Simulation of five intracellular C2+-regulation mechanisms in response to voltage-clamp pulses

We report the simulation of five C2+ regulation mechanisms in response to voltage-clamp pulses applied at the membrane. We obtain the currents generated and also record the C2+ concentration changes in the whole cell during and after the pulse. Our simulator includes models for the L-type voltage-dependent C2+ channels, the C2+ pump, the Na+/C2+ exchanger and a fast buffering system, along with diffusion assuming a spheric cell shape.     

The construction and validation of a new scale for measuring emotional response style in adolescents

BACKGROUND: Research on children's emotional behaviour has been hampered by the lack of psychometric assessment scales. The present study reports on the construction and validation of a new self-report instrument to measure the emotional response styles of adolescents. METHOD: Exploratory and confirmatory factor analyses were carried out on the responses of three samples of adolescents aged between 11 and 14 years (total = 609) to an item pool derived from a scenario study. In addition to assessing reliability, the final form of the questionnaire was concurrently validated using a variety of related psychometric instruments as well as teacher ratings of pupils' behaviour. RESULTS: The factor analyses of the item pool yielded three independent factors labelled social anxiety, malevolent aggression, and social self-esteem. These categories correspond closely to the three dimensions that have emerged consistently from studies of children's temperament, labelled inhibited, undercontrolled and well-adjusted. The three factors comprising the final Emotional Behaviour Scale (EBS) all demonstrated satisfactory internal (coefficient alpha) and re-test reliability. Concurrent validation showed that the three factors were related in predictable ways to other related constructs, and comparisons with teacher ratings of pupils confirmed the relationship between the EBS subscales and children's actual social and emotional behaviour. CONCLUSION: The new scale offers a valid and reliable instrument for assessing adolescent emotional behaviour, and current work is aimed at extending the research to the assessment of young offenders.     

Adaptive evolution of cytochrome c oxidase: Infrastructure for a carnivorous plant radiation

Much recent attention in the study of adaptation of organismal form has centered on developmental regulation. As such, the highly conserved respiratory machinery of eukaryotic cells might seem an unlikely target for selection supporting novel morphologies. We demonstrate that a dramatic molecular evolutionary rate increase in subunit I of cytochrome c oxidase (COX) from an active-trapping lineage of carnivorous plants is caused by positive Darwinian selection. Bladderworts (Utricularia) trap plankton when water-immersed, negatively pressured suction bladders are triggered. The resetting of traps involves active ion transport, requiring considerable energy expenditure. As judged from the quaternary structure of bovine COX, the most profound adaptive substitutions are two contiguous cysteines absent in approximately 99.9% of databased COX I sequences from Eukaryota, Archaea, and Bacteria. This motif lies directly at the docking point of COX I helix 3 and cytochrome c, and modeling of bovine COX I suggests the possibility of an unprecedented helix-terminating disulfide bridge that could alter COX/cytochrome c dissociation kinetics. Thus, the key adaptation in Utricularia likely lies in molecular energetic changes that buttressed the mechanisms responsible for the bladderworts' radical morphological evolution. Along with evidence for COX evolution underlying expansion of the anthropoid neocortex, our findings underscore that important morphological and physiological innovations must often be accompanied by specific adaptations in proteins with basic cellular functions.     

Characterization of a CD46 transgenic pig and protection of transgenic kidneys against hyperacute rejection in non-immunosuppressed baboons

Human membrane cofactor protein (CD46) controls complement activation and when expressed sufficiently as a transgene protects xenografts against complement-mediated rejection, as shown here using non-immunosuppressed baboons and heterotopic CD46 transgenic pig kidney xenografts. This report is of a carefully engineered transgene that enables high-level CD46 expression. A novel CD46 minigene was validated by transfection and production of a transgenic pig line. Pig lymphocytes were tested for resistance to antibody and complement-mediated lysis, transgenic tissues were characterized for CD46 expression, and kidneys were transplanted to baboons without immunosuppression. Absorption of anti-Galalpha(1,3)Gal epitope (anti-GAL) serum antibodies was measured. Transgenic pigs expressed high levels of CD46 in all tissues, especially vascular endothelium, with stable expression through three generations that was readily monitored by flow cytometry of transgenic peripheral blood mononuclear cells (PBMC). Transgenic PBMC pre-sensitized with antibody were highly resistant to human complement-mediated lysis which readily lysed normal pig PBMC. Normal pig kidneys transplanted without cold ischemia into non-immunosuppressed adult baboons survived a median of 3.5 h (n = 7) whereas transgenic grafts (n = 9), harvested at approximately 24-h intervals, were either macroscopically normal (at 29, 48 and 68 h) or showed limited macroscopic damage (median > 50 h). Microscopic assessment of transplanted transgenic kidneys showed only focal tubular infarcts with viable renal tissue elsewhere, no endothelial swelling or polymorph adherence and infiltration by lymphocytes beginning at 3 days. Coagulopathy was not a feature of the histology in four kidneys not rejected and assessed at 48 h or later after transplantation. Baboon anti-GAL serum antibody titers were high before transplantation and, in one extensively analyzed recipient, reduced approximately 8-fold within 5.5 h. The data demonstrate that a single CD46 transgene controls hyperacute kidney graft rejection in untreated baboons despite the presence of antibody and complement deposition. The expression levels, tissue distribution and in vitro functional tests indicate highly efficient CD46 function, controlling both classical and alternative pathway complement activation, which suggests it might be the complement regulator of choice to protect xenografts.     

Transcriptional activators Cat8 and Sip4 discriminate between sequence variants of the carbon source-responsive promoter element in the yeast<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The structural genes for gluconeogenesis in the yeast Saccharomyces cerevisiae are activated by the carbon source-responsive element (CSRE) found in the respective upstream regions. Regulatory genes CAT8 and SIP4 both encode zinc-cluster proteins which can bind to CSRE motifs and activate target genes under conditions of glucose deprivation. In this work, we describe a functional analysis of sequence variants containing single mutations within the strongly activating CSRE(ICL1) motif. While the sequence CCNNNNNNCCG was required as the minimal UAS for gene activation by both Cat8 and Sip4, the activators responded differently to sequence variations in the central part of the CSRE. Our results allowed us to derive a consensus sequence for efficient gene activation by Cat8 (YCCNYTNRKCCG), while a more specific motif is required for activation by Sip4 (TCCATTSRTCCGR). Although their zinc cluster domains are clearly related, Cat8 and Sip4 are not isofunctional. This conclusion is further supported by the finding that biosynthetic derepression of Cat8 in the presence of a nonfermentable carbon source precedes that of Sip4 by about 90 min.     

Regulatory volume increase after secretory volume decrease in colonic epithelial cells under muscarinic stimulation

To address the question of whether colonic secretory cells change their volume in response to carbachol (CCh) stimulation and, if so, the mechanisms involved therein, we used two-photon laser scanning microscopy to measure the volume of individual epithelial cells in the fundus region of crypts isolated from the guinea-pig distal colon. We also measured the volume of human colonic epithelial T84 cells using an electronic sizing technique. Both types of colonocytes responded to stimulation by CCh with shrinkage and then underwent a regulatory volume increase (RVI), even during continued stimulation by CCh. The secretory volume decrease (SVD) induced by CCh was antagonized by atropine, BAPTA loading and niflumic acid, a blocker of Ca²⁺-activated Cl^– channels. An increase in the intracellular free [Ca²⁺] was observed with fura-2 during these volume responses to CCh. Removal of all Na⁺ or K⁺ or of most of the Cl^– from the extracellular solution abolished the RVI, but not the preceding SVD. The RVI, but not the preceding SVD, was abolished by bumetanide, a blocker of the Na⁺-K⁺-2Cl^– cotransporter. We conclude that guinea-pig crypt colonocytes and human T84 cells exhibit a cytosolic Ca²⁺-dependent SVD and undergo a subsequent RVI that is dependent on the operation of Na⁺-K⁺-2Cl^– cotransporters.     

Behaviour in the elevated plus-maze predicts coping after subchronic mild stress in mice

This study was aimed at investigating the coping style of mice subjected to a subchronic unpredictable mild stress procedure and its relationship to initial emotional reactivity. Two inbred strains of mice, the BALB/c ByJ and the C57BL/6 J, known to exhibit distinct emotionality, have been used. They were first observed in the elevated plus-maze and the free exploratory paradigm, each provides a separation of the population in high and low emotional mice. Half of the mice of each strain were then confronted to a 2-week subchronic unpredictable mild stress and tested for their responses in different behavioural situations (consumption of a palatable food, physical state, grooming behaviours and reactivity to a conflict situation). Mice were also tested in the light/dark procedure to assess the effect of the subchronic stress on emotional reactivity. First, a relationship between initial emotional reactivity in the elevated plus-maze and behavioural coping style in response to stress was found, high emotional mice (i.e., BALB mice) displaying inhibited behaviours and less emotional mice (i.e., BL/6 mice) exhibiting few behavioural changes. Furthermore, emotional reactivity was increased in stressed mice compared with nonstressed ones.     

A Spatially Explicit Nanomechanical Model of the Half-Sarcomere: Myofilament Compliance Affects Ca2+-Activation

The force exerted by skeletal muscle is modulated by compliance of tissues to which it is connected. Force of the muscle sarcomere is modulated by compliance of the myofilaments. We tested the hypothesis that myofilament compliance influences Ca2+ regulation of muscle by constructing a computational model of the muscle half sarcomere that includes compliance of the filaments as a variable. The biomechanical model consists of three half-filaments of myosin and 13 thin filaments. Initial spacing of motor domains of myosin on thick filaments and myosin-binding sites on thin filaments was taken to be that measured experimentally in unstrained filaments. Monte-Carlo simulations were used to determine transitions around a three-state cycle for each cross-bridge and between two-states for each thin filament regulatory unit. This multifilament model exhibited less "tuning" of maximum force than an earlier two-filament model. Significantly, both the apparent Ca(2+)-sensitivity and cooperativity of activation of steady-state isometric force were modulated by myofilament compliance. Activation-dependence of the kinetics of tension development was also modulated by filament compliance. Tuning in the full myofilament lattice appears to be more significant at submaximal levels of thin filament activation.