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The archaeology of apoptosis.

Human death has been recognised as a significant personal and social event for many thousands of years, and classical archaeologists have revealed the changing complexity of rituals associated with it. The study of cell death, however, is a much more recent event, although many of the molecular pathways involved have now been identified, at least in mammalian systems. In studying the loss of cells, the use of the term 'death' is, perhaps, not altogether appropriate both since it carries the cultural resonance associated with bodily death, and because we do not study cell death itself, but rather the processes that lead up to it. Mammalian cell death processes are complex and involve a dynamic equilibrium between death promoting and death inhibiting factors, suggesting that some components of death pathways may have a paradoxical survival function. Since parasites must survive an often hostile environment, they may be a useful model to study whether component molecules of mammalian death pathways originally formed modules of parasite survival strategies, and whether survival and death pathways coevolved.

Animals↗

Play as physical flexibility training in five ceboid primates.

Instantaneous focal animal sampling of positional behavior (posture and locomotion) during the play and nonplay of adults and juveniles of 5 ceboid monkeys (Alouatta palliata, Ateles geoffroyi, Cacajao calvus, Cebus capucinus, and Saimiri boliviensis) in an optimal design field study of the function of play as physical flexibility training produced a sample of 98,261 observations. Analyses of 67 distinct modes of positional behavior demonstrated that significantly enhanced rates of suspension and nonquadrupedal locomotion plus reduced rates of quadrupedal locomotion (especially quadrupedal walking) typified play in all 5 species. These findings support the hypothesis that play promotes the development of dynamic and static flexibility in arboreal primates. This argument from design that play functions as physical flexibility training was discussed as fully compatible with other functional interpretations.

Animals↗

Sticky issue of adherence.

The phenomenon of adherence, also known as compliance, is the vital link that allows effective medications to have the desired clinical effect when self-administered. It is often assumed that the population is generally adherent, but more than 50% of people with chronic illness do not take their medication as prescribed. We highlight how the terminology and language of non-adherence act to conceptualize adherence as a patient problem in a manner that is inadvertently judgmental, narrowly focused and clinically unhelpful. In contrast, knowledge of the dynamic nature of adherence promotes the conceptualization of adherence as the common problem that it is, where the responsibility for improving it lies primarily with the health professional. The example of asthma is used to highlight how individually focused clinical strategies can fit within a population perspective that, in its entirety, can be conceptualized as a framework of adherence-promoting strategies.

Adolescent↗

Visualization of synaptotagmin I oligomers assembled onto lipid monolayers.

Neuronal exocytosis is mediated by Ca(2+)-triggered rearrangements between proteins and lipids that result in the opening and dilation of fusion pores. Synaptotagmin I (syt I) is a Ca(2+)-sensing protein proposed to regulate fusion pore dynamics via Ca(2+)-promoted binding of its cytoplasmic domain (C2A-C2B) to effector molecules, including anionic phospholipids and other copies of syt. Functional studies indicate that Ca(2+)-triggered oligomerization of syt is a critical step in excitation-secretion coupling; however, this activity has recently been called into question. Here, we show that Ca(2+) does not drive the oligomerization of C2A-C2B in solution. However, analysis of Ca(2+).C2A-C2B bound to lipid monolayers, using electron microscopy, revealed the formation of ring-like heptameric oligomers that are approximately 11 nm long and approximately 11 nm in diameter. In some cases, C2A-C2B also assembled into long filaments. Oligomerization, but not membrane binding, was disrupted by neutralization of two lysine residues (K326,327) within the C2B domain of syt. These data indicate that Ca(2+) first drives C2A-C2B.membrane interactions, resulting in conformational changes that trigger a subsequent C2B-mediated oligomerization step. Ca(2+)-mediated rearrangements between syt subunits may regulate the opening or dilation kinetics of fusion pores or may play a role in endocytosis after fusion.

Animals↗

BRCA1-A and LIG4 complexes mediate ecDNA biogenesis and cancer drug resistance.

Extrachromosomal circular DNA (ecDNA) is frequently generated within the nucleus, contributing to genome dynamics and heterogeneity, thereby promoting cancer cell evolution and adaptation. However, the mechanisms underlying ecDNA biogenesis remain poorly understood. Here, using genome-wide CRISPR screening in human cells, we identified the BRCA1-A and the LIG4 complexes as key drivers of ecDNA production. Following DNA segmentation, the upstream BRCA1-A complex protects DNA ends from excessive resection, promoting end-joining for circularization. Conversely, the MRN complex, which mediates end resection and thus antagonizes the BRCA1-A complex, suppresses ecDNA formation. Downstream, LIG4 conservatively mediates ecDNA production by joining the free ends of the DNA fragments. Furthermore, ecDNA from patient tumors harbors junction sites with a LIG4 signature. Notably, disruption of either LIG4 or the BRCA1-A complex in cancer cells impairs ecDNA-mediated adaptation, hindering the development of resistance to both chemotherapy and targeted therapies. Together, our study reveals the roles of the LIG4 and BRCA1-A complexes in ecDNA biogenesis, and uncovers therapeutic targets to block ecDNA-mediated adaptation for cancer treatment.

Humans↗

Specific interaction of mutant p53 with regions of matrix attachment region DNA elements (MARs) with a high potential for base-unpairing.

Mutant, but not wild-type p53 binds with high affinity to a variety of MAR-DNA elements (MARs), suggesting that MAR-binding of mutant p53 relates to the dominant-oncogenic activities proposed for mutant p53. MARs recognized by mutant p53 share AT richness and contain variations of an AATATATTT "DNA-unwinding motif," which enhances the structural dynamics of chromatin and promotes regional DNA base-unpairing. Mutant p53 specifically interacted with MAR-derived oligonucleotides carrying such unwinding motifs, catalyzing DNA strand separation when this motif was located within a structurally labile sequence environment. Addition of GC-clamps to the respective MAR-oligonucleotides or introducing mutations into the unwinding motif strongly reduced DNA strand separation, but supported the formation of tight complexes between mutant p53 and such oligonucleotides. We conclude that the specific interaction of mutant p53 with regions of MAR-DNA with a high potential for base-unpairing provides the basis for the high-affinity binding of mutant p53 to MAR-DNA.

Animals↗

Subcellular localization and mechanisms of nucleocytoplasmic trafficking of steroid receptor coactivator-1.

Steroid hormone receptors are ligand-stimulated transcription factors that modulate gene transcription by recruiting coregulators to gene promoters. Subcellular localization and dynamic movements of transcription factors have been shown to be one of the major means of regulating their transcriptional activity. In the present report we describe the subcellular localization and the dynamics of intracellular trafficking of steroid receptor coactivator 1 (SRC-1). After its synthesis in the cytoplasm, SRC-1 is imported into the nucleus, where it activates transcription and is subsequently exported back to the cytoplasm. In both the nucleus and cytoplasm, SRC-1 is localized in speckles. The characterization of SRC-1 nuclear localization sequence reveals that it is a classic bipartite signal localized in the N-terminal region of the protein, between amino acids 18 and 36. This sequence is highly conserved within the other members of the p160 family. Additionally, SRC-1 nuclear export is inhibited by leptomycin B. The region involved in its nuclear export is localized between amino acids 990 and 1038. It is an unusually large domain differing from the classic leucine-rich NES sequences. Thus SRC-1 nuclear export involves either an alternate type of NES or is dependent on the interaction of SRC-1 with a protein, which is exported through the crm1/exportin pathway. Overall, the intracellular trafficking of SRC-1 might be a mechanism to regulate the termination of hormone action, the interaction with other signaling pathways in the cytoplasm and its degradation.

Amino Acid Sequence↗

Integrin dynamics and matrix assembly: tensin-dependent translocation of alpha(5)beta(1) integrins promotes early fibronectin fibrillogenesis.

Fibronectin matrix assembly is a multistep, integrin-dependent process. To investigate the role of integrin dynamics in fibronectin fibrillogenesis, we developed an antibody-chasing technique for simultaneous tracking of two integrin populations by different antibodies. We established that whereas the vitronectin receptor alpha(v)beta(3) remains within focal contacts, the fibronectin receptor alpha(5)beta(1) translocates from focal contacts into and along extracellular matrix (ECM) contacts. This escalator-like translocation occurs relative to the focal contacts at 6.5 +/- 0.7 microm/h and is independent of cell migration. It is induced by ligation of alpha(5)beta(1) integrins and depends on interactions with a functional actin cytoskeleton and vitronectin receptor ligation. During cell spreading, translocation of ligand-occupied alpha(5)beta(1) integrins away from focal contacts and along bundles of actin filaments generates ECM contacts. Tensin is a primary cytoskeletal component of these ECM contacts, and a novel dominant-negative inhibitor of tensin blocked ECM contact formation, integrin translocation, and fibronectin fibrillogenesis without affecting focal contacts. We propose that translocating alpha(5)beta(1) integrins induce initial fibronectin fibrillogenesis by transmitting cytoskeleton-generated tension to extracellular fibronectin molecules. Blocking this integrin translocation by a variety of treatments prevents the formation of ECM contacts and fibronectin fibrillogenesis. These studies identify a localized, directional, integrin translocation mechanism for matrix assembly.

Biological Transport↗

The rate-limiting enzyme in phosphatidylcholine synthesis regulates proliferation of the nucleoplasmic reticulum.

The nucleus contains a network of tubular invaginations of the nuclear envelope (NE), termed the nucleoplasmic reticulum (NR), implicated in transport, gene expression, and calcium homeostasis. Here, we show that proliferation of the NR, measured by the frequency of NE invaginations and tubules, is regulated by CTP:phosphocholine cytidylyltransferase-alpha (CCTalpha), the nuclear and rate-limiting enzyme in the CDP-choline pathway for phosphatidylcholine (PtdCho) synthesis. In Chinese hamster ovary (CHO)-K1 cells, fatty acids triggered activation and translocation of CCTalpha onto intranuclear tubules characteristic of the NR. This was accompanied by a twofold increase in NR tubules quantified by immunostaining for lamin A/C or the NE. CHO MT58 cells expressing a temperature-sensitive CCTalpha allele displayed reduced PtdCho synthesis and CCTalpha expression and minimal proliferation of the NR in response to oleate compared with CHO MT58 cells stably expressing CCTalpha. Expression of CCTalpha mutants in CHO58 cells revealed that both enzyme activity and membrane binding promoted NR proliferation. In support of a direct role for membrane binding in NR tubule formation, recombinant CCTalpha caused the deformation of liposomes into tubules in vitro. This demonstrates that a key nuclear enzyme in PtdCho synthesis coordinates lipid synthesis and membrane deformation to promote formation of a dynamic nuclear-cytoplasmic interface.

Animals↗

Disability as a public health issue: findings and reflections from the Massachusetts survey of secondary conditions.

Public health researchers and practitioners have begun to recognize the dynamic nature of disability, promote the health of people with disabilities, and develop strategies to prevent secondary conditions among them. To understand the epidemiology of secondary conditions, the authors developed the Massachusetts Survey of Secondary Conditions, a longitudinal study of adults with major disabilities (n = 656) based on a conceptual framework linking disability, mediating factors, and health outcomes. This paper reports baseline data on the number of secondary conditions experienced by survey respondents. Respondents experienced a mean of 5.3 of 17 secondary conditions. More numerous secondary conditions were associated with fair or poor general health and number of days unable to do routine activities. Factors amenable to public health interventions included difficulty with weight and exercise maintenance, tobacco and marijuana use, and experiencing assault. Disability should be a focus in all public health research, policy, and programs.

Adolescent↗

Temperature and concentration-controlled dynamics of rhizobial small heat shock proteins.

A hallmark of alpha-crystallin-type small heat shock proteins (sHsps) is their highly dynamic oligomeric structure which promotes intermolecular interactions involved in subunit exchange and substrate binding (chaperone-like activity). We studied the oligomeric features of two classes of bacterial sHsps by size exclusion chromatography and nanoelectrospray mass spectrometry. Proteins of both classes formed large complexes that rapidly dissociated upon dilution and at physiologically relevant heat shock temperatures. As the secondary structure was not perturbed, temperature- and concentration-dependent dissociations were fully reversible. Complexes formed between sHsps and the model substrate citrate synthase were stable and exceeded the size of sHsp oligomers. Small Hsps, mutated in a highly conserved glycine residue at the C-terminal end of the alpha-crystallin domain, formed labile complexes that disassembled more readily than the corresponding wild-type proteins. Reduced complex stability coincided with reduced chaperone activity.

Chromatography↗

Dynamic protein-DNA architecture of a yeast heat shock promoter.

Here we present an in vivo footprinting analysis of the Saccharomyces cerevisiae HSP82 promoter. Consistent with current models, we find that yeast heat shock factor (HSF) binds to strong heat shock elements (HSEs) in non-heat-shocked cells. Upon heat shock, however, additional binding of HSF becomes apparent at weak HSEs of the promoter as well. Recovery from heat shock results in a dramatic reduction in HSF binding at both strong and weak HSEs, consistent with a model in which HSF binding is subject to a negative feedback regulation by heat shock proteins. In vivo KMnO4 footprinting reveals that the interaction of the TATA-binding protein (TBP) with this promoter is also modulated: heat shock slightly increases TBP binding to the promoter and this binding is reduced upon recovery from heat shock. KMnO4 footprinting does not reveal a high density of polymerase at the promoter prior to heat shock, but a large open complex between the transcriptional start site and the TATA box is formed rapidly upon activation, similar to that observed in other yeast genes.

Base Sequence↗

On the emergence of multifocal cancers.

Several tumors can exist as multiple lesions within a tissue. The lesions may either arise independently, or they may be monoclonal. The importance of multiple lesions for tumor staging, progression, and treatment is subject to debate. Here we use mathematical models to analyze the emergence of multiple, clonally related lesions within a single tissue. We refer to them as multi-focal cancers. We find that multifocal cancers can arise through a dynamical interplay between tumor promoting and inhibiting factors. This requires that tumor promoters act locally, while tumor inhibitors act over a longer range. An example of such factors may be angiogenesis promoters and inhibitors. The model further suggests that multifocal cancers represent an intermediate stage in cancer progression as the tumor evolves away from inhibition and towards promotion. Different patterns of progression can be distinguished: (i) If tumor inhibition is strong, the initial growth occurs as a unifocal and self contained lesion; progression occurs through bifurcation of the lesion and this gives rise to multiple lesions. As the tumor continues to evolve and pushes the balance between inhibition and promotion further towards promotion, the multiple lesions eventually give rise to a single large mass which can invade the entire tissue. (ii) If tumor inhibition is weaker upon initiation, growth can occur as a single lesion without the occurrence of multiple lesions, until the entire tissue is invaded. The model suggests that the sum of the tumor sizes across all lesions is the best characteristic which correlates with the stage and metastatic potential of the tumor.

Journal Article↗

Studies on the ejection properties of asters: astral microtubule turnover influences the oscillatory behavior and positioning of mono-oriented chromosomes.

The position of a mono-oriented chromosome changes as it oscillates to and from the pole to which it is attached. Such oscillatory behavior reveals that the net force on a mono-oriented chromosome is constantly changing. Fluctuations may occur in both the polewardly directed force acting at the kinetochore and the opposing outwardly directed force associated with the aster. We have examined the ejection properties of the aster--as well as the oscillatory behavior and positioning of mono-oriented chromosomes--in relation to astral microtubule turnover. We treated cells containing monopolar spindles with drugs that affect microtubule turnover, either by promoting the depletion of dynamically unstable astral microtubules (nocodazole and colcemid) or by augmenting their numbers and stability (taxol). Both types of drugs stopped the oscillatory behavior of mono-oriented chromosomes within seconds. The final position of the chromosomes depended on how microtubule turnover was affected. In the case of nocodazole and colcemid, non-kinetochore astral microtubules were depleted first and the kinetochore-to-pole distance shortened. In these cells chromosome fragments generated by laser microsurgery were no longer expelled from the center of the aster. By contrast, with taxol the number of non-kinetochore microtubules increased and the astral ejection force became stronger as shown by the finding that the chromosomes moved away from the pole to the periphery of the monaster. Moreover, arms severed from chromosomes at the periphery of the taxol monaster failed to move further away from the aster's center. From these observations we conclude that the oscillatory movements and changing position of a mono-oriented chromosome relative to the pole are mediated by changes in the number of astral microtubules. The dynamic instability of astral microtubules that leads to a rapid turnover may contribute to the astral ejection force by allowing the continual growth of microtubules out from the aster. Growing astral microtubules may exert a pushing force that their rigidity maintains until their depolymerization.

Alkaloids↗

A multiscale model for avascular tumor growth.

The desire to understand tumor complexity has given rise to mathematical models to describe the tumor microenvironment. We present a new mathematical model for avascular tumor growth and development that spans three distinct scales. At the cellular level, a lattice Monte Carlo model describes cellular dynamics (proliferation, adhesion, and viability). At the subcellular level, a Boolean network regulates the expression of proteins that control the cell cycle. At the extracellular level, reaction-diffusion equations describe the chemical dynamics (nutrient, waste, growth promoter, and inhibitor concentrations). Data from experiments with multicellular spheroids were used to determine the parameters of the simulations. Starting with a single tumor cell, this model produces an avascular tumor that quantitatively mimics experimental measurements in multicellular spheroids. Based on the simulations, we predict: 1), the microenvironmental conditions required for tumor cell survival; and 2), growth promoters and inhibitors have diffusion coefficients in the range between 10(-6) and 10(-7) cm2/h, corresponding to molecules of size 80-90 kDa. Using the same parameters, the model also accurately predicts spheroid growth curves under different external nutrient supply conditions.

Animals↗

[The role of 5'proximal fragment of POMC gene in the regulation of transcription in AtT20 cells].

OBJECTIVE: Identification of the vital regions in 5'-flanking sequence of POMC gene in order to determine the characteristics of POMC gene promoter and provide the basis for stadying the effects of multiple hormones and other factors in the POMC gene expression. METHODS: Construction of four kinds of plasmid recombined by rat POMC gene promoter regions and eukarytic expression reporter gene vector pG12-Basic. Each kind of them was identified by restriction maps analysis and DNA sequencing. All recombinant plasmids were transfected into AtT20 cells respectively by using a lipofectamine-based method. Luciferase activity in each cell-lytate was measured by monolight 2010 luminometer. RESULTS: Four recombinant plasmids were constructed, including pGL2-POMC480 (rPOMC -480/+63), pGL2-POMC323 (rPOMC -323/+63), pGL2-POMC165 (rPOMC -165/+63). pGL2-POMC34 (rPOMC -34/+63) Among them, PGL2-POMC34 contains the core promoter which consist of TATA box at -31/-25 bp and transcription start site at +1 bp of POMC gene. It showed that, comparing with the pGL2-POMC34, the relative luciferase activity of pGL2-POMC165 was 2.1 +/- 0.3, pGL2-POMC323 was 3.3 +/- 0.3, pGL2-POMC480 was 3.7 +/- 0.5, and the positive control pGL2-promoter was 4.8 +/- 0.8. CONCLUSIONS: (1) A model system to study the direct effects of various ACTH secretagogues on the dynamics of the POMC gene promoter activity in vitro was established. (2) The studying of POMC promoter basal transcriptional activities identified that the POMC gene has a weak core promoter. The proximal sequence elements were concentrated in the fragment of -34/-165 bp, and the transcriptional activities need POMC 5' promoter -480 bp or a longer region.

Adrenal Cortex Neoplasms↗

[Lesions of zone II flexor tendons: splints and rehabilitation].

Rehabilitation of the hand after flexor tendon injuries in zone 2 is a challenge for both surgeons and therapists. Prevention of adherences is based on early controlled motion programs. Two different and therefore often complementary protocols are presented: 1. Kleinert: an active extension--passive flexion method; 2. Duran: a controlled passive method, as well as the splints required at this early stage of reeducation. The angles of flexion for the wrist and the metacarpal joints are discussed. When the splint is removed, active exercises and activities are proposed according to the interval from the operation and up to return to work. Static splints used to promote analytic movements, and dynamic ones used against stiffness are shown. Treatment of scar is also demonstrated.

Combined Modality Therapy↗

[The outpatient psychiatric clinic for adults at Saint-Jean-sur-Richelieu. A new approach for a clientele in an acute phase.].

The objective of day hospitals is to treat a psychiatric adult clientele, in the acute phase, in a state of crisis. Control of the situation is taken immediately in an intensive way with the aim of avoiding or shortening the usual 24-hour hospitalization period. A systemic approach that takes into consideration the bio-psycho-social aspects, allows a transfer of responsibility from the institution to the individual and his/her system which, in turn, promotes an active and dynamic resolution of the state of crisis.

English Abstract↗