Proton nuclear-magnetic-resonance and resonance Raman studies of thermophilic cytochrome c-552 from Thermus thermophilus HB8

The pH and temperature dependences of the 270-MHz proton nuclear magnetic resonance and resonance Raman spectra of Thermus thermophilus cytochrome c-552 were studied. Observation of the NMR methyl signal of the iron-bound methionine indicates that a methionine residue is the sixth ligand of heme iron in both ferric and ferrous states, although the environment of this methionine is not similar to that in mitochondrial cytochrome c. The NMR methyl signal of the coordinated methionine in the ferrous state was observed even at 87 degrees C, indicating the retention of the methionine ligand at the sixth coordination position. None of resonance Raman lines in ferrous cytochrome c-552 at higher temperatures showed a prominant temperature-dependent frequency shift, which implies that the heme iron was still bound with strong ligands and retained the low-spin state. In either redox state overall thermal denaturation did not occur even at 87 degrees C, although the ferric form existed in thermal spin mixture of the low-spin and high-spin species at higher temperatures. The hyperfine-shifted NMR resonances of the ferric form indicated rapid exchange of the sixth ligand at alkaline pH in the process of a single-step alkaline isomerization.     

Inhibition by polyamines of lipid peroxide formation in rat liver microsomes.

Both NADPH- and ascorbic acid-dependent lipid peroxidations were inhibited by spermine, the degree of inhibition being greater with the former peroxidation. The effective concentration of spermine required for inhibition was higher when larger amounts of microsomes were used. However, the activities of NADPH-cytochrome c reductase and NADPH-peroxidase were not influenced by spermine. These results suggest that spermine inhibits lipid peroxidation by binding to phospholipids in the microsomes.     

Regulatory mechanism of reagin production in mice at the T cell level. I. Suggestive evidence for the generation of class specific PPD-reactive helper T cell population in Mycobacterium-primed cells.

Helper T cell activities specific for purified protein derivative (PPD) generated by immunization with Mycobacterium tuberculosis (Tbc) or PPD were investigated concerning adoptive IgE and IgG antibody responses. It is interesting that preferential triggering activity of IgG antibody response was observed when PPD-reactive cells from mice immunized with Tbc were used as a helper cell source. The selective triggering of IgG B cells by Tbc-primed cells was consistently observed using DNP-primed B cell populations from mice immunized with DNP-carrier conjugate in either ICFA or alum. T cell dependency of helper activity was demonstrated by the fact that treatment of Tbc-primed cells with anti-Thy 1 antiserum plus complement abolished their helper activity. We also demonstrated that purified T cell populations selectively triggerred IgG B cells. Selective triggering of IgG B lymphocytes by Tbc-primed T cells may not be due to the influence of suppressor T cells supposedly present in Tbc-primed cells since this selectivity was not affected by X-irradiation of Tbc-primed T cell populations which may inactivate suppressor T cells. Furthermore, passive transfer of Tbc-primed cells into normal recipient mice, the condition which may detect the suppressor T cell effect much more sensitively in IgE production, or preimmunization with Tbc 2 weeks before, did not suppress primary anti-DNP IgE antibody response to DNP-PPD. Thus, the observations presented here are favorable to the concept of the presence of IgG class-specific helper T lymphocytes. Furthermore, PPD-reactive T cells from mice immunized with PPD itself exerted their helper function for triggering B cells of both IgE and IgG classes. This may also indicate that some of the components associated with Tbc other than PPD might negatively affect the development of PPD-reactive helper T cells specific to the IgE class. The generation of such IgG-specific T cell activity in the presence of Tbc will be discussed in the light of the T cell population involved in the regulation of antibody responses of different immunoglobulin classes.     

Alkaline isomerization of thermoresistant cytochrome c-552 and horse heart cytochrome c studied by absorption and resonance Raman spectroscopy.

The structure of the thermoresistant cytochrome c (552, Thermus thermophilus) has been investigated at neutral and alkaline pH by absorption and resonance Raman spectroscopy and compared with that of horse heart cytochrome c. The ligands of the ferricytochrome c-552 at neutral pH are considered to be histidine and methionine, whereas the ligands of ferrocytochrome c-552 are histidine and another nitrogen base, histidine or lysine. Ferric cytochrome c-552 undergoes an alkaline isomerization with a pK of 12.3 (25 degrees C), accompanied by a ligand exchange. Horse heart cytochrome c has at least three isomerization states at alkaline pH (pK 9.3, 12.9 and greater than 13.5 at 25 degrees C). The replacement of the sixth ligand may not be involved in the second isomerization. The thermodynamic parameters for the isomerization were also estimated. The entropy change upon isomerization of cytochrome c-552 is negative, whereas for that of horse heart cytochrome c the entropy change is positive.     

Plant growth regulators and Axl and immune checkpoint inhibitors from the edible mushroom Leucopaxillus giganteus

ABSTRACT

A novel compound, (R)-4-ethoxy-2-hydroxy-4-oxobutanoic acid (1), and six known compounds (2–7) were isolated from the fruiting bodies of the wild edible mushroom Leucopaxillus giganteus. The planar structure of 1 was determined by the interpretation of spectroscopic data analysis. The absolute configuration of 1 was determined by comparing specific rotation of the synthetic compounds. In the plant regulatory assay, the isolated compounds (1–7) and the chemically prepared compounds (8–10) were evaluated their biological activity against the lettuce (Lactuca sativa) growth. Compounds 1 and 3–10 showed the significant regulatory activity of lettuce growth. 1 showed the strongest inhibition activity among the all the compounds tested. In the lung cancer assay, all the compounds were assessed the mRNA expression of Axl and immune checkpoints (PD-L1, PD-L2) in the human A549 alveolar epithelial cell line by RT-PCR. Compounds 1–10 showed significant inhibition activity against Axl and/or immune checkpoint.     

Functional activity of RLIM/Rnf12 is regulated by phosphorylation-dependent nucleocytoplasmic shuttling

The X-linked gene Rnf12 encodes the ubiquitin ligase really interesting new gene (RING) finger LIM domain–interacting protein (RLIM)/RING finger protein 12 (Rnf12), which serves as a major sex-specific epigenetic regulator of female mouse nurturing tissues. Early during embryogenesis, RLIM/Rnf12 expressed from the maternal allele is crucial for the development of extraembryonic trophoblast cells. In contrast, in mammary glands of pregnant and lactating adult females RLIM/Rnf12 expressed from the paternal allele functions as a critical survival factor for milk-producing alveolar cells. Although RLIM/Rnf12 is detected mostly in the nucleus, little is known about how and in which cellular compartment(s) RLIM/Rnf12 mediates its biological functions. Here we demonstrate that RLIM/Rnf12 protein shuttles between nucleus and cytoplasm and this is regulated by phosphorylation of serine S214 located within its nuclear localization sequence. We show that shuttling is important for RLIM to exert its biological functions, as alveolar cell survival activity is inhibited in cells expressing shuttling-deficient nuclear or cytoplasmic RLIM/Rnf12. Thus regulated nucleocytoplasmic shuttling of RLIM/Rnf12 coordinates cellular compartments during mammary alveolar cell survival.     

Comparison of the host-handling behavior of Eretmocerus mundus on Bemisia tabaci B biotype, Q biotype and Trialeurodes vaporariorum

Host-handling behaviors of Eretmocerus mundus (Mercet) (Hymenoptera: Aphelinidae) toward first to fourth instar nymphs of the Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae) B and Q biotypes and of Trialeurodes vaporariorum (Westwood) (Homoptera: Aleyrodidae) were compared under laboratory conditions. The host acceptance rates differed among host stages but not among host species or biotypes. In all host species and biotypes, the parasitoids stopped their host-handling behavior before oviposition more often with older hosts. The total host-handling time was longer for the fourth instars than for younger instars, mainly due to the longer oviposition times and probing times. E. mundus showed marking behavior for the B. tabaci B and Q biotypes, but not for T. vaporariorum. In conclusion, E. mundus could parasitize all whitefly biotypes and species used in this study, but both B. tabaci biotypes were more suitable hosts than T. vaporariorum. The younger instars were good oviposition targets in all hosts.