Convenient Assay for Settlement Inducing Substances of Barnacles

A convenient assay method for estimation of barnacle, Balanus amphitrite, settlement inductive activity was developed. To avoid the inductive effect by comrades and laborious observation requirements, cyprid larvae were put individually into wells of a 96-well plate. The settlement ratio from the experiment without any inducers was quite low; therefore this assay allowed easy estimation of settlement inductive activities. Some known inductive agents, such as serotonin and barnacle extracts, clearly showed inductive activities. This assay method is proven to be suitable for estimation of barnacle settlement-inducing activities of both water-soluble and -insoluble compounds. Received October 31, 1997; accepted July 10, 1998.     

Calcium ions trigger the exposure of phosphatidylserine on the surface of necrotic cells

Intracellular Ca²⁺ level is under strict regulation through calcium channels and storage pools including the endoplasmic reticulum (ER). Mutations in certain ion channel subunits, which cause mis-regulated Ca²⁺ influx, induce the excitotoxic necrosis of neurons. In the nematode Caenorhabditis elegans, dominant mutations in the DEG/ENaC sodium channel subunit MEC-4 induce six mechanosensory (touch) neurons to undergo excitotoxic necrosis. These necrotic neurons are subsequently engulfed and digested by neighboring hypodermal cells. We previously reported that necrotic touch neurons actively expose phosphatidylserine (PS), an “eat-me” signal, to attract engulfing cells. However, the upstream signal that triggers PS externalization remained elusive. Here we report that a robust and transient increase of cytoplasmic Ca²⁺ level occurs prior to the exposure of PS on necrotic touch neurons. Inhibiting the release of Ca²⁺ from the ER, either pharmacologically or genetically, specifically impairs PS exposure on necrotic but not apoptotic cells. On the contrary, inhibiting the reuptake of cytoplasmic Ca²⁺ into the ER induces ectopic necrosis and PS exposure. Remarkably, PS exposure occurs independently of other necrosis events. Furthermore, unlike in mutants of DEG/ENaC channels, in dominant mutants of deg-3 and trp-4, which encode Ca²⁺ channels, PS exposure on necrotic neurons does not rely on the ER Ca²⁺ pool. Our findings indicate that high levels of cytoplasmic Ca²⁺ are necessary and sufficient for PS exposure. They further reveal two Ca²⁺-dependent, necrosis-specific pathways that promote PS exposure, a “two-step” pathway initiated by a modest influx of Ca²⁺ and further boosted by the release of Ca²⁺ from the ER, and another, ER-independent, pathway. Moreover, we found that ANOH-1, the worm homolog of mammalian phospholipid scramblase TMEM16F, is necessary for efficient PS exposure in thapsgargin-treated worms and trp-4 mutants, like in mec-4 mutants. We propose that both the ER-mediated and ER-independent Ca²⁺ pathways promote PS externalization through activating ANOH-1.     

Three-dimensional reconstructions of haustoria in two parasitic plant species in the Orobanchaceae

Parasitic plants infect other plants by forming haustoria, specialized multicellular organs consisting of several cell types, each of which has unique morphological features and physiological roles associated with parasitism. Understanding the spatial organization of cell types is, therefore, of great importance in elucidating the functions of haustoria. Here, we report a three-dimensional (3-D) reconstruction of haustoria from two Orobanchaceae species, the obligate parasite Striga hermonthica infecting rice (Oryza sativa) and the facultative parasite Phtheirospermum japonicum infecting Arabidopsis (Arabidopsis thaliana). In addition, field-emission scanning electron microscopy observation revealed the presence of various cell types in haustoria. Our images reveal the spatial arrangements of multiple cell types inside haustoria and their interaction with host roots. The 3-D internal structures of haustoria highlight differences between the two parasites, particularly at the xylem connection site with the host. Our study provides cellular and structural insights into haustoria of S. hermonthica and P. japonicum and lays the foundation for understanding haustorium function.

Three-dimensional image reconstruction visualized the spatial organization of cell types in the haustoria of the Orobanchaceae parasitic plants Striga hermonthica and Phtheirospermum japonicum.     

Evolutionary optimization of a modular ligase ribozyme: a small catalytic unit and a hairpin motif masking an element that could form an inactive structure

The YFL ribozyme is an artificial ligase ribozyme isolated by a ‘design and selection’ strategy, in which a modular catalytic unit was generated on a rationally designed modular scaffold RNA. This ligase ribozyme has a versatile catalytic unit that accepts not only β-nicotinamide mononucleotide (β-NMN) but also inorganic pyrophosphate as leaving groups for template-dependent RNA ligation. Although this property is interesting from an evolutionary viewpoint regarding primitive RNA ligation/polymerization systems in the RNA world, structural analysis of the YFL ribozyme has not been continued due to apparent structural nonuniformity of its folded state. To elucidate the active structure of the YFL ribozyme, we performed in vitro evolution experiments to improve its folding ability. Biochemical and phylogenetic analyses of evolved variants indicated that the catalytic unit of the YFL ribozyme is compact and the 3′ single-stranded region of the parent YFL-1 ribozyme contributes to mask an element that could form an inactive structure.     

Seasonal dynamics of the fish parasite Neoplagioporus ayu (Digenea) in its definitive host, Plecoglossus altivelis, in the Chikugo River, Kyushu, Japan

Seasonal dynamics of the fish parasite Neoplagioporus ayu (Digenea; Opecoelidae; Plagioporinae) in its definitive host, the ayu Plecoglossus altivelis, in the Chikugo River, Kyushu, Japan, was examined. Natural ayu are amphidromous. In the upper Chikugo River, ayu fry raised in hatcheries are released into the river every spring because the migration of ayu is completely blocked by a dam that has no fish-pass channel. Ayu collected in April 2003 (within 1 month after release) harbored no N. ayu, but the prevalence of N. ayu rose to about 80% in May. The prevalence, abundance, and maturity of N. ayu were maintained at high levels from June to September. These finding indicate that the life cycle of N. ayu is completed in a freshwater area, although its intermediate hosts have not yet been identified despite the intense examination of invertebrates.     

Spontaneous formation of helical strands from nucleic acid-lipid conjugates

We have developed a new class of helical strands that self-assembled spontaneously in aqueous solution. Phospholipid-deoxynucleoside conjugates containing two myristoyl groups and a deoxynucleosidyl group spontaneously assembled to form different types of helical strands.     

Cardiovascular characterization of SD-3211, a novel benzothiazine calcium channel blocker, in isolated rabbit hearts

The cardiovascular effects of SD-3211, a novel benzothiazine Ca++ channel blocker, were compared with those of diltiazem and nicardipine in Langendorff-perfused rabbit hearts. SD-3211 was more potent in increasing coronary artery flow than in depressing cardiac function (i.e., contractile force, heart rate and conduction time). The relative specificity of SD-3211 for coronary vasodilation to cardiodepression was clearly greater than that of diltiazem, but less than that of nicardipine. Thus, the present study demonstrates that SD-3211, despite a non-dihydropyridine type of Ca++ channel blocker, can be characterized as a potent coronary vasodilator with a little effect on cardiac function.