Telomere-binding protein TRF2 binds to and stimulates the Werner and Bloom syndrome helicases

Werner syndrome is a human premature aging disorder displaying cellular defects associated with telomere maintenance including genomic instability, premature senescence, and accelerated telomere erosion. The yeast homologue of the Werner protein (WRN), Sgs1, is required for recombination-mediated lengthening of telomeres in telomerase-deficient cells. In human cells, we report that WRN co-localizes and physically interacts with the critical telomere maintenance protein TRF2. This interaction is mediated by the RecQ conserved C-terminal region of WRN. In vitro, TRF2 demonstrates high affinity for WRN and for another RecQ family member, the Bloom syndrome protein (BLM). TRF2 interaction with either WRN or BLM results in a notable stimulation of their helicase activities. Furthermore, the WRN and BLM helicases, partnered with replication protein A, actively unwind long telomeric duplex regions that are pre-bound by TRF2. These results suggest that TRF2 functions with WRN, and possibly BLM, in a common pathway at telomeric ends.     

Tail regression in Ciona intestinalis (Prochordate) involves a Caspase-dependent apoptosis event associated with ERK activation

Two apoptotic events take place during embryonic development of Ciona intestinalis. The first concerns extra-embryonic cells and precedes hatching. The second controls tail regression at metamorphosis, occurs through a polarized wave originating from tail extremity, and is caspase dependent. This was shown by: (1) in vivo incorporation of a fluorescent marker of caspase activation in different cell types of the tail; (2) detection of an activated form of caspase 3-like protein by western blotting; and (3) failure of 30% of larvae to undergo metamorphosis after treatment of fertilized eggs with a pan-caspase inhibitor. In addition, Ciona embryos express a single ERK protein, specifically phosphorylated at metamorphosis. ERK activation was shown to be located in cells of the tail. Addition of MEK inhibitor in the culture medium prevented ERK activation and metamorphosis. In silico analysis of Ciona genome pointed to 15 caspases with high homology with humans, and a single ERK gene with high homology to both mammalian ERK1 and ERK2. It is concluded that the sequence of events leading to metamorphosis includes ERK phosphorylation followed by caspase-dependent apoptosis and tail regression.     

Genome informatics for data-driven biology

A report on the 12th International Conference on Genome Informatics, Tokyo, Japan, 17-19 December 2001.     

PRC1 is a microtubule binding and bundling protein essential to maintain the mitotic spindle midzone

Midzone microtubules of mammalian cells play an essential role in the induction of cell cleavage, serving as a platform for a number of proteins that play a part in cytokinesis. We demonstrate that PRC1, a mitotic spindle-associated Cdk substrate that is essential to cell cleavage, is a microtubule binding and bundling protein both in vivo and in vitro. Overexpression of PRC1 extensively bundles interphase microtubules, but does not affect early mitotic spindle organization. PRC1 contains two Cdk phosphorylation motifs, and phosphorylation is possibly important to mitotic suppression of bundling, as a Cdk phosphorylation-null mutant causes extensive bundling of the prometaphase spindle. Complete suppression of PRC1 by siRNA causes failure of microtubule interdigitation between half spindles and the absence of a spindle midzone. Truncation mutants demonstrate that the NH2-terminal region of PRC1, rich in alpha-helical sequence, is important for localization to the cleavage furrow and to the center of the midbody, whereas the central region, with the highest sequence homology between species, is required for microtubule binding and bundling activity. We conclude that PRC1 is a microtubule-associated protein required to maintain the spindle midzone, and that distinct functions are associated with modular elements of the primary sequence.     

From autopoiesis to neurophenomenology: Francisco Varela's exploration of the biophysics of being

This paper reviews in detail Francisco Varela's work on subjectivity and consciousness in the biological sciences. His original approach to this "hard problem" presents a subjectivity that is radically intertwined with its biological and physical roots. It must be understood within the framework of his theory of a concrete, embodied dynamics, grounded in his general theory of autonomous systems. Through concepts and paradigms such as biological autonomy, embodiment and neurophenomenology, the article explores the multiple levels of circular causality assumed by Varela to play a fundamental role in the emergence of human experience. The concept of biological autonomy provides the necessary and sufficient conditions for characterizing biological life and identity as an emergent and circular self-producing process. Embodiment provides a systemic and dynamical framework for understanding how a cognitive self--a mind--can arise in an organism in the midst of its operational cycles of internal regulation and ongoing sensorimotor coupling. Global subjective properties can emerge at different levels from the interactions of components and can reciprocally constrain local processes through an ongoing, recursive morphodynamics. Neurophenomenology is a supplementary step in the study of consciousness. Through a rigorous method, it advocates the careful examination of experience with first-person methodologies. It attempts to create heuristic mutual constraints between biophysical data and data produced by accounts of subjective experience. The aim is to explicitly ground the active and disciplined insight the subject has about his/her experience in a biophysical emergent process. Finally, we discuss Varela's essential contribution to our understanding of the generation of consciousness in the framework of what we call his "biophysics of being."     

Adhesion of enteropathogenic Escherichia coli to host cells

Enteropathogenic Escherichia coli (EPEC) adhere to the intestinal mucosa and to tissue culture cells in a distinctive fashion, destroying microvilli, altering the cytoskeleton and attaching intimately to the host cell membrane in a manner termed the attaching and effacing effect. Typical EPEC strains also form three-dimensional microcolonies in a pattern termed localized adherence. Attaching and effacing, and in particular intimate attachment requires an outer membrane adhesin called intimin, which binds to the translocated intimin receptor, Tir. Tir is produced by the bacteria and delivered to the host cell via a type III secretion system. In addition to this well-established adhesin-receptor pair, numerous other adhesin interactions between EPEC and host cells have been described including those between intimin and cellular receptors and those involving a bundle-forming pilus and flagella and unknown receptors. Much additional work is needed before a full understanding of EPEC adhesion to host cells comes to light.     

A rare localization in right-sided endocarditis diagnosed by echocardiography: A case report

Background

Right-sided endocarditis occurs predominantly in intravenous drug users, patients with pacemakers or central venous lines and with congenital heart diseases. The vast majority of cases involve the tricuspid valve.

Case presentation

A case of a 31-year-old woman with intravenous drug abuse who had a right-sided vegetation attached to the muscular bundle of the right ventricle is presented. Transthoracic echocardiography revealed a vegetation in the right ventricular outflow tract. Transesophageal echocardiography clearly showed that the 1.8 cm vegetation was not adherent to the pulmonary valve but attached to a muscular bundle.

Conclusions

Our case points to an unusual location of right-sided endocarditis in intravenous drug users. It confirms that TTE remains an easy and highly sensitive first-line examination for the diagnosis of right-sided endocarditis.     

Alteration of poly(ADP-ribose) glycohydrolase nucleocytoplasmic shuttling characteristics upon cleavage by apoptotic proteases

Poly(ADP-ribosyl)ation is an important post-translational modification which mostly affects nuclear proteins. The major roles of poly(ADP-ribose) synthesis are assigned to DNA damage signalling during base excision repair, apoptosis and excitotoxicity. The transient nature and modulation of poly(ADP-ribose) levels depend mainly on the activity of poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG), the key catabolic enzyme of poly(ADP-ribose). Given the fact that PARG substrate, poly(ADP-ribose), is found almost exclusively in the nucleus and that PARG is mainly localized in the cytoplasm, we wanted to have a closer look at PARG subcellular localization in order to better understand the mechanism by which PARG regulates intracellular poly(ADP-ribose) levels. We examined the subcellular distribution of PARG and of its two enzymatically active C-terminal apoptotic fragments both biochemically and by fluorescence microscopy. Green fluorescent protein (GFP) fusion proteins were constructed for PARG (GFP-PARG), its 74 kDa (GFP-74) and 85 kDa (GFP-85) apoptotic fragments and transiently expressed in COS-7 cells. Localization experiments reveal that all three fusion proteins localize predominantly to the cytoplasm and that a fraction also co-localizes with the Golgi marker FTCD. Moreover, leptomycin B, a drug that specifically inhibits nuclear export signal (NES)-dependent nuclear export, induces a redistribution of GFP-PARG from the cytoplasm to the nucleus and this nuclear accumulation is even more pronounced for the GFP-74 and GFP-85 apoptotic fragments. This observation confirms our hypothesis for the presence of important regions in the PARG sequence that would allow the protein to engage in CRM1-dependent nuclear export. Moreover, the altered nuclear import kinetics found for the apoptotic fragments highlights the importance of PARG N-terminal sequence in modulating PARG nucleocytoplasmic trafficking properties.     

Analysis of ADP-ribose polymer sizes in intact cells

Poly(ADP-ribose) is a polymer (pADPr) that is synthesized by poly (ADP-ribose) polymerases in response to DNA damaging agents. For instance, chemical alkylating agents such as MNNG [1] or physical stimulation of cells by -rays [2] are well known to induce pADPr synthesis. PARPs are members of a growing family of enzymes which includes PARP-1, PARP-2, S-PARP-1, tankyrase and V-PARP [3]. The association of PARP-1 and PARP-2 in DNA damage signaling pathways has been characterized, but tankyrase and V-PARP seem to be independent of DNA repair mechanisms.Poly(ADP-ribosyl)ation leads to heterogenous chain lengths of up to 200 units (mers) in vitro [3]. While most of these will be covalently bound to proteins, they may be released under alkaline conditions for analysis. Previous immunological methods such as immunoblots [4] showed that about 60–70% of the 6–8 mers pADPr were lost during fixation and that the very short pADPr (2–5 mers) were very weakly bound to the membrane [5]. Furthermore, detection of cellular pADPr using enzyme-linked immunosorbent assay (ELISA) revealed that some molecules of pADPr are also lost during fixation and washings. This phenomenon leads to underestimation of the short pADPr population in cells. Thus, evaluating which pADPr sizes are present in cells and tissues becomes critical.We report here the development of a new highly sensitive immunological method to detect synthesized pADPr sizes distribution in intact cells.     

Proliferation in murine epidermis after minor mechanical stimulation. Part 1. Sustained increase in keratinocyte production and migration

It was our objective to obtain an insight into the details and dynamics of the cell proliferative changes following minor barrier disruption, the mechanisms of recovery, and their regulation. Hair of the dorsal area of DBA2-mice was removed and the epidermis was tape stripped. Tritiated thymidine was injected into groups of mice at daily intervals thereafter. Labelling and nuclear densities were measured at several time intervals later in the various epidermal strata to characterize cell production and cell fluxes through the tissue. A dramatic proliferative response was observed at 24 h when the labelling density increased more than sixfold in the basal layer. Labelled cells rapidly appeared in suprabasal layers within a few hours in large quantities while this process took over 2 days in normal skin. Some cycling cells were also found in the suprabasal layer (pulse labelling at 24 h) in contrast with the controls. The cellular flux through the suprabasal layers was drastically (20-fold) increased and the transit time was shortened. Although the nuclear density in the basal layer showed only moderate changes it increased four-fold in the suprabasal layer within 5 days. A kinetic model analysis suggested that the cell cycle time of proliferative cells dropped from a normal value of about 200 h to less than 12 h post tape strip. After 7 days, the proliferative activation still persisted, even though at 3 days post tape strip the stratum corneum had been re-established. Hence, a mild mechanical alteration with removal of some parts of the cornified layer in mouse backskin epidermis triggers a huge proliferative response with massive overproduction of cells that lasts at least 7 days. Our findings suggest that the re-establishment of the cornified layer does not immediately shut down cell proliferation and that more complex, slower (long-term) regulatory processes are involved.