Identification of Cdk targets that control cytokinesis

The final event of the eukaryotic cell cycle is cytokinesis, when two new daughter cells are born. How the timing and execution of cytokinesis is controlled is poorly understood. Here, we show that downregulation of cyclin-dependent kinase (Cdk) activity, together with upregulation of its counteracting phosphatase Cdc14, controls each of the sequential steps of cytokinesis, including furrow ingression, membrane resolution and cell separation in budding yeast. We use phosphoproteome analysis of mitotic exit to identify Cdk targets that are dephosphorylated at the time of cytokinesis. We then apply a new and widely applicable tool to generate conditionally phosphorylated proteins to identify those whose dephosphorylation is required for cytokinesis. This approach identifies Aip1, Ede1 and Inn1 as cytokinetic regulators. Our results suggest that cytokinesis is coordinately controlled by the master cell cycle regulator Cdk together with its counteracting phosphatase and that it is executed by concerted dephosphorylation of Cdk targets involved in several cell biological processes.     

A phenological scale for the development of Gladiolus

A staging system for development of gladiola (Gladiolus × grandiflorus) that relies on simple, visual, non‐destructive criteria is proposed. Four field trials were conducted during the spring 2010, autumn/winter 2011 and winter 2011 at Santa Maria, RS, Brazil, with different gladiola cultivars, in order to observe the developmental stages of the above‐ground parts and their dry matter. The developmental cycle, which starts at dormant corm and ends with plant senescence, is divided into four developmental phases: dormancy phase, sprouting phase (from filiform roots appearance to sheaths appearance), vegetative phase (from emergence of the first leaf tip to emergence of the final leaf tip on the stem) and reproductive phase (from heading to plant senescence). The developmental stages that were identified during the dormancy phase and during the sprouting phases are coded as S stages: S0 = dormant corm, S1 = appearance of roots, S2.1 = first sheath, S2.2 = second sheath and S2.3 = third sheath. Vegetative phase is coded as V stages: VE = emergence of the sheaths above ground, V1 = first leaf, V2 = second leaf, Vn = nth leaf and VF = flag leaf. Leaf tip is the marker for V1–VF. The developmental stages during the reproductive phases are coded as R stages: R1 = heading, R2 = blooming, R3 = onset of flowering, R4 = end of anthesis, R5 = end of florets senescence and R6 = plant senescence (leaves and floret axis are brown). Sub‐stages have also been assigned between R1 and R2 and between R3 and R4. Illustrations (photographs) of each developmental stage taken from field pot‐grown plants are provided and the proposed scale was tested with field observations. These criteria are straight forward and allow for quick determination of development stage. This system can be used by both farmers and for experimental trials.     

Potent inhibitory activity of chimeric oligonucleotides targeting two different sites of human telomerase

Suppression of telomerase activity in tumor cells has been considered as a new anticancer strategy. Here, we present chimeric oligonucleotides (chimeric ODNs) as a new type of telomerase inhibitor that contains differently modified oligomers to address two different sites of telomerase: the RNA template and a suggested protein motif. We have shown previously that phosphorothioate-modified oligonucleotides (PS ODNs) interact in a length-dependent rather than in a sequence-dependent manner, presumably with the protein part of the primer-binding site of telomerase, causing strong inhibition of telomerase. In the present study, we demonstrate that extensions of these PS ODNs at their 3'-ends with an antisense oligomer partial sequence covering 11 bases of the RNA template cause significantly increased inhibitory activity, with IC(50) values between 0.60 and 0.95 nM in a Telomeric Repeat Amplification Protocol (TRAP) assay based on U-87 cell lysates. The enhanced inhibitory activity is observed regardless of whether the antisense part is modified (phosphodiester, PO; 2'-O-methylribosyl, 2'-OMe/PO; phosphoramidate, PAM). However, inside intact U-87 cells, these modifications of the antisense part proved to be essential for efficient telomerase inhibition 20 hours after transfection. In particular, the chimeric ODNs containing PAM or 2'-OMe/PO modifications, when complexed with lipofectin, were most efficient telomerase inhibitors (ID(50) = 0.04 and 0.06 microM, respectively). In conclusion, ODNs of this new type emerged as powerful inhibitors of human telomerase and are, therefore, promising candidates for further investigations of the anticancer strategy of telomerase inhibition.     

The mechanism of sister chromatid cohesion

Each of our cells inherit their genetic information in the form of chromosomes from a mother cell. In order that we obtain the full genetic complement, cells need to ensure that replicated chromosomes are accurately split and distributed during cell division. Mistakes in this process lead to aneuploidies, cells with supernumerous or missing chromosomes. Most aneuploid human embryos are not viable, and if they are, they develop severe birth defects. Aneuploidies later in human life are frequently found associated with the development of malignant cancer. DNA replication during S-phase is linked to segregation of the sister copies in mitosis by sister chromatid cohesion. A chromosomal protein complex, cohesin, holds replicated sister DNA strands together after their synthesis. This allows pairs of replication products to be recognised by the spindle apparatus in mitosis for segregation into opposite direction. At anaphase onset, cohesin is destroyed by a site-specific protease, separase. Here I review what we have learned about the molecular mechanism of sister chromatid cohesion. Cohesin forms a large proteinaceous ring that may hold sister chromatids by encircling and topological trapping. To understand how cohesin links newly synthesised replication products, biochemical assays to study the enzymology of cohesin will be required.     

Mortality of discards from southeastern Australian beach seines and gillnets

Two experiments were done in an Australian estuary to quantify the mortalities and contributing factors for key species discarded during 8 and 9 deployments of commercial beach (or shore) seines and gillnets, respectively. In both experiments, bycatches (2347 individuals comprising 16 species) were handled according to conventional practices and assessed for immediate mortalities before live samples of selected species were discarded into replicate cages along with appropriate controls, and monitored for short-term mortalities (< or =10 d). All of the seined or gilled fish were alive prior to discarding. During the beach seine experiment, 20% of caged seined-and-discarded surf bream Acanthopagrus australis (n = 290) were dead after 5 d, with most mortalities occurring between the second and fifth day. In the gillnet experiment, 42 and 11% of gilled-and-discarded A. australis (n = 161) and lesser salmon catfish Neoarius graeffei (n = 67), respectively, died during a 10 d monitoring period, mostly within the first 5 d. There were no deaths in any controls for these fish. Mixed-effects logistic models revealed that the mortality of A. australis discarded from both gears was significantly (p < 0.01) and negatively correlated with their total length, while N. graeffei had a significantly (p < 0.05) greater (5-fold) probability of dying when jellyfish Catostylus sp. were present in the gillnet. Simple modifications to the operations of beach seines and gillnets and/or post-capture handling procedures, such as close regulation of size selectivity for the target species, careful removal of fish from meshes, and abstention from setting during high abundances of jellyfish will maximise the survival of discarded bycatch.     

Silver as a Residual Disinfectant To Prevent Biofilm Formation in Water Distribution Systems

Biofilms can have deleterious effects on drinking water quality and may harbor pathogens. Experiments were conducted using 100 μg/liter silver to prevent biofilm formation in modified Robbins devices with polyvinyl chloride and stainless steel surfaces. No significant difference was observed on either surface between the silver treatment and the control.     

Secured cutting: controlling separase at the metaphase to anaphase transition

The final irreversible step in the duplication and distribution of genomes to daughter cells takes place at the metaphase to anaphase transition. At this point aligned sister chromatid pairs split and separate. During metaphase, cohesion between sister chromatids is maintained by the chromosomal multi-subunit cohesin complex. Here, I review recent findings as to how anaphase is initiated by proteolytic cleavage of the Scc1 subunit of cohesin. Scc1 is cleaved by a site-specific protease that is conserved in all eukaryotes, and is now called ‘separase’. As a result of this cleavage, the cohesin complex is destroyed, allowing the spindle to pull sister chromatids into opposite halves of the cell. Because of the final and irreversible nature of Scc1 cleavage, this reaction is tightly controlled. Several independent mechanisms seem to impose regulation on Scc1 cleavage, acting on both the activity of separase and the susceptibility of the substrate.