Role of G1 phase cyclins and cyclin dependent kinases during hypertrophic growth of adult rat cardiomyocytes

Cell cycle regulatory molecules are implicated in cardiomyocyte hypertrophy. We have investigated protein expression of cyclins A, D1–3, and E and cyclin-dependent kinases (CDKs) 2, 4, 5, and 6 in left ventricular (LV) tissues during the development of LV hypertrophy in rats following aortic constriction (AC). Compared with their expression in sham-operated controls (SH), expression of cyclins D2 and D3 and of CDK4 and CDK6 increased significantly fromday 3 to day 21 after AC concomitant with increased LV mass. However, no significant difference was observed for CDK2 or CDK5. Cyclins A, D1, and E were undetectable. In vitro kinase activities of CDK4 and CDK6 increased ∼70% from day 7 to day 14 in AC myocytes compared with SH myocytes (P< 0.03). Fluorescence-activated cell sorter analysis revealed a G0/G1to G2/M phase progression in AC myocyte nuclei (22.0 ± 1.1% in G2/M) by day 7 postoperation compared with progression in SH myocyte nuclei (14.0 ± 0.8% in G2/M;P < 0.01). Thus an upregulation of certain cell cycle regulators is associated with cardiomyocyte hypertrophy.

Dietary supplementation with Bifidobacterium lactis NCC2818 from weaning reduces local immunoglobulin production in lymphoid-associated tissues but increases systemic antibodies in healthy neonates

Weaning is associated with a major shift in the microbial community of the intestine, and this instability may make it more acquiescent than the adult microbiota to long-term changes. Modulation achieved through dietary interventions may have potentially beneficial effects on the developing immune system, which is driven primarily by the microbiota. The specific aim of the present study was to determine whether immune development could be modified by dietary supplementation with the human probiotic Bifidobacterium lactis NCC2818 in a tractable model of weaning in infants. Piglets were reared by their mothers before being weaned onto a solid diet supplemented with B. lactis NCC2818, while sibling controls did not receive supplementation. Probiotic supplementation resulted in a reduction in IgA (P,0·0005) and IgM (P,0·009) production by mucosal tissues but had no effect on IgG production (P.0·05). Probiotic- supplemented pigs had more mast cells than unsupplemented littermates (P,0·0001), although numbers in both groups were low. In addition, the supplemented piglets made stronger serum IgG responses to fed and injected antigens (P,0·05). The present findings are consistent with B. lactis NCC2818 reducing intestinal permeability induced by weaning, and suggest that the piglet is a valuable intermediate between rodent models and human infants. The results also strongly suggest that measures of the effect of probiotic supplementation on the immune system need to be interpreted carefully as proxy measures of health benefit. However, they are useful in developing an understanding of the mechanism of action of probiotic strains, an important factor in predicting favourable health outcomes of nutritional intervention.

Effect of dietary supplementation of different oils during the first or second half of pregnancy on the glucose tolerance of the sow

Poor glucose tolerance may be an under-researched contributory factor in the high (10% to 20%) pre-weaning mortality rate observed in pigs. Insulin resistance commences at around week 12 of gestation in the sow, although there are conflicting reports in the literature about the extent to which insulin resistance is modulated by maternal diet. The aim of the study was to determine the effects of supplementing the maternal diet with different dietary oils during either the first half or the second half of gestation on the glucose tolerance of the sow. Sows were offered the control (C: n = 5) diet as pellets or the C diet plus 10% extra energy (h = 16 per group) derived from either. (i) extra pellets; (ii) palm oil; (iii) olive oil; (iv) sunflower oil; or (v) fish oil. Experimental diets were fed during either the first (G1) or second (G2) half of gestation. A glucose tolerance test (GTT) was conducted on day 108 of gestation by administering 0.5g/kg glucose i.v. Blood samples were taken every 5 to 10 min for 90 min post administration. The change in body weight and backfat thickness during gestation was similar but both type and timing of dietary supplementation influenced litter size and weight. With the exception of the sunflower oil group, supplementing the maternal diet in G1 resulted in larger and heavier litters, particularly in mothers offered palm oil. Basal blood glucose concentrations tended to be more elevated in G1 than G2 groups, whilst plasma insulin concentrations were similar Following a GTT, the adjusted area under the curve was greater in G1 compared to G2 sows, despite no differences in glucose clearance. Maternal diet appeared to influence the relationship between glucose curve characteristics following a GTT and litter outcome. In conclusion, the degree of insulin sensitivity can be altered by both the period during which maternal nutritional supplementation is offered and the fatty acid profile of the diet.

Intergenerational effects of birth weight on glucose tolerance and reproductive performance

Women who were themselves small-for-gestational age (SGA) are at a greater risk of adulthood diseases such as non-insulin-dependent diabetes mellitus (NIDDM), and twice at risk of having an SGA baby themselves. The aim of this study was to examine the intergenerational pig. Low (L) and normal (N) birth weight female piglets were followed throughout their first pregnancy (generation 1 (0)). After they had given birth, the growth and development of the lightest (I) and heaviest (n) female piglet from each litter were monitored until approximately 5 months of age (generation 2 (G2)). A glucose tolerance test (GTT) was conducted on G1 pig at similar to 6 months of age and again during late pregnancy; a GTT was also conducted on G2 pigs at similar to 4 months of age. G1 L offspring exhibited impaired glucose metabolism in later life compared to their G1 N sibling but in the next generation a similar scenario was only observed between I and n offspring born to G1 L mothers. Despite G1 L mothers showing greater glucose intolerance in late pregnancy and a decreased litter size, average piglet birth weight was reduced and there was also a large variation in litter weight; this suggests that they were, to some extent, prioritising their nutrient intake towards themselves rather than promoting their reproductive performance. There were numerous relationships between body shape at birth and glucose curve characteristics in later life, which can, to some extent, be used to predict neonatal outcome. In conclusion, intergenerational effects are partly seen in the pig. It is likely that some of the intergenerational influences may be masked due to the pig being a litter-bearing species.

Effect of type and timing of oil supplements to sows during pregnancy on the growth performance and endocrine profile of low and normal birth weight offspring

Eighty-eight multiparous sows were used to evaluate whether type and timing of oil supplementation during gestation influences the incidence of low birth weight (LBW). Sows were allocated (eight per treatment) commercial sow pellets (3 kg/d; control diet) or an experimental diet consisting of control diet plus 10 % extra energy in the form of excess pellets, palm oil, olive oil (OO), sunflower oil (SO) or fish oil; experimental diets were fed during either the first half (G1) or second half (G2) of gestation. Growth performance and endocrine profile of LBW ( < 1·09 kg) and normal birth weight (NBW; 1·46–1·64 kg) offspring were compared. Maternal dietary supplementation altered the distribution curve for piglet birth weight. SOG1 sows had a greater proportion of LBW piglets (P < 0·05), whilst it was reduced in the OOG1 group (P < 0·05). Growth rate of LBW piglets was lower compared with their NBW siblings (P < 0·05) when dietary supplementation was offered in G2 but were similar for G1. At birth, LBW offspring of supplemented animals possessed more fat compared with the control group (P < 0·05); LBW offspring of control animals exhibited a more rapid decline in fat free mass/kg prior to weaning. Plasma metabolites and insulin concentrations were influenced by maternal diet and birth weight. In conclusion, maternal dietary supplementation altered the distribution of piglet birth weights and improved the energy status of LBW piglets. Supplementation with MUFA during G1 reduced the incidence of LBW, whereas PUFA had the reverse effect.

Cell and nuclear degradation in sweetpotato [Ipomoea batatas (L.) Lam.] root meristems following exposure to salinity

Sodium chloride-induced cell and nuclear degradation in the root meristems of sweetpotato [Ipomoea batatas (L.) Lam.] were determined using fluorescent microscopy and flow cytometry analysis. Two sweetpotato cultivars were grown in liquid Murashige and Skoog medium and subjected to 0 mM and 500 mM NaCl, with or without 15 mM CaCl2, for periods up to 24 h. Changes to the nuclei of root meristematic cells showed a similar pattern of damage to the nuclei using both fluorescent microscopy and flow cytometry analysis. Damage occurring after only a few hours was followed by nuclear degradation at 24 h. Flow cytometry histograms showed a reduction in G1 and G2 nuclei and an increase in degraded nuclei in NaCl-stressed roots. Salinity-induced nuclear degradation was alleviated by the addition of CaCl2.

Caf1A usher possesses a Caf1 subunit-like domain that is crucial for Caf1 fibre secretion

The chaperone/usher pathway controls assembly of fibres of adhesive organelles of Gram-negative bacteria. The final steps of fibre assembly and fibre translocation to the cell surface are co-ordinated by the outer membrane proteins, ushers. Ushers consist of several soluble periplasmic domains and a single transmembrane beta-barrel. Here we report isolation and structural/functional characterization of a novel middle domain of the Caf1A usher from Yersinia pestis. The isolated UMD (usher middle domain) is a highly soluble monomeric protein capable of autonomous folding. A 2.8 angstrom (1 angstrom = 0.1 nm) resolution crystal structure of UMD revealed that this domain has an immunoglobulin-like fold similar to that of donor-strand-complemented Caf1 fibre subunit. Moreover, these proteins displayed significant structural similarity. Although UMD is in the middle of the predicted amphipathic beta-barrel of Caf1A, the usher still assembled in the membrane in the absence of this domain. UMD did not bind Caf1M-Caf1 complexes, but its presence was shown to be essential for Caf1 fibre secretion. The study suggests that UMD may play the role of a subunit-substituting protein (dummy subunit), plugging or priming secretion through the channel in the Caf1A usher. Comparison of isolated UMD with the recent strcture of the corresponding domain of PapC usher revealed high similarity of the core structures, suggesting a universal structural adaptation of FGL (F(1)G(1) long) and FGS (F(1)G(1) short) chaperone/usher pathways for the secretion of different types of fibres. The functional role of two topologically different states of this plug domain suggested by structural and biochemical results is discussed.

Developmental expression of myostatin in cardiomyocytes and its effect on foetal and neonatal rat cardiomyocyte proliferation

Objectives: Myostatin, a member of the transforming growth factor-beta (TGF-beta) family, plays a key role in skeletal muscle myogenesis by limiting hyperplastic and hypertrophic muscle growth. In cardiac muscle, myostatin has been shown to limit agonist-induced cardiac hypertrophic growth. However, its role in cardiac hyperplastic growth remains undetermined. The aim of this study was to characterise the expression of myostatin in developing myocardium, determine its effect on cardiomyocyte proliferation, and explore the signalling mechanisms affected by myostatin in dividing cardiomyocytes. Methods: We used quantitative PCR and Western blotting to study the expression of myostatin in cardiomyocytes isolated from rat myocardium at different developmental ages. We. determined the effect of recombinant myostatin on proliferation and cell viability in dividing cardiomyocytes in culture. We analysed myostatin's effect on cardiomyocyte cell cycle progression by flow cytometry and used Western blotting to explore the signalling mechanisms involved. Results: Myostatin is expressed differentially in cardiomyocytes during cardiac development such that increasing expression correlated with a low cardiomyocyte proliferation index. Proliferating foetal cardiomyocytes, from embryos at 18 days of gestation, expressed low levels of myostatin mRNA and protein, whereas isolated cardiomyocytes from postnatal day 10 hearts, wherein the majority of cardiomyocytes have lost their ability to proliferate, displayed a 6-fold increase in myostatin expression. Our in vitro studies demonstrated that myostatin inhibited proliferation of dividing foetal and neonatal cardiomyocytes. Flow cytometric analysis showed that this inhibition occurs mainly via a block in the G1-S phase transition of the cardiomyocyte cell cycle. Western blot analysis showed that part of the mechanism underpinning the inhibition of cardiomyocyte proliferation by myostatin involves phosphorylation of SMAD2 and altered expressions of the cell cycle proteins p21 and CDK2. Conclusions: We conclude that myostatin is an inhibitor of cardiomyocyte proliferation with the potential to limit cardiomyocyte hyperplastic growth by altering cardiac cell cycle progression. (c) 2007 European Society of Cardiology. Published by Elsevier B.V. All fights reserved.

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit vascular smooth muscle cell proliferation via differential effects on the cell cycle

Abnormal vascular smooth muscle cell (VSMC) proliferation plays an important role in the pathogenesis of both atherosclerosis and restenosis. Recent studies suggest that high-dose salicylates, in addition to inhibiting cyclooxygenase activity, exert an antiproliferative effect on VSMC growth both in-vitro and in-vivo. However, whether all non-steroidal anti-inflammatory drugs (NSAIDs) exert similar anti proliferative effects on VSMCs, and do so via a common mechanism of action, remains to be shown. In this study, we demonstrate that the NSAIDs aspirin, sodium salicylate, diclofenac, ibuprofen, indometacin and sulindac induce a dose-dependent inhibition of proliferation in rat A10 VSMCs in the absence of significant cytotoxicity. Flow cytometric analyses showed that exposure of A10 cells to diclofenac, indometacin, ibuprofen and sulindac, in the presence of the mitotic inhibitor, nocodazole, led to a significant G0/G1 arrest. In contrast, the salicylates failed to induce a significant G1 arrest since flow cytometry profiles were not significantly different from control cells. Cyclin A levels were elevated, and hyperphosphorylated p107 was present at significant levels, in salicylate-treated A10 cells, consistent with a post-G1/S block, whereas cyclin A levels were low, and hypophosphorylated p107 was the dominant form, in cells treated with other NSAIDs consistent with a G1 arrest. The ubiquitously expressed cyclin-dependent kinase (CDK) inhibitors, p21 and p27, were increased in all NSAID-treated cells. Our results suggest that diclofenac, indometacin, ibuprofen and sulindac inhibit VSMC proliferation by arresting the cell cycle in the G1 phase, whereas the growth inhibitory effect of salicylates probably affects the late S and/or G2/M phases. Irrespective of mechanism, our results suggest that NSAIDs might be of benefit in the treatment of certain vasculoproliferative disorders.

A novel Bayesian approach to drug design with simple and complex enzymes: Use with lens aldehyde dehydrogenase and the glyoxalase pathway

Purpose: Acquiring details of kinetic parameters of enzymes is crucial to biochemical understanding, drug development, and clinical diagnosis in ocular diseases. The correct design of an experiment is critical to collecting data suitable for analysis, modelling and deriving the correct information. As classical design methods are not targeted to the more complex kinetics being frequently studied, attention is needed to estimate parameters of such models with low variance. Methods: We have developed Bayesian utility functions to minimise kinetic parameter variance involving differentiation of model expressions and matrix inversion. These have been applied to the simple kinetics of the enzymes in the glyoxalase pathway (of importance in posttranslational modification of proteins in cataract), and the complex kinetics of lens aldehyde dehydrogenase (also of relevance to cataract). Results: Our successful application of Bayesian statistics has allowed us to identify a set of rules for designing optimum kinetic experiments iteratively. Most importantly, the distribution of points in the range is critical; it is not simply a matter of even or multiple increases. At least 60 % must be below the KM (or plural if more than one dissociation constant) and 40% above. This choice halves the variance found using a simple even spread across the range.With both the glyoxalase system and lens aldehyde dehydrogenase we have significantly improved the variance of kinetic parameter estimation while reducing the number and costs of experiments. Conclusions: We have developed an optimal and iterative method for selecting features of design such as substrate range, number of measurements and choice of intermediate points. Our novel approach minimises parameter error and costs, and maximises experimental efficiency. It is applicable to many areas of ocular drug design, including receptor-ligand binding and immunoglobulin binding, and should be an important tool in ocular drug discovery.

Inhibition of vascular smooth muscle cell proliferation by non-steroidal anti-inflammatory drugs

Abnormal vascular smooth muscle cell (VSMC) proliferation is known to play an important role in the pathogenesis of atherosclerosis, restenosis and instent stenosis. Recent studies suggest that salicylates, in addition to inhibiting cyclooxygenase activity, exert an antiproliferative effect on VSMC growth both in vitro and in vivo. However, whether all non-steroidal anti-inflammatory drugs (NSAID) exert similar antiproliferative effects on VSMCs, and do so via a common mechanism of action, remains unknown. In the present study, we demonstrated that the NSAIDs, aspirin, ibuprofen and sulindac induced a dose-dependent inhibition of proliferation in rat A10 VSMCs (IC50 = 1666 mumol/L, 937 mumol/L and 520 mumol/L, respectively). These drugs did not show significant cytotoxic effects as determined by LDH release assay, even at the highest concentrations tested (aspirin, 5000 mumol/L; ibuprofen, 2500 mumol/L; and sulindac, 1000 mumol/L). Flow cytometric analyses showed that a 48 h exposure of A10 VSMCs to ibuprofen (1000 mumol/L) and sulindac (750 mumol/L) led to a significant G1 arrest (from 68.7 +/- 2.0% of cells in G1 to 76.6 +/- 2.2% and 75.8 +/- 2.2%, respectively, p < 0.05). In contrast, aspirin (2500 mumol/L) failed to induce a significant G1 arrest (68.1 +/- 5.2%). Clearer evidence of a G1 block was obtained by treatment of cells with the mitotic inhibitor, nocodazole (40 ng/ml), for the final 24 h of the experiment. Under these conditions, aspirin still failed to induce a G1 arrest (from 25.9 +/- 10.9% of cells in G1 to 19.6 +/- 2.3%) whereas ibuprofen and sulindac led to a significant accumulation of cells in G1(51.8% +/- 17.2% and 54.1% +/- 10.6%, respectively, p < 0.05). These results indicate that ibuprofen and sulindac inhibit VSMC proliferation by arresting the cell cycle in the G1 phase whereas the effect of aspirin appears to be independent of any special phase of the cell cycle. Irrespective of mechanism, our results suggest that NSAIDs might be of benefit to the treatment of vascular proliferative disorders.

Incorporation of a sodium ion guest in the host of copper(II)-Schiff-base complexes: Structural characterization and magnetic study

Three copper(II) complexes, [CuL1], [CuL2] and [CuL3] where L-1, L-2 and L-3 are the tetradentate di-Schiff-base ligands prepared by the condensation of acetylacetone and appropriate diamines (e.g. 1,2-diaminoethane, 1,2-diaminopropane and 1,3-diaminopropane, respectively) in 2:1 ratios, have been prepared. These complexes act as host molecules and include a guest sodium ion by coordinating through the oxygen atoms to result in corresponding new trinuclear complexes, [(CuL1)(2)Na(ClO4)(H2O)][CuL1], [(CuL2)(2)Na(ClO4)(H2O)] (2) and [(CuL3)(2)Na(ClO4)(H2O)] (3) when crystallized from methanol solution containing sodium perchlorate. All three complexes have been characterized by single crystal X-ray crystallography. In all the complexes, the sodium cation has a six-coordinate distorted octahedral environment being bonded to four oxygen atoms from two Schiff-base complexes of Cu(II) in addition to a perchlorate anion and a water molecule. The copper atoms are four coordinate in a square planar environment being bonded to two oxygen atoms and two nitrogen atoms of the Schiff-base ligand. The variable temperature susceptibilities for complexes 1-3 were measured over the range 2-300 K. The isotropic Hamiltonian, H = g(1)beta HS1 + g(2)beta HS2 + J(12)S(1)S(2) + g(3)beta HS3 for complex 1 and H = g(1)beta HS1 + g2 beta HS2 +J(12)S(1)S(2) for complexes 2 and 3 has been used to interpret the magnetic data. The best fit parameters obtained are: g(1) = g(2) = 2.07(0), J = - 1.09(1) cm(-1) for complex 1, g(1) = g(2) = 2.06(0), J = -0.55(1) cm(-1) for complex 2 and g1 = g2 = 2.07(0).J = -0.80(1) cm(-1) for 3. Electrochemical studies displayed an irreversible Cu(II)/Cu(I) one-electron reduction process. (C) 2008 Elsevier Ltd. All rights reserved.

Solid-phase synthesis of an A-B loop mimetic of the C epsilon 3 domain of human IgE: macrocyclization by Sonogashira coupling

The solid-phase synthesis of a cyclic peptide containing the 21-residue epitope found in the A-B loop of the Cepsilon3 domain of human immunoglobulin E has been carried out. The key macrocyclization step to form the 65-membered ring is achieved in similar to15% yield via an "on-resin" Sonogashira coupling reaction which concomitantly installs a diphenylacetylene amino acid conformational constraint within the loop.

Molecular cloning and comparative analysis of four beta-galactosidase genes from Bifidobacterium bifidum NCIMB41171

Bifidobacterium bifidum NCIMB41171 carries four genes encoding different beta-galactosidases. One of them, named bbgIII, consisted of an open reading frame of 1,935 amino acid (a.a.) residues encoding a protein with a multidomain structure, commonly identified on cell wall bound enzymes, having a signal peptide, a membrane anchor, FIVAR domains, immunoglobulin Ig-like and discoidin-like domains. The other three genes, termed bbgI, bbgII and bbgIV, encoded proteins of 1,291, 689 and 1,052 a.a. residues, respectively, which were most probably intracellularly located. Two cases of protein evolution between strains of the same species were identified when the a.a. sequences of the BbgI and BbgIII were compared with homologous proteins from B. bifidum DSM20215. The homologous proteins were found to be differentiated at the C-terminal a.a. part either due to a single nucleotide insertion or to a whole DNA sequence insertion, respectively. The bbgIV gene was located in a gene organisation surrounded by divergently transcribed genes putatively for sugar transport (galactoside-symporter) and gene regulation (LacI-transcriptional regulator), a structure that was found to be highly conserved in B. longum, B. adolescentis and B. infantis, suggesting optimal organisation shared amongst those species.

Selective separation of beta-lactoglobulin from sweet whey using CGAs generated from the cationic surfactant CTAB

The selective separation of whey proteins was studied using colloidal gas aphrons generated from the cationic surfactant cetyl trimethyl ammonium bromide (CTAB). From the titration curves obtained by zeta potential measurements of individual whey proteins, it was expected to selectively adsorb the major whey proteins, i.e., bovine serum albumin, alpha-lactalbumin, and beta-lactoglobulin to the aphrons and elute the remaining proteins (lactoferrin and lactoperoxidase) in the liquid phase. A number of process parameters including pH, ionic strength, and mass ratio of surfactant to protein (M-CTAB/M-TP) were varied in order to evaluate their effect on protein separation. Under optimum conditions (2 mmol/l CTAB, M-CTAB/M-TP = 0.26-0.35, pH 8, and ionic strength = 0.018 mol/l), 80-90% beta-lactoglobulin was removed from the liquid phase as a precipitate, while about 75% lactoferrin and lactoperoxidase, 80% bovine serum albumin, 95% immunoglobulin, and 65% alpha-lactalbumin were recovered in the liquid fraction. Mechanistic studies using zeta potential measurements and fluorescence spectroscopy proved that electrostatic interactions modulate only partially the selectivity of protein separation, as proteins with similar surface charges do not separate to the same extent between the two phases. The selectivity of recovery of beta-lactoglobulin probably occurs in two steps: the first being the selective interaction of the protein with opposite-charged surfactant molecules by means of electrostatic interactions, which leads to denaturation of the protein and subsequent formation and precipitation of the CTAB-beta-lactoglobulin complex. This is followed by the separation of CTAB-beta-lactoglobulin aggregates from the bulk liquid by flotation in the aphron phase. In this way, CGAs act as carriers which facilitate the removal of protein precipitate. (c) 2005 Wiley Periodicals, Inc.