Calcium- and Cyclic AMP-independent,Labile Protein Kinase Appearing during Starfish Oocyte Maturation: its Extraction and Partial Characterization*

A burst of protein phosphorylation and an appearance of maturation-promoting factor have been reported to occur shortly before germinal vesicle (nucleus) breakdown (GVBD) in 1-methyladenine-induced oocyte maturation of starfish. To detect if a protein kinase is activated before GVBD, protein kinase activity was compared in maturing oocytes which were just undergoing GVBD and immature oocytes of Asterina pectinifera. The oocytes were homogenized in a buffer modified from that used for extracting amphibian maturation-promoting factor. When the supernatant protein of homogenized immature oocytes was used as a substrate, protein kinase activity in the supernatant of the maturing oocytes was 7-fold higher than that of immature oocytes. The protein kinase in the supernatant of the maturing oocytes showed a high substrate specificity for histone H1 among the exogenous substrates examined, and the activity of the maturing oocytes for histone H1 was 6- to 7-fold higher than that of immature oocytes. The protein kinase detected in the maturing oocytes was very labile and was inhibited neither by ethylene glycol bis(β-aminoethyl ether)N, N, N′, N′-tetraacetic acid nor by the heat-stable inhibitor protein of cyclic AMP-dependent protein kinase. These results indicate that a calcium- and cyclic AMP-independent, labile “maturation-specific protein kinase” appeared before GVBD in maturing oocytes, and suggest its participation in the phosphorylation burst in vivo. The possible correlation of this kinase with maturation-promoting factor and chromosome condensation was discussed.     

Biosynthesis of folic acid compounds in plants VI. The occurrence and properties of the dihydrofolate-synthesizing enzyme in pea seedlings

An enzyme, which catalyzes the formation of dihydrofolate fromdihydropteroic acid and L-glutamic acid, was found in pea seedlings.The enzyme was purified approximately 25-fold from the crudeextracts of pea seedlings, and its some properties were investigated.Optimum pH for the enzyme activity was found to be 8.8. Pteroicand tetrahydropteroic acids were not active as substrate. Theenzymatic reaction required as cofactors ATP, divalent (Mg²⁺or Mn²⁺) and univalent (K⁺, NH₄⁺ or Rb⁺) cations. The productwas characterized as dihydrofolic acid by bioautography. MICHAELIS constants for L-glutamic acid, ATP, dihydropteroicacid and Mg²⁺ were 7.0x10^–4, 9.0x10^–5, 3.5x10^–6and 1.2x10^–3 M, respectively. The MICHAELIS constant forMn²⁺ was 3.0x10^–4. The enzyme was inhibited by PCMB orsilver nitrate and, to some extent, by L-aspartic acid. Inhibitionby PCMB was completely reversed by addition of 2-mercaptoethanol.Enzyme activity was distributed widely among plants. The importanceof magnesium and potassium ions for enzyme catalysis is discussed. ¹For the previous paper, Part V, see Reference (30). (Received March 28, 1970; )     

Stimulation of Spermatid Phosphofructokinase by Fructose 2,6-Bisphosphate from Rat Testes

Effect of fructose 2, 6-bisphosphate on 6-phosphofructokinase (ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) in spermatid extract from rat testes was studied. Fructose 2, 6-bisphosphate stimulated the enzyme greatly by increasing its affinity for fructose 6-phosphate and relieving the inhibition by ATP. Fructose 2, 6-bisphosphate (0.8 μM) was required for 50% activation of 6-phosphofructokinase (PFK). In addition, fructose 2, 6-bisphosphate, AMP and fructose 6-phosphate acted cooperatively to stimulate the activity of PFK. This stimulation may play an important role in the regulation of glycolysis in spermatids of rat testes.     

A portable integrated automatic apparatus for the real-time monitoring of bioluminescence in plants

Real-time monitoring of gene expression by a bioluminescence reporter gene is a powerful method for large-scale, detailed analysis of gene expression in living cells and large-scale screening of mutants. We have developed a portable, compact, integrated automatic bioluminescence-monitoring apparatus that can continuously monitor 960 individual plant seedlings or micro-organism colonies under uniform light conditions at temperatures up to 50 °C. The apparatus gave reproducible and reliable results for both bioluminescence photon counts and period length of bioluminescence rhythms of Arabidopsis reporter strain. Using the apparatus, we measured bioluminescence rhythms in the thermophilic cyanobacterium Thermosynechococcus at temperature up to 43 °C. We also monitored the expression of the flowering regulator gene CONSTANS in Arabidopsis as bioluminescence in high time resolution under different photoperiodic conditions. The high-throughput bioluminescence-monitoring apparatus developed here is a powerful tool for real-time monitoring of gene expression and gene function.     

A RANKL-Inducible Gene Znf216 in Osteoclast Differentiation

Osteoclasts possess catabolic activity in mineralized tissues and are involved in bone remodeling coordinating with osteoblasts. Although the pathway using receptor and activator of NF-κ B (RANK) and its ligand, RANKL, is known to be essential for osteoclast differentiation, their precise mechanisms are not fully understood. Using DNA microarray technology, we searched for genes that were up-regulated after RANKL stimulation in the macrophage cell line, RAW264.7 cells. A gene, Znf216, which encodes a zinc-finger protein, was detected among those genes up-regulated after RANKL stimulation. Expression of Znf216 was also induced by other cytokines such as TNFα and IL-1β. Although ectopic expression of full-length ZNF216 abrogated osteoclast differentiation, its truncated forms accelerated it. No significant inhibitory effect on the NF-κ B pathway was observed, however. These results suggest that ZNF216 is a potent inhibitory factor for osteoclast differentiation and that the mechanism is unlikely due to direct attenuation of the NF-κ B pathway.     

Isolation of Zn2+ as an Endogenous Agonist of GPR39 from Fetal Bovine Serum

We attempted to determine natural agonists of GPR39 in fetal bovine serum (FBS). FBS was conditioned to extract peptides and fractionated by two types of HPLC. The activity of each fraction was monitored by intracellular calcium mobilization. Then the purified active ingredient was analyzed by inductively coupled plasma mass spectrometry. In this fashion, Zn^{2 +} ion was identified as an agonist of GPR39, though no peptidergic molecules were found. The calcium-mobilizing activity of Zn^{2 +} was not abolished by pertussis toxin but was by a phospholipase C (PLC) inhibitor, U73122, indicating that the activity of GPR39 is mediated through the G_qα -PLC pathway. In addition, Zn^{2 +} also activated mouse and rat GPR39, showing that the function of GPR39 as a Zn^{2 +} receptor is conserved across species. This study is the first exploration of GPR39 agonists in FBS and indicates that GPR39 functions as a Gq-coupled Zn^{2 +}-sensing receptor.     

Natural selection on PHYE by latitude in the Japanese archipelago: insight from locus specific phylogeographic structure in Arcterica nana (Ericaceae)

Phytochromes play a key role in allowing plants to monitor their surrounding environment and, conversely, adaptation to local environments has driven the evolutionary history of phytochromes. As a result of natural selection, polymorphisms in phytochrome genes would thus be expected to exhibit locus‐specific phylogeographic structure. To evaluate this hypothesis, we conducted a phylogeographic investigation based on four nuclear genes, including two phytochrome genes (PHYB and PHYE) using 155 samples of Arcterica nana from the entire range of the Japanese archipelago. Bayesian clustering revealed geographic differentiation between northern and southern Japan when all four genes were included. However, this geographic differentiation is inconsistent with previously reported genetic structure of genome‐wide polymorphisms based on amplified fragment length polymorphisms, as these did not show geographic differentiation throughout the Japanese archipelago. In contrast, the north–south differentiation was not apparent when PHYE was excluded. This indicates that PHYE alone could be responsible for the north–south differentiation (F_CT = 0.15, P < 0.001). Furthermore, a single nonsynonymous polymorphism (C360T) strongly contributed to geographic differentiation (F_CT = 0.57, P < 0.001) and its corresponding amino acid replacement (P120L) was significantly under positive selection based on maximum likelihood analysis (P = 0.98). Consequently, the locus‐specific geographic differentiation in PHYE could be caused by natural selection, suggesting the involvement of PHYE in local adaptation between populations of A. nana in northern and southern Japan. This finding is consistent with a previous study on Cardamine nipponica, indicating the importance of PHYE for local adaptation in Japanese alpine plants.     

Diverse diet compositions among harpaline ground beetle species revealed by mixing model analyses of stable isotope ratios

1. Species diversities of some insect lineages have been attributed to differentiation of feeding habits among species. Our objective was to determine variation in diet composition among harpaline ground beetle species occurring in a riverside grassland. 2. We examined the diet compositions of 14 species from six genera in the spring and 10 species from two genera in the autumn. We performed measurements of nitrogen and carbon stable isotope ratios in consumers and in their potential food items, and estimated relative contributions of different food items with two mixing models, IsoSource and MixSIR. 3. IsoSource and MixSIR software gave similar results, but IsoSource tended to calculate higher contributions of principal food items and smaller percentile ranges than MixSIR. Among harparine beetle species, there were diverse food utilisation patterns among four food categories (detritivorous invertebrates, herbivorous invertebrates, C3 plants, and C4 plants). Detritivores comprised the main diets of abundant harpaline species in the spring, whereas abundant harpaline species in the autumn were primarily herbivores feeding on C4 plants, or omnivores feeding on herbivorous invertebrates and C3 plants. Seasonal changes in food use were related to seasonal changes in the abundance of each food resource. 4. Mixing model analysis of stable isotope ratios is a convenient and effective method for roughly estimating diets of many species with diverse food habits (such as ground beetles). This method can contribute to determining the trophic relationships of related insects in one ecosystem.     

Diversification process of stag beetles belonging to the genus Platycerus Geoffroy (Coleoptera: Lucanidae) in Japan based on nuclear and mitochondrial genes

Molecular phylogenies of the genus Platycerus in Japan were characterized based on the nuclear 28S ribosomal RNA and mitochondrial cytochrome oxidase subunit I (COI) genes. These analyses, combined with our previous morphological information, revealed a detailed diversification process of Platycerus in Japan, as well as estimates of their divergence times. Japanese species were monophyletic and were inferred to have diverged to the acuticollis species group and all other species ca 1.69 Mya. The acuticollis species group then appeared to split into P. viridicuprus and a group including P. takakuwai, P. albisomni and P. acuticollis ca 1.26 Mya. Other specific divergences have occurred primarily since ca 0.33 Mya. Comparing the molecular trees and the morphological tree, we also found introgression of the COI gene in some species. Genetic divergence of Platycerus has occurred intensely in southwestern Japan.     

Food web structure of the fungivorous insect community on bracket fungi in a Bornean tropical rain forest

1. If fungivorous insect diversity is maintained by host specialisation on particular fungi, it should be higher in the tropics than in temperate or boreal regions owing to high macrofungus species diversity. 2. To reveal the community and food web structure of fungivorous insects on bracket fungi, fungivorous insects were collected from 427 fruiting bodies belonging to 22 genera throughout the development and deterioration process in a 3‐ha plot of lowland dipterocarp tropical rain forest on Borneo Island. 3. Eight hundred and twenty‐nine individuals of 82 coleopteran species in 13 families from 111 fruiting bodies of 15 fungal genera were collected. Tenebrionidae and Staphylinidae were most common. Fifty‐three and 19 insect species were observed on Ganoderma and Phellinus, respectively. The numbers of insect species and individuals on a particular fungal genus were positively correlated with the abundance of that fungal genus. 4. Quantitative food web analysis revealed a high degree of specialisation at the whole‐community level. At least 65% of insect individuals were observed on Ganoderma at every stage of development and deterioration. Diverse insects coexist on one dominant fungal genus, Ganoderma, in contrast to our hypothesis. 5. The high abundance of Ganoderma fruiting bodies, which lack obvious defences against insect feeding, probably influences the bracket fungus–insect food web in this tropical rainforest.