Cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus replication

In eukaryotic cells, cell growth and division occur in a stepwise, orderly fashion described by a process known as the cell cycle. The relationship between positive-strand RNA viruses and the cell cycle and the concomitant effects on virus replication are not clearly understood. We have shown that infection of asynchronously replicating and synchronized replicating cells with the avian coronavirus infectious bronchitis virus (IBV), a positive-strand RNA virus, resulted in the accumulation of infected cells in the G(2)/M phase of the cell cycle. Analysis of various cell cycle-regulatory proteins and cellular morphology indicated that there was a down-regulation of cyclins D1 and D2 (G(2) regulatory cyclins) and that a proportion of virus-infected cells underwent aberrant cytokinesis, in which the cells underwent nuclear, but not cytoplasmic, division. We assessed the impact of the perturbations on the cell cycle for virus-infected cells and found that IBV-infected G(2)/M-phase-synchronized cells exhibited increased viral protein production when released from the block when compared to cells synchronized in the Go phase or asynchronously replicating cells. Our data suggested that IBV induces a G(2)/M phase arrest in infected cells to promote favorable conditions for viral replication.

Antioxidant and anti-atherogenic activities of olive oil phenolics

The aim of the current study was to investigate the antioxidant and cellular activity of the olive oil phenolics oleuropein, tyrosol, hydroxytyrosol, and homovanillic alcohol (which is also a major metabolite of hydroxytyrosol). Well-characterized chemical and biochemical assays were used to assess the antioxidant potential of the compounds. Further experiments investigated their influence in cell culture on cytotoxic effects of hydrogen peroxide and oxidized low-density lipoprotein (LDL), nitric oxide production by activated macrophages, and secretion of chemoattractant and cell adhesion molecules by the endothelium. Inhibitory influences on in vitro platelet aggregation were also measured. The antioxidant assays indicated that homovanillic alcohol was a significantly more potent antioxidant than the other phenolics, both in chemical assays and in prolonging the lag phase of LDL oxidation. Cell culture experiments suggested that the olive oil phenolics induce a significant reduction in the secretion of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 (and a trend towards a reduced secretion of monocyte chemoattractant protein-1), and protect against cytotoxic effects of hydrogen peroxide and oxidized LDL. However, no influence on nitric oxide production or platelet aggregation was evident. The data show that olive oil phenolics have biochemical and cellular actions, which, if also apparent in vivo, could exert cardioprotective effects.

Role of oxygen radicals in DNA damage and cancer incidence

The development of cancer in humans and animals is a multistep process. The complex series of cellular and molecular changes participating in cancer development are mediated by a diversity of endogenous and exogenous stimuli. One type of endogenous damage is that arising from intermediates of oxygen (dioxygen) reduction - oxygen-free radicals (OFR), which attacks not only the bases but also the deoxyribosyl backbone of DNA. Thanks to improvements in analytical techniques, a major achievement in the understanding of carcinogenesis in the past two decades has been the identification and quantification of various adducts of OFR with DNA. OFR are also known to attack other cellular components such as lipids, leaving behind reactive species that in turn can couple to DNA bases. Endogenous DNA lesions are genotoxic and induce mutations. The most extensively studied lesion is the formation of 8-OH-dG. This lesion is important because it is relatively easily formed and is mutagenic and therefore is a potential biomarker of carcinogenesis. Mutations that may arise from formation of 8-OH-dG involve GC. TA transversions. In view of these findings, OFR are considered as an important class of carcinogens. The effect of OFR is balanced by the antioxidant action of non-enzymatic antioxidants as well as antioxidant enzymes. Non-enzymatic antioxidants involve vitamin C, vitamin E, carotenoids (CAR), selenium and others. However, under certain conditions, some antioxidants can also exhibit a pro-oxidant mechanism of action. For example, beta-carotene at high concentration and with increased partial pressure of dioxygen is known to behave as a pro-oxidant. Some concerns have also been raised over the potentially deleterious transition metal ion-mediated (iron, copper) pro-oxidant effect of vitamin C. Clinical studies mapping the effect of preventive antioxidants have shown surprisingly little or no effect on cancer incidence. The epidemiological trials together with in vitro experiments suggest that the optimal approach is to reduce endogenous and exogenous sources of oxidative stress, rather than increase intake of anti-oxidants. In this review, we highlight some major achievements in the study of DNA damage caused by OFR and the role in carcinogenesis played by oxidatively damaged DNA. The protective effect of antioxidants against free radicals is also discussed.

Antioxidant and anti-atherogenic activities of olive oil phenolics

The aim of the current study was to investigate the antioxidant and cellular activity of the olive oil phenolics oleuropein, tyrosol, hydroxytyrosol, and homovanillic alcohol (which is also a major metabolite of hydroxytyrosol). Well-characterized chemical and biochemical assays were used to assess the antioxidant potential of the compounds. Further experiments investigated their influence in cell culture on cytotoxic effects of hydrogen peroxide and oxidized low-density lipoprotein (LDL), nitric oxide production by activated macrophages, and secretion of chemoattractant and cell adhesion molecules by the endothelium. Inhibitory influences on in vitro platelet aggregation were also measured. The antioxidant assays indicated that homovanillic alcohol was a significantly more potent antioxidant than the other phenolics, both in chemical assays and in prolonging the lag phase of LDL oxidation. Cell culture experiments suggested that the olive oil phenolics induce a significant reduction in the secretion of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 (and a trend towards a reduced secretion of monocyte chemoattractant protein-1), and protect against cytotoxic effects of hydrogen peroxide and oxidized LDL. However, no influence on nitric oxide production or platelet aggregation was evident. The data show that olive oil phenolics have biochemical and cellular actions, which, if also apparent in vivo, could exert cardioprotective effects.

Conjugated linoleic acid and human health: a critical evaluation of the evidence

Purpose of review: This review critically evaluates studies investigating the effects of conjugated linoleic acid on human health, including effects on body composition, blood lipids, liver metabolism, insulin sensitivity and immune function. It focuses mainly on human intervention studies, but includes some reference to animal and cellular studies which provide insight into potential molecular mechanisms of action of conjugated linoleic acid. Recent findings: Human studies continue to report inconsistent effects of conjugated linoleic acid on human health. Some of these reports are based on overinterpretation of marginal effects of supplementation. Recent data suggest that the effects of the substance may be isomer dependent and that cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acids have opposing effects on blood lipids and on metabolism in adipocytes and hepatic cells. Summary: Claims that conjugated linoleic acid is beneficial for health remain as yet unconvincing. Human studies investigating the effects of conjugated linoleic acid supplements have tended to use mixtures of isomers and have been inconsistent. More recent studies have attempted to use relatively pure preparations of single isomers and these studies suggest that the effects of conjugated linoleic acid may be isomer-specific. These recent data suggest a relative detrimental effect of trans-10, cis-12 conjugated linoleic acid on blood lipids. There appears to be little effect of conjugated linoleic acid on immune function and the effects on insulin sensitivity remain unclear.

Incorporation of cis-9, trans-11 conjugated linoleic acid and vaccenic acid (trans-11 18 : 1) into plasma and leucocyte lipids in healthy men consuming dairy products naturally enriched in these fatty acids

The present study investigated whether consuming dairy products naturally enriched in cis-9, trans-11 (c9,t11) conjugated linoleic acid (CLA) by modification of cattle feed increases the concentration of this isomer in plasma and cellular lipids in healthy men. The study had a double-blind cross-over design. Subjects aged 34-60 years consumed dairy products available from food retailers for 1 week and then either control (0.17 g c9,t11 CLA/d; 0.31 g trans-vaccenic acid (tVA)/d) or CLA-enriched (1.43 g c9,t11 CLA/d; 4.71 g tVA/d) dairy products for 6 weeks. After 7 weeks washout, this was repeated with the alternate products. c9,t11 CLA concentration in plasma lipids was lower after consuming the control products, which may reflect the two-fold greater c9,t11 CLA content of the commercial products. Consuming the CLA-enriched dairy products increased the c9,t11 CLA concentration in plasma phosphatidylcholine (PC) (38 %; P=0.035), triacylglycerol (TAG) (22 %; P < 0.0001) and cholesteryl esters (205 %; P < 0.0001), and in peripheral blood mononuclear cells (PBMC) (238 %; P < 0.0001), while tVA concentration was greater in plasma PC (65 %; P=0.035), TAG (98 %; P=0.001) and PBMC (84 %; P=0.004). Overall, the present study shows that consumption of naturally enriched dairy products in amounts similar to habitual intakes of these foods increased the c9,t11 CLA content of plasma and cellular lipids.

Role of quercetin and its in vivo metabolites in protecting H9c2 cells against oxidative stress

The aim of this study was to investigate the potential of quercetin and two of its "in vivo" metabolites, 3'-O-methyl quercetin and 4'-O-methyl quercetin, to protect H9c2 cardiomyoblasts against H2O2-induced oxidative stress. As limited data are available regarding the potential uptake and cellular effects of quercetin and its metabolites in cardiac cells, we have evaluated the cellular association/uptake of the three compounds and their involvement in the modulation of two pro-survival signalling pathways: ERK1/2 signalling cascade and PI3K/Akt pathway. The three flavonols associated with cells to differing extents. Quercetin and its two O-methylated metabolites were able to reduce intracellular ROS production but only quercetin was able to counteract H2O2 cell damage, as measured by MTT reduction assay, caspase-3 activity and DNA fragmentation assays. Furthermore, only quercetin was observed to modulate pro-survival signalling through ERK1/2 and PI3K/Akt pathway. In conclusion we have demonstrated that quercetin, but not its O-methylated metabolites, exerts protective effects against H2O2 cardiotoxicity and that the mechanism of its action involves the modulation of PI3K/Akt and ERK1/2 signalling pathways. (c) 2006 Elsevier Masson SAS. All rights reserved.

Can drug-bearing liposomes penetrate intact skin?

Using liposomes to deliver drugs to and through human skin is controversial, as their function varies with type and composition. Thus they may act as drug carriers controlling release of the medicinal agent. Alternatively, they may provide a localized depot in the skin so minimizing systemic effects or can be used for targeting delivery to skin appendages (hair follicles and sweat glands). Liposomes may also enhance transdermal drug delivery, increasing systemic drug concentrations. With such a multiplicity of functions, it is not surprising that mechanisms of liposomal delivery of therapeutic agents to and through the skin are unclear. Accordingly, this article provides an overview of the modes and mechanisms of action of different vesicles as drug delivery vectors in human skin. Our conclusion is that vesicles, depending on the composition and method of preparation, can vary with respect to size, lamellarity, charge, membrane fluidity or elasticity and drug entrapment. This variability allows for multiple functions ranging from local to transdermal effects. Application to dissimilar skins (animal or human) via diverse protocols may reveal different mechanisms of action with possible vesicle skin penetration reaching different depths, from surface assimilation to (rarely) the viable tissue and subsequent systemic absorption.

Expressions and activities of cell cycle regulatory molecules during the transition from myocyte hyperplasia to hypertrophy

The role of cell cycle dependent molecules in controlling the switch from cardiac myocyte hyperplasia to hypertrophy remains unclear, although in the rat this process occurs between day 3 and 4 after birth. In this study we have determined (1) cell cycle profiles by fluorescence activated cell sorting (FACS); and (2) expressions, co-expressions and activities of a number of cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors by reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting andin vitrokinase assays in freshly isolated rat cardiac myocytes obtained from 2, 3, 4 and 5-day-old animals. The percentage of myocytes found in the S phase of the cell cycle decreased significantly during the transition from hyperplasia to hypertrophy (5.5, 3.5, 2.3 and 1.9% of cells in 2-, 3-, 4- and 5-day-old myocytes, respectively,P<0.05), concomitant with a significant increase in the percentage of G0/G1phase cells. At the molecular level, the expressions and activities of G1/S and G2/M phase acting cyclins and CDKs were downregulated significantly during the transition from hyperplasia to hypertrophy, whereas the expressions and activities of G1phase acting cyclins and CDKs were upregulated significantly during this transition. In addition, p21CIP1- and p27KIP1- associated CDK kinase activities remained relatively constant when histone H1 was used as a substrate, whereas phosphorylation of the retinoblastoma protein was upregulated significantly during the transition from hyperplasia to hypertrophy. Thus, there is a progressive and significant G0/G1phase blockade during the transition from myocyte hyperplasia to hypertrophy. Whilst CDK2 and cdc2 may be pivotal in the withdrawal of cardiac myocytes from the cell cycle, CDK4 and CDK6 may be critical for maintaining hypertrophic growth of the myocyte during development.

Expression and activities of cyclins and cyclin-dependent kinases in developing rat ventricular myocytes

The molecular mechanisms responsible for the alterations in proliferative capacity of cardiac myocytes during development remain unknown; however, cell cycle dependent molecules may be involved. We have determined the expression of cyclins A, D1–3and E, and cyclin-dependent kinases (CDKs) 2, 4, 5 and 6 and cdc2 in freshly isolated rat cardiac myocytes from fetal (18 days gestation), neonatal (2 days post-natal) and adult animals by immunoblotting. Our results show a dramatic decrease in expression of these proteins during normal cardiac development, such that levels are highest in fetal myocytes but are significantly down-regulated in adult cells (P<0.05, in each case). We also have determined thein vitrokinase activities of cdc2, CDK2, CDK4, CDK5 and CDK6 immunocomplexes in fetal, neonatal and adult myocytes. There was a consistent and significant loss of cdc2, CDK2, CDK4 and CDK6 kinase activities in adult cardiac cell lysates (5.3-, 10.6-, 1.5- and 1.9-fold decreases, respectively) when compared to neonatal samples (P<0.05); CDK5 activity showed a similar trend but failed to reach significance. In conclusion, our results show that the expression and activities of various positive regulators of the cell cycle are down-regulated significantly during development of the cardiac myocyte, concomitant with the loss of proliferative capacity in adult myocytes. Down-regulation of these proteins may be pivotal in the withdrawal of the cardiac myocyte from the cell cycle.

Expression and regulation of 80K/MARCKS, a major substrate of protein kinase C, in the developing rat heart

Objective: Protein kinase C (PKC) plays a pivotal role in modulating the growth and differentiation of many cell types including the cardiac myocyte. However, little is known about molecules that act immediately downstream of PKC in the heart. In this study we have investigated the expression of 80K/MARCKS, a major PKC substrate, in whole ventricles and in cardiac myocytes from developing rat hearts. Methods: Poly A+ RNA was prepared from neonatal (2-day) and adult (42-day) cardiac myocytes and whole ventricular tissue and mRNA expression determined by reverse transcription-polymerase chain reaction (RT-PCR) using primers designed to identify a 420 bp fragment in the 80K/MARCKS gene. Protein extracts were prepared from either 2-day and 42-day cardiac myocytes or from whole ventricular tissue at 2, 5–11, 14, 17, 21, 28 and 42 days of age. Protein expression was determined by immunoblotting with an 80K/MARCKS antipeptide antibody and PKC activity was determined by measuring the amount of γ32P-ATP transferred to a specific peptide substrate. Results: RT-PCR analysis of 80K/MARCKS mRNA in neonatal (2-day) and adult (42-day) cardiac myocytes showed the expression of this gene in both cell types. Immunoblotting revealed maximum 80K/MARCKS protein expression in whole ventricular tissue at 5 days (a 75% increase above values at 2 days), followed by a transient decrease in expression during the 6–8-day period (61% of the protein expressed at 2 days for 8-day tissue) with levels returning to 5 day levels by 11 days of age. 80K/MARCKS protein was present in cardiac myocytes at 2 days of age whereas it was not detectable in adult cells. In addition, PKC activity levels increased to 160% of levels present at 2 days in 8-day-old ventricles with PKC activity levels returning to 5-day levels by 9 days of age. This was then followed by a steady decline in both 80K/MARCKS protein expression and PKC activity through to adulthood. Conclusions: Expression of the PKC substrate, 80K/MARCKS, in cardiac myocytes changes significantly during development and the transient loss of immunoreactive protein during the 6–8-day developmental period may reflect 80K/MARCKS phosphorylation and subsequent down-regulation as a result of the concomitant up-regulation of PKC activity at this time.

Increased levels of microparticles originating from endothelial cells, platelets and erythrocytes in subjects with metabolic syndrome: relationship with oxidative stress

Background and aims The Metabolic Syndrome (MetS) is associated with increased cardiovascular risk. Circulating microparticles (MP) are involved in the pathogenesis of atherothrombotic disorders and are raised in individual with CVD. We measured their level and cellular origin in subjects with MetS and analyzed their associations with 1/anthropometric and biological parameters of MetS, 2/inflammation and oxidative stress markers. Methods and results Eighty-eight subjects with the MetS according to the NCEP-ATPIII definition were enrolled in a bicentric study and compared to 27 healthy controls. AnnexinV-positive MP (TMP), MP derived from platelets (PMP), erythrocytes (ErMP), endothelial cells (EMP), leukocytes (LMP) and granulocytes (PNMP) were determined by flow cytometry. MetS subjects had significantly higher counts/μl of TMP (730.6 ± 49.7 vs 352.8 ± 35.6), PMP (416.0 ± 43.8 vs 250.5 ± 23.5), ErMP (243.8 ± 22.1 vs 73.6 ± 19.6) and EMP (7.8 ± 0.8 vs 4.0 ± 1.0) compared with controls. LMP and PNMP were not statistically different between groups. Multivariate analysis demonstrated that each criterion for the MetS influenced the number of TMP. Waist girth was a significant determinant of PMP and EMP level and blood pressure was correlated with EMP level. Glycemia positively correlated with PMP level whereas dyslipidemia influenced EMP and ErMP levels. Interestingly, the oxidative stress markers, plasma glutathione peroxydase and urinary 8-iso-prostaglandin F2 α, independently influenced TMP and PMP levels whereas inflammatory markers did not, irrespective of MP type. Conclusion Increased levels of TMP, PMP, ErMP and EMP are associated with individual metabolic abnormalities of MetS and oxidative stress. Whether MP assessment may represent a marker for risk stratification or a target for pharmacological intervention deserves further investigation.

Implications and consequences of robots with biological brains

In this paper a look is taken at the relatively new area of culturing neural tissue and embodying it in a mobile robot platform—essentially giving a robot a biological brain. Present technology and practice is discussed. New trends and the potential effects of and in this area are also indicated. This has a potential major impact with regard to society and ethical issues and hence some initial observations are made. Some initial issues are also considered with regard to the potential consciousness of such a brain.