Differences in the regulation of microtubule stability by the pro-rich region variants of microtubule-associated protein 4

We have recently reported a neural variant of microtubule-associated protein 4 with a short pro-rich region (MAP4-SP). Here, we show that the neural MAP4 has reduced microtubule-stabilizing activity, compared to the ubiquitous MAP4 with a long pro-rich region (MAP4-LP), both in vitro and in vivo. Fluorescence recovery after photobleaching analyses revealed that the interaction of MAP4-SP with the microtubules is very rapid, with a half-time of fluorescence recovery of 7 +/- 2.36 s, compared to 19.5 +/- 3.03 s in case of MAP4-LP. The dynamic interaction of MAP4-SP with microtubules in neural cells may contribute to the dynamic behaviors of extending neurites.     

A model of the evolution of dichogamy incorporating sex-ratio selection, anther-stigma interference, and inbreeding depression

Historically, explanations for the evolution of floral traits that reduce self-fertilization have tended to focus on selection to avoid inbreeding depression. However, there is growing support for the hypothesis that such traits also play a role in promoting efficient pollen dispersal by reducing anther-stigma interference. The relative importance of these two selective pressures is currently a popular topic of investigation. To date, there has been no theoretical exploration of the relative contributions of selection to avoid the genetic costs of self-fertilization and selection to promote efficient pollen dispersal on the evolution of floral traits. We developed a population genetic model to examine the influence of these factors on the evolution of dichogamy: the temporal separation of anther maturation and stigma receptivity. Our analysis indicates that anther-stigma interference can favor dichogamy even in the absence of in-breeding depression. Although anther-stigma interference and inbreeding depression are the key forces driving the initial evolution of dichogamy, selection to match the timing of pollen dispersal to the availability of ovules at the population level becomes a more potent force opposing the further evolution of dichogamy as the extent of temporal separation increases. This result may help to explain otherwise puzzling phenomena such as why dichogamy is rarely complete in nature and why dichogamy tends to be associated with asynchronous flower presentation.     

The fission yeast Map4 protein is a novel adhesin required for mating

Cell adhesion is required for many cellular processes. In fungi, cell-cell contact during mating, flocculation or virulence is mediated by adhesins, which typically are glycosyl phosphatidyl inositol (GPI)-modified cell wall glycoproteins. Proteins with internal repeats (PIR) are surface proteins involved in the response to stress. In Schizosaccharomyces pombe no adhesins or PIR proteins have been described. Here we study the S. pombe Map4p, which defines a new class of surface protein that is not GPI-modified and has a serine/threonine rich domain and internal repeats that differ from those present in PIR proteins. Map4p is a mating type-specific adhesin required for mating in h(+) cells and enhances cell adhesion when overexpressed.     

Phenotypic screening of hepatocyte nuclear factor (HNF) 4-gamma receptor knockout mice

Using the mouse as a model organism in pharmaceutical research presents unique advantages as its physiology in many ways resembles the human physiology, it also has a relatively short generation time, low breeding and maintenance costs, and is available in a wide variety of inbred strains. The ability to genetically modify mouse embryonic stem cells to generate mouse models that better mimic human disease is another advantage. In the present study, a comprehensive phenotypic screening protocol is applied to elucidate the phenotype of a novel mouse knockout model of hepatocyte nuclear factor (HNF) 4-gamma. HNF4-gamma is expressed in the kidneys, gut, pancreas, and testis. The first level of the screen is aimed at general health, morphologic appearance, normal cage behaviour, and gross neurological functions. The second level of the screen looks at metabolic characteristics and lung function. The third level of the screen investigates behaviour more in-depth and the fourth level consists of a thorough pathological characterisation, blood chemistry, haematology, and bone marrow analysis. When compared with littermate wild-type mice (HNF4-gamma(+/+)), the HNF4-gamma knockout (HNF4-gamma(-/-)) mice had lowered energy expenditure and locomotor activity during night time that resulted in a higher body weight despite having reduced intake of food and water. HNF4-gamma(-/-) mice were less inclined to build nest and were found to spend more time in a passive state during the forced swim test.     

G protein-coupled receptor 12 deficiency results in dyslipidemia and obesity in mice

Obesity has been proposed to be a result of an imbalance in the physiological system that controls and maintains the body energy homeostasis. Several G-protein coupled receptors (GPCRs) are involved in the regulation of energy homeostasis. To investigate the importance of GPCR12, mice deficient of this receptor (GPCR12 KO) were studied regarding metabolism. Expression of GPCR12 was found primarily in the limbic and sensory systems, indicating its possible involvement in motivation, emotion together with various autonomic functions, and sensory information processing. GPCR12 KO mice were found to have higher body weight, body fat mass, lower respiratory exchange ratio (RER), hepatic steatosis, and were dyslipidemic. Neither food intake nor energy in faeces was affected in the GPCR12 KO mice. However, lower energy expenditure was found in the GPCR12 KO mice, which may explain the obesity. In conclusion, GPCR12 is considered important for the energy balance since GPCR12 KO mice develop obesity and have lower energy expenditure. This may be important for future drugs that target this receptor.     

Ultrastructural comparison of developing mouse embryonic stem cell- and in vivo-derived cardiomyocytes

Embryonic stem cells (ESCs) are expected to become a powerful tool for future regenerative medicine and developmental biology due to their capacity for self-renewal and pluripotency. The present study involves characterization and particularly, the ultrastructure of ESC-derived cardiomyocytes (ESC-CMs). Spontaneously differentiated murine (C57BL/6) ESC-CMs were cultured for 21 days. At different stages, growth characteristics of the CMs were assessed by immunocytochemistry, RT-PCR, transmission electron microscopy, and by addition of chronotropic drugs. EB-derived spontaneously beating cells expressed markers characteristic of CMs including alpha-actinin, desmin, troponin I, sarcomeric myosin heavy chain (MHC), pan-cadherin, connexin 43, cardiac alpha-MHC, cardiac beta-MHC, atrial natriuretic factor (ANF), and myosin light chain isoform-2V (MLC-2V) and responded to drugs in a maturation- and dose-dependent manner. At the ultrasructural level, maturation proceeded with increasing time in culture. In 7+21 days CMs, all sarcomeric components, such as Z-discs, A-, I- and H-bands as well as M-lines, T-tubules, intercalated discs, and the sarcoplasmic reticulum were present. Our data suggest that ESCs can differentiate into functional mature CMs in vitro. Furthermore, ESC-CMs may provide an ideal model for the study of cardiomyocytic development and may be useful for cell therapy of various cardiac diseases.     

A new hypothesis for Ca2+ flows in skeletal muscle and its implications for other cell types

We offer a new hypothesis to explain calcium flows in skeletal muscle cells. Our model accounts for the uptake of Ca²⁺ from the extracellular fluid, and the release of Ca²⁺ from the sarcoplasmic reticulum (SR/ER) (the endoplasmic reticulum in muscle is named sarcoplasmic reticulum); this has engendered difficulty in reviews encompassing both muscle and nonmuscle cells. Here we will typically refer to the organelle as ER, except when specifically discussing muscle cells. The broad consideration of two major, still unexplained properties of skeletal muscle function, namely excitation contraction coupling and capacitative calcium entry are accounted for in a unitary hypothesis. This model allows a reinterpretation of existing data, and points to areas where new investigation may be fruitful. While primarily aimed at explaining Ca²⁺ flows in skeletal muscle, we consider findings of other systems to explore the implications of this hypothesis for other cell types.     

In silico pharmacogenetics of warfarin metabolism

Pharmacogenetic approaches can be instrumental for predicting individual differences in response to a therapeutic intervention. Here we used a recently developed murine haplotype-based computational method to identify a genetic factor regulating the metabolism of warfarin, a commonly prescribed anticoagulant with a narrow therapeutic index and a large variation in individual dosing. After quantification of warfarin and nine of its metabolites in plasma from 13 inbred mouse strains, we correlated strain-specific differences in 7-hydroxywarfarin accumulation with genetic variation within a chromosomal region encoding cytochrome P450 2C (Cyp2c) enzymes. This computational prediction was experimentally confirmed by showing that the rate-limiting step in biotransformation of warfarin to its 7-hydroxylated metabolite was inhibited by tolbutamide, a Cyp2c isoform-specific substrate, and that this transformation was mediated by expressed recombinant Cyp2c29. We show that genetic variants responsible for interindividual pharmacokinetic differences in drug metabolism can be identified by computational genetic analysis in mice.     

A three-level problem-centric strategy for selecting NMR precursor labeling and analytes

We have developed a sequential set of computational screens that may prove useful for evaluating analyte sets for their ability to accurately report on metabolic fluxes. The methodology is problem-centric in that the screens are used in the context of a particular metabolic engineering problem. That is, flux bounds and alternative flux routings are first identified for a particular problem, and then the information is used to inform the design of nuclear magnetic resonance (NMR) experiments. After obtaining the flux bounds via MILP, analytes are first screened for whether the predicted NMR spectra associated with various analytes can differentiate between different extreme point (or linear combinations of extreme point) flux solutions. The second screen entails determining whether the analytes provide unique flux values or multiple flux solutions. Finally, the economics associated with using different analytes is considered in order to further refine the analyte selection process in terms of an overall utility index, where the index summarizes the cost-benefit attributes by quantifying benefit (contrast power) per cost (e.g., NMR instrument time required). We also demonstrate the use of an alternative strategy, the Analytical Hierarchy Process, for ranking analytes based on the individual experimentalist's-generated weights assigned for the relative value of flux scenario contrast, unique inversion of NMR data to fluxes, etc.