Design and synthesis of Rho kinase inhibitors (II)

In a previous study, we identified several structurally unrelated scaffolds of the Rho kinase inhibitor using pharmacophore information obtained from the results of a high-throughput screening and structural information from a homology model of Rho kinase. 1H-Indazole is one of the candidate scaffolds on which a new series of potent Rho kinase inhibitors could be developed. In this study, the detailed structure-activity relationship of 1H-indazole analogues was studied. During this study, we found that the cell-free enzyme inhibitory potential of Rho kinase inhibitors having the 1H-indazole scaffold did not necessarily correlate with their inhibitory potential toward the chemotaxis of cultured cells. The choice of the linker substructure was shown to be an important factor for the 1H-indazole analogues to inhibit the chemotaxis of cells. Optimization of the 1H-indazole inhibitors with respect to the in vitro inhibition of monocyte chemotaxis induced by MCP-1 was carried out. The inhibitory potential was improved both in the cell-free enzyme assay and in the chemotaxis assay.     

Structure-activity studies on diphenylpyrazine derivatives: a novel class of prostacyclin receptor agonists

To develop nonprostanoid prostacyclin receptor agonists with a high degree of metabolic resistance and an extended duration of action, a novel series of diphenylpyrazine derivatives was synthesized and evaluated for their inhibition of ADP-induced human platelet aggregation. Structure-activity relationship studies on the side chain containing the carboxylic acid moiety of the lead compound 5c showed that the length of the linker and the presence of the concatenating nitrogen atom adjacent to the pyrazine ring are critical for the antiaggregatory activity. This study led to the discovery of 2-amino-5,6-diphenylpyrazine derivatives 8c, 15a, and 15b, which showed potent inhibition of platelet aggregation with IC(50) values of 0.2 microM. Among these compounds, 15b is an orally available and long-lasting prostacyclin receptor agonist which is promising for the treatment of various vascular diseases.     

Self-assembly of double-stranded DNA molecules at nanomolar concentrations

Some proteins have the property of self-assembly, known to be an important mechanism in constructing supramolecular architectures for cellular functions. However, as yet, the ability of double-stranded (ds) DNA molecules to self-assemble has not been established. Here we report that dsDNA molecules also have a property of self-assembly in aqueous solutions containing physiological concentrations of Mg2+. We show that DNA molecules preferentially interact with molecules with an identical sequence and length even in a solution composed of heterogeneous DNA species. Curved DNA and DNA with an unusual conformation and property also exhibit this phenomenon, indicating that it is not specific to usual B-form DNA. Atomic force microscopy (AFM) directly reveals the assembled DNA molecules formed at concentrations of 10 nM but rarely at 1 nM. The self-assembly is concentration-dependent. We suggest that the attractive force causing DNA self-assembly may function in biological processes such as folding of repetitive DNA, recombination between homologous sequences, and synapsis in meiosis.     

Involvement of Ca(2+)/calmodulin-dependent protein kinases in mycelial growth of the basidiomycetous mushroom, Coprinus cinereus

Although multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) are widely distributed in animal cells, the occurrence of CaM-kinases in the basidiomycetous mushroom has not previously been documented. When the extracts from various developmental stages from mycelia to the mature fruiting body of Coprinus cinereus were analyzed by Western blotting using Multi-PK antibodies, which had been generated to detect a wide variety of protein serine/threonine kinases (Ser/Thr kinases), a variety of stage-specific Ser/Thr kinases was detected. Calmodulin (CaM) overlay assay using digoxigenin-labeled CaM detected protein bands of 65 kDa, 58 kDa, 46 kDa, 42 kDa, and 38 kDa only in the presence of CaCl(2), suggesting that these bands were CaM-binding proteins. When the CaM-binding fraction was prepared from mycelial extract of C. cinereus by CaM-Sepharose and analyzed with Multi-PK antibodies, two major immunoreactive bands corresponding to 65 kDa and 46 kDa were detected. CaM-binding fraction, thus obtained, exhibited Ca(2+)/CaM-dependent protein kinase activity toward protein substrates such as histones. These CaM-kinases were found to be highly expressed in the actively growing mycelia, but not in the resting mycelial cells. Mycelial growth was enhanced by the addition of CaCl(2) in the culture media, but inhibited by the addition of EGTA or trifluoperazine, a potent CaM inhibitor. This suggested that CaM-dependent enzymes including CaM-kinases play crucial roles in mycelial growth of basidiomycete C. cinereus.     

Molecular assembly formation of cyclic hexa-beta-peptide composed of acetylated glycosamino acids

A novel cyclic hexamer of acetylated beta-glycosamino acid was synthesized and its conformation and molecular assembly formation was investigated. Variable temperature NMR study indicated that the cyclic hexapeptide took a C(3) symmetric conformation at room temperature, but at elevated temperatures a C(6) symmetric one, which was not due to averaging of the C(3) symmetric conformation, appeared. Computational geometry optimization showed that the C(6) symmetric conformation was a highly planar structure with amide groups orienting perpendicular to the ring plane. The cyclic hexa-beta-peptide formed rod-shaped crystals from an N,N-dimethyl formamide solution at elevated temperature. The optical microscopy observation with a sensitive tint plate under cross-nicol configuration and electron diffraction analysis of the crystals revealed that the cyclic hexa-beta-peptides were stacked one after the other to form a regular nanotube structure.     

BAF as a caspase-dependent mediator of nuclear apoptosis in Drosophila

BAF is a double-stranded DNA binding protein required for proper nuclear morphology and function in Drosophila development. Imaginal discs of Drosophila baf-null mutants were found to exist only in younger larvae as small degenerative tissues. Immunohistochemical analyses showed diffuse lamin distribution, DNA fragmentation, and activation of caspase drICE in these tissues, suggesting that apoptotic events can be induced by the loss of baf. We therefore investigated the fate of BAF after induction of the pro-apoptotic hid transgene, and found that the loss of DNA binding forms of BAF preceded that of non-DNA binding forms of BAF. Furthermore, the DNA binding forms of BAF disappeared from nuclei before DNA fragmentation and NPC clustering were detected, showing that the loss of BAF occurs at the initial stages of nuclear apoptosis. This BAF loss was not detected before drICE activation and was inhibited by Ac-DEVD-CHO caspase inhibitors. In summary, BAF disappears at an early stage due to caspase activity when apoptosis is induced by hid, and its depletion in mutants is sufficient in itself to induce cell death, suggesting it is an apoptotic mediator.     

Description of a fugu CXC chemokine and two CXC receptor genes, and characterization of the effects of different stimulators on their expression

The primary structures of a CXC chemokine (CXCL8) and two CXC receptors (CXCR) have been characterized in fugu, Takifugu rubripes. Unlike mammalian and avian species, CXCL8 of teleosts including fugu lacks the ELR motif that appears to be important in ligand/receptor interactions on neutrophils. Genomic organization shows that fugu CXCL8 gene consists of four exons and three introns. As in other vertebrates, two CXCR genes isolated from fugu encode proteins CXCR1 and CXCR2 that possess characteristic seven transmembrane domains. Each receptor consists of two exons separated by an intron. Synteny analysis indicates that these two CXCRs were derived from whole genome duplication in teleosts, differing from mammalian CXCR1 and CXCR2. All of these genes are primarily expressed in the lymphoid tissues. Immune stimulation with PHA showed that the expression of both CXCL8 and CXCRs in PBL are upregulated even after only a short time period, but downregulated by LPS stimulation, implying that these genes are involved in the regulation of the immune response in fugu.     

Mesenchymal progenitor cells in adult human dental pulp and their ability to form bone when transplanted into immunocompromised mice

The technique of tissue engineering is developing for the restoration of lost tissues. This new technique requires cells that fabricate tissue. Mesenchymal stem cells in bone marrow have been used as the cell source for this technique; however, dental pulp cells have recently been shown to possess stem-cell-like properties. We earlier demonstrated that dental pulp cells proliferate and produce an extracellular matrix that subsequently becomes mineralized in vitro. We now report that such dental pulp cells (first to eighth passage) produced bone instead of dentin when those cells were implanted into subcutaneous sites in immunocompromised mice with HA/TCP powder as their carrier. This evidence shows that dental pulp cells are the common progenitors of odontoblasts and osteoblasts, or dental pulp cells are mesenchymal stem cells themselves. It is expected that dental pulp cells can be a useful candidate cell source for tissue engineering, and contain the potential of new therapeutic approaches for the restoration of damaged or diseased tissue.     

Preliminary evaluation of greenhouses employing positive-pressure forced ventilation to prevent invasion by insect pests

We conducted a preliminary comparison of greenhouses using positive-pressure forced ventilation (PFV) systems and natural ventilation (NV) systems, and assessed the effectiveness of both systems for preventing the invasion of greenhouses used to cultivate tomatoes by insect pests. In Trial 1 (August–December 2006), greenhouses using a PFV system and an insect-proof screen (mesh size 1.0 mm) had fewer sweetpotato whiteflies and Bemisia tabaci (Gennadius), and more onion thrips, Thrips tabaci (Lindeman), than greenhouses that employed an NV system fitted with the same screen. Tomato leafminers, Liriomyza sativae Blanchard, were not observed in the greenhouse using the PFV system, but some were observed in the greenhouse using the NV system. In Trial 2 (August–December 2007), the greenhouse using the PFV system combined with an insect-proof screen (mesh size 0.4 mm) had higher whitefly densities after late October compared to the greenhouse using the NV system and the same screen. However, there were more thrips in the greenhouse using the PFV system compared to the greenhouse using the NV system. In both trials, Tomato yellow leaf curl virus, which is transmitted by B. tabaci, was recorded in the greenhouse using the NV system but not in the greenhouse using the PFV system. The results showed that the PFV system was effective for preventing invasion by leafminers and partially effective for preventing invasion by whiteflies, but not effective for preventing invasion by thrips.     

Synthesis and evaluation of a radioiodinated peptide probe targeting αvβ6 integrin for the detection of pancreatic ductal adenocarcinoma

Introduction

Pancreatic ductal adenocarcinoma (PDAC) remains a major cause of cancer-related death. Since significant upregulation of αvβ6 integrin has been reported in PDAC, this integrin is a promising target for PDAC detection. In this study, we aimed to develop a radioiodinated probe for the imaging of αvβ6 integrin-positive PDAC with single-photon emission computed tomography (SPECT).

Methods

Four peptide probes were synthesized and screened by competitive and saturation binding assays using 2 PDAC cell lines (AsPC-1, αvβ6 integrin-positive; MIA PaCa-2, αvβ6 integrin-negative). The probe showing the best affinity was used to study the biodistribution assay, an in vivo blocking study, and SPECT imaging using tumor bearing mice. Autoradiography and immunohistochemical analysis were also performed.

Results

Among the 4 probes examined in this study, ¹²⁵I-IFMDV2 showed the highest affinity for αvβ6 integrin expressed in AsPC-1 cells and no affinity for MIA PaCa-2 cells. The accumulation of ¹²⁵I-IFMDV2 in the AsPC-1 xenograft was 3–5 times greater than that in the MIA PaCa-2 xenograft, consistent with the expression of αvβ6 integrin in each xenograft, and confirmed by immunohistochemistry. Pretreatment with excess amounts of A20FMDV2 significantly blocked the accumulation of ¹²⁵I-IFMDV2 in the AsPC-1 xenograft, but not in the MIA PaCa-2 xenograft. Furthermore, ¹²³I-IFMDV2 enabled clear visualization of the AsPC-1 xenograft.

Conclusion

¹²³I-IFMDV2 is a potential SPECT probe for the imaging of αvβ6 integrin in PDAC.