Studies on Japanese Botryllid Ascidians. III. A new species of the genus Botryllus with a vivid colony color from the vicinity of Shimoda

The morphology and life history of a new species of the genus Botryllus belonging to the family Botryllidae are described in detail. This ascidian was collected from the stony shore in the cove near Shimoda Marine Research Center, University of Tsukuba (Shimoda, Shizuoka Prefecture, Japan). The ascidian colony was easily distinguished from colonies of other botryllids because it was very thin and bright pink in color. The arrangement of ovary and testis in this ascidian was the same as that in other species of the genus Botryllus. This ascidian was prolific, with 1-5 embryos on each side of a zooid, and the embryos of this ascidian developed in the peribranchial cavity without any brooding organs as in Botryllus scalaris. We observed the processes and features of the allorecognition reaction in colony specificity and found that allorejection occurred after fusion of the vascular system between two incompatible colonies. This manner of allorejection is also shown in B. scalaris and Botryllus delicatus; however, the reaction speed of allorejection is faster than that of B. delicatus and similar to that of B. scalaris. These results indicate that this ascidian might be closely related to B. scalaris.     

A potential mechanism for the impairment of nitric oxide formation caused by prolonged oral exposure to arsenate in rabbits

We have recently found evidence for impairment of nitric oxide (NO) formation and induction of oxidative stress in residents of an endemic area of chronic arsenic poisoning in Inner Mongolia, China. To investigate the underlying mechanisms responsible for these phenomena, a subchronic animal experiment was conducted using male New Zealand White rabbits. After 18 weeks of continuous exposure of rabbits to 5 mg/l of arsenate in drinking water, a significant decrease in systemic NO production occurred, as shown by significantly reduced plasma NO metabolites levels (76% of control) and a tendency towards decreased serum cGMP levels (81.4% of control). On the other hand, increased oxidative stress, as shown by significantly increased urinary hydrogen peroxide (H(2)O(2)) (120% of control), was observed in arsenate-exposed rabbits. In additional experiments measuring aortic tension, the addition of either the calcium ionophore A23187 or acethylcholine (ACh) induced a transient vasoconstriction of aortic rings prepared from arsenate-exposed rabbits, but not in those prepared from control animals. This calcium-dependent contractility action observed in aorta rings from arsenate-exposed rabbits was markedly attenuated by the superoxide (O2(.-)) scavenging enzyme Cu, Zn-SOD, as well as diphenyleneiodonium (DPI) or N(G)-nitro-L-arginine methyl ester (L-NAME), which are inhibitors for nitric oxide synthase (NOS). However, the cyclooxygenase inhibitor indomethacin or the xanthine oxidase blocker allopurinol had no effect on this vasoconstriction. These results suggest that arsenate-mediated reduction of systemic NO may be associated with the enzymatic uncoupling reaction of NOS with a subsequent enhancement of reactive oxygen species such as O2(.-), an endothelium-derived vasoconstricting factor. Furthermore, hepatic levels of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH(4)), a cofactor for NOS, were markedly reduced in arsenate-exposed rabbits to 62% of control, while no significant change occurred in cardiac L-arginine levels. These results suggest that prolonged exposure of rabbits to oral arsenate may impair the bioavailability of BH(4) in endothelial cells and, as a consequence, disrupt the balance between NO and O2(.-) produced from endothelial NOS, such that enhanced free radicals are produced at the expense of NO.     

Presence of free D-amino acids in microalgae

Several species of microalgae (phytoplankton), 4 species of freshwater algae and 4 species of marine diatoms, were cultured germ-free in the laboratory. The presence of free D-amino acids was verified in these species by a reversed-phase HPLC analysis. D-Aspartate was detected in all the microalgae examined, but D-alanine was only present in the marine diatoms. The D-amino acid content in Asterionella sp. of the marine diatoms increased from the exponential phase to the stationary phase and then decreased to the phase of decline.     

Simple synthesis of mite pheromone beta-acaridial and its analogs in the secretion of Caloglyphus polyphyllae (Acari: Acaridae)

A simple synthesis of beta-acaridial [(E)-1], the active principle of the sex, alarm and aggregation pheromone among astigmatid mites, was achieved in 5 steps from 1,2,4-butanetriol 2 in a 19% overall yield. Its analog, beta-acariolal 8, was also prepared in a 63% yield by oxidation of the intermediate, beta-acaridiol [(E)-7], with pyridinium dichromate (PDC). This synthetic route also gave beta-(Z)-acaridiol [(Z)-7] by using a Z-selective base in the Wittig reaction. (Z)-7 was oxidized to give a new monoterpene, beta-(Z)-acaridial [(Z)-1], which was detected as a trace component in the secretion of Caloglyphus polyphyllae, together with 8.     

(E)-2-(2-Hydroxyethylidene)-6-methyl-5-heptenal (alpha-acariolal) and (E)-2-(2-hydroxyethyl)-6-methyl-2,5-heptadienal (beta-acariolal), two new types of isomeric monoterpenes from Caloglyphus polyphyllae (Acari: Acaridae)

The opisthonotal gland secretion of the acarid mite, Caloglyphus polyphyllae, contained two new monoterpenes, (E)-2-(2-hydroxyethylidene)-6-methyl-5-heptenal (1) and (E)-2-(2-hydroxyethyl)-6-methyl-2,5-heptadienal (2), to which we have given the trivial names alpha- and beta-acariolal in relation to alpha- and beta-acaridial (3 and 4), respectively. Elucidation of the structure of 1 was established mainly from 1H-NMR and GC/MS spectral data after partial purification, together with the fact that 1 was recovered in the more-polar fraction from a silica gel column than alpha- and beta-acaridial (3 and 4) present in the secretion. Compound 2 was obtained in the same fraction as a mixture with 1. Based on the facts that 2 had the same molecular weight by GC/MS and the same polarity as that of 1, compound 2 was assumed to be a structural analog of 1. The structures of compounds 1 and 2 were confirmed by their synthesis in nine and ten respective steps starting from alpha-bromo-gamma-butyrolactone.     

Induction of oxidatively modified proteins in skeletal muscle by electrical stimulation and its suppression by dietary supplementation of (-)-epigallocatechin gallate

The oxidative stress produced by electrical stimulation-induced muscle contraction was examined in the skeletal muscle proteins of rats that had been fed on the dietary flavonoid, (-)-epigallocatechin gallate (EGCg). Electrical stimulation of the rat leg muscle every second day for a two-week period resulted in an increased (p < 0.05) muscle weight and accumulation of oxidatively induced modified proteins. Similar stimulation conducted every day for only one week had no effect on the muscle weight or protein oxidation, although the rate of protein degradation increased. Rats fed on a 20% casein diet supplemented with 0.1% EGCg for 2 weeks responded to the electrical stimulation of muscle contraction by reducing the increased muscle protein carbonyl content when compared to their counterparts fed on a control diet. There was no change in activity of antioxidative enzymes in muscle tissue of the EGCg-fed rats receiving electrical stimulation. The results of this study show that the antioxidative property of EGCg was effective for suppressing oxidative modification of the skeletal muscle protein induced by electrical stimulation. This finding demonstrates that EGCg has a beneficial effect in vivo on the free radical-mediated oxidative damage to muscle proteins.     

Interaction of Cytotoxic Bicyclic Peptides, Theonellamides A and F, with Glutamate Dehydrogenase and 17β-Hydroxysteroid Dehydrogenase IV

Theonellamide A, a bicyclic peptide isolated from a Theonella sponge, was fixed on hydrazide-containing gel beads and screened for its binding proteins from rabbit liver tissues. Analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis revealed that two major proteins of 80 kDa and 55 kDa interacted with theonellamide A. The interaction between theonellamide A and two proteins was confirmed by competition experiments in which these two proteins failed to bind to theonellamide A–conjugated gel beads in the presence of theonellamide A or F. Amino-terminal amino acid sequence analysis of peptide fragments derived from the binding proteins by lysylendopeptidase digestion demonstrated that the 80-kDa and 55-kDa proteins were 17β-hydroxysteroid dehydrogenase IV and glutamate dehydrogenase, respectively. In an in vitro assay system, amination of α-ketoglutarate by glutamate dehydrogenase was activated with theonellamide F, although this effect was weaker than that with adenosine diphosphate, a well-known activator. Received October 15, 1999; accepted January 4, 2000.     

Nutrient dynamics and lignocellulose degradation in decomposing Quercus serrata leaf litter

The litter mass loss, concentration and mass of some major nutrient elements, degradation of lignin and cellulose in decomposing Quercus serrata Murray leaf litter were monitored for 3 years using the litterbag method. The mobility of elements during the course of the study was in the order of: K > P > C > Mg > Ca > N. Three patterns of nutrient dynamics were observed: (i) concentration increased while mass decreased (N, Mg and Ca); (ii) concentration and nutrient mass decreased (K and C); and (iii) both concentration and mass had fluctuated (P). The C to element ratio tended to increase as the element was released, and decreased as the element was retained. Nitrogen mobility in relation to carbon was characterized by three phases: (i) initial release; (ii) accumulation and (iii) final release. The decay rate (k) calculated from 0–6 months period was overestimated for an average annual rate while those of 0–36 months fit the negative single exponential model (Adj. r² = 0.99) better than shorter periods. For lignin, the concentration had increased then decreased but tended to stabilize after 1 year while the lignin mass had continuously decreased throughout the study period. During the first 9 months, both the concentrations and mass of cellulose had fluctuated but declined thereafter. The amounts of N had initially increased but declined after 1 year; P had fluctuated while K, Ca, Mg and C had decreased throughout the study. N and C/N ratio exerted strong influence on mass loss during the first24 months but the influence of lignin emerged after 24 months.     

Identification of Potential Regulatory Elements for the Transport of Emp24p

To examine the possibility of active recycling of Emp24p between the endoplasmic reticulum (ER) and the Golgi, we sought to identify transport signal(s) in the carboxyl-terminal region of Emp24p. Reporter molecules were constructed by replacing parts of a control invertase-Wbp1p chimera with those of Emp24p, and their transport rates were assessed. The transport of the reporter was found to be accelerated by the presence of the cytoplasmic domain of Emp24p. Mutational analyses revealed that the two carboxyl-terminal residues, leucine and valine (LV), were necessary and sufficient to accelerate the transport. The acceleration was sequence specific, and the terminal valine appeared to be more important. The LV residues accelerated not only the overall transport to the vacuole but also the ER to cis-Golgi transport, suggesting its function in the ER export. Hence the LV residues are a novel anterograde transport signal. The double-phenylalanine residues did not affect the transport by itself but attenuated the effect of the anterograde transport signal. On the other hand, the transmembrane domain significantly slowed down the ER to cis-Golgi transport and effectively counteracted the anterograde transport signal at this step. It may also take part in the retrieval of the protein, because the overall transport to the vacuole was more evidently slowed down. Consistently, the mutation of a conserved glutamine residue in the transmembrane domain further slowed down the transport in a step after arriving at the cis-Golgi. Taken together, the existence of the anterograde transport signal and the elements that regulate its function support the active recycling of Emp24p.