Purification and characterization of aβ-galactosidase fromTreponema phagedenis (Reiter strain)

A β-galactosidase was highly purified from a cellular extract ofTreponema phagedenis (Reiter strain) by ammonium sulfate precipitation and successive chromatography on Sepharose 6B and DEAE-Sephadex. The purified enzyme was homogeneous in SDS-polyacrylamide gel electrophoresis. The molecular weight estimated was 580,000. The optimal pH, ionic strength, and temperature were 6.5, 0.1, and 50°C, respecitvely. The enzyme was stable only at around pH 6.5 and at temperatures lower than 35°C. The enzyme was irreversibly inhibited byp-chloromercuribenzoate and divalent cations such as Hg²⁺, Cu²⁺, Zn²⁺, Cd²⁺, and Pb²⁺. The Km values forp-nitrophenyl-β-D-galactopyranoside,o-nitrophenyl-β-D-galactopyranoside, and lactose were 0.29, 0.36, and 5.4 mM, respectively.     

Lack of bone resorption in osteosclerotic (oc/oc) mice is due to a defect in osteoclast progenitors rather than the local microenvironment provided by osteoblastic cells.

In a co-culture system of mouse spleen cells and osteoblastic cells, we have demonstrated that a suitable microenvironment must be provided by osteoblastic cells in order for osteoclast-like multinucleated cell (MNC) formation. Using this co-culture system, we examined the pathogenetic mechanism underlying the lack of bone resorption in osteosclerotic oc/oc mice. Numerous tartrate-resistant acid phosphatase (TRAP, an osteoclast marker enzyme)-positive MNCs were formed in response to 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] both in co-cultures of oc/oc spleen cells and normal osteoblastic cells and in those of normal spleen cells and oc/oc osteoblastic cells. TRAP-positive MNCs derived from normal spleen cells tended to spread out on culture dishes, whereas those from oc/oc spleen cells remained as small, compact MNCs. When TRAP-positive MNCs enriched from co-cultures of normal spleen cells and oc/oc osteoblastic cells were cultured on dentine slices, they formed numerous resorption pits with ruffled borders and clear zones. In contrast, none of the TRAP-positive MNCs derived from oc/oc spleen cells formed either ruffled borders or resorption pits. These results indicate that the lack of bone resorption in oc/oc mice is due to a defect in osteoclast progenitors rather than the local microenvironment provided by osteoblastic cells.     

A method of statistical neurodynamics

A method of statistical neurodynamics is presented for treating ensembles of nets of randomly connected neuron-like elements. The concept of a macrostate plays a fundamental role in statistical neurodynamics and a criterion is given for ascertaining that given macroscopic quantities together constitute a macrostate. The activity of a nerve net is shown to be a macrostate and the equation of the dynamics of the activity is elucidated for various ensembles of random nerve nets. It is shown that the distance between two microstates can also be treated as a macrostate in a generalized sense. The equation of its dynamics represents how the distance between two states changes in the course of state transitions. The dynamics of distance reveals interesting microscopic properties of random nerve nets, such as the stability of state-transition, the transient lengths, etc.     

Dynamics of pattern formation in lateral-inhibition type neural fields

The dynamics of pattern formation is studied for lateral-inhibition type homogeneous neural fields with general connections. Neural fields consisting of single layer are first treated, and it is proved that there are five types of pattern dynamics. The type of the dynamics of a field depends not only on the mutual connections within the field but on the level of homogeneous stimulus given to the field. An example of the dynamics is as follows: A fixed size of localized excitation, once evoked by stimulation, can be retained in the field persistently even after the stimulation vanishes. It moves until it finds the position of the maximum of the input stimulus. Fields consisting of an excitatory and an inhibitory layer are next analyzed. In addition to stationary localized excitation, fields have such pattern dynamics as production of oscillatory waves, travelling waves, active and dual active transients, etc.This research was supported in part by a Sloan Foundation grant to the Center for Systems Neuroscience, University of Massachusetts at AmherstOn sabbatical leave from the University of Tokyo     

A circa-decadal change in the gastropod fauna on a tidal flat in an island mangrove estuary

An investigation on gastropod fauna was carried out on a tidal flat in the Nagura Estuary on Ishigaki Island, the Ryukyu Islands in 1989 and 1998 using similar methods. 470-480 quadrats covering ca. 1900 m2 were surveyed during low tides from February to April in each year. Of the total 19 species recorded, the ranges of eight species had varied significantly between the two surveys, with six species expanding their range and two species contracting their range. Percentage in abundance of muddy-bottom species and tropical (<29 degrees N) species increased significantly between the two years. Topography of the flat also changed: the mouth of the river was narrowed and the elevated sections of the tidal flat expanded. During the period from 1984 to 1998, the farmland development around the study site caused influxes of soil into the estuary and the sea-water temperature was rising. These results suggest that the topographical changes due to soil influx and the rising temperature affected the gastropod assemblage at the study site, by increasing the abundance of muddy-bottom species and tropical species. The methodology used in this study, i.e. surface observation at low tides, includes more than 95% of the gastropod fauna, demonstrating the usefulness of surface counts for the study of soft-bottom fauna.     

Prostaglandin E2 receptors EP2 and EP4 are down-regulated during differentiation of mouse osteoclasts from their precursors

Prostaglandin E2 (PGE2) has been proposed to be a potent stimulator of bone resorption. However, PGE2 itself has been shown to directly inhibit bone-resorbing activity of osteoclasts. We examined the role of PGE2 in the function of mouse osteoclasts formed in vitro. Bone marrow macrophage osteoclast precursors expressed PGE2 receptors EP1, EP2, EP3beta, and EP4, and the expression of EP2 and EP4 was down-regulated during osteoclastic differentiation induced by receptor activator of NF-kappaB ligand and macrophage colony-stimulating factor. In contrast, functional EP1 was continuously expressed in mature osteoclasts. PGE2 as well as calcitonin caused intracellular Ca2+ influx in osteoclasts. However, PGE2 and 17-phenyltrinol-PGE2 (an EP1 agonist) failed to inhibit actin-ring formation and pit formation by osteoclasts cultured on dentine slices. When EP4 was expressed in osteoclasts using an adenovirus carrying EP4 cDNA, both actin-ring and pit-forming activities of osteoclasts were inhibited in an infectious unit-dependent manner. Treatment of EP4-expressing osteoclasts with PGE2 further inhibited their actin-ring and pit-forming activities. Such inhibitory effects of EP4-mediated signals on osteoclast function are similar to those that are calcitonin receptor-mediated. Thus, osteoclast precursors down-regulate their own EP2 and EP4 levels during their differentiation into osteoclasts to escape inhibitory effects of PGE2 on bone resorption.     

Transcytosis of calcium from bone by osteoclast-like cells evidenced by direct visualization of calcium in cells

'Transcytosis' of calcium (Ca) from bone by osteoclasts was identified by using a newly developed method that uses fixed or living osteoclast-like cells previously differentiated in vitro, a Ca-specific cell-membrane-impermeable fluorescent dye, and confocal laser scanning microscopy. This method, called the cell-membrane-impermeable dye method, revealed that in fixed osteoclast-like cells, a large quantity of Ca was confined within vacuoles and transported toward the apical cell membrane in the cells. These Ca-confined vacuoles were co-localized with marker proteins of both ruffled border and lysosome. The vacuoles were disrupted when treated with an inhibitor of ruffled border ATPase. In living osteoclast-like cells, Ca-confined vacuoles were again preferentially located at the central region and near the apical cell membrane. These results suggest actual transcytosis of Ca from bone by osteoclasts, and are the first direct evidence of the significant role of osteoclasts in the entire process of Ca metabolism in bone.     

Evidence for the translation initiation of leaderless mRNAs by the intact 70 S ribosome without its dissociation into subunits in eubacteria

In eubacteria, the dissociation of the 70 S ribosome into the 30 S and 50 S subunits is the essential first step for the translation initiation of canonical mRNAs that possess 5'-leader sequences. However, a number of leaderless mRNAs that start with the initiation codon have been identified in some eubacteria. These have been shown to be translated efficiently in vivo. Here we investigated the process by which leaderless mRNA translation is initiated by using a highly reconstituted cell-free translation system from Escherichia coli. We found that leaderless mRNAs bind preferentially to 70 S ribosomes and that the leaderless mRNA.70 S.fMet-tRNA complex can transit from the initiation to the elongation phase even in the absence of initiation factors (IFs). Moreover, leaderless mRNA translation proceeds more efficiently if the intact 70 S ribosome is involved compared with the 30 S subunit. Furthermore, excess amounts of IF3 inhibit leaderless mRNA translation, probably because it promotes the disassembly of the 70 S ribosome into subunits. Finally, excess amounts of fMet-tRNA facilitate the IF-independent translation of leaderless mRNA. These observations strongly suggest that leaderless mRNA translation is initiated by the assembled 70 S ribosome and thereby bypasses the dissociation process.