Cytotoxic labdane alkaloids from an ascidian Lissoclinum sp.: isolation, structure elucidation, and structure-activity relationship

Four labdane alkaloids, haterumaimides N-Q (1-4), were isolated from an ascidian Lissoclinum sp. and their structures were elucidated by chemical and spectral analyses. Investigation of the structure-activity relationships of haterumaimides J-K, N-Q, and 14 related compounds suggested that the presence of hydroxyl groups at C-6, C-7, C-12, and C-18, a chlorine atom at C-2, and an imido NH in ring C should be essential for cytotoxicity against P388 cells.     

Real‐time imaging of single cancer‐cell dynamics of lung metastasis

We have developed a new in vivo mouse model to image single cancer‐cell dynamics of metastasis to the lung in real‐time. Regulating airflow volume with a novel endotracheal intubation method enabled controlling lung expansion adequate for imaging of the exposed lung surface. Cancer cells expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm were injected in the tail vein of the mouse. The right chest wall was then opened in order to image metastases on the lung surface directly. After each observation, the chest wall was sutured and the air was suctioned in order to re‐inflate the lung, in order to keep the mice alive. Observations have been carried out for up to 8 h per session and repeated up to six times per mouse thus far. The seeding and arresting of single cancer cells on the lung, accumulation of cancer‐cell emboli, cancer‐cell viability, and metastatic colony formation were imaged in real‐time. This new technology makes it possible to observe real‐time monitoring of cancer‐cell dynamics of metastasis in the lung and to identify potential metastatic stem cells. J. Cell. Biochem. 109: 58–64, 2010. © 2009 Wiley‐Liss, Inc.     

Effects of compressed hydrocarbon gases on the growth activity of Saccharomyces cerevisiae

The inhibitory action of compressed hydrocarbon gases on the growth of the yeast Saccharomyces cerevisiae was investigated quantitatively by microcalorimetry. Both the 50% inhibitory pressure (IP(50)) and the minimum inhibitory pressure (MIP), which are regarded as indices of the toxicity of hydrocarbon gases, were determined from growth thermograms. Based on these values, the inhibitory potency of the hydrocarbon gases increased in the order methane < ethane < propane < i-butane < n-butane. The toxicity of these hydrocarbon gases correlated to their hydrophobicity, suggesting that hydrocarbon gases interact with some hydrophobic regions of the cell membrane. In support of this, we found that UV absorbing materials at 260 nm were released from yeast cells exposed to compressed hydrocarbon gases. Additionally, scanning electron microscopy indicated that morphological changes occurred in these cells.     

PAC1 Gene Knockout Reveals an Essential Role of Chaperone-Mediated 20S Proteasome Biogenesis and Latent 20S Proteasomes in Cellular Homeostasis

The 26S proteasome, a central enzyme for ubiquitin-dependent proteolysis, is a highly complex structure comprising 33 distinct subunits. Recent studies have revealed multiple dedicated chaperones involved in proteasome assembly both in yeast and in mammals. However, none of these chaperones is essential for yeast viability. PAC1 is a mammalian proteasome assembly chaperone that plays a role in the initial assembly of the 20S proteasome, the catalytic core of the 26S proteasome, but does not cause a complete loss of the 20S proteasome when knocked down. Thus, both chaperone-dependent and -independent assembly pathways exist in cells, but the contribution of the chaperone-dependent pathway remains unclear. To elucidate its biological significance in mammals, we generated PAC1 conditional knockout mice. PAC1-null mice exhibited early embryonic lethality, demonstrating that PAC1 is essential for mammalian development, especially for explosive cell proliferation. In quiescent adult hepatocytes, PAC1 is responsible for producing the majority of the 20S proteasome. PAC1-deficient hepatocytes contained normal amounts of the 26S proteasome, but they completely lost the free latent 20S proteasome. They also accumulated ubiquitinated proteins and exhibited premature senescence. Our results demonstrate the importance of the PAC1-dependent assembly pathway and of the latent 20S proteasomes for maintaining cellular integrity.The 26S proteasome is a eukaryotic ATP-dependent protease responsible for the degradation of proteins tagged with polyubiquitin chains (21). The ubiquitin-dependent proteolysis by the proteasome plays a pivotal role in various cellular processes by catalyzing the selective degradation of short-lived regulatory proteins as well as damaged proteins. Thus, the proteasome is essential for the viability of all eukaryotic cells.The 26S proteasome is a large protein complex consisting of two portions; one is the catalytic 20S proteasome of approximately 700 kDa (also called the 20S core particle), and the other is the 19S regulatory particle (RP; also called PA700) of approximately 900 kDa, both of which are composed of a set of multiple distinct subunits (70). The 20S proteasome is a cylindrically shaped stack of four heptameric rings, where the outer and inner rings each are composed of seven homologous α subunits (α1 to α7) and seven homologous β subunits (β1 to β7), respectively (5). The proteolytic active sites reside within the central chamber enclosed by the two inner β-rings, while a small channel formed by the outer α-ring, which is primarily closed, restricts the access of native proteins to the catalytic chamber. Thus, the 20S proteasome is a latent enzyme. Appending 19S RP, which consists of 19 different subunits, to the α-ring enables the 20S proteasome to degrade native proteins; 19S RP accepts ubiquitin chains of substrate proteins, removes ubiquitin chains while unfolding the substrates, and feeds the substrates into the interior proteolytic chamber of the 20S proteasome through the α-ring that is opened when the C-terminal tails of the ATPase subunits of 19S RP are inserted into the intersubunit spaces of the α-ring (24, 62, 74). However, it also has been reported that some denatured or unstructured proteins can be degraded directly by the 20S proteasome even in the absence of 19S RP and ubiquitination (37, 39).Much attention has been focused on how such a highly elaborate structure is achieved. Recent studies have identified various proteasome-dedicated chaperones that assist in the assembly of the proteasome in eukaryotic cells (23, 40, 56, 57, 65, 66). In yeast, while most of the proteasome subunits are essential for viability, the deletion of any of these chaperones does not cause lethality. In fact, many, if not all, of the deletions exhibit subtle phenotypes. In mammalian cells, although the knockdown of the assembly chaperones reduced proteasome assembly and thus proteasome activity, leading to slow cell growth, the degree of reduction was much lower than that which occurred following the knockdown of the proteasome subunit itself (33, 35, 40). These results indicate that the assembly chaperones play an auxiliary role in proteasome biogenesis.Proteasome assembly chaperone 1 (PAC1) is one of the assembly chaperones originally identified in mammalian cells (34). PAC1 plays a role in α-ring formation that occurs during the initial assembly of the 20S proteasome; it also prevents the aberrant dimerization of the α-ring. As is the case for most assembly chaperones, the knockdown of PAC1 in mammalian cells decreases proteasome activity but to a lesser extent than that in, for example, β2 knockdown (34, 35). Therefore, both PAC1-dependent and -independent assembly pathways exist in cells, but the importance of the PAC1-dependent pathway remains elusive. To further elucidate the biological significance of PAC1 and PAC1-dependent proteasome biogenesis, we generated conditional mouse mutants carrying an inactivating mutation in Psmg1, the gene coding for PAC1 protein, in the whole body, the nervous system, and in the liver. Our results demonstrate that PAC1 is essential for the development of a mouse, and that it plays important roles in maintaining cellular integrity in quiescent tissue. Our study revealed for the first time the importance of chaperone-mediated proteasome biogenesis in a whole-body mammalian system and may provide valuable knowledge in medical drug development targeting proteasomes.     

Novel pyridobenzimidazole derivatives exhibiting antifungal activity by the inhibition of β-1,6-glucan synthesis

Based on the HTS hit compound 1a, an inhibitor of β-1,6-glucan synthesis, we synthesized novel pyridobenzimidazole derivatives and evaluated their antifungal activity. Among the compounds synthesized, we identified the potent compound 15e, which exhibits excellent activity superior to fluconazole against both Candida glabrata and Candida krusei. From the SAR study, we revealed essential moieties for antifungal activity.     

1,3-Benzoxazole-4-carbonitrile as a novel antifungal scaffold of β-1,6-glucan synthesis inhibitors

Synthesis and in vitro antifungal evaluations of 1,3-benzoxazole-7-carbonitrile 3, 1,3-benzoxazole-4-carbonitrile 4, benzofuran 5, benzoxazine 7, and benzimidazole 8 were reported. Among them, 1,3-benzoxazole-4-carbonitrile was found to be a superior scaffold structure with moderate growth inhibition against Candida species. 1,3-Benzoxazole-4-carbonitrile 6 showed potent activity against Candida species compared to 5-desmethyl compound 4 and triazolopyridine 2. Compound 6 was efficiently prepared from versatile intermediate 24, which possessed six different substituents on the benzene ring. Conversion of benzene 24 into various 1,3-benzoxazole derivatives such as 2-aliphatic 34, 2-amino 35, and lactone 38 was demonstrated.     

Regulation of IL-8 by Irsogladine maleate is involved in abolishment of Actinobacillus actinomycetemcomitans-induced reduction of gap-junctional intercellular communication

Our previous report has shown that Irsogladine maleate (IM) counters and obviates the reduction in gap junction intercellular communication (GJIC) and the increase in IL-8 levels, respectively, induced by outer membrane protein 29 from Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) in cultured human gingival epithelial cells (HGEC). In addition, IM suppresses the increase in the secretion of IL-8 caused by whole live A. actinomycetemcomitans. These findings implicate the modulation of IL-8 levels by IM in abolishment of the reduction of GJIC in HGEC. Tight junctions are also responsible for cell-cell communication. Zonula occludens protein-1 (ZO-1) is a major tight junction protein. To investigate the regulatory mechanism of intercellular communication mediated by IM, in the present study, we focused on the involvement of IL-8 in A. actinomycetemcomitans-induced change in GJIC and ZO-1 expression in HGEC. IM countered the A. actinomycetemcomitans-induced reduction in levels of Connexin (CX) 43, suggesting that it could abolish the A. actinomycetemcomitans-induced reduction in GJIC in HGEC. CXCR-1 is a receptor of IL-8. The simultaneous addition of A. actinomycetemcomitans and anti-CXCR-1 antibody also abrogated the repression of GJIC and CX43 expression by A. actinomycetemcomitans in HGEC, although the anti-CXCR-1 antibody was less effective than IM. IM inhibited the IL-8-induced reduction in CX43 levels and GJIC in HGEC. IM countered the A. actinomycetemcomitans-induced reduction in the expression of ZO-1, although anti-CXCR-1 antibody did not influence the decrease in ZO-1 mRNA levels caused by A. actinomycetemcomitans. Furthermore, IL-8 had little effect on the mRNA levels of ZO-1. These findings suggest that IL-8 mediates the A. actinomycetemcomitans-induced reduction of GJIC and CX43 expression in HGEC. The regulation of IL-8 levels by IM in HGEC is partially involved in abrogation of the reduction of GJIC and CX43 expression by A. actinomycetemcomitans. Furthermore, the regulatory effect of IM on the expression of CX43 and ZO-1 is different.     

Selection of novel structural zinc sites from a random peptide library

Zinc ion (Zn(2+)) can be coordinated with four or three amino acid residues to stabilize a protein's structure or to form a catalytic active center. We used phage display selection of a dodecamer random peptide library with Zn(2+) to identify structural zinc sites. The binding specificity for Zn(2+) of selected sequences was confirmed using enzyme-linked immunosorbent and competitive inhibition assays. Circular dichroism spectra indicated that the interaction with Zn(2+) induced a change in conformation, which means the peptide acts as a structural zinc site. Furthermore, a search of protein databases revealed that two selected sequences corresponded to parts of natural zinc sites of copper/zinc superoxide dismutase and zinc-containing ferredoxin. We demonstrated that Zn(2+)-binding sequences selected from the random combinatorial library would be candidates for artificial structural zinc sites.     

Changes in rabbit skeletal myosin and its subfragments under high hydrostatic pressure

The pressure-induced denaturation of rabbit skeletal myosin and its subfragments under hydrostatic pressure were investigated. Four nanometer of red shift of the intrinsic fluorescence spectrum was observed in myosin under a pressure of 400 MPa. The ANS fluorescence of myosin increased with elevating pressure. Changes in the intrinsic fluorescence spectra of myosin and its subfragments were quantified and expressed as the center of spectral mass. The center of spectral mass of myosin and its subfragments linearly decreased with elevating pressure, and increased with lowering pressure. The fluorescence intensity of the ANS-labeled rod did not change during pressure treatment. The present results indicate that the most pressure-sensitive portion of myosin molecule is the head. Hysteresis of the center of spectral mass of S1 appeared under pressures above 300 MPa. Changes in the center of spectral mass of S1 above 350 MPa showed stronger hysteresis. The center of spectral mass did not decrease above 350 MPa during the compression process, indicating that S1 was stable in a partially denatured state at 350 MPa under pressure. The changes in the relative intensities of ANS fluorescence of S1 were measured under pressures up to 400 MPa, and the ANS fluorescence intensity increased with elevating pressure but it did not change after pressure release. The ANS fluorescence intensity increased under constant pressure suggesting that the pressure-induced denaturation of myosin was accelerated during pressurization.