Contractility assessment in enzymatically isolated cardiomyocytes

The use of enzymatically isolated cardiac myocytes is ubiquitous in modern cardiovascular research. Parallels established between cardiomyocyte shortening responses and those of intact tissue make the cardiomyocyte an invaluable experimental model of cardiac function. Much of our understanding regarding the fundamental processes underlying heart function is owed to our increasing capabilities in single-cell stimulation and direct or indirect observation, as well as quantitative analysis of such cells. Of the many important mechanisms and functions that can be readily assessed in cardiomyocytes at all stages of development, contractility is the most representative and one of the most revealing. The purpose of this review is to provide a survey of various methodological approaches in the literature used to assess adult and neonatal cardiomyocyte contractility. The various methods employed to evaluate the contractile behavior of enzymatically isolated mammalian cardiac myocytes can be conveniently divided into two general categories??those employing optical (image)-based systems and those that use transducer-based technologies. This survey is by no means complete, but we have made an effort to include the most popular methods in terms of reliability and accessibility. These techniques are in constant evolution and hold great promise for the next generation of breakthrough studies in cell biology for the prevention, treatment, and cure of cardiovascular diseases.     

Impact of high-fat diet and obesity on energy balance and fuel utilization during the metabolic challenge of lactation

The effects of obesity and a high-fat (HF) diet on whole body and tissue-specific metabolism of lactating dams and their offspring were examined in C57/B6 mice. Female mice were fed low-fat (LF) or HF diets before and throughout pregnancy and lactation. HF-fed mice were segregated into lean (HF-Ln) and obese (HF-Ob) groups before pregnancy by their weight gain response. Compared to LF-Ln dams, HF-Ln, and HF-Ob dams exhibited a greater positive energy balance (EB) and increased dietary fat retention in peripheral tissues (P < 0.05). HF-Ob dams had greater dietary fat retention in liver and adipose compared to HF-Ln dams (P < 0.05). De novo synthesized fat was decreased in tissues and milk from HF-fed dams compared to LF-Ln dams (P < 0.05). However, less dietary and de novo synthesized fat was found in the HF-Ob mammary glands compared to HF-Ln (P < 0.05). Obesity was associated with reduced milk triglycerides relative to lean controls (P < 0.05). Compared to HF diet alone obesity has additional adverse affects, impairing both lipid metabolism as well as milk fat production. Growth rates of LF-Ln litters were lower than HF-Ln and HF-Ob litters (P < 0.05). Total energy expenditure (TEE) of HF-Ob litters was reduced relative to HF-Ln litters, whereas their respiratory exchange ratios (RERs) were increased (P < 0.05). Collectively these data show that consumption of a HF diet significantly affects maternal and neonatal metabolism and that maternal obesity can independently alter these responses.     

Male inclusion in randomized controlled trials of lifestyle weight loss interventions

The prevalence of obesity is similar for men (32.2%) and women (35.5%). It has been assumed that lifestyle weight loss interventions have been developed and tested in predominately female samples, but this has not been systematically investigated. The aim of this review was to investigate total and ethnic male inclusion in randomized controlled trials of lifestyle interventions. PUBMED, MEDLINE, and PSYCHINFO were searched for randomized controlled trials of lifestyle weight loss interventions (N = 244 studies with a total of 95,207 participants) published in the last 10 years (1999-2009). A trial must be in English, included weight loss as an outcome, and tested a dietary, exercise, and/or other behavioral intervention for weight loss. Results revealed samples were on average 27% male vs. 73% female (P < 0.001). Trials recruiting a diseased sample included a larger proportion of males than those not targeting a disease (35% vs. 21%; P < 0.001). About 32% of trials used exclusively female samples, whereas only 5% used exclusively male samples (P < 0.001). No studies in the past 10 years specifically targeted minority males. Ethnic males identified composed 1.8% of total participants in US studies. Only 24% of studies that underrepresented males provided a reason. Males, especially ethnic males, are underrepresented in lifestyle weight loss trials.     

The effects of H-bonding and sterics on the photoreactivity of a trimethyl butyrophenone derivative

The irradiation of ester 1 in methanol and chloroform does not yield any photoproducts, whereas the photolysis of 1 in dry argon-saturated benzene produces cyclobutanol 4, which is converted to lactone 5 by the addition of HCl. Laser-flash photolysis of ester 1 demonstrates that 1 undergoes intramolecular H-atom abstraction to form the biradical 2 (λ(max)～ 310 nm, τ = 200 ns, benzene), which intersystem crosses to photoenols, Z-3 (λ(max)～ 380 nm, τ = 30-60 μs, benzene) and E-3 (λ(max)～ 380 nm, τ = 11 ms, benzene). Density functional theory calculations were performed to support the proposed mechanism for forming cyclobutanol 4 and to explain how steric demand facilitates photoenol E-3 to form cyclobutanol 4 rather than lactone 5.     

Designed ankyrin repeat proteins as scaffolds for multivalent recognition

Ankyrin repeat (AR) proteins are composed of tandem repeats of a basic structural motif of ca. 33 amino acid residues that form a β-turn followed by two antiparallel α-helices. Multiple repeats stack together in a modular fashion to form a scaffold that is ideally suited for the presentation of multiple functional groups and/or recognition elements. Here we describe a biosynthetic strategy that takes advantage of the modular nature of these proteins to generate multivalent ligands that are both chemically homogeneous and structurally well-defined. Glycosylated AR proteins cluster the tetrameric lectin concanavalin A (Con A) at a rate that is comparable to the rate of Con A aggregation mediated by globular protein conjugates and variable density linear polymers. Thus, AR proteins define a new class of multivalent ligand scaffolds that have significant potential application in the study and control of a variety of multivalent interactions.     

Biosynthesis of Poly[(R)-3-hydroxyalkanoate] Copolymers with Controlled Repeating Unit Compositions and Physical Properties

As applications for biodegradable and biologically produced poly[(R)-3-hydroxyalkanoates] (PHAs) grow into more specialized areas, the need to precisely control the repeating unit composition and consequently the physical properties of these polymers has become essential. A previous study reported our development of Escherichia coli LSBJ in order to produce PHA polymers composed of single repeating units ranging from 4 to 12 carbon atoms. This investigation expands the scope of our effort toward controlling the repeating unit composition of a variety of PHA copolymers. The sizes for the repeating units within the copolymers were modulated by feeding specific ratios of fatty acids with defined carbon lengths to E. coli LSBJ, which resulted in defined mole ratios for the repeating units. Various physical properties of the copolymers (including the Young's modulus, elongation to break, and glass-transition temperature) were shown to be strongly dependent upon the mole ratios of repeating units. This work demonstrates that copolymers of PHAs with repeating units from 4 to 12 carbons can be incorporated accurately to obtain any desired mole ratio within the PHA copolymers. Our methodology may thus be extended to generate tailor-made PHA copolymers with prescribed values for key sets of physical properties.     

Chitin-Like Molecules Associate with Cryptococcus neoformans Glucuronoxylomannan To Form a Glycan Complex with Previously Unknown Properties

In prior studies, we demonstrated that glucuronoxylomannan (GXM), the major capsular polysaccharide of the fungal pathogen Cryptococcus neoformans, interacts with chitin oligomers at the cell wall-capsule interface. The structural determinants regulating these carbohydrate-carbohydrate interactions, as well as the functions of these structures, have remained unknown. In this study, we demonstrate that glycan complexes composed of chitooligomers and GXM are formed during fungal growth and macrophage infection by C. neoformans. To investigate the required determinants for the assembly of chitin-GXM complexes, we developed a quantitative scanning electron microscopy-based method using different polysaccharide samples as inhibitors of the interaction of chitin with GXM. This assay revealed that chitin-GXM association involves noncovalent bonds and large GXM fibers and depends on the N-acetyl amino group of chitin. Carboxyl and O-acetyl groups of GXM are not required for polysaccharide-polysaccharide interactions. Glycan complex structures composed of cryptococcal GXM and chitin-derived oligomers were tested for their ability to induce pulmonary cytokines in mice. They were significantly more efficient than either GXM or chitin oligomers alone in inducing the production of lung interleukin 10 (IL-10), IL-17, and tumor necrosis factor alpha (TNF-α). These results indicate that association of chitin-derived structures with GXM through their N-acetyl amino groups generates glycan complexes with previously unknown properties.     

Secreted VAPB/ALS8 major sperm protein domains modulate mitochondrial localization and morphology via growth cone guidance receptors

The VAPB/ALS8 major sperm protein domain (vMSP) is implicated in amyotrophic lateral sclerosis and spinal muscular atrophy, yet its function in the nervous system is not well understood. In Caenorhabditis elegans and Drosophila, the vMSP is cleaved from its transmembrane anchor and secreted in a cell type-specific fashion. We show that vMSPs secreted by neurons act on Lar-like protein-tyrosine phosphatase and Roundabout growth cone guidance receptors expressed in striated muscle. This signaling pathway promotes Arp2/3-dependent actin remodeling and mitochondrial localization to actin-rich muscle I-bands. C. elegans VAPB mutants have mitochondrial localization, morphology, mobility, and fission/fusion defects that are suppressed by Lar-like receptor or Arp2/3 inactivation. Hence, growth cone guidance receptor pathways that remodel the actin cytoskeleton have unanticipated effects on mitochondrial dynamics. We propose that neurons secrete vMSPs to promote striated muscle energy production and metabolism, in part through the regulation of mitochondrial localization and function. VIDEO ABSTRACT: