Roles of creatine in the regulation of cardiac protein synthesis

The observation that increased muscular activity leads to muscle hypertrophy is well known, but identification of the biochemical and physiological mechanisms by which this occurs remains an important problem. Experiments have been described (5, 6) which suggest that creatine, an end product of contraction, is involved in the control of contractile protein synthesis in differentiating skeletal muscle cells and may be the chemical signal coupling increased muscular activity and the increased muscular mass. During contraction, the creatine concentration in muscle transiently increases as creatine phosphate is hydrolyzed to regenerate ATP. In isometric contraction in skeletal muscle for example, Edwards and colleagues (3) have found that nearly all of the creatine phosphate is hydrolyzed. In this case, the creatine concentration is increased about twofold, and it is this transient change in creatine concentration which is postulated to lead to increased contractile protein synthesis. If creatine is found in several intracellular compartments, as suggested by Lee and Vissher (7), local changes in concentration may be greater then twofold. A specific effect on contractile protein synthesis seems reasonable in light of the work of Rabinowitz (13) and of Page et al. (11), among others, showing disproportionate accumulation of myofibrillar and mitochondrial proteins in response to work-induced hypertrophy and thyroxin-stimulated growth. Previous experiments (5, 6) have shown that skeletal muscles cells which have differentiated in vitro or in vivo synthesize myosin heavy-chain and actin, the major myofibrillar polypeptides, faster when supplied creatine in vitro. The stimulation is specific for contractile protein synthesis since neither the rate of myosin turnover nor the rates of synthesis of noncontractile protein and DNA are affected by creatine. The experiments reported in this communication were undertaken to test whether creatine selectively stimulates contractile protein synthesis in heart as it does in skeletal muscle.     

Effect of growth rate on the amounts of ribosomal and transfer ribonucleic acids in yeast.

The steady-state growth rate of Saccharomyces cerevisiae was varied by growing the cells in different media. The total amount of ribonucleic acid (RNA) per cell was found to decrease as a nonlinear function of decreasing growh rate. The RNA from cells growing in different media was analyzed by polyacrylamide gel electrophoresis. Although the amounts of both ribosomal RNA and transfer RNA decreased with decreasing growth rate, the ratio of ribosomal to transfer RNA was not constant. As the growth rate was reduced the ribosomal RNA fraction decreased slightly, whereas the transfer RNA fraction increased slightly. Thus the levels of ribosomal and transfer RNA were regulated to similar yet different extents. The levels of the different ribosomal RNA species were more closely coordinated. At all growth rates the ribosomal RNAs (including 5S RNA) were present in equimolar amounts. The rate of protein synthesis in yeast cells also decreased with decreasing growth rate. The low rates of protein synthesis did not appear to be due to limiting numbers of ribosomes or transfer RNA molecules.     

Microengineered systems with iPSC-derived cardiac and hepatic cells to evaluate drug adverse effects

Hepatic and cardiac drug adverse effects are among the leading causes of attrition in drug development programs, in part due to predictive failures of current animal or in vitro models. Hepatocytes and cardiomyocytes differentiated from human induced pluripotent stem cells (iPSCs) hold promise for predicting clinical drug effects, given their human-specific properties and their ability to harbor genetically determined characteristics that underlie inter-individual variations in drug response. Currently, the fetal-like properties and heterogeneity of hepatocytes and cardiomyocytes differentiated from iPSCs make them physiologically different from their counterparts isolated from primary tissues and limit their use for predicting clinical drug effects. To address this hurdle, there have been ongoing advances in differentiation and maturation protocols to improve the quality and use of iPSC-differentiated lineages. Among these are in vitro hepatic and cardiac cellular microsystems that can further enhance the physiology of cultured cells, can be used to better predict drug adverse effects, and investigate drug metabolism, pharmacokinetics, and pharmacodynamics to facilitate successful drug development. In this article, we discuss how cellular microsystems can establish microenvironments for these applications and propose how they could be used for potentially controlling the differentiation of hepatocytes or cardiomyocytes. The physiological relevance of cells is enhanced in cellular microsystems by simulating properties of tissue microenvironments, such as structural dimensionality, media flow, microfluidic control of media composition, and co-cultures with interacting cell types. Recent studies demonstrated that these properties also affect iPSC differentiations and we further elaborate on how they could control differentiation efficiency in microengineered devices. In summary, we describe recent advances in the field of cellular microsystems that can control the differentiation and maturation of hepatocytes and cardiomyocytes for drug evaluation. We also propose how future research with iPSCs within engineered microenvironments could enable their differentiation for scalable evaluations of drug effects.     

Segregation in the African wintering ranges of English and Swiss Swallow Hirundo rustica populations: a stable isotope study

Capsule Stable isotope analysis of Swallow feathers, grown in Africa, revealed significant differences between populations breeding in Switzerland and England.

Aims To investigate the extent to which Swallow populations breeding in Switzerland and England are separated on their African wintering grounds.

Methods Swallows were caught at breeding colonies, biometric measurements were taken and feathers, grown in Africa, were collected. Feathers were combusted in a Carlo Erba C/N/S analyser and the δ¹³C and δ¹⁵N signatures were measured using a mass spectrometer.

Results The δ¹³C signatures of Swiss birds were significantly more depleted than those of birds from England. The δ¹⁵N signatures did not differ between the two populations.

Conclusion Birds from Switzerland and England probably winter in geographically distinct parts of Africa. The Swiss birds probably feed on prey that are more reliant on C₃ vegetation, from woodlands, than the prey of English birds, which are more reliant on C₄ vegetation, from grasslands.     

The changing incidence of human papillomavirus-associated oropharyngeal cancer using multiple imputation from 2000 to 2010 at a Comprehensive Cancer Centre

Introduction Human papillomavirus (HPV) is a risk and prognostic factor for oropharyngeal cancer (OPC). Determining whether the incidence of HPV-associated OPC is rising informs health policy. Methods HPV status was ascribed using p16 immunohistochemistry in 683/1474 OPC patients identified from the Princess Margaret Hospital's Cancer Registry (from 2000 to 2010). Missing p16 data was estimated using multiple (n = 100) imputation (MI) and validated using an independent OPC cohort (n = 214). Non-OPC head and neck squamous cell carcinoma (HNSCC) (n = 3262) were also used for time-trend comparison. Regression was used to compare HNSCC subsets and time-trends. The c-index was used to measure the predictive ability of MI. Results The incidence of OPC rose from 23.3% of all HNSCC in 2000 to 31.2% in 2010 (p = 0.002). In the subset of OPC tested for p16, there was no change in p16 positivity over time (p = 0.9). However, p16 testing became more frequent over time (p < 0.0001), but was nonetheless biased, favouring never-smokers [OR 1.87 (95% CI 1.29–2.70)] and tumors of the tonsil [OR 2.30 (1.52–3.47)] or base-of-tongue [OR 1.72 (1.10–2.70)]. These same factors were also associated with p16-positivity [ORs 3.22 (1.27–8.16), 7.26 (3.50–15.1), 5.83 (2.70–12.7), respectively]. Following MI and normalization, the proportion of OPC that was p16-associated rose from 39.8% in 2000 to 65.0% in 2010, p = 0.002, fully explaining the rise in OPC in our patient population. Conclusion The rise in HNSCC referrals seen from 2000 to 2010 at our institution was driven primarily by p16-associated OPC. MI was necessary to derive reliable conclusions when cases with missing data are considerable.     

Arabinoxylan and mono- and dimeric ferulic acid release from brewer’s grain and wheat bran by feruloyl esterases and glycosyl hydrolases from<Emphasis Type="Italic"> Humicola insolens</Emphasis>

An enzyme preparation from the thermophilic fungus Humicola insolens, Ultraflo L, was able to solubilise more than half of the biomass of brewers grain and wheat bran, two agro-industrial co-products. While almost all of the ferulic acid was released in the free form, the majority of diferulates were released still attached to soluble feruloylated oligosaccharides, except for the 8,5 benzofuran form, which remained mostly in the residue. H. insolens also produced an esterase capable of releasing over 50% of p-coumaric acid present in wheat bran, but only 9% from the brewers grain. The polysaccharide content in the residues after enzyme treatment comprised mostly cellulose and arabinoxylan, which suggests that part of the arabinoxylan in these residues is inaccessible to the xylanases of H. insolens. Differences in the solubilised arabinose-to-xylose ratio coupled to high free ferulate release suggest that the structure of feruloylated arabinoxylan in barley and wheat may differ.     

Stable isotope ratios indicate that body condition in migrating passerines is influenced by winter habitat

Although predicted some time ago, there has been little success in demonstrating that the overall fitness of migratory birds depends on the combined influences of their experiences over all seasons. We used stable carbon isotope signatures (delta13C) in the claws of migrating black-throated blue warblers Dendroica caerulescens to infer their wintering habitats and investigated whether winter habitat selection can be linked to condition during migration. Resident bird species with low delta13C corresponded to selection of more mesic habitats, and migrating birds with low delta13C were in better condition than conspecifics with higher delta13C signatures. These findings concur with empirical observations on the wintering grounds, where dominants (mostly males) tend to exclude subordinates from mesic areas (considered to be high-quality habitats). We believe that variation in condition during migration may be one of the key factors determining differences in arrival times and condition at the breeding areas, which in turn have a major influence on reproductive success.     

Anion binding to a protein-protein complex lacks dependence on net charge

The binding of anions to proteins occurs in numerous physiological and metabolic processes. In an effort to understand the factors important in these interactions, we have studied the weak binding of phosphate and sulfate to a protein-protein complex using isothermal titration calorimetry. To our knowledge, this is the first system in which the thermodynamics of anion binding have been determined calorimetrically. By studying both phosphate and sulfate binding and using a range of pH values, the charge on the anion was varied from approximately -1 to -2. Surprisingly, no dependence of the binding energetics on the charge of the anion was observed. This result indicates that charge-charge interactions are not the dominant factor in binding and suggests the importance of hydrogen bonding in specifically recognizing and coordinating anions.     

Stabilization of proteins by ligand binding: application to drug screening and determination of unfolding energetics

The observed stability of a protein is altered when ligands bind, which results in a shift in the melting temperature (T(m)). Binding to the native state in the absence of binding to the denatured state will necessarily lead to an increase in the T(m), while binding to the unfolded state in the absence of native state binding will decrease the T(m) relative to that of the protein in the absence of ligand. These effects are required by the thermodynamics of reversible folding. However, the relationship between binding affinity and the magnitude of the observed temperature shift is not a simple correlation (i.e., a larger shift in T(m) does not necessarily mean tighter binding) and is complicated by interaction with the denatured state. Using exact simulations, the range of behavior for the dependence of the observed T(m) shift on the energetics of ligand binding is investigated here. Specifically, differential scanning calorimetry (DSC) curves are simulated for protein unfolding in the presence of ligands binding to both the native and denatured states. The results have implications for drug screening and the determination of heat capacity changes for protein unfolding.     

The effect of physical and chemical treatment on the mechanical properties of the cell wall of the alga Chara corallina

Single large internode cells of the charophyte (giant alga) Chara corallina were dissected to give sheets of cell wall, which were then notched and their mechanical properties in tension determined. The cells were subjected to a thermal treatment in excess water (cf. cooking), which had little effect on strength but increased the stiffness, contrasting with the behaviour of higher-plant tissues. Extraction in CDTA (cyclohexane-trans-1,2-diamine-N,N,N',N'-tetraacetate) or 4 M KOH reduced the strength from 17 MPa to 10 MPa, although sequential extraction in CDTA and 4 M KOH reduced the strength further to 4 MPa. The stiffness decreased from 500 MPa to 300 MPa on extraction in CDTA or 4 M KOH, while falling to 70 MPa after extraction in CDTA followed by 4 M KOH. Conventional sequential extraction in CDTA, Na2CO3 at 1 degrees C and 20 degrees C, and KOH at 0.5 M, 1 M, 2 M and 4 M caused a gradual decrease in stiffness and strength after the CDTA treatment to the same lower values. This result is in keeping with mechanical properties for plant tissues, but in contrast to the removal of pectic polysaccharides from model cell wall systems, which does not reduce the stiffness.