Differential regulation of TIMP-1, -2, and -3 mRNA and protein expressions during mouse incisor development

Tissue inhibitors of metalloproteinases (TIMPs) possess multiple functions, in addition to their matrix metalloproteinase (MMP) inhibitory activity. The continuously growing incisor of mouse possesses a stem cell compartment at the apical end of the epithelium (the apical loop) and thus provides an excellent tool to analyze the mechanisms of organogenesis and cytodifferentiation. To understand the functions of TIMPs in tooth development, we have analyzed the gene expression and protein localization of TIMP-1, -2, and -3 during mouse incisor development, from embryonic day 13 (E13) to postnatal day 3 (P3). TIMP-1 was present on the basement membrane during early developmental stages. At P2, TIMP-1 was strongly detected along the apical loop, transiently disappeared from the basement membrane in the cytodifferentiation zone, and later reappeared at the distal end of functional ameloblasts. Expression of TIMP-2 protein was restricted to the outer part of the apical loop throughout the examined stages. At P2, TIMP-2 was present on the basement membrane at the outer part of the apical loop. The dental follicle also expressed Timp-2, and the corresponding protein was abundant within the extracellular matrix. Timp-3 mRNA was highly expressed in the mesenchyme surrounding the apical loop. During matrix formation, Timp-3 was expressed by subodontoblasts, and the protein was detected in this layer and between odontoblasts. Distinct temporal and spatial expression patterns of TIMPs suggest divergent functions of these factors in incisor organogenesis. This work was supported by INSERM, CNRS, ARC, French Ministry of Research (ACI), Japanese Ministry of Education, Culture, Sports, Science, and Technology, and Niigata University Research Projects.     

Quantification of human angiotensinogen by a novel sandwich ELISA

The urinary angiotensinogen excretion rates show a clear relationship to kidney angiotensin II content, suggesting that urinary angiotensinogen may serve as an index of angiotensin II-dependent hypertensive rats. However, simple and accurate methods to measure human angiotensinogen are unavailable at this time. We have developed two antibodies and a sensitive and specific quantification ELISA system for human angiotensinogen to be applicable to human subjects. The ELISA is able to detect human angiotensinogen at range of 0.01-1 microg/well (R(2)=0.9945) using standard ELISA plates. This ELISA will be a useful tool to investigate the relationship between urinary angiotensinogen excretion rates and reactivity to antihypertensive drugs in hypertensive human subjects.     

Mechanism of tail-mediated inhibition of kinesin activities studied using synthetic peptides

We used a truncated form of human conventional kinesin (K560) and a set of synthetic tail-derived peptides to investigate the mechanism by which the kinesin tail domain inhibits the protein's ATPase and motor activities. A peptide that spans residues 904-933 (C3) exhibited the strongest inhibitory effect on steady-state motility and ATPase activity. This inhibition reflected diminished binding of the ADP-bound kinesin head to the microtubule. Although peptide C3 bound to both K560 and microtubules, gliding assays using subtilisin-treated microtubules suggested that the binding to the microtubule contributes only little to the inhibition if there is sufficient affinity between the peptide and kinesin. We suggest that tail-mediated inhibition of kinesin activity is mainly the product of allosteric inhibition induced by the intramolecular binding of the kinesin tail domain to the motor domain, but simultaneous binding of the tail to the microtubule also may exert a minor effect.     

rAAV-mediated shRNA ameliorated neuropathology in Huntington disease model mouse

Huntington disease (HD) is a fatal progressive neurodegenerative disorder associated with expansion of a CAG repeat in the first exon of the gene coding the protein huntingtin (htt). Although the feasibility of RNA interference (RNAi)-mediated reduction of htt expression to attenuate HD-associated symptoms is suggested, the effects of post-symptomatic RNAi treatment in the HD model mice have not yet been certified. Here we show the effects of recombinant adeno-associated virus (rAAV)-mediated delivery of RNAi into the HD model mouse striatum after the onset of disease. Neuropathological abnormalities associated with HD, such as insoluble protein accumulation and down-regulation of DARPP-32 expression, were successfully ameliorated by the RNAi transduction. Importantly, neuronal aggregates in the striatum were reduced after RNAi transduction in the animals comparing to those at the time point of RNAi transduction. These results suggest that the direct inhibition of mutant gene expression by rAVV would be promising for post-symptomatic HD therapy.     

Amino-acid Type Identification in 15N-HSQC Spectra by Combinatorial Selective 15N-labelling

The efficiency of cell-free protein synthesis combined with combinatorial selective ¹⁵N-labelling provides a method for the rapid assignment of ¹⁵N-HSQC cross-peaks to the 19 different non-proline amino-acid types from five ¹⁵N-HSQC spectra. This strategy was explored with two different constructs of the C-terminal domain V of the τ subunit of the Escherichia coli DNA polymerase III holoenzyme, τ_C16 and τ_C14. Since each of the five ¹⁵N-HSQC spectra contained only about one third of the cross-peaks present in uniformly labelled samples, spectral overlap was much reduced. All ¹⁵N-HSQC cross-peaks of the backbone amides could be assigned to the correct amino-acid type. Availability of the residue-type information greatly assisted the evaluation of the changes in chemical shifts observed for corresponding residues in τ_C16 vs. those in τ_C14, and the analysis of the structure and mobility of the C-terminal residues present in τ_C16 but not in τ_C14.     

Structure–activity relationship studies of 5-benzylaminoimidazo[1,2-c]pyrimidine-8-carboxamide derivatives as potent,highly selective ZAP-70 kinase inhibitors

Zeta-associated protein, 70 kDa (ZAP-70), a spleen tyrosine kinase (Syk) family kinase, is normally expressed on T cells and natural killer cells and plays a crucial role in activation of the T cell immunoresponse. Thus, selective ZAP-70 inhibitors might be useful not only for treating autoimmune diseases, but also for suppressing organ transplant rejection. In our recent study on the synthesis of Syk family kinase inhibitors, we discovered that novel imidazo[1,2-c]pyrimidine-8-carboxamide derivatives possessed potent ZAP-70 inhibitory activity with good selectivity for ZAP-70 over other kinases. In particular, compound 26 showed excellent ZAP-70 kinase inhibition and high selectivity for ZAP-70 over structurally related Syk. The discovery of a potent, highly selective ZAP-70 inhibitor would contribute a new therapeutic tool for autoimmune diseases and organ transplant medication.     

Pearl bodies of Cayratia japonica (Thunb.) Gagnep. (Vitaceae) as alternative food for a predatory mite Euseius sojaensis (Ehara) (Acari: Phytoseiidae)

On the young leaves, shoots, and buds of Cayratia japonica (Thunb.) Gagnep. (Vitaceae), we observed nutritious bodies called pearl bodies and hypothesized that they are utilized by generalist predators as alternative foods. Some ambulate organisms consume pearl bodies in the wild and the predatory mite Euseius sojaensis (Ehara) (Acari: Phytoseiidae) was considered as a primary candidate. Pearl bodies promoted E. sojaensis settlement on C. japonica leaves and E. sojaensis could prey on the phytophagous mite Tetranychus kanzawai Kishida (Acari: Tetranychidae) when the predators settle on a leaf before the prey. In addition, the presence of pearl bodies did not reduce predation of E. sojaensis on T. kanzawai. This was seemingly because food quality of T. kanzawai was higher than pearl bodies. These results implied that pearl bodies on C. japonica leaves are utilized by E. sojaensis as alternative foods.