PROTON GRADIENT REGULATION 5 contributes to ferredoxin-dependent cyclic phosphorylation in ruptured chloroplasts

Antimycin A-sensitive cyclic electron flow (CEF) was discovered as cyclic phosphorylation by Arnon et al. (1954). Because of its sensitivity to antimycin A, PROTON GRADIENT REGULATION 5 (PGR5)/PGR5-like Photosynthetic Phenotype 1 (PGRL1)-dependent CEF has been considered identical to the CEF of Arnon et al. However, this conclusion still needs additional supportive evidence, mainly because of the absence of definitive methods of evaluating CEF activity. In this study, we revisited the classical method of monitoring cyclic phosphorylation in ruptured chloroplasts to characterize two Arabidopsis mutants: pgr5, which is defective in antimycin A-sensitive CEF, and chlororespiratory reduction 2-1 (crr2-1), which is defective in chloroplast NDH-dependent CEF. We observed a significant reduction in CEF-dependent pmf formation and consequently ATP synthesis in the pgr5 mutant, although LEF-dependent pmf formation and ATP synthesis were not impaired at photosynthetic photon flux densities below 130?μmol?m^?2?s^?1. In contrast, the contribution of chloroplast NDH complex to pmf formation and ATP synthesis was not significant. Antimycin A partially inhibited CEF-dependent pmf formation, although there may be further inhibition sites. Unlike in the observation in leaves, the proton conductivity of ATP synthase, monitored as g_H⁺, was not enhanced in ruptured chloroplasts of the pgr5 mutant.     

Synthesis of 1,5-Anhydro-d-fructose derivatives and evaluation of their inflammasome inhibitors

Synthesis of several 1,5-Anhydro-d-fructose (1,5-AF) derivatives to evaluate inhibitory activities of the inflammasome was carried out. Recently, 1,5-AF reported to suppress the inflammasome, although with only low activity. We focused on the hydration of 2-keto form of 1,5-AF and speculated that this hydration was the cause of low activity. Therefore, we synthesized some 1,5-AF derivatives that would not be able to form the dimer conformation and can be expected to have high activity against inflammasome, and then evaluated their inhibitory activities with respect to the NLRP3 inflammasome by using mouse bone marrow-derived macrophages and human THP-1 cells. As a result, some synthesized 2-keto form compounds had much higher inhibitory activities with respect to the NLRP3 inflammasome than did 1,5-AF.     

Unconventional Myosin ID is Involved in Remyelination After Cuprizone-Induced Demyelination

Myelin, which is a multilamellar structure that sheathes the axon, is essential for normal neuronal function. In the central nervous system (CNS), myelin is produced by oligodendrocytes (OLs), which wrap their plasma membrane around axons. The dynamic membrane trafficking system, which relies on motor proteins, is required for myelin formation and maintenance. Previously, we reported that myosin ID (Myo1d) is distributed in rat CNS myelin and is especially enriched in the outer and inner cytoplasm-containing loops. Further, small interfering RNA (siRNA) treatment highlighted the involvement of Myo1d in the formation and maintenance of myelin in cultured OLs. Myo1d is one of the unconventional myosins, which may contribute to membrane dynamics, either in the wrapping process or transport of myelin membrane proteins during myelination. However, the function of Myo1d in myelin formation in vivo remains unclear. In the current study, to clarify the function of Myo1d in vivo, we surgically injected siRNA in the corpus callosum of a cuprizone-treated demyelination mouse model via stereotaxy. Knockdown of Myo1d expression in vivo decreased the intensities of myelin basic protein and myelin proteolipid protein immunofluorescence staining. However, neural/glial antigen 2-positive signals and adenomatous polyposis coli (APC/CC1)-positive cell numbers were unchanged by siRNA treatment. Furthermore, Myo1d knockdown treatment increased pro-inflammatory microglia and astrocytes during remyelination. In contrast, anti-inflammatory microglia were decreased. The percentage of caspase 3-positive cells in total CC1-positive OLs were also increased by Myo1d knockdown. These results indicated that Myo1d plays an important role during the regeneration process after demyelination.

     

Effect of N-Methyl-4-Phenylpyridinium Ion on Monoamine Oxidase in a Clonal Rat Pheochromocytoma Cell Line, PC 12h

The effects of the neurotoxin N-methyl-4-phenylpyridinium ion (MPP+) on the enzymes involved in synthesis and catabolism of catecholamines were examined using a clonal rat pheochromocytoma cell line, PC12h, as a model of dopaminergic neurons. MPP+ added in the culture medium was found to be accumulated in PC12h cells after 30-min incubation. Monoamine oxidase (MAO) activity in PC12h cells was inhibited by MPP+ in a dose-dependent way from 10 nM to 10 microM, but concentrations of MPP+ higher than 100 microM were found to increase the MAO activity. At the lower concentrations MPP+ inhibited MAO noncompetitively with respect to the substrate, kynuramine, and at the higher concentrations it increased both the Km and the Vmax values of MAO toward the substrate. On the other hand, tyrosine hydroxylase activity and the dopamine concentrations in PC12 cells were not changed by incubation with MPP+ for 30 min, 60 min, or 24 h.     

Old-Culture Flowering of Lemna paucicostata 6746 in Aged Hutner's Medium

Lemna paucicostata 6746, a short-day plant, flowers in agedHutner's medium even under continuous light, when the endogenousnitrogen level decreases to below 1.6 µmg fr wt. At thesenitrogen levels, daylength-independent flowering of the plantcan be induced even in fresh Hutner's medium. Thus, old-cultureflowering in Hutner's medium is due to nitrogen deficiency inthe plants. ¹Present address: Biological Institute, Faculty of Science,Shizuoka University, Shizuoka 422, Japan. (Received February 12, 1987; Accepted August 28, 1987)     

Two types of liver-specific F antigen are encoded by a locus located on chromosome 5 in mice

Two types of liver-specific F antigen in mice were distinguished by an immunoblotting technique after IEF of liver extracts. The IEF banding patterns consist of several bands whose pI vary from 7.57 to 8.15. One type of antigen (designated F2 antigen) showed a pattern that lacked some basic bands which are present in the pattern of the other type of antigen (designated F1 antigen). The latter type was found in AKR, CBA, C3H, DBA/2, and SM strains, while the former type was found in A, C57BL, and many other strains. Breeding experiments indicated that this variation is controlled by a single autosomal locus designated Laf (liver antigen F). Linkage analysis showed that the Laf locus is linked to the Pgm-1 locus on chromosome 5. The recombination frequency between these two loci is estimated to be 0.173 ± 0.037. The distribution of F1 and F2 antigen types among inbred strains is concordant with that of type 1 and type 2 F antigens, which have been previously distinguished by their immunogenic differences, i. e., whether alloimmunization with the liver extracts from a given strain of mice can produce the antibody to F antigen in certain strains of mice. It is suggested, therefore, that the Laf locus may encode an allogeneic moiety of F antigen molecules.Abbreviations used in this paper IEF isoelectric focusing - RI recombinant inbred - ICLAS International Council for Laboratory Animal Science     

Proton computed tomography applied to small biomedical samples

The computed tomography (CT) using the high-energy proton beam has the potential advantage of superior density resolution in the soft tissue analysis as compared to the conventional X-ray CT. We have developed the “first generation” type proton CT system using a 25-MeV proton beam to study the density distributions in small biomedical samples (<6 mm ϕ). The specimens were fixed in a cylindrical sample holder that was immersed in a water bath. The sample holder was scanned in front of the external semimicro beam of protons with a width of 0.06 mm at full width of half maximum (FWHM). The residual energy of the penetrated proton beam was analyzed with a solid state detector (SSD). The obtained integral density projection views were reconstructed into the density image by the filtered back projection method using the Shepp-Logan filter function. The spatial resolution of 0.1 mm and the density resolution of 0.1% were obtained by our system. The technique was applied to the eyes of small animals to study the changes in the density distributions of lenses, which develop with lens opacification in cataracts.     

Rapid Determination of Norepinephrine, Dopamine, Serotonin, Their Precursor Amino Acids, and Related Metabolites in Discrete Brain Areas of Mice Within Ten Minutes by HPLC with Electrochemical Detection

A rapid and simple chromatographic procedure using HPLC with electrochemical detection is described for simultaneous determination of the substrates from precursor amino acids to metabolites related to synthesis and metabolism of three monoamine neurotransmitters--norepinephrine (NE), dopamine (DA), and 5-hydroxytryptamine (5-HT, serotonin)--in discrete brain areas of the mouse. Under the present instrumental and mobile phase conditions, the procedure permits simultaneous determination of three monoamines (NE, DA, and 5-HT), two precursor amino acids (tyrosine and tryptophan), and four respective metabolites (3-methoxy-4-hydroxyphenylglycol, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid) within 10 min in one chromatographic run. By varying column temperature, this procedure also permits simultaneous determination of 10-14 monoamine-related substrates including the nine substrates described above within 15-21 min. The validity of the present procedure is demonstrated by analyzing the effect of an alpha 2-adrenergic agonist (clonidine) and an alpha 2-antagonist (yohimbine) in mouse hypothalamus.     

Extragenic suppressor mutations of a β-tubulin mutation in the basidiomyceteCoprinus cinereus: Isolation and genetic and biochemical analyses

In total, 111 revertants were isolated from the oidia of a heat-sensitive, -tubulin mutant BEN193 (benA193) of the basidiomyceteCoprinus cinereus after mutagenesis by ultraviolet. Of the 111 revertants, 48 were genetically analyzed. In 15 of the 48 revertants, reversion was due to mutations at loci unlinked tobenA, whereas in the remaining 33 revertants, reversion was due to mutations withinbenA or at loci closely linked tobenA. The 15 extragenic suppressor mutations comprised three groups in terms of genetic linkage; two of them were designated asmipA andmipB. Suppressor mutations in the third group were found to bebenC, one of the four loci we have previously identified as genes conferring benomyl resistance. Biochemical analysis revealed thatbenC ⁺ is a structural gene for a major -tubulin in vegetative hyphae.     

Induction of flowering by nitrogen deficiency in Lemna paucicostata 441

Lemna paucicostata 441, a short-day plant, flowered even undercontinuous light in nitrogen-deficient, half-strength Hutner’smedium, when the endogenous level of nitrogen was decreasedto 1.4µg/mg fr wt or lower, but no flowering occurredin nitrogen-deficient, modified Hoagland medium when the endogenouslevel of nitrogen was similarly reduced. The failure to flowerin this medium can be ascribed to the presence of high concentrationsof calcium, the absence of EDTA, and low pH. Daylength-independent flowering induced by nitrogen deficiencywas greatly enhanced by the addition to the growth medium ofmicronutrients and EDTA at high concentrations. Among the micronutrients,zinc seemed to be the most important. (Received May 30, 1988; Accepted September 22, 1988)