Downregulation of cyclin-dependent kinase inhibitors p21 and p27 in pressure-overload hypertrophy

We postulated that the cyclin-dependent kinase inhibitors p21 and p27 could regulate the alterations in growth potential of cardiomyocytes during left ventricular hypertrophy (LVH). LVH was induced in adult rat hearts by aortic constriction (AC) and was monitored at days 0, 1, 3, 7, 14, 21, and 42 postoperation. Relative to sham-operated controls (SH), left ventricle (LV) weight-to-body weight ratio in AC increased progressively with time without significant differences in body weight or right ventricle weight-to-body weight ratio. Atrial natriuretic factor mRNA increased significantly in AC to 287% at day 42 compared with SH (P < 0.05), whereas p21 and p27 mRNA expression in AC rats decreased significantly by 58% (P < 0.03) and 40% (P < 0.05) at day 7, respectively. p21 and p27 protein expression decreased significantly from days 3 to 21 in AC versus SH, concomitant with LV adaptive growth. Immunocytochemistry showed p21 and p27 expression in cardiomyocyte nuclei. Thus downregulation of p21 and p27 may modulate the adaptive growth of cardiomyocytes during pressure overload-induced LVH.

Heterosis for yield and its physiological determinants in wheat

Heterosis in hybrid wheat varieties produced using a chemical hybridising agent was assessed in field experiments. Hyno Esta and its parents were compared in factorial combinations of four-seed rates (25-300 seeds m(-2)) and two nitrogen fertilizer rates (0 and 200 kg N ha(-1)) in 2001/02 and again in 2002/03. Hyno Rista and Hyno Renta and their parents were compared at two-seed rates in 2001/02. Hyno Rista and its parents were added factorially to the Hyno Esta experiment in 2002/03, while Hyno Renta and Hybred and their parents were compared at two seed rates in 2002/03. Mid parent heterosis for grain yield was found in three hybrids and two of these showed high parent heterosis. High parent heterosis in Hyno Esta over a range of sowing densities was mostly exhibited in total biomass but also, in one of two years, in harvest index. High parent heterosis in Hyno Renta was associated more with harvest index than with biomass. The heterosis for biomass in Hyno Esta resulted from greater interception of photosynthetically active radiation (PAR) than the male parent, with better radiation use efficiency than the female parent. In both seasons Hyno Esta achieved grain numbers per ear at least as high as the high parent for this trait (Audace), and combined this with mean grain weights at least as heavy as the high parent for mean grain weight (Estica). Much of the increased biomass and grain yield in the hybrid came late in the season as high parent heterosis was expressed for both maximum grain filling rate and grain filling duration. Heterosis was higher when nitrogen was applied than when withheld; only greater at lower seed rates when expressed in proportionate terms (e.g. as a percentage of the parents), rather than in absolute terms (e.g. t ha(-1)); and greater in the year with the cooler and wetter summer.

Molecular, cellular and physiological investigation of myostatin propeptide-mediated muscle growth in adult mice

Inhibition of myostatin signalling or its biological activity has recently emerged as a potential remedial approach against muscle wasting and degenerative diseases such as muscular dystrophies. In the present study we systemically administered a recombinant AAV8 vector expressing a mutated myostatin propeptide (AAV8ProMyo) to healthy mice in order to assess its impact on the histological, cellular and physiological properties of the skeletal muscle, exploiting the fact that myostatin is naturally inhibited by its own propeptide. We report that a single intravenous administration of AAV8ProMyo leads to increases in muscle mass of tibialis anterior, extensor digitorum longus and gastrocnemius muscles 8 weeks post-injection and tibialis anterior, gastrocnemius and rectus femoris muscles 17 weeks post-injection. Moreover, treatment resulted in muscle fibre hypertrophy but not hyperplasia, with IIB myofibres responding to the greatest extent following propeptide-induced myostatin inhibition. Additionally, myofibre nuclear: cytoplasmic ratio was decreased in the AAV8ProMyo treated animals. Importantly, the hypertrophic EDL muscle 8 weeks after AAV8ProMyo treatment did not show the dramatic decrease in specific force displayed by the germline myostatin null mice. (C) 2009 Elsevier B.V. All rights reserved.

Skeletal muscle fibre plasticity in response to selected environmental and physiological stimuli

Skeletal muscle constitutes a highly adaptable and malleable tissue that responds to environmental and physiological challenges by changing its phenotype in terms of size and composition, outcomes that are brought about by changes in gene expression, biochemical and metabolic properties. Both the short- and long-term effects of nutritional alterations on skeletal muscle homeostasis have been defined as the object of intensive research over the last thirty years. This review focuses predominantly on assimilating our understanding of the changes in muscle fibre phenotype and functional properties induced by either food restriction or alternatively existing on a high fat diet. Firstly, food restriction has been shown in a number of studies to decrease the myofibre cross sectional area and consistently, it has been found that glycolytic type IIB fibres are more prone to atrophy than oxidative fibres. Secondly, in rodents, a high fat diet has been shown to induce an oxidative profile in skeletal muscle, although obese humans usually show higher numbers of glycolytic type IIB fibres. Moreover, attention is paid to the effect of prenatal maternal food restriction on muscle development of the offspring in various species. A key point related to these experiments is the timing of food restriction for the mother. Furthermore, we explore extensively the seemingly species-specific response to maternal malnutrition. Finally, key signalling molecules that play a pivotal role in energy metabolism, fibre type transitions and muscle hypertrophy are discussed in detail.

Shoot dieback in clipped young Golden Leyland (Cupressocyparis leylandii) trees - a physiological mechanism?

Shoot dieback is a problem in frequently trimmed Leyland hedges and is increasingly affecting gardeners’ choice of hedge trees, having a negative effect on a conifer nursery industry. Some damage can be attributed to the feeding by aphids, but it is unclear if there are also underlying physiological causes. In this study, we tested the hypothesis that shoot-clipping of conifer trees during adverse growing conditions (i.e. high air temperature and low soil moisture) could be leading to shoot ‘dieback’. Three-year-old Golden Leyland Cypress (x Cupressocyparis leylandii ‘Excalibur Gold’) plants were subjected to either a well-watered or droughted irrigation regime and placed in either a ‘hot’ (average day temperature = 40°C) or a ‘cool’ (average day temperature = 27°C) glasshouse compartment. Half of the plants from each glasshouse were clipped on Day 14 and again on Day 50. Measurements of soil moisture content (SMC), net CO2 assimilation rate (A), stomatal conductance (gs), branchlet xylem water potential (XWP), plant height and foliage colour were made. Within the clipped and unclipped treatments of both glasshouse compartments, plants from the droughted regime had significantly lower values for A, gs and XWP than those from the well-watered regime. However, there was no difference in these parameters between the hot and cool glasshouse compartments. The trends seen for A, gs and XWP of all treatments generally mirrored changes in SMC indicating a direct effect of water supply on these parameters. By the end of the experiment the overall foliage colour of plants from the hot glasshouse was darker than that of plants from the cool glasshouse and the overall foliage colour was also darker following shoot clipping. In general, shoot clipping led to increases in A, gs XWP and SMC. This may be due to the reduction in total leaf area leading to a greater supply of water for the remaining leaves. No shoot ‘dieback’ was observed in any treatment in response to drought stress or shoot-clipping.

Glycogen phosphorylase inhibitors: a free energy perturbation analysis of glucopyranose spirohydantoin analogues

GP catalyzes the phosphorylation of glycogen to Glc-1-P. Because of its fundamental role in the metabolism of glycogen, GP has been the target for a systematic structure-assisted design of inhibitory compounds, which could be of value in the therapeutic treatment of type 2 diabetes mellitus. The most potent catalytic-site inhibitor of GP identified to date is spirohydantoin of glucopyranose (hydan). In this work, we employ MD free energy simulations to calculate the relative binding affinities for GP of hydan and two spirohydantoin analogues, methyl-hydan and n-hydan, in which a hydrogen atom is replaced by a methyl- or amino group, respectively. The results are compared with the experimental relative affinities of these ligands, estimated by kinetic measurements of the ligand inhibition constants. The calculated binding affinity for methyl-hydan (relative to hydan) is 3.75 +/- 1.4 kcal/mol, in excellent agreement with the experimental value (3.6 +/- 0.2 kcal/mol). For n-hydan, the calculated value is 1.0 +/- 1.1 kcal/mol, somewhat smaller than the experimental result (2.3 +/- 0.1 kcal/mol). A free energy decomposition analysis shows that hydan makes optimum interactions with protein residues and specific water molecules in the catalytic site. In the other two ligands, structural perturbations of the active site by the additional methyl- or amino group reduce the corresponding binding affinities. The computed binding free energies are sensitive to the preference of a specific water molecule for two well-defined positions in the catalytic site. The behavior of this water is analyzed in detail, and the free energy profile for the translocation of the water between the two positions is evaluated. The results provide insights into the role of water molecules in modulating ligand binding affinities. A comparison of the interactions between a set of ligands and their surrounding groups in X-ray structures is often used in the interpretation of binding free energy differences and in guiding the design of new ligands. For the systems in this work, such an approach fails to estimate the order of relative binding strengths, in contrast to the rigorous free energy treatment.

Kinetic and crystallographic studies of glucopyranose spirohydantoin and glucopyranosylamine analogs inhibitors of glycogen phosphorylase

Glycogen phosphorylase (GP) is currently exploited as a target for inhibition of hepatic glycogenolysis under high glucose conditions. Spirohydantoin of glucopyranose and N-acetyl-beta-D-glucopyranosylamine have been identified as the most potent inhibitors of GP that bind at the catalytic site. Four spirohydantoin and three beta-D-glucopyranosylamine analogs have been designed, synthesized and tested for inhibition of GP in kinetic experiments. Depending on the functional group introduced, the K(i) values varied from 16.5 microM to 1200 microM. In order to rationalize the kinetic results, we determined the crystal structures of the analogs in complex with GP. All the inhibitors bound at the catalytic site of the enzyme, by making direct and water-mediated hydrogen bonds with the protein and by inducing minor movements of the side chains of Asp283 and Asn284, of the 280s loop that blocks access of the substrate glycogen to the catalytic site, and changes in the water structure in the vicinity of the site. The differences observed in the Ki values of the analogs can be interpreted in terms of variations in hydrogen bonding and van der Waals interactions, desolvation effects, ligand conformational entropy, and displacement of water molecules on ligand binding to the catalytic site.

Cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors: detection methods and activity measurements

The cyclin/cyclin-dependent kinase (Cdk) complexes and the Cdk inhibitors (CDKI) are crucial regulators of cell cycle progression in all eukaryotic cells. Using rat cardiac myocytes as a model system, this chapter provides a detailed account of methods that can be employed to measure both cyclin/Cdk activity in cells and the extent of CDKI inhibitory activity present in a particular cell type.

The role of polyphenolic compounds in the diet as inhibitors of platelet function

Platelets play a substantial role in cardiovascular disease, and for many years there has been a search for dietary components that are able to inhibit platelet function and therefore decrease the risk of cardiovascular disease. Platelets can be inhibited by alcohol, dietary fats and some antioxidants, including a group of compounds, the polyphenols, found in fruits and vegetables. A number of these compounds have been shown to inhibit platelet function both in vitro and in vivo. In the present study the effects of the hydroxycinnamates and the flavonoid quercetin on platelet activation and cell signalling in vitro were investigated. The hydroxycinnamates inhibited platelet function, although not at levels that can be achieved in human plasma by dietary intervention. However, quercetin inhibited platelet aggregation at levels lower than those previously reported. Quercetin was also found to inhibit intracellular Ca mobilisation and whole-cell tyrosine protein phosphorylation in platelets, which are both processes essential for platelet activation. The effect of polyphenols on platelet aggregation in vivo was also investigated. Twenty subjects followed a low-polyphenol diet for 3 d before and also during supplementation. All subjects were supplemented with a polyphenol-rich meal every lunchtime for 5 d. Platelet aggregation and plasma flavonols were measured at baseline and after 5 d of dietary supplementation. Total plasma flavonoids increased significantly after the dietary intervention period (P = 0.001). However, no significant changes in ex vivo platelet aggregation were observed. Further investigation of the effects of individual polyphenolic compounds on platelet function, both in vitro and in vivo, is required in order to elucidate their role in the relationship between diet and the risk of cardiovascular disease.

Effect of fluid mechanical stresses and plasma constituents on aggregation of LDL

LDL aggregates when exposed to even moderate fluid mechanical stresses in the laboratory, yet its half-life in the circulation is 2-3 days, implying that little aggregation occurs. LDL may be protected from aggregation in vivo by components of plasma, or by a qualitative difference in flows. Previous studies have shown that HDL and albumin inhibit the aggregation induced by vortexing. Using a more reproducible method of inducing aggregation and assessing aggregation both spectrophotometrically and by sedimentation techniques, we showed that at physiological concentrations, albumin is the more effective inhibitor, and that aggregation is substantially but not completely inhibited in plasma. Heat denatured and fatty-acid-stripped albumin were more effective inhibitors than normal albumin, supporting the idea that hydrophobic interactions are involved. Aggregation of LDL in a model reproducing several aspects of flow in the circulation was 200-fold slower, but was still inhibited by HDL and albumin, suggesting similar mechanisms are involved. Within the sensitivity of our technique, LDL aggregation did not occur in plasma exposed to these flows.jlr Thus, as a result of the characteristics of blood flow and the inhibitory effects of plasma components, particularly albumin, LDL aggregation is unlikely to occur within the circulation.

Application of the asymmetric hetero Diels-Alder reaction for synthesising carbohydrate derivatives and glycosidase inhibitors

This review provides a discussion of recent developments in the asymmetric hetero Diels-Alder reaction (AHDAR), with particular emphasis on the synthesis of carbohydrates, their derivatives, and inhibitors of carbohydrate processing enzymes.

The impact of flavonoids on memory: physiological and molecular considerations

Emerging evidence suggests that a group of dietary-derived phytochemicals known as flavonoids are able to induce improvements in memory acquisition, consolidation, storage and retrieval. These low molecular weight polyphenols are widespread in the human diet, are absorbed to only a limited degree and localise in the brain at low concentration. However, they have been found to be highly effective in reversing age-related declines in memory via their ability to interact with the cellular and molecular architecture of the brain responsible for memory. These interactions include an ability to activate signalling pathways, critical in controlling synaptic plasticity, and a potential to induce vascular effects capable of causing new nerve cell growth in the hippocampus. Their ability to activate the extracellular signal-regulated kinase (ERK1/2) and the protein kinase B (PKB/Akt) signalling pathways, leading to the activation of the cAMP response element-binding protein (CREB), a transcription factor responsible for increasing the expression of a number of neurotrophins important in de. ning memory, will be discussed. How these effects lead to improvements in memory through induction of synapse growth and connectivity, increases in dendritic spine density and the functional integration of old and new neurons will be illustrated. The overall goal of this critical review is to emphasize future areas of investigation as well as to highlight these dietary agents as promising candidates for the design of memory-enhancing drugs with relevance to normal and pathological brain ageing (161 references).

Physiological changes in Campylobacter jejuni on entry into stationary phase

Campylobacter jejuni NCTC 11168 does not exhibit the general increase in cellular stress resistance on entry into stationary phase that is seen in most other bacteria. This is consistent with the lack of global stationary phase regulatory elements in this organism. deduced from an analysis of its genome sequence. We now show that C. jejuni NCTC 11168 does undergo certain changes in stationary phase, of a pattern not previously described. As cells entered stationary phase there was a change in membrane fatty acid composition, principally a decrease in the proportion of unsaturated fatty acids and an increase in the content of cyclopropane and short-chain fatty acids. These changes in membrane composition were accompanied by an increase in the resilience of the cell membrane towards loss of integrity caused by pressure and an increase in cellular pressure resistance. By contrast. there were no major changes in resistance to acid or heat treatment. A similar pattern of changes in stress resistance on entry, into stationary phase was seen in C. jejuni NCTC 11351, the type strain. These changes appear to represent a restricted Physiological response to the conditions existing in stationary phase cultures, in an organism having limited capacity for genetic regulation and adaptation to environment. © 2004 Elsevier B.V. All rights reserved.