Mitotic phosphatase activity is required for MCC maintenance during the spindle checkpoint

The spindle checkpoint prevents activation of the anaphase-promoting complex (APC/C) until all chromosomes are correctly attached to the mitotic spindle. Early in mitosis, the mitotic checkpoint complex (MCC) inactivates the APC/C by binding the APC/C activating protein CDC20 until the chromosomes are properly aligned and attached to the mitotic spindle, at which point MCC disassembly releases CDC20 to activate the APC/C. Once the APC/C is activated, it targets cyclin B and securin for degradation, and the cell progresses into anaphase. While phosphorylation is known to drive many of the events during the checkpoint, the precise molecular mechanisms regulating spindle checkpoint maintenance and inactivation are still poorly understood. We sought to determine the role of mitotic phosphatases during the spindle checkpoint. To address this question, we treated spindle checkpoint-arrested cells with various phosphatase inhibitors and examined the effect on the MCC and APC/C activation. Using this approach we found that 2 phosphatase inhibitors, calyculin A and okadaic acid (1 μM), caused MCC dissociation and APC/C activation leading to cyclin A and B degradation in spindle checkpoint-arrested cells. Although the cells were able to degrade cyclin B, they did not exit mitosis as evidenced by high levels of Cdk1 substrate phosphorylation and chromosome condensation. Our results provide the first evidence that phosphatases are essential for maintenance of the MCC during operation of the spindle checkpoint.     

Aminothiazoles inhibit RANKL‐ and LPS‐mediated osteoclastogenesis and PGE2 production in RAW 264.7 cells

Periodontitis is characterized by chronic inflammation and osteoclast‐mediated bone loss regulated by the receptor activator of nuclear factor‐κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG). The aim of this study was to investigate the effect of aminothiazoles targeting prostaglandin E synthase‐1 (mPGES‐1) on RANKL‐ and lipopolysaccharide (LPS)‐mediated osteoclastogenesis and prostaglandin E₂ (PGE₂) production in vitro using the osteoclast precursor RAW 264.7 cells. RAW 264.7 cells were treated with RANKL or LPS alone or in combination with the aminothiazoles 4‐([4‐(2‐naphthyl)‐1,3‐thiazol‐2‐yl]amino)phenol (TH‐848) or 4‐(3‐fluoro‐4‐methoxyphenyl)‐N‐(4‐phenoxyphenyl)‐1,3‐thiazol‐2‐amine (TH‐644). Aminothiazoles significantly decreased the number of multinucleated tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclast‐like cells in cultures of RANKL‐ and LPS‐stimulated RAW 264.7 cells, as well as reduced the production of PGE₂ in culture supernatants. LPS‐treatment induced mPGES‐1 mRNA expression at 16 hrs and the subsequent PGE₂ production at 72 hrs. Conversely, RANKL did not affect PGE₂ secretion but markedly reduced mPGES‐1 at mRNA level. Furthermore, mRNA expression of TRAP and cathepsin K (CTSK) was reduced by aminothiazoles in RAW 264.7 cells activated by LPS, whereas RANK, OPG or tumour necrosis factor α mRNA expression was not significantly affected. In RANKL‐activated RAW 264.7 cells, TH‐848 and TH‐644 down‐regulated CTSK but not TRAP mRNA expression. Moreover, the inhibitory effect of aminothiazoles on PGE₂ production was also confirmed in LPS‐stimulated human peripheral blood mononuclear cell cultures. In conclusion, the aminothiazoles reduced both LPS‐ and RANKL‐mediated osteoclastogenesis and PGE₂ production in RAW 264.7 cells, suggesting these compounds as potential inhibitors for treatment of chronic inflammatory bone resorption, such as periodontitis.     

Muskoxen Modify Plant Abundance,Phenology, and Nitrogen Dynamics in a High Arctic Fen

Ecosystems - Herbivores are key drivers of vegetation dynamics in most ecosystems. However, the effect of high arctic herbivores on vegetation dynamics throughout a growing season is not well...     

Dual Role of Mitochondrial Porin in Metabolite Transport across the Outer Membrane and Protein Transfer to the Inner Membrane

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Comparative analysis of complete plastid genome reveals powerful barcode regions for identifying wood of Dalbergia odorifera and D. tonkinensis (Leguminosae)

Dalbergia odorifera T. C. Chen (Leguminosae), a rare and endangered tree species endemic to Hainan Island of China, produces the most expensive and rarest wood in China. The wood characteristics of D. odorifera are remarkably similar to those of D. tonkinensis (a much less sought-after species from Vietnam), and the DNA from wood is often highly degraded, making it very difficult to identify the two species using anatomical features or DNA barcoding based on regular DNA markers. To solve the confusion of identifying wood reliably from the two species, we built and analyzed the plastome library of 26 samples from 18 Dalbergia species, of which 12 samples from eight closely related species of D. odorifera are newly sequenced in this study. Phylogenomic analysis suggested that the relationships among the 26 samples are mostly well resolved, and conspecific individuals from different populations of D. odorifera and D. tonkinensis clustered together. Between the plastid genomes of the two species, we identified 129 indels and 114 single nucleotide polymorphisms. By assessing a subset of 20 nucleotide polymorphisms and 10 indels using 37 population-level samples (20 samples of D. odorifera and 17 samples of D. tonkinensis), we recovered eight species-specific barcode regions that could be suitable for identifying the wood D. odorifera and D. tonkinensis. To examine their utility in wood identification, we amplified the eight DNA barcodes using six wood samples and recovered an amplification success rate of 83.3%, demonstrating a reliable method for precise wood identification of the two species.     

Synthesis of fused bicyclic thioglycosides of N-acylated glucosamine as analogues of mycothiol

The synthesis of a fused bicyclic thioglycoside analogue of mycothiol, (3R)-3-acetylamino-4-one-6,7-dihydro-(1',2'-dideoxy-beta-D-glucopyranoso)[2',1'-f]-1,5-thiazepane (5), is reported. Treatment of phthalimido-protected peracetylated glucosamine with N-acetyl-cysteine and boron trifluoride-etherate gave the beta-linked thioglycoside, which was deprotected and cyclized, using HOBt and EDCl to form the lactam and giving the target structure. This mycothiol mimic and its tri-O-acetate will be investigated as potential inhibitors of enzymes involved in the biosynthesis of mycothiol. The protected derivative also has the potential to be an alpha-selective N-cysteinyl glucosamine donor; however, initial glycosylation attempts failed due to the apparent stability of the fused bicyclic system.     

The importance of clutch characteristics and learning for antiparasite adaptations in hosts of avian brood parasites

There is considerable variation in rejection rates of parasitic eggs among hosts of avian brood parasites. In this article, we develop a model that can be used to predict host egg rejection behavior in brood parasite-host systems in general, by considering both intra- and interclutch variation in host egg appearance; clutch characteristics that may be important in calculating the fitness of individuals adopting rejecter or acceptor strategies. In addition, we consider the importance of learning the appearance of own eggs during the first breeding attempt and host probability of survival between breeding seasons on evolution of rejection behavior. Based on this model we can predict at which level of parasitism fitness of rejecter individuals is higher than that of acceptor individuals and vice versa. The model analyses show that variation in egg appearance can be a key factor for the evolution of host defense against parasitism. In more detail, analyses show that we should expect to find a prolonged learning period only in hosts that have a high intraclutch variation in egg appearance, because such hosts may potentially experience high costs in terms of recognition errors. Furthermore, learning is in general more adaptive in parasite-host systems in which hosts do have some reproductive success even when parasitized, and when parasitism rates are moderate. By including variables that have not been considered in previous models, our model represents a useful tool in investigations of host rejection behavior in various host-parasite systems.     

Expression of the RAI gene is conducive to apoptosis: studies of induction and interference

The RAI gene is also known as iASPP and PPP1R13L. Recent investigations have shown that the region encompassing RAI is important for the development of cancer in young and middle-aged persons. It has been speculated that the RAI product induces apoptosis by blocking NF-kappaB or inhibits apoptosis by blocking p53. Either way the gene could influence the survival of precancerous lesions. Here we report that the expression of RAI mRNA was increased in non-transformed lymphocytes and fibroblasts induced to undergo apoptosis by various means, such as treatment with etoposide, calcium ions, or interleukin-2 and/or serum deprivation. Treatment with etoposide increased the content of RAI protein, too, and caused it to translocate to the nucleus. Inhibition of RAI expression in lymphocytes and fibroblasts with siRNA reduced apoptosis, but treatment with the NF-kappaB-inhibiting substance sulfasalazine relieved this dependence. In the transformed cell line HEK-293 the association between RAI induction and apoptosis seemed broken. Thus, we hypothesize that RAI induction is necessary but not sufficient for apoptosis induction in non-transformed cells. Our results could be explained by a NF-kappaB mediated mechanism.     

Plasticity of proton pathway structure and water coordination in cytochrome c oxidase

Cytochrome c oxidase (CytcO) is a redox-driven, membrane-bound proton pump. One of the proton transfer pathways of the enzyme, the D pathway, used for the transfer of both substrate and pumped protons, accommodates a network of hydrogen-bonded water molecules that span the distance between an aspartate (Asp(132)), near the protein surface, and glutamate Glu(286), which is an internal proton donor to the catalytic site. To investigate how changes in the environment around Glu(286) affect the mechanism of proton transfer through the pathway, we introduced a non-hydrogen-bonding (Ala) or an acidic residue (Asp) at position Ser(197) (S197A or S197D), located approximately 7 A from Glu(286). Although Ser(197) is hydrogen-bonded to a water molecule that is part of the D pathway "proton wire," replacement of the Ser by an Ala did not affect the proton transfer rate. In contrast, the S197D mutant CytcO displayed a turnover activity of approximately 35% of that of the wild-type CytcO, and the O(2) reduction reaction was not linked to proton pumping. Instead, a fraction of the substrate protons was taken from the positive ("incorrect") side of the membrane. Furthermore, the pH dependence of the proton transfer rate was altered in the mutant CytcO. The results indicate that there is plasticity in the water coordination of the proton pathway, but alteration of the electrostatic potential within the pathway results in uncoupling of the proton translocation machinery.