A novel role of group VIB calcium-independent phospholipase A2 (iPLA2gamma) in the inducible expression of group IIA secretory PLA2 in rat fibroblastic cells

Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) is a prototypic sPLA(2) enzyme that may play roles in modification of eicosanoid biosynthesis as well as antibacterial defense. In several cell types, inducible expression of sPLA(2) by pro-inflammatory stimuli is attenuated by group IVA cytosolic PLA(2) (cPLA(2)alpha) inhibitors such as arachidonyl trifluoromethyl ketone, leading to the proposal that prior activation of cPLA(2)alpha is required for de novo induction of sPLA(2). However, because of the broad specificity of several cPLA(2)alpha inhibitors used so far, a more comprehensive approach is needed to evaluate the relevance of this ambiguous pathway. Here, we provide evidence that the induction of sPLA(2)-IIA by pro-inflammatory stimuli requires group VIB calcium-independent PLA(2) (iPLA(2)gamma), rather than cPLA(2)alpha, in rat fibroblastic 3Y1 cells. Results with small interfering RNA unexpectedly showed that the cytokine induction of sPLA(2)-IIA in cPLA(2)alpha knockdown cells, in which cPLA(2)alpha protein was undetectable, was similar to that in replicate control cells. By contrast, knockdown of iPLA(2)gamma, another arachidonyl trifluoromethyl ketone-sensitive intracellular PLA(2), markedly reduced the cytokine-induced expression of sPLA(2)-IIA. Supporting this finding, the R-enantiomer of bromoenol lactone, an iPLA(2)gamma inhibitor, suppressed the cytokine-induced sPLA(2)-IIA expression, whereas (S)-bromoenol lactone, an iPLA(2)beta inhibitor, failed to do so. Moreover, lipopolysaccharide-stimulated sPLA(2)-IIA expression was also abolished by knockdown of iPLA(2)gamma. These findings open new insight into a novel regulatory role of iPLA(2)gamma in stimulus-coupled sPLA(2)-IIA expression.     

Oligomerization of N-tosylindole with aluminum chloride

The reactivity of N-tosylindole (4) in the presence of aluminum chloride was studied, and two types of oligomerization of 4 were observed. One type was condensation between both pyrrole parts (dimers 5 and 6 and trimer 7) and the other was between a pyrrole part and a benzene part of each indole nucleus (dimers 8 and 9).     

Dietary polyunsaturated fatty acids suppress acute hepatitis, alter gene expression and prolong survival of female Long-Evans Cinnamon rats, a model of Wilson disease

In the Long-Evans Cinnamon rat, copper accumulates in the liver because of a mutation in the copper-transporting ATPase gene, and peroxidative stresses are supposed to be augmented. We examined the effects of dietary fatty acids on hepatitis, hepatic gene expression, and survival. Rats were fed a conventional, low-fat diet (CE2), a CE2 diet supplemented with 10 wt% of lard (Lar), high-linoleic soybean oil (Soy), or a mixture of docosahexaenoic acid (DHA)-rich fish oil and soybean oil (DHA/Soy). Among female rats, the mean survival times of the DHA/Soy and the Soy groups were longer by 17 approximately 20% than in the Lar and the CE2 groups. Among male rats, the survival times were much longer than in the females, but no significant difference in survival was observed among the dietary groups. Serum ceruloplasmin levels in female and male rats of all of the dietary groups were similar. Serum transaminase levels of the DHA/Soy group tended to be lower than in the CE2 group. Histological examinations revealed a marked degeneration in hepatic tissue integrity in the Lar and CE2 groups but not in the DHA/Soy group. Hepatic levels of metal-related genes, transferrin and ceruloplasmin, as well as those related to bile acid synthesis were up-regulated, and an inflammation-related gene (cyclooxygenase [COX]-2) was down-regulated in the DHA/Soy group. Some proliferation-related genes were also affected by the dietary fatty acids. These results indicate that polyunsaturated fatty acids suppress the development of acute hepatitis and prolong survival in females, regardless of whether they are of the n-6 or n-3 type, which are associated with altered gene expressions.     

Patch establishment and development of a clonal plant,Polygonum cuspidatum,on Mount Fuji

Microsatellite analysis was used to investigate the patch establishment and development of Polygonum cuspidatum Sieb. et Zucc, a clonal herbaceous plant that dominates the primary succession on the southeast slope of Mount Fuji. Genotypes of P. cuspidatum in 155 patches at the study site differed from each other. This indicates that P. cuspidatum patches are initially established by seed dispersed on the bare scoria field, and not by clonal rhizome extension. Genetic differentiation was estimated using the FST values between subpopulations at the study site. There was almost no genetic differentiation between subpopulations, indicating the presence of massive gene flow. The pollen fathers of seeds and maternal genets of current-year seedlings were inferred from the microsatellite allele composition by a simple exclusion method. The wide, random distribution of pollen fathers suggests that pollen dispersal occurs over a broad area. Maternal analysis showed a tendency for seed dispersal to be biased to the area nearby and down slope from the mother plants. Patch establishment under massive gene flow may result from such pollen and seed dispersal. To understand the process of patch development, aerial photographs taken from 1962 to 1999 were compared, and then genets in each of 36 patches were identified from the microsatellite genotypes of P. cuspidatum shoots. The comparison of aerial photographs showed that most of the patches enlarged each year and that some neighbouring patches combined during growth. Genet analysis demonstrated a high correlation between patch area and the area of the largest genet within it, and that new genets were recruited at the patch periphery. These findings indicate that both vegetative and sexual reproduction, i.e. rhizome extension and the establishment of new seedlings, contribute to the development of P. cuspidatum patches.     

Japanese quail (Coturnix japonica) inseminations are more likely to fertilize eggs in a context predicting mating opportunities

Theoretical developments in behavioural ecology have generated increased interest in the proximate mechanisms underlying fertilization, but little is known about how fertilization success is regulated by cues from the external or social environment in males and females. Here, we use a Pavlovian conditioning paradigm to show that inseminations resulting from mating male and female Japanese quail (Coturnix japonica) are more likely to fertilize eggs when they occur in a context predicting that an opposite-sex bird will appear than when they occur in a context predicting that an opposite-sex bird will not appear. This effect occurs when either the male or the female is the target of the conditioning. Thus, processes occurring during or after mating that contribute to fertilization success are subject to the influence of distal cues, confirming control by brain-level mechanisms. Conditioning is a widespread property of the nervous system and the demonstration that context conditioning can influence male and female reproductive success, and not simply mating success, has widespread implications for the fertilization successes of different types of copulation in natural mating systems.     

Diurnal changes in needle gas exchange in alpine Pinus pumila during snow-melting and summer seasons

Pinus pumila (Pallas) Regel. is a dominant dwarf tree in alpine regions of Japan. The possible factors limiting the net photosynthetic rate (P_n) of the needles of P. pumila were examined in the snow-melting (May) and the summer (August) seasons. In August, in situ maximum P_n was 20 mol kg^–1 needle s^–1 in the current-year needles and 25 mol kg^–1 needle s^–1 in the 1-year-old needles. Diurnal trends of P_n in August were positively related to fluctuations in photosynthetic photon flux density (PPFD) and no midday depression of P_n was found, indicating that a decrease in PPFD rather than an increase in needle-to-air vapor pressure deficit (W) might cause the reduction of P_n. Both stomatal conductance (g_s) and P_n were lower in May than in August. In May, P_n and g_s were almost zero in the morning, but gradually increased with decreasing W, reaching maximum P_n values (4 mol kg^–1 needle s^–1) and g_s (60 mmol kg^–1 needle s^–1) at 16.00 hours. The daytime P_n in May was positively related to g_s. Relative water content in the exposed needles above the snow in May was 83%, which was far above the lethal level. This indicates that the water flow from stems or soils to needles was enough to compensate for a small amount of water loss due to the low g_s in May, although the water supplied to needles would be impeded by the low temperatures. Thus, the reduced g_s in May would be important for avoiding needle desiccation, and would result in a reduced P_n.     

Optimum modification for the highest cytotoxicity of cationized ribonuclease

Cationization of a protein is considered to be a powerful strategy for internalizing a functional protein into cells. Cationized proteins appear to adsorb to the cell surface by electrostatic interactions, then enter the cell in a receptor- and transporter-independent fashion. Thus, in principle, all cell types appear to take up cationized proteins. Since ribonucleases (RNases) have a latent cytotoxic potential, cationized RNases could be useful cancer chemotherapeutics. In this study, we investigated the effect of the degree of cationization on the cytotoxicity of RNase A by modifying carboxyl groups with ethylenediamine. We found that there is an optimum degree of modification for cytotoxicity, in which 5 to 7 out of 11 carboxyl groups in RNase A are modified, toward MCF-7 and 3T3-SV-40 cells. More interestingly, the cytotoxicity of cationized RNase As correlates well with the value of [RNase activity] x [estimated concentration of RNase free from RNase inhibitor], mimicking the practical enzymatic activity of cationized RNase As in cytosol. The results indicate that cationization of a protein to an optimum level is important for maintaining protein function in the cytosol. Sophisticated protein cationization techniques will help to advance protein transduction technology.     

Two flavonoid glucosyltransferases from Petunia hybrida: molecular cloning,biochemical properties and developmentally regulated expression

Two flavonoid glucosyltransferases, UDP-glucose:flavonoid 3-O-glucosyltransferase (3-GT) and UDP-glucose: anthocyanin 5-O-glucosyltransferase (5-GT), are responsible for the glucosylation of anthocyani(di)ns to produce stable molecules in the anthocyanin biosynthetic pathway. The cDNAs encoding 3-GT and 5-GT were isolated from Petunia hybrida by hybridization screening with heterologous probes. The cDNA clones of 3-GT, PGT8, and 5-GT, PH1, encode putative polypeptides of 448 and 468 amino acids, respectively. A phylogenetic tree based on amino acid sequences of the family of glycosyltransferases from various plants shows that PGT8 belongs to the 3-GT subfamily and PH1 belongs to the 5-GT subfamily. The function of isolated cDNAs was identified by the catalytic activities for 3-GT and 5-GT exhibited by the recombinant proteins produced in yeast. The recombinant PGT8 protein could convert not only anthocyanidins but also flavonols into the corresponding 3-O-glucosides. In contrast, the recombinant PH1 protein exhibited a strict substrate specificity towards anthocyanidin 3-acylrutinoside, comparing with other 5-GTs from Perilla frutescens and Verbena hybrida, which showed broad substrate specificities towards several anthocyanidin 3-glucosides. The mRNA expression of both 3-GT and 5-GT increased in the early developmental stages of P. hybrida flower, reaching the maximum at the stage before flower opening. Southern blotting analysis of genomic DNA indicates that both 3-GT and 5-GT genes exist in two copies in P. hybrida, respectively. The results are discussed in relation to the molecular evolution of flavonoid glycosyltransferases.