The regulation of death-associated protein (DAP) kinase in apoptosis

DAP-kinase is a calcium/calmodulin (Ca2+/CaM) serine/threonine kinase which positively mediates programmed cell death in a variety of cell systems. The kinase is localized to the actin microfilament and has a unique, multidomain structure consisting of ankyrin repeats and a death domain. One of the substrates of DAP-kinase was identified as myosin light chain (MLC), the phosphorylation of which mediates membrane blebbing. Another arm in its mode of action leads to the formation of autophagic vesicles. Recent work addressed its mode of regulation and identified a mechanism which restrains its apoptotic function in growing cells and enables its activation during cell death. It involves an inhibitory type of autophosphorylation on serine 308 within the CaM regulatory domain. This negative phosphorylation takes place in growing cells and is strongly reduced upon their exposure to the apoptotic stimulus of C6-ceramide. The substitution of serine 308 to alanine, which mimics the ceramide-induced dephosphorylation at this site, increases Ca2+/CaM-independent substrate phosphorylation, as well as binding and overall sensitivity of the kinase to CaM. At the cellular level, it strongly enhances the death-promoting activity of the kinase. These results are consistent with a molecular model in which phosphorylation on serine 308 stabilizes a locked conformation of the CaM regulatory domain within the catalytic cleft and, simultaneously, also interferes with CaM binding. We propose that this unique mechanism of auto-inhibition evolved to impose a locking device which keeps DAP-kinase silent in healthy cells and ensures its activation only in response to apoptotic signals.     

Cell Wall Structure in Cells Adapted to Growth on the Cellulose-Synthesis Inhibitor 2,6-Dichlorobenzonitrile : A Comparison between Two Dicotyledonous Plants and a Graminaceous Monocot

Our previous work (E. Shedletzky, M. Shmuel, D.P. Delmer, D.T.A. Lamport [1990] Plant Physiol 94:980-987) showed that suspension-cultured tomato cells adapted to growth on the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB) have a markedly altered cell wall composition, most notably a markedly reduced level of the cellulose-xyloglucan network. This study compares the adaptation to DCB of two cell lines from dicots (tomato [Lycopersicon esculentum] and tobacco [Nicotiana tabacum]) and a Graminaceous monocot (barley [Hordeum bulbosum] endosperm). The difference in wall structures between the dicots and the monocot is reflected in the very different types of wall modifications induced by growth on DCB. The dicots, having reduced levels of cellulose and xyloglucan, possess walls the major integrity of which is provided by Ca²⁺-bridged pectates because protoplasts can be prepared from these cells simply by treatment with divalent cation chelator and a purified endopolygalacturonase. The tensile strength of these walls is considerably less than walls from nonadapted cells, but wall porosity is not altered. In contrast, walls from adapted barley cells contain very little pectic material and normal to elevated levels of noncellulosic polysaccharides compared with walls from nonadapted cells. Surprisingly, they have tensile strengths higher than their nonadapted counterpart, although cellulose levels are reduced by 70%. Evidence is presented that these walls obtain their additional strength by an altered pattern of cross-linking of polymers involving phenolic components. Such cross-linking may also explain the observation that the porosity of these walls is also considerably reduced. Cells of adapted lines of both the dicots and barley are resistant to plasmolysis, suggesting that they possess very strong connections between the wall and the plasma membrane.     

Characterization of a novel murine retrovirus mixture that facilitates hematopoiesis

A new virus previously arose in BALB/c females mated repeatedly to C57BL/6 (B6) males and then injected with fixed, activated B6 male spleen cells (V. S. Ter-Grigorov, O. Krifuks, E. Liubashevsky, A. Nyska, Z. Trainin, and V. Toder, Nat. Med. 3:37-41, 1997). In the present study, BALB/cJ mice inoculated with virus-containing plasma from affected mice developed splenomegaly, which was caused by increased numbers of Sca-1(+) Lin(-) hematopoietic stem cells (HSC) and their differentiated progeny. Biological and molecular analyses of a new virus revealed a mixture of murine leukemia viruses (MuLVs). These MuLVs comprised ecotropic and mink lung cell focus-forming (MCF) virus classes and are termed Rauscher-like MuLVs because they bear numerous similarities to the ecotropic and MCF viruses of the Rauscher MuLV complex but do not include a spleen focus-forming virus. The ecotropic virus component alone transferred some disease characteristics, while MCF virus alone did not. Thus, we have described a novel virus mixture, termed Rauscher-like MuLV, that causes an increase in hematopoiesis due to activation of pluripotent HSC. Experiments using mice and a protocol that replicated the pregnancy and immunization strategy of the original experiment demonstrated that endogenous BALB/c mouse ecotropic and xenotropic MuLVs are activated by these treatments. Emv1 was expressed in the spleens of multiparous mice but not in those of virgin mice, and Bxv1Emv1-pseudotyped MuLVs were recovered following injection of fixed, activated B6 cells. Thus, multiple pregnancies and allostimuli appear to have provided the signals required for activation of and recombination among endogenous viruses and could have resulted in generation of the Rauscher-like MuLV mixture.     

Using the T-Coffee package to build multiple sequence alignments of protein, RNA, DNA sequences and 3D structures

T-Coffee (Tree-based consistency objective function for alignment evaluation) is a versatile multiple sequence alignment (MSA) method suitable for aligning most types of biological sequences. The main strength of T-Coffee is its ability to combine third party aligners and to integrate structural (or homology) information when building MSAs. The series of protocols presented here show how the package can be used to multiply align proteins, RNA and DNA sequences. The protein section shows how users can select the most suitable T-Coffee mode for their data set. Detailed protocols include T-Coffee, the default mode, M-Coffee, a meta version able to combine several third party aligners into one, PSI (position-specific iterated)-Coffee, the homology extended mode suitable for remote homologs and Expresso, the structure-based multiple aligner. We then also show how the T-RMSD (tree based on root mean square deviation) option can be used to produce a functionally informative structure-based clustering. RNA alignment procedures are described for using R-Coffee, a mode able to use predicted RNA secondary structures when aligning RNA sequences. DNA alignments are illustrated with Pro-Coffee, a multiple aligner specific of promoter regions. We also present some of the many reformatting utilities bundled with T-Coffee. The package is an open-source freeware available from http://www.tcoffee.org/.     

A Mutation Causes MuSK Reduced Sensitivity to Agrin and Congenital Myasthenia

Congenital myasthenic syndromes (CMSs) are a heterogeneous group of genetic disorders affecting neuromuscular transmission. The agrin/muscle-specific kinase (MuSK) pathway is critical for proper development and maintenance of the neuromuscular junction (NMJ). We report here an Iranian patient in whom CMS was diagnosed since he presented with congenital and fluctuating bilateral symmetric ptosis, upward gaze palsy and slowly progressive muscle weakness leading to loss of ambulation. Genetic analysis of the patient revealed a homozygous missense mutation c.2503A>G in the coding sequence of MUSK leading to the p.Met835Val substitution. The mutation was inherited from the two parents who were heterozygous according to the notion of consanguinity. Immunocytochemical and electron microscopy studies of biopsied deltoid muscle showed dramatic changes in pre- and post-synaptic elements of the NMJs. These changes induced a process of denervation/reinnervation in native NMJs and the formation, by an adaptive mechanism, of newly formed and ectopic NMJs. Aberrant axonal outgrowth, decreased nerve terminal ramification and nodal axonal sprouting were also noted. In vivo electroporation of the mutated MuSK in a mouse model showed disorganized NMJs and aberrant axonal growth reproducing a phenotype similar to that observed in the patient’s biopsy specimen. In vitro experiments showed that the mutation alters agrin-dependent acetylcholine receptor aggregation, causes a constitutive activation of MuSK and a decrease in its agrin- and Dok-7-dependent phosphorylation.     

A finite element model of cell-matrix interactions to study the differential effect of scaffold composition on chondrogenic response to mechanical stimulation

Mechanically induced cell deformations have been shown to influence chondrocyte response in 3D culture. However, the relationship between the mechanical stimulation and cell response is not yet fully understood. In this study a finite element model was developed to investigate cell-matrix interactions under unconfined compression conditions, using a tissue engineered encapsulating hydrogel seeded with chondrocytes. Model predictions of stress and strain distributions within the cell and on the cell boundary were shown to exhibit space-dependent responses that varied with scaffold mechanical properties, the presence of a pericellular matrix (PCM), and the cell size. The simulations predicted that when the cells were initially encapsulated into the hydrogel scaffolds, the cell size hardly affected the magnitude of the stresses and strains that were reaching the encapsulated cells. However, with the inclusion of a PCM layer, larger cells experienced enhanced stresses and strains resulting from the mechanical stimulation. It was also noted that the PCM had a stress shielding effect on the cells in that the peak stresses experienced within the cells during loading were significantly reduced. On the other hand, the PCM caused the stresses at the cell-matrix interface to increase. Based on the model predictions, the PCM modified the spatial stress distribution within and around the encapsulated cells by redirecting the maximum stresses from the periphery of the cells to the cell nucleus. In a tissue engineered cartilage exposed to mechanical loading, the formation of a neo-PCM by encapsulated chondrocytes appears to protect them from initially excessive mechanical loading. Predictive models can thus shed important insight into how chondrocytes remodel their local environment in order to redistribute mechanical signals in tissue engineered constructs.     

A test of the relationship between seasonal rainfall and saguaro cacti branching patterns

Reproductive output, as well as photosynthetically active radiation interception and CO₂ uptake, increase as saguaro cacti Carnegiea gigantea (Engelm.) Britt. and Rose branch, and branching increases with increasing moisture. The Sonoran Desert experiences distinct summer and winter precipitation regimes that vary in both geography and scale. Many aspects of saguaro ecology are known to depend on the summer rains, which has resulted in an emphasis on summer rains in the literature. Similarly, branching studies have been limited geographically to areas that receive relatively high amounts of summer rainfall. These studies, therefore, attribute branching patterns to the summer (or possibly annual) rains, and conclusions reflect the summer precipitation bias.
Environmental variability in space was explored in the present study to investigate saguaro branching patterns. I collected height and branching data in thirty saguaro populations across their American range. Stepwise regression was used to determine which climate, vegetation and soil variables best predict branching. Contrary to the literature, this study found that winter precipitation, particularly from January to April, was the best predictor of branching, not summer or annual rain. Surprisingly, the relationship between the summer monsoons (July and August precipitation) and branching was negative. This is likely due to the fact that summer and winter rainfall patterns are geographically distinct. Winter precipitation appears to play a key role in branching, and thus in seed production. This suggests that saguaros benefit from moisture during the winter, possibly utilizing cold-season rains for increasing their reproductive output through branching, and challenging the view that the summer rains dominate virtually every aspect of the saguaro life-cycle, and creating a more balanced view of saguaro ecology.     

Systematic identification of gene annotation errors in the widely used yeast mutation collections

The baker's yeast mutation collections are extensively used genetic resources that are the basis for many genome-wide screens and new technologies. Anecdotal evidence has previously pointed to the putative existence of a neighboring gene effect (NGE) in these collections. NGE occurs when the phenotype of a strain carrying a particular perturbed gene is due to the lack of proper function of its adjacent gene. Here we performed a large-scale study of NGEs, presenting a network-based algorithm for detecting NGEs and validating software predictions using complementation experiments. We applied our approach to four datasets uncovering a similar magnitude of NGE in each (7-15%). These results have important consequences for systems biology, as the mutation collections are extensively used in almost every aspect of the field, from genetic network analysis to functional gene annotation.     

Detecting biomarkers with microdroplet technology

The efficient analysis and noninvasive detection of molecules such as DNA, mRNA, and miRNA for clinical diagnostics requires sensitive, high-throughput methods. By segregating individual sequences within separate compartments, digital procedures allow identification of very rare sequences. These procedures are based on the limiting dilution of biological samples in individual compartments such as droplets of a water-in-oil emulsion, and relies on the discrete counting of a given event, providing an absolute value and quantitative data. Coupled with microfluidic systems, digital procedures could become an essential diagnostic tool for the study of diseases as well as patient management.     

Variation in age and height of onset of reproduction in the saguaro cactus (<Emphasis Type="Italic">Carnegiea gigantea</Emphasis>) in the Sonoran Desert

The objective of this study is to determine the height and age at which reproduction begins (i.e., production of flowers and fruits; the transition to adulthood) in the giant saguaro cactus (Carnegiea gigantea) in four geographically and environmentally distinct populations, and to relate observed variability to environmental differences. The onset of reproduction has been estimated at a height of 2.2 m in near optimal conditions. This value has been widely accepted and applied to populations in less optimal conditions, although variations under less ideal conditions have not been investigated. In addition, previous research has demonstrated that Carnegiea growth rates are highly variable over their range. Thus, even if 2.2 m is a consistent transition height to adulthood over their range, the age of individuals in different populations would be different. I investigate the age and height at which this transition occurs. The author sampled the heights of the shortest reproductive individuals and the tallest non-reproductive individuals to estimate the mean height of the onset of flowering in each of four locales in the northern Sonoran Desert. Using a previously published age-height-growth model, the mean age of the start of reproduction was also computed for the four sites. ANOVA and t-tests were used to compare the average transition to adulthood across sites by both age and height. Statistical results are robust and significant variations in the onset of reproduction are observed by both age and height across the four sites. Saguaro National Park and Organ Pipe Cactus National Monument individuals are transitioning to adulthood, on average, at younger ages and shorter heights than the other two locales. At the arid and marginal Kofa site, individuals that established during the regeneration peak of the late 1800s-early 1900s are only now becoming reproductive (individuals that established around 1899), while at Saguaro National Park, on average, individuals that established in the 1950s are already transitioning to adulthood. These results have implications for regeneration, particularly in marginal locales where regeneration is already limited.