 16. Indications were present in the difference Fourier results to suggest that the loop containing tyrosine 41 had undergone a slight conformational rearrangement to accommodate this interaction.     

大鼠中脑导水管壁及周围灰质在针刺镇痛时的~3H-5-羟色胺含量变化

本研究用~3H-5-羟色胺作示踪,由大鼠侧脑室注入后用冰冻微观放射自显影和组织固定微观放射自显影平行探讨了针刺镇痛时中脑导水管壁及周围灰质部位5-羟色胺的含量定位变化。研究结果发现,当电针达到镇痛时,在中脑导水管壁及周围灰质部位~3H-5-羟色胺的放射自显影象都呈明显增高,这表明在针刺镇痛条件下,~3H-5-羟色胺可迅速被中脑导水管壁及周围灰质部位摄取和储存,从而提示上述部位与针刺镇痛作用有密切关系。     

琼脂糖平板制备聚焦电泳法进一步提纯胸腺素组分五

用琼脂糖平板等电聚焦电泳法,由胸腺素组分五中分离出三种在聚焦电泳谱上是单一谱线的多肽成份——CP1、CP2和CP3,等电点分别为4.3、4.9和5.6。测定了这些多肽对脐带血中淋巴细胞形成羊红细胞玫瑰花的影响。与对照相比,CP1(2微克/0.6毫升),和CP3(0.2-2微克/毫升)分别在统计学上呈显著和非常显著差异。在相同测定条件下,这三种多肽成份的活性均高于化学合成的胸腺多肽——胸腺素α_1。     

Induction of autolysis of staphylococci by the basic peptide antibiotics Pep 5 and nisin and their influence on the activity of autolytic enzymes

Pep 5 and nisin are cationic bactericidal peptides which were shown to induce autolysis in Staphylococcus cohnii 22. In contrast to nisin, Pep 5 induced lysis could be stimulated in the presence of glucose. Addition of lipoteichoic acids (LTA) (d-alanine:phosphorus=0.475:1) inhibited all effects of Pep 5 on susceptible cells in a molar ratio LTA:Pep 5 of 10:1. Treatment of S. cohnii 22 with Pep 5 or nisin for 20 min and subsequent washing with 2.5 M NaCl released autolysin activity. Crude preparations of the hydrolyzing enzymes produced free amino groups as well as polysaccharide fragments from the murein backbone, suggesting the presence of a muramidase or glucosamidase, and endopeptidase or amidase. Both enzyme activities were inhibited by lipoteichoic acid; they could be fully reactivated by addition of Pep 5 in sufficient concentrations. The velocity of hydrolysis was not influenced by nisin, whereas it was doubled in presence of Pep 5. The results are discussed in view of a possible mechanism of induction of lysis by Pep 5 and nisin.Abbreviations A.U. arbitrary unit - CCCP carbonylcyanide-m-chlorophenyl hydrazone - DNase deoxyribonuclease - CYG casein yeast extract glucose - IT initial turbidity - LTA lipoteichoic acid - RNase ribonuclease - TSB Tryptone Soy Broth     

Macrophage mediation of the inhibitory effects of elevated intracellular levels of adenosine-3':5' cyclic monophosphate (cAMP) on macrophage-Trypanosoma cruzi association

Presence of theophylline and dibutyryl-cAMP—agents which cause elevation of intracellular levels of cAMP—in in vitro systems in which murine macrophages interact with virulent blood forms of Trypanosoma cruzi resulted in a marked inhibition of cell-parasite association (i.e., decreased surface binding and/or internalization). This effect was evidenced in terms of significant reductions in both the percentage of infected macrophages and the average number of trypanosomes per 100 macrophages. Pretreatment of the macrophages with these agents produced a similar inhibition whereas pretreatment of the parasites had no significant consequence on the interaction. The inhibitory effect was transient since it was no longer seen after 30 min of incubation of the treated macrophages in fresh medium. Thus, the inhibitory effect is exerted through a transient effect on the macrophage.     

Lectin-induced modulation of the antibody response to type III pneumococcal polysaccharide

Several lectins were tested for their capacity to alter the antibody response to type III pneumococcal polysaccharide (SSS-III). The antibody response was enhanced by concanavalin A (Con A), phytohemagglutinin (PHA), as well as lectins from Phytolacca americana (Pa-2), Pisum sativum (PSA), and Lens culinaris (LCH), when these lectins were given 2 days after immunization with SSS-III; however, suppression was obtained when Con A and Pa-2 were given at the time of immunization. By contrast the lectins from Vicia villosa (VVL) and Bauhinia purpurea (BPA) did not alter the antibody response. Since the lectins PSA and LCH bind to the same monosaccharide as Con A, whereas the other lectins bind to different monosaccharides, these findings indicate that there is no relationship between nominal monosaccharide specificity and the capacity to modulate the antibody response. Substantial increases in the magnitude of the IgG₁ antibody response was noted after the administration of Con A whereas profound enhancement of IgG_2a antibody response was noted after PHA was given.     

T-cell-mediated regression of "spontaneous" and of Epstein-Barr virus-induced B-cell transformation in vitro: studies with cyclosporin A

The regression of Epstein-Barr (EB) virus-transformed B-cell outgrowth which is seen in experimentally-infected cultures of blood mononuclear (UM) cells from healthy seropositive donors can be abolished in medium containing the T-cell-suppressive agent cyclosporin A (CSA) at concentrations of 0.05 microgram/ml and above. CSA mediates its effect within the first 4 days post-infection of the UM cells and this prevents subsequent in vitro generation of the EB virus-specific cytotoxic-T-cell response which normally brings about regression. Regression can be fully restored by supplementing the CSA-treated culture with interleukin 2 (IL-2)-containing culture supernatants or indeed with purified IL-2 itself, suggesting that CSA mediates its effect in this system through inhibiting the endogenous production of IL-2 which is required to amplify the virus-specific cytotoxic response. "Spontaneous transformation" to EB virus genome-positive lymphoblastoid cell lines in noninfected cultures of UM cells from healthy seropositive donors, though rare in normal medium, is enhanced to such a degree in the presence of CSA that, for many donors, the phenomenon becomes titratable against input cell dose across the 2.0 X 10(6)-2.5 X 10(5) cells/culture range. Cell mixing experiments suggest that the spontaneously transformed cell lines which arise with such efficiency under these conditions do so not by direct in vitro outgrowth of progenitor cells transformed by the virus in vivo, but by a two-step mechanism involving virus release and secondary infection in vitro.     

Inhibition of macrophage DNA synthesis by immunomodulators. II. Characterization of the suppression by muramyl dipeptide or lipopolysaccharide [3H]thymidine incorporation into macrophages

Guinea pig peritoneal exudate macrophages actively incorporated [3H]thymidine into trichloroacetic acid-insoluble fraction in vitro. The incorporation of [3H]thymidine was almost completely inhibited by aphidicolin, an inhibitor of DNA polymerase alpha and an autoradiograph showed heavy labeling in nuclei of 15% of macrophage populations. These results indicate that the observed thymidine incorporation was due to a nuclear DNA synthesis. The [3H]thymidine incorporation was markedly suppressed when macrophages were activated by immunoadjuvants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The suppression of [3H]thymidine incorporation by MDP was neither due to the decrease in thymidine transport through the cell membrane, nor due to dilution by newly synthesized "cold" thymidine. An autoradiograph revealed that MDP markedly decreased the number of macrophages the nuclei of which were labeled by [3H]thymidine. These results suggest that the suppression of [3H]thymidine incorporation by the immunoadjuvants reflects a true inhibition of DNA synthesis. The inhibition of DNA synthesis by MDP was also observed in vivo. Further, it was strongly suggested that the inhibition was not caused by some mediators, such as prostaglandin E2, released from macrophages stimulated by the immunoadjuvants but caused by a direct triggering of the adjuvants at least at the early stage of activation. Cyclic AMP appears to be involved in the inhibitory reaction.     

Effects of glucagon on the redox states of cytochromes in mitochondria in situ in perfused rat liver

The effects of glucagon on the respiratory function of mitochondria in situ were investigated in isolated perfused rat liver. Glucagon at the concentrations higher than 20 pM and cyclic AMP (75 microM) stimulated hepatic respiration, and shifted the redox state of pyridine nucleotide (NADH/NAD) in mitochondria in situ to a more reduced state as judged by organ fluorometry and beta-hydroxybutyrate/acetoacetate ratio. The organ spectrophotometric study revealed that glucagon and cyclic AMP induced the reduction of redox states of cytochromes a(a3), b and c+c1. Atractyloside (4 micrograms/ml) abolished the effects of glucagon on these parameters and gluconeogenesis from lactate. These observations suggest that glucagon increases the availability of substrates for mitochondrial respiration, and this alteration in mitochondrial function is crucial in enhancing gluconeogenesis.