Development of lateral line organs in leptocephali of the freshwater eel Anguilla japonica (Teleostei,Anguilliformes)

A study of the ontogeny of the lateral line system in leptocephali of the Japanese eel Anguilla japonica reveals the existence of three morphologically different types of lateral line organs. Type I is a novel sensory organ with hair cells bearing a single kinocilium, lacking stereocilia, distributed mainly on the head of larvae, and morphologically different from typical superficial neuromasts of the lateral line system. Its developmental sequence suggests that it may be a presumptive canal neuromast. Type II is an ordinary superficial neuromast, common in other teleost larvae, which includes presumptive canal neuromasts that first appear on the trunk and accessory superficial neuromasts that later appear on the head and trunk. Type III is a very unusual neuromast located just behind the orbit, close to the otic vesicle, with radially oriented hair cells, suggesting that these serve as multiple axes of sensitivity for mechanical stimuli. The behavior of larval eels suggests that the radially oriented neuromasts may act as the sole mechanosensory organ until the ordinary superficial neuromasts develop. The finding that larval eels possess a well-developed mechanosensory system suggests the possibility that they are also capable of perceiving weak environmental mechanical stimuli, like other teleost larvae.     

New highly active taxoids from 9 beta-dihydrobaccatin-9,10-acetals

To synthesize new highly active taxoids, we designed and synthesized 9 beta-dihydro-9,10-acetal taxoids. In vitro study of these analogues clearly showed them to be more potent than docetaxel.     

An Insight into the pathway of the amyloid fibril formation of hen egg white lysozyme obtained from a small-angle X-ray and neutron scattering study

It is known that hen egg white lysozyme (HEWL) forms amyloid fibrils. Since HEWL is one of the proteins that have been studied most extensively and is closely related to human lysozyme, the variants of which form the amyloid fibrils that are related to hereditary systemic amyloidosis, this protein is an ideal model to study the mechanism of amyloid fibril formation. In order to gain an insight into the mechanism of amyloid fibril formation, systematic and detailed studies to detect and characterize various structural states of HEWL were conducted. Since HEWL forms amyloid fibrils in highly concentrated ethanol solutions, solutions of various concentrations of HEWL in various concentrations of ethanol were prepared, and the structures of HEWL in these solutions were investigated by small-angle X-ray and neutron scattering. It was shown that the structural states of HEWL were distinguished as the monomer state, the state of the dimer formation, the state of the protofilament formation, the protofilament state, and the state towards the formation of amyloid fibrils. A phase diagram of these structural states was obtained as a function of protein, water and ethanol concentrations. It was found that under the monomer state the structural changes of HEWL were not gross changes in shape but local conformational changes, and the dimers, formed by the association at the end of the long axis of HEWL, had an elongated shape. Circular dichroism measurements showed that the large changes in the secondary structures of HEWL occurred during dimer formation. The protofilaments were formed by stacking of the dimers with their long axis (nearly) perpendicular to and rotated around the protofilament axis to form a helical structure. These protofilaments were characterized by their radius of gyration of the cross-section of 2.4nm and the mass per unit length of 16,000(+/-2300)Da/nm. It was shown that the changes of the structural states towards the amyloid fibril formation occurred via lateral association of the protofilaments. A pathway of the amyloid fibril formation of HEWL was proposed from these results.     

Proteases involved in long-term potentiation

Much attention has been paid to proteases involved in long-term potentiation (LTP). Calpains, Ca-dependent cysteine proteases, have first been demonstrated to be the mediator of LTP by the proteolytic cleavage of fodrin, which allows glutamate receptors located deep in the postsynaptic membrane to move to the surface. It is now generally considered that calpain activation is necessary for LTP formation in the cleavage of substrates such as protein kinase Czeta, NMDA receptors, and the glutamate receptor-interacting protein. Recent studies have shown that serine proteases such as tissue-type plasminogen activator (tPA), thrombin, and neuropsin are involved in LTP. tPA contributes to LTP by both receptor-mediated activation of cAMP-dependent protein kinase and the cleavage of NMDA receptors. Thrombin induces a proteolytic activation of PAR-1, resulting in activation of protein kinase C, which reduces the voltage-dependent Mg2+ blockade of NMDA receptor-channels. On the other hand, neuropsin may act as a regulatory molecule in LTP via its proteolytic degradation of extracellular matrix protein such as fibronectin. In addition to such neuronal proteases, proteases secreted from microglia such as tPA may also contribute to LTP. The enzymatic activity of each protease is strictly regulated by endogenous inhibitors and other factors in the brain. Once activated, proteases can irreversibly cleave peptide bonds. After cleavage, some substrates are inactivated and others are activated to gain new functions. Therefore, the issue to identify substrates for each protease is very important to understand the molecular basis of LTP.     

Changes in mutagenicity of herbicide chlornitrofen during biodegradation

We used the Ames assay to investigate changes in the mutagenicity of chlornitrofen during its aerobic biodegradation. Although a mixed culture of bacteria obtained from river water degraded chlornitrofen and reduced its concentration from 39 to 6 microg/l in 21 days, the indirect mutagenicity of the solution to Salmonella strains TA98, YG1021, and YG1026 increased gradually. This finding suggests that mutagenic metabolites were produced during the aerobic biodegradation. The increase in the mutagenicity was, however, much smaller under aerobic than under anaerobic conditions. The differing sensitivities of our test strains to the functional groups on the mutagens showed that the mutagenic metabolites were indirect frameshift-type mutagens that might have neither nitro nor amino groups.     

In vivo photochemical micronucleus induction due to certain quinolone antimicrobial agents in the skin of hairless mice

The skin micronucleus test combined with irradiation due to a sunlight simulator having a spectrum almost identical to solar irradiation was used as a novel in vivo testing method for detecting or comparing the photochemical chromosome damage of quinolone antibacterial agents (quinolones). Eight-week-old male SKH1 hairless mice were orally administered once lomefloxacin (LFLX), a strong in vitro photochemical clastogen, at 25 or 50 mg/kg, followed by light irradiation at 7.9-9.4J/cm2 of ultraviolet A (UVA). Animals were killed on Days 2, 3, 4, 5 or 8 (the dosing day was designated as Day 1), and the incidence of micronucleus in the epidermis was determined. As results, LFLX at either dose caused significant increases in the micronucleus frequency, which peaked on Day 4. These changes tended to return to the control level on Day 8. Then, the micronucleus induction potential of the quinolone derivatives levofloxacin (LVFX) and clinafloxacin (CLFX) at 10, 20 or 40 mg/kg was assessed on Day 4 under the same experimental conditions as for LFLX. Although LVFX was negative even at 40 mg/kg, CFLX dose-dependently induced significant increases in micronucleus frequency at all doses. The correlation of magnitude among the three quinolones in the skin micronucleus test with light irradiation was similar to that in our previous in vitro photochemical clastogenicity study. No significant increase in micronucleus frequency was observed in any of three quinolones employed without light irradiation. In conclusion, the experimental method presented here would be a useful tool for detecting in vivo photochemical chromosome damage and for research on photochemical carcinogenesis of chemicals.     

An attempt to promote neo-vascularization by employing a newly synthesized inhibitor of protein tyrosine phosphatase

Vascular endothelial growth factor (VEGF) and its receptors play a key role in angiogenesis. VEGF receptor-2 (VEGFR-2) has a tyrosine kinase domain, and, once activated, induces the phosphorylation of cytoplasmic signaling proteins. The phosphorylated VEGFR-2 may be a substrate for intracellular protein tyrosine phosphatases (PTPs) which prevent VEGF signaling. We synthesized a series of alpha,alpha-difluoro(phenyl)methylphosphonic acids (DFPMPAs) which inhibit the action of PTP. In this study, we test their effects on VEGF-induced angiogenesis. DFPMPA-3, the most effective inhibitor of human PTP-1B, promoted tube formation by human umbilical vein endothelial cells (HUVEC) on Matrigel more effectively than any other DFPMPAs. The inhibitor promoted the VEGF-induced proliferation and migration of HUVEC by inhibiting the dephosphorylation of VEGFR-2. Its effectiveness was proven through neo-vascularization in mice. The present findings suggest that targeting PTP to promote therapeutic neo-vascularization may be a potential strategy.