3,5-Di-tert-butyl-4-hydroxybenzylidenemalononitrile. Effects of pH on its binding to liposomes and evidence for formation of a ternary complex with valinomycin and potassium ion

1. The properties of 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847) were studied chemically and spectroscopically. Two molecular species of SF6847 were identified: the undissociated form (SFH; ?₃₆₃, 10 mM^?1) and the dissociated form (SF^?; ?₄₅₄, 35 mM^?1). The pK_a value of the molecule was determined to be 6.9.2. On the basis of these properties the interactions of SF6847 with liposomes and valinomycin · K⁺ were studied. The partition constants of SFH (Kⁿ_p and SF^? (K^?_p) to liposomes were determined separately; Kⁿ_p was 56 mM^?1 and was independent of the pH of the medium, whereas K^?_p dependend greatly on the pH, being 1.2 mM^?1 at pH 7.0 and 2.9 mM^?1 at pH 8.0. Using these values, the partition constant of total SF6847 (K_p) was calculated and found to be essentially the same as that calculated from the kinetics of proton uptake. It was concluded that the amount of SF^? bound to liposomes is rate limiting for proton uptake.3. The effects of membrane potential on partition constants were studied. The K^?_p decreased greatly upon generation of a membrane potential negative inside the liposomes but increased upon generation of a membrane potential positive inside the liposomes.4. The interaction of SF6847 with valinomycin in aqueous solution and in liposomes was demonstrated only in the presence of potassium ion. Potassium ion could not be replaced by sodium ion. Evidence was obtained for the formation of the ternary complex valinomycin · K⁺ · SF^? in liposomes and in hexane. It was concluded that SF^? became more soluble in the liposomal membranes on formation of this ternary complex. All these results support our proposed mechanism for the proton uptake cycle (Yamaguchi, A. and Anraku, Y. (1978) Biochim. Biophys. Acta 501, 136–149).     

Repression of porin synthesis by salicylate in Escherichia coli, Klebsiella pneumoniae and Serratia marcescens

The synthesis of the OmpF porin in Escherichia coli K-12 was highly and reversibly inhibited by 5 mM salicylate in the bacterial growth medium, and salicylate also inhibited the OmpC porin synthesis, although only weakly. The full expression of the salicylate effect was presumed to require the ompB gene product on comparison between the wild type and ompB mutant strains. The salicylate effect was also observed for the porin protein synthesis in Klebsiella pneumoniae and Serratia marcescens, although an ompB-like gene remains to be identified in both species.     

Mutations affecting early distribution of primordial germ cells in Medaka (Oryzias latipes) embryo

The development of germ cells has been intensively studied in Medaka (Oryzias latipes). We have undertaken a large-scale screen to identify mutations affecting the development of primordial germ cells (PGCs) in Medaka. Embryos derived from mutagenized founder fish were screened for an abnormal distribution or number of PGCs at embryonic stage 27 by RNA in situ hybridization for the Medaka vasa homologue (olvas). At this stage, PGCs coalesce into two bilateral vasa-expressing foci in the ventrolateral regions of the trunk after their migration and group organization. Nineteen mutations were identified from a screen corresponding to 450 mutagenized haploid genomes. Eleven of the mutations caused altered PGC distribution. Most of these alterations were associated with morphological abnormalities and could be grouped into four phenotypic classes: Class 1, PGCs dispersed into bilateral lines; Class 2, PGCs dispersed in a region more medial than that in Class 1; Class 3, PGCs scattered laterally and over the yolk sac area; and Class 4, PGCs clustered in a single median focus. Eight mutations caused a decrease in the number of PGCs. This decrease was observed in the offspring of heterozygous mothers, indicating the contribution of a maternal factor in determining PGC abundance. Taken together, these mutations should prove useful in identifying molecular mechanisms underlying the early PGC development and migration.     

Essential role of the PSI–LHCII supercomplex in photosystem acclimation to light and/or heat conditions by state transitions

Light and temperature affect state transitions through changes in the plastoquinone (PQ) redox state in photosynthetic organisms. We demonstrated that light and/or heat treatment induced preferential photosystem (PS) I excitation by binding light-harvesting complex II (LHCII) proteins. The photosystem of wheat was in state 1 after dark overnight treatment, wherein PQ was oxidized and most of LHCII was not bound to PSI. At the onset of the light treatment [25 °C in the light (100 µmol photons m^?2 s^?1)], two major LHCIIs, Lhcb1 and Lhcb2 were phosphorylated, and the PSI–LHCII supercomplex formed within 5 min, which coincided with an increase in the PQ oxidation rate. Heat treatment at 40 °C of light-adapted wheat led to further LHCII protein phosphorylation of, resultant cyclic electron flow promotion, which was accompanied by ultrafast excitation of PSI and structural changes of thylakoid membranes, thereby protecting PSII from heat damage. These results suggest that LHCIIs are required for the functionality of wheat plant PSI, as it keeps PQ oxidized by regulating photochemical electron flow, thereby helping acclimation to environmental changes.     

A 38 nt region and its flanking sequences within gag of Friend murine leukemia virus are crucial for splicing at the correct 5′ and 3′ splice sites

The genome of the Friend murine leukemia virus (Fr‐MLV) contains a 5′ splice site (5′ss) located at 205 nt and a 3′ss located at 5489 nt. In our previous studies, it was shown that if the HindIII–BglII (879–1904 bp) fragment within gag is deleted from the proA8m1 vector, which carries the entire Fr‐MLV sequence, then cryptic splicing of env‐mRNA occurs. Here, attempts were made to identify the genomic segment(s) in this region that is/are essential to correct splicing. First, vectors with a serially truncated HindIII–BglII fragment were constructed. The vector, in which a 38 bp fragment (1612–1649 bp) is deleted or reversed in proA8m1, only produced splice variants. It was found that a 38 nt region within gag contains important elements that positively regulate splicing at the correct splice sites. Further analyses of a series of vectors carrying the 38 bp fragment and its flanking sequences showed that a region (1183–1611 nt) upstream of the 38 nt fragment also contains sequences that positively or negatively influence splicing at the correct splice sites. The SphI–NdeI (5140–5400 bp) fragment just upstream of the 3′ss was deleted from vectors that carried the 38 bp fragment and its flanking sequences, which yielded correctly spliced mRNA; interestingly, these deleted vectors showed cryptic splicing. These findings suggest that the 5140–5400 nt region located just upstream of the 3′ss is required for the splicing function of the 38 nt fragment and its flanking sequences.     

Characterization of recombinant amyloidogenic chicken cystatin mutant I66Q expressed in yeast

Amyloidogenic chicken cystatin mutant I66Q (cC I66Q) was successfully secreted by yeasts Pichia pastoris and Saccharomyces cerevisiae. The soluble monomer and dimer forms of amyloidogenic cC I66Q were found in the culture medium, while large amounts of insoluble aggregate and polymeric form cC I66Q besides the monomer and dimer forms were secreted into the culture medium. The amyloidogenic cC I66Q showed a comparable circular dichroism spectrum to that of the wild cystatin, and the monomer form exhibited a similar level of inhibitory activity toward papain, but the dimmer form did not. During storage of amyloidogenic cC I66Q under physiological and acidic conditions, typical binding with Congo red and thioflavin T, and the formation of amyloid fibrils were observed, whereas the characteristic of similar amyloidosis was hardly detected for the wild recombinant cystatin.     

Specific induction of a 72-kDa heat shock protein protects esophageal mucosa from reflux esophagitis

AimsThe aim of this study is to investigate the expression and cytoprotective function of a 72-kDa heat shock protein (HSP72) using a reflux esophagitis model in rats.Main methodsExpression of HSP60, HSP72, and HSP90 in rat esophageal mucosa was evaluated by Western blot analysis before and after hyperthermia (42.5 °C, 20 min). Rats received the operation to produce reflux esophagitis with or without pretreatment with hyperthermia to induce HSPs. The esophageal mucosal damage was evaluated 12 h after the operation.Key findingsExpression of HSP72 was significantly increased by hyperthermia in rat esophageal mucosa. Reflux esophagitis was dramatically prevented when HSP72 was preinduced by hyperthermia. Furthermore, activation of TNF-α and IL-1β in esophageal mucosa was also suppressed.SignificanceThese results suggested that hyperthermia protects the esophageal mucosa in reflux esophagitis model by inducing HSP72 and suppressing proinflammatory cytokine activation. These findings might suggest that HSP-inducing therapy could be a novel and unique therapy for reflux esophagitis.