DNA content contributes to nuclear size control in Xenopus laevis

Cells adapt to drastic changes in genome quantity during evolution and cell division by adjusting the nuclear size to exert genomic functions. However, the mechanism by which DNA content within the nucleus contributes to controlling the nuclear size remains unclear. Here, we experimentally evaluated the effects of DNA content by utilizing cell-free Xenopus egg extracts and imaging of in vivo embryos. Upon manipulation of DNA content while maintaining cytoplasmic effects constant, both plateau size and expansion speed of the nucleus correlated highly with DNA content. We also found that nuclear expansion dynamics was altered when chromatin interaction with the nuclear envelope or chromatin condensation was manipulated while maintaining DNA content constant. Furthermore, excess membrane accumulated on the nuclear surface when the DNA content was low. These results clearly demonstrate that nuclear expansion is determined not only by cytoplasmic membrane supply but also by the physical properties of chromatin, including DNA quantity and chromatin structure within the nucleus, rather than the coding sequences themselves. In controlling the dynamics of nuclear expansion, we propose that chromatin interaction with the nuclear envelope plays a role in transmitting chromatin repulsion forces to the nuclear membrane.     

Effect of osmolarity and viscosity on the motility of pathogenic and saprophytic Leptospira

The motility of bacteria is an important factor in their infectivity. In this study, the motility of Leptospira, a member of the spirochete family that causes a zoonotic disease known as leptospirosis, was analyzed in different viscous or osmotic conditions. Motility assays revealed that both pathogenic and saprophytic strains increase their swimming speeds with increasing viscosity. However, only pathogenic Leptospira interrogans maintained vigorous motility near physiological osmotic conditions. This suggests that active motility in physiological conditions is advantageous when Leptospira enters hosts and when it migrates toward target tissues.     

Macrophage lipoprotein lipase modulates the development of atherosclerosis but not adiposity

The role of macrophage lipoprotein lipase (LpL) in the development of atherosclerosis and adiposity was examined in macrophage LpL knockout (MLpLKO) mice. MLpLKO mice were generated using cre-loxP gene targeting. Loss of LpL in macrophages did not alter plasma LpL activity or lipoprotein levels. Incubation of apolipoprotein E (ApoE)-deficient β-VLDL with peritoneal macrophages from ApoE knockout mice lacking macrophage LpL (MLpLKO/ApoEKO) led to less cholesteryl ester formation than that found with ApoEKO macrophages. MLpLKO/ApoEKO macrophages had reduced intracellular triglyceride levels, with decreased CD36 and carnitine palmitoyltransferase-1 mRNA levels compared with ApoEKO macrophages, when incubated with VLDL. Although both MLpLKO/ApoEKO and ApoEKO mice developed comparable hypercholesterolemia in response to feeding with a Western-type diet for 12 weeks, atherosclerosis was less in MLpLKO/ApoEKO mice. Epididymal fat mass and gene expression levels associated with inflammation did not differ between the two groups. In conclusion, macrophage LpL plays an important role in the development of atherosclerosis but not adiposity.     

CCN3 Protein Participates in Bone Regeneration as an Inhibitory Factor

CCN3, a member of the CCN protein family, inhibits osteoblast differentiation in vitro. However, the role of CCN3 in bone regeneration has not been well elucidated. In this study, we investigated the role of CCN3 in bone regeneration. We identified the Ccn3 gene by microarray analysis as a highly expressed gene at the early phase of bone regeneration in a mouse bone regeneration model. We confirmed the up-regulation of Ccn3 at the early phase of bone regeneration by RT-PCR, Western blot, and immunofluorescence analyses. Ccn3 transgenic mice, in which Ccn3 expression was driven by 2.3-kb Col1a1 promoter, showed osteopenia compared with wild-type mice, but Ccn3 knock-out mice showed no skeletal changes compared with wild-type mice. We analyzed the bone regeneration process in Ccn3 transgenic mice and Ccn3 knock-out mice by microcomputed tomography and histological analyses. Bone regeneration in Ccn3 knock-out mice was accelerated compared with that in wild-type mice. The mRNA expression levels of osteoblast-related genes (Runx2, Sp7, Col1a1, Alpl, and Bglap) in Ccn3 knock-out mice were up-regulated earlier than those in wild-type mice, as demonstrated by RT-PCR. Bone regeneration in Ccn3 transgenic mice showed no significant changes compared with that in wild-type mice. Phosphorylation of Smad1/5 was highly up-regulated at bone regeneration sites in Ccn3 KO mice compared with wild-type mice. These results indicate that CCN3 is up-regulated in the early phase of bone regeneration and acts as a negative regulator for bone regeneration. This study may contribute to the development of new strategies for bone regeneration therapy.     

Human acidic mammalian chitinase as a novel target for anti-asthma drug design using in silico screening

Human acidic mammalian chitinase (hAMCase) was recently shown to be involved in the development of asthma, suggesting a possible application for hAMCase inhibitors as novel therapeutic agents for asthma. We therefore initiated drug discovery research into hAMCase using a combination of in silico methodologies and a hAMCase assay system. We first selected 23 candidate hAMCase inhibitors from a database of four million compounds using a multistep screening system combining Tripos Topomer Search technology, a docking calculation and two-dimensional molecular similarity analysis. We then measured hAMCase inhibitory activity of the selected compounds and identified seven compounds with IC₅₀ values ?100 μM. A model describing the binding modes of these hit compounds to hAMCase was constructed, and we discuss the structure–activity relationships of the compounds we identified, suggested by the model and the actual inhibitory activities of the compounds.     

Kinetic Properties of a Dipeptidase from Streptococcus cremoris

Some kinetic properties of a dipeptidase purified from a cell-free extract of Streptococcus cremoris H 61 were investigated. The Km values of this enzyme for various dipeptides were divided into 3 groups. Group 1 comprised mainly of neutral dipeptides, such as Leu-Gly, Leu-Leu and Leu-Ala, which had relatively low Km values (in the range 4.0-6.6 mm). Group 2 consisted of dipeptides with aromatic large amino acids either at the N- or C-terminal positions, like Leu-Phe, Phe-Ala and Leu-Tyr, which had very low Km values (in the range 1.0-2.4 mm). Group 3 was made up by dipeptides with acidic or basic amino acids at the N-terminals; His-Ala and Glu-Val were typical of this group. These had very high Km values (in the range 10–20 mm). Substantial substrate competition was found to exist in the presence of His-Ala. Bestatin inhibited the enzyme competitively with Leu-Gly and was found to have an apparent Ki value of 3.0 × 10^?8 m for the enzyme. Further, the enzyme was completely inhibited by EDTA at a concentration of 2.0 × 10^?5 m. On the other hand, once the activity was inhibited by EDTA, it could be restored by Co²⁺ and Zn²⁺ in the acidic pH side, and by Ca²⁺ and Mn²⁺ in the alkaline pH side.     

Involvement of meso-α,∊-Diamino-pimelate D-Dehydrogenase in Lysine Biosynthesis in Corynebacterium glutamicum

To characterize aspartyl aminopeptidase from Aspergillus oryzae, the recombinant enzyme was expressed in Escherichia coli. The enzyme cleaves N-terminal acidic amino acids. About 30% activity was retained in 20% NaCl. Digestion of defatted soybean by the enzyme resulted in an increase in the glutamic acid content, suggesting that the enzyme is potentially responsible for the release of glutamic acid in soy sauce mash.     

Studies on Chlorosis-Inducing Activities and Plant Growth Inhibition of Benzophenone Derivatives

Some benzophenones substituted with methyl, methoxy, hydroxy or halogen groups inhibited growth and induced chlorosis in various plants. The structure activity relationships about the chlorosis-inducing activity and the growth inhibitory activity of the 3-methyl-benzophenones were well expressed by use of hydrophobic contant π, and Hammett’s σ. The highest selectivity for phytotoxic activities against barnyardgrass and the rice plant was with 3,3′-dimethyl-4-methoxybenzophenone (Methoxyphenone).