Chaperonin-affected refolding of alpha-lactalbumin: effects of nucleotides and the co-chaperonin GroES.

We have studied how nucleotides (ADP, AMP-PNP, and ATP) and the co-chaperonin GroES influence the GroEL-affected refolding of apo-alpha-lactalbumin. The refolding reactions induced by stopped-flow pH jumps were monitored by alpha-lactalbumin tryptophan fluorescence. The simple single-exponential character of the free-refolding kinetics of the protein allowed us to quantitatively analyze the kinetic traces of the GroEL-affected refolding with the aid of computer simulations, and to obtain the best-fit parameters for binding between GroEL and the refolding intermediate of alpha-lactalbumin by the non-linear least-squares method. When GroES was absent, the interaction between GroEL and alpha-lactalbumin could be well represented by a "cooperative-binding" model in which GroEL has two binding sites for alpha-lactalbumin with the affinity of the second site being tenfold weaker than that of the first, so that there is negative cooperativity between the two sites. The affinity between GroEL and alpha-lactalbumin was significantly reduced when ATP was present, while ADP and AMP-PNP did not alter the affinity. A comparison of this result with those reported previously for other target proteins suggests a remarkable adjustability of the GroEL 14-mer with respect to the nucleotide-induced reduction of affinity. When GroES was present, ATP as well as ADP and AMP-PNP were effective in reducing the affinity between GroEL and the refolding intermediate of alpha-lactalbumin. The affinity at a saturating concentration of ADP or AMP-PNP was about ten times lower than with GroEL alone. The ADP concentration at which the acceleration of the GroEL/ES-affected refolding of alphaLA was observed, was higher than the concentration at which the nucleotide-induced formation of the GroEL/ES complex took place. These results indicate that GroEL/ES complex formation itself is not enough to reduce the affinity for alpha-lactalbumin, and that further binding of the nucleotide to the GroEL/ES complex is required to reduce the affinity.     

Isolation and characterization of outer and inner envelope membranes of cyanelles from a glaucocystophyte, Cyanophora paradoxa

Envelope membranes were isolated by sucrose density gradient floatation centrifugation from the homogenate of cyanelles prepared from Cyanophora paradoxa. Two yellow bands were separated after 40 h of centrifugation. The buoyant density of one of the two fractions (fraction Y2) coincided with that of inner envelope membranes of spinach or plasma membranes of cyanobacteria. The other yellow fraction (fraction Y1) migrated to top of sucrose-gradient even at 0% sucrose. Pigment analysis revealed that the heavy yellow fraction was rich in zeaxanthin while the light fraction was rich in β-carotene, and the both fractions contained practically no chlorophylls. Another yellow fraction (fraction Y3) was isolated from the phycobiliprotein fraction, which was the position where the sample was placed for gradient centrifugation. Its buoyant density and absorption spectra were similar to outer membranes of cyanobacteria. We have assigned fractions Y2 and Y3 as inner and outer envelope membrane fractions of cyanelles, respectively. Protein compositions were rather different between the two envelope membranes indicating little cross-contamination among the fractions. H. Koike and Y. Ikeda contributed equally.     

Effects of extending the light phase on diapause induction in a Japanese population of the two-spotted spider mite, <Emphasis Type="Italic">Tetranychus urticae</Emphasis>

Artificial lighting is a merit of a ‘plant factory’, which might be utilized to suppress an increase in pest population. We investigated the effects of extending the light phase on diapause induction in the two-spotted spider mite (TSSM), Tetranychus urticae. TSSM were reared at 18°C under light phases ranging from 2 to 64 h combined with a constant dark phase of 16 h in aluminum bottles, with white light emitting diodes attached inside to minimize fluctuations in air temperature between the light and dark phases. Diapause was induced in adult TSSM females when the light phase was 24 h or shorter, and diapause induction was inhibited when the light phase extended over 32 h. The development of deutonymphs was delayed under a diapause-inducing photoperiod. Diapause inducing photoperiods may suppress an increase in the TSSM population, by slowing down development and reproduction.     

Toxicity of bifenazate and its principal active metabolite, diazene, to Tetranychus urticae and Panonychus citri and their relative toxicity to the predaceous mites, Phytoseiulus persimilis and Neoseiulus californicus

Bifenazate is a novel carbazate acaricide discovered by Uniroyal Chemical (now Chemtura Corporation) for the control of phytophagous mites infesting agricultural and ornamental crops. Its acaricidal activity and that of its principal active metabolite, diazene, were characterized. Bifenazate and diazene had high toxicity and specificity both orally and topically to all life stages of Tetranychus urticae and Panonychus citri. Acute poisoning was observed with no temperature dependency. No cross-resistance was found to mites resistant to several other classes of acaricides, such as tebufenpyrad, etoxazole, fenbutatin oxide and dicofol. Bifenazate remained effective for a long time with only about a 10% loss of efficacy on T. urticae after 1 month of application in the field. All stages of development of the predatory mites, Phytoseiulus persimilis and Neoseiulus californicus, survived treatment by both bifenazate and diazene. When adult females of the two predatory mite species were treated with either bifenazate or diazene, they showed a normal level of fecundity and predatory activity in the laboratory, effectively suppressing spider mite population growth. Even when the predators were fed spider mite eggs that had been treated previously with bifenazate, they survived. These findings indicate that bifenazate is a very useful acaricide giving high efficacy, long-lasting activity and excellent selectivity for spider mites. It is, therefore, concluded that bifenazate is an ideal compound for controlling these pest mites.     

Isolation and characterization of oxygen-evolving thylakoid membranes and photosystem II particles from a marine diatom Chaetoceros gracilis

Thylakoid membranes retaining high oxygen-evolving activity (about 250 micromol O(2)/mg Chl/h) were prepared from a marine centric diatom, Chaetoceros gracilis, after disruption of the cells by freeze-thawing. We also succeeded in purification of Photosystem II (PSII) particles by differential centrifugation of the thylakoid membranes after treatment with 1% Triton X-100. The diatom PSII particles showed an oxygen-evolving activity of 850 and 1045 micromol O(2)/mg Chl/h in the absence and presence of CaCl(2), respectively. The PSII particles contained fucoxanthin chlorophyll a/c-binding proteins in addition to main intrinsic proteins of CP47, CP43, D2, D1, cytochrome b559, and the antenna size was estimated to be 229 Chl a per 2 molecules of pheophytin. Five extrinsic proteins were stoichiometrically released from the diatom PSII particles by alkaline Tris-treatment. Among these five extrinsic proteins, four proteins were red algal-type extrinsic proteins, namely, PsbO, PsbQ', PsbV and PsbU, whereas the other one was a novel, hypothetical protein. This is the first report on isolation and characterization of diatom PSII particles that are highly active in oxygen evolution and retain the full set of extrinsic proteins including an unknown protein.     

Evidence for two vitellogenin-related genes in Leucophaea maderae: the protein primary structure and its processing

We previously reported a cDNA for vitellogenin (Vg) from the cockroach, Leucophaea maderae (Lm). In the present study, we identified another cDNA encoding a second Vg (Vg2) having stretches of amino acid sequences different from the first one, Vg1, reported earlier. The complete nucleotide sequence of Vg2 consisted of 5,915 bp, which encoded a primary protein of 1,911 residues including a 16-residue putative signal peptide. The regions different in both Vg precursors (Pro-Vg1 and pro-Vg2) were four in number, and two, relatively longer, existed at the carboxy terminal. The presence of two Vg-related cDNAs was confirmed by sequencing of RT-PCR products generated using primers designed based on the common sequences flanking the regions different in amino acid sequences. Both forms were transcribed since they could be amplified on mRNA from fat bodies of different individual females. Southern blot analysis of digested genomic DNA revealed the existence of two Vg-related genes in L. maderae indicating that each Vg cDNA originated from a separate gene. Also, the immunoblot analysis using antibodies generated against peptides unique to both Vg1 and Vg2 probed the same antigen in the same individual, suggesting LmVg to be a product coded by two different Vg precursors. Both Vg primary products showed 96% similarity at an amino acid level. Compared to other insect Vgs, Vg2 showed a slightly higher (1-2%) similarity than Vg1. We previously reported, based on amino-terminal sequence analysis, that L. maderae pro-Vg was cleaved into four subunit polypeptides (112-, 100-, 92-, and 55-kD), which were deposited in the egg as four respective vitellin (Vn) polypeptides. We show now based on immunoblot analysis that the 112-kD polypeptide is further cleaved, near the C-terminus, to an 87-kD polypeptide before it is secreted into the hemolymph. Both the L. maderae Vgs were compared with each other and with other insect Vgs and the processing pattern is discussed.     

Interleukin-1β-Induced Autophagy-Related Gene 5 Regulates Proliferation of Embryonic Stem Cell-Derived Odontoblastic Cells

We previously established a method for the differentiation of induced pluripotent stem cells and embryonic stem cells into α2 integrin-positive odontoblast-like cells. We also reported that Wnt5 in response to interleukin (IL)-1β induces matrix metalloproteinase (MMP)-3-regulated cell proliferation in these cells. Our findings suggest that MMP-3 plays a potentially unique physiological role in the generation of odontoblast-like cells under an inflammatory state. Here, we examined whether up-regulation of autophagy-related gene (Atg) 5 by IL-1β was mediated by Wnt5 signaling, thus leading to increased proliferation of odontoblast-like cells. IL-1β increased the mRNA and protein levels of Atg5, microtubule-associated protein 1 light chain (LC3, a mammalian homolog of yeast Atg8) and Atg12. Treatment with siRNAs against Atg5, but not LC3 and Atg12, suppressed the IL-1β-induced increase in MMP-3 expression and cell proliferation. Our siRNA analyses combined with western blot analysis revealed a unique sequential cascade involving Atg5, Wnt5a and MMP-3, which resulted in the potent increase in odontoblastic cell proliferation. These results demonstrate the unique involvement of Atg5 in IL-1β-induced proliferation of embryonic stem cell-derived odontoblast-like cells.