Hinge-mediated dimerization of SMC protein is essential for its dynamic interaction with DNA

Structural maintenance of chromosomes (SMC) proteins play central roles in regulating higher order chromosome dynamics from bacteria to humans. As judged by electron microscopy, the SMC homodimer from Bacillus subtilis (BsSMC) is composed of two antiparallel, coiled-coil arms with a flexible hinge. Site-directed cross-linking experiments show here that dimerization of BsSMC is mediated by a hinge-hinge interaction between self-folded monomers. This architecture is conserved in the eukaryotic SMC2-SMC4 heterodimer. Analysis of different deletion mutants of BsSMC unexpectedly reveals that the major DNA-binding activity does not reside in the catalytic ATPase domains located at the ends of a dimer. Instead, point mutations in the hinge domain that disturb dimerization of BsSMC drastically reduce its ability to interact with DNA. Proper hinge function is essential for BsSMC to recognize distinct DNA topology, and mutant proteins with altered hinge angles cross-link double-stranded DNA in a nucleotide-dependent manner. We propose that the hinge domain of SMC proteins is not a simple dimerization site, but rather it acts as an essential determinant of dynamic SMC-DNA interactions.     

Resistance against ricin-induced apoptosis in a brefeldin A-resistant mutant cell line (BER-40) of Vero cells

We have found that a brefeldin A (BFA)-resistant mutant cell line derived from Vero cells (BER-40) is highly resistant to ricin-induced apoptosis as compared with parental Vero cells. In BER-40 cells, all apoptotic events caused by ricin including cytolysis, nuclear morphological changes, and DNA fragmentation occur to a lesser extent than in Vero cells, even though both cell lines show similar sensitivities to ricin-mediated inhibition of protein synthesis. Furthermore, no significant apoptotic signaling events, such as increases in caspase-3 and -9-like activities, release of cytochrome c from mitochondria, or the cleavage of PARP, were observed in BER-40 cells under the conditions at which these changes were evident in Vero cells. Intracellular biochemical changes associated with ricin-induced apoptosis, such as the depletion of glutathione and an increase in free Zn2+, were also less apparent in BER-40 cells than in Vero cells. BER-40 cells were also found to be highly resistant to apoptosis induced by other toxins with different intoxication mechanisms such as diphtheria toxin, modeccin, and anisomycin. These results suggest that the entire apoptotic signal transduction mechanism in BER-40 cells, which may be triggered after the inhibition of protein synthesis by toxins, becomes resistant. Since MDCK cells, a naturally BFA resistant cell line, are highly sensitive to ricin-induced apoptosis, it seems likely that the BFA resistance phenotype may not necessarily lead to resistance to apoptotic cell death. Probably the underlaying BFA-resistance mechanism in BER-40 cells is distinct from that in MDCK cells, and the resistance to ricin-induced apoptosis of BER-40 cells may be a unique phenotype acquired concomitantly with BFA-resistance.     

Phosphoinositide 3-kinase accelerates calpain-dependent proteolysis of fodrin during hypoxic cell death

We have shown recently that phosphoinositide 3-kinase (PI 3-kinase) accelerates the hypoxia-induced necrotic cell death of H9c2, derived from rat cardiomyocytes, by enhancing metabolic acidosis. Here we show the downstream events of acidosis that cause hypoxic cell death. Hypoxia induces the proteolysis of fodrin, a substrate of calpain. Intracellular Ca(2+) chelation by BAPTA, and the addition of SJA6017, a specific peptide inhibitor of calpain, also reduces cell death and fodrin proteolysis, indicating that Ca(2+) influx and calpain activation might be involved in these events. The overexpression of wild type PI 3-kinase accelerates fodrin proteolysis, while dominant-negative PI 3-kinase reduces it. Both (N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na(+)/H(+) exchanger, and KB-R7943, an inhibitor of the Na(+)/Ca(2+) exchanger, reduce hypoxic cell death and fodrin proteolysis. The depletion of intracellular Ca(2+ )stores by thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase, also reduces cell death and fodrin proteolysis, indicating that Ca(2+ )release from intracellular Ca(2+ )stores might be also involved. These results indicate that PI 3-kinase might accelerate hypoxic cell death by enhancing the calpain-dependent proteolysis of fodrin.     

Seed dispersal by Japanese marten <Emphasis Type="Italic">Martes melampus</Emphasis> in the subalpine shrubland of northern Japan

The functions of carnivorous mammals as seed dispersers were studied during a snow-free period of 2 years in the subalpine shrubland of northern Japan. Mammal appearances, which were recorded using three automatic cameras, were dominated by Japanese marten, Martes melampus (Wagner), followed by three carnivores (ermine, red fox, and raccoon dog) and two other mammals (field mouse and Japanese hare). Fecal analysis revealed that the martens dispersed seeds of 11 species (eight woody and three herbaceous plants), which included a vine species that is an inhabitant of lower elevation sites. Fruit weight and size, and flesh weight and volume of the fleshy-fruited species foraged by the martens were significantly greater than those that were not foraged. These facts indicate that the marten plays a key role as a relatively long-distance seed disperser for major component species with large and flesh-rich fruits in the subalpine shrubland.     

OsRALyase1, a putative F-box protein identified in rice,Oryza sativa,with enzyme activity identical to that of wheat RALyase

A rice gene, OsRALyase1, encoding a product similar to wheat ribosomal RNA apurinic site specific lyase (RALyase), was isolated and expressed in vitro. An open reading frame of the gene predicted a protein of 476 amino acid residues with 75% identity to RALyase and contained an F-box-like motif in its amino terminal region. The rice gene product expressed in a wheat-germ protein expression system had the same characteristics as its wheat counterpart, cleaving a specific depurinated site of the 28S rRNA sarcin-ricin domain.     

Apoptosis induction by wheat-flour sphingoid bases in DLD-1 human colon cancer cells

The apoptotic effects of plant sphingoid bases prepared from wheat-flour cerebroside on human colorectal cancer DLD-1 cells were examined. The viability of DLD-1 cells treated with such plant sphingoid bases was reduced in a dose-dependent manner and was similar to that of cells treated with sphingosine. Morphological changes such as condensed chromatin fragments were found, so those sphingoid bases reduced cell viability through causing apoptosis in these cells.     

PTHrP and Indian hedgehog control differentiation of growth plate chondrocytes at multiple steps

In developing murine growth plates, chondrocytes near the articular surface (periarticular chondrocytes) proliferate, differentiate into flat column-forming proliferating cells (columnar chondrocytes), stop dividing and finally differentiate into hypertrophic cells. Indian hedgehog (Ihh), which is predominantly expressed in prehypertrophic cells, stimulates expression of parathyroid hormone (PTH)-related peptide (PTHrP) which negatively regulates terminal chondrocyte differentiation through the PTH/PTHrP receptor (PPR). However, the roles of PTHrP and Ihh in regulating earlier steps in chondrocyte differentiation are unclear. We present novel mouse models with PPR abnormalities that help clarify these roles. In mice with chondrocyte-specific PPR ablation and mice with reduced PPR expression, chondrocyte differentiation was accelerated not only at the terminal step but also at an earlier step: periarticular to columnar differentiation. In these models, upregulation of Ihh action in the periarticular region was also observed. In the third model in which the PPR was disrupted in about 30% of columnar chondrocytes, Ihh action in the periarticular chondrocytes was upregulated because of ectopically differentiated hypertrophic chondrocytes that had lost PPR. Acceleration of periarticular to columnar differentiation was also noted in this mouse, while most of periarticular chondrocytes retained PPR signaling. These data suggest that Ihh positively controls differentiation of periarticular chondrocytes independently of PTHrP. Thus, chondrocyte differentiation is controlled at multiple steps by PTHrP and Ihh through the mutual regulation of their activities.     

Possible factors responsible for the toxicity of Cochlodinium polykrikoides,a red tide phytoplankton

Cochlodinium polykrikoides, a harmful red tide dinoflagellate, is highly toxic to fish, but the toxic mechanism is still unknown. Recent study has suggested that C. polykrikoides generates reactive oxygen species (ROS) such as superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)), and the ROS-mediated ichthyotoxicity has been proposed. In this study, we found that the levels of O(2)(-) and H(2)O(2) detected in C. polykrikoides were trace levels as compared with those of Chattonella marina which is well-known to produce ROS. Furthermore, no significant increase in O(2)(-) generation by C. polykrikoides was observed in the presence of lectins such as concanavalin A (Con A) and wheat germ agglutinin (WGA) or fish mucus prepared from skin and gill of yellowtail, whereas C. marina generated increased level of O(2)(-) responding to these stimuli. Interestingly, the cell-free aqueous extract prepared from C. polykrikoides showed toxic effect on the HeLa cells, but the extract of C. marina had no significant effect. Furthermore, gradual accumulation of polysaccharides in the medium was observed during the growth of C. polykrikoides, and the medium gradually became viscous, but no such changes were observed in the medium of C. marina. These results suggest that multiple factors may be responsible for the toxic mechanism of C. polykrikoides.     

Transforming growth factor-beta1 is responsible for maturation-dependent spontaneous apoptosis of cultured gastric pit cells

In this study, we established a system of high concentration serum-dependent spontaneous apoptosis of guinea pig gastric pit cells in primary culture, which seems to mimic the spontaneous apoptosis of matured gastric pit cells at gastric surface in vivo. In addition to induction of the spontaneous apoptosis, cell growth was inhibited in the presence of 10% serum compared with 0.5% serum. Transforming growth factor-beta1 (TGF-beta1), which is known to cause both apoptosis and growth inhibition in mammalian cells, was present in serum of both fetal calf and guinea pig. The addition of recombinant TGF-beta1 to the culture medium containing 0.5% fetal calf serum caused both induction of apoptosis and inhibition of cell growth. On the other hand, immunodepletion of TGF-beta1 from fetal calf serum caused inability to induce both the spontaneous apoptosis and inhibition of cell growth. These data suggest that TGF-beta1 is involved in the spontaneous apoptosis of guinea pig gastric pit cells in primary culture.     

Transgenic pigs expressing human decay-accelerating factor regulated by porcine MCP gene promoter

Porcine membrane cofactor protein (pMCP) is abundantly expressed throughout the body with particularly strong expression on the vascular endothelia. Previous studies demonstrated that the promoter of the pMCP gene induced efficient expression of a human complement regulatory protein, decay-accelerating factor (DAF; CD55), in transgenic mice. In the present study, we tried to produce transgenic pigs with two hybrid genes, 0.9/hDAF and 5.4/hDAF, which were composed of human DAF (hDAF) gene regulated under pMCP promoters of different lengths (0.9 and 5.4 kb). Five live founder transgenic pigs were obtained only with the 0.9/hDAF construct. Although, four founder pigs transmitted the transgene to the second generation, the transmission rates varied among founders. We examined the expression of hDAF in tissues of descendants of two lines (Dm1 and Dm4). Human DAF specific RNAs were confirmed by an RT-PCR analysis in all organs examined. Levels of hDAF protein in the organs from the descendants of Dm1 line were higher than those in the corresponding human organs as determined by enzyme-linked immunosorbent assay. Immunohistochemical studies showed that the tissue distribution of hDAF in the descendants of both lines was similar to that of endogenous pMCP. The expression level of hDAF on the vascular endothelial cells in Dm1 line was twice that on the corresponding human cells. We tested whether proinflammatory cytokines upregulate an efficiency of pMCP promoter on hDAF expression in transgenic pigs. Although the expression of hDAF on the human endothelial cells increased with a combination of cytokines, tumor necrosis factor alpha and interferon-gamma, no cytokine-induced upregulation was seen in the cells of transgenic pigs. The endothelial cells from transgenic pigs exhibited high resistance to the human serum-mediated cytolysis.