Identification of differentially expressed genes in a porcine in vivo model of adipogenesis using suppression subtractive hybridization

Although they provide valuable information, in vitro models of adipocyte development often require high doses of hormones and growth factors, which may influence gene expression and adipocyte differentiation patterns. To overcome these problems, a novel in vivo model of adipose tissue development was used to characterize genes involved in adipogenesis. The suppression subtractive hybridization technique was used to identify genes showing differential expression between the adipose tissue of a day 90 gestating sow, which is enriched in adipocytes, and day 90 fetal adipose tissue, which is enriched in preadipocytes. A total of 149 expressed sequence tags corresponding to identified genes and tentative consensus sequences emerged. Thirty-seven clones matched expressed sequence tags or genomic DNA sequences and six novel sequences were also identified. Adipogenesis-related genes were identified, many of which have never been reported to be expressed in mammalian adipose tissue, and may play a role in regulation of adipose tissue differentiation. Validation of differentially expressed genes was confirmed for perilipin, monocyte to macrophage differentiation-associated, myocilin, paraoxonase 3, stearoyl-CoA desaturase, angiotensinogen and adiponectin genes using real-time RT-PCR.     

Seasonal water availability predicts the relative abundance of C3 and C4 grasses in Australia

Aim Numerous studies have examined the climatic factors that influence the abundance of C₄ species within the grass flora (C₄ relative species richness) in various regions throughout the world, but very few have examined the relative abundance of C₄ vs. C₃ grasses (C₄ relative abundance). We sought to determine the climatic factors that influence C₄ relative abundance throughout Australia. Location Australia (including Tasmania). Methods We measured C₄ relative abundance at 168 locations and measured δ¹³C (the abundance of ¹³C relative to ¹²C) of the bone collagen of 779 kangaroos collected throughout Australia, as bone collagen δ¹³C was assumed to be proportional to the relative abundance of C₄ grasses in the diet. Results Both C₄ relative abundance and kangaroo bone collagen δ¹³C were found to have a strong positive relationship with seasonal water availability, i.e. the distribution of rainfall in the C₄ vs. C₃ growing seasons (76% and 69% of deviance explained, respectively). There was clear evidence that seasonal water availability was a better predictor of both C₄ relative abundance and bone collagen δ¹³C than other climate variables such as mean annual temperature and January daily minimum temperature. However, seasonal water availability appeared to be a relatively poor predictor of C₄ relative species richness, which was most closely related to January daily minimum temperature (90% of deviance explained). Main conclusions Our results highlight the relatively poor relationship between C₄ relative abundance and C₄ relative species richness, and suggest that these two variables may be related to different climatic factors. They also suggest that caution is required when using C₄ relative species richness to infer the relative biomass and productivity of C₄ grasses on a global scale.     

Mitochondrial lipid abnormality and electron transport chain impairment in mice lacking alpha-synuclein

The presynaptic protein alpha-synuclein, implicated in Parkinson disease (PD), binds phospholipids and has a role in brain fatty acid (FA) metabolism. In mice lacking alpha-synuclein (Snca-/-), total brain steady-state mass of the mitochondria-specific phospholipid, cardiolipin, is reduced 22% and its acyl side chains show a 51% increase in saturated FAs and a 25% reduction in essential n-6, but not n-3, polyunsaturated FAs. Additionally, 23% reduction in phosphatidylglycerol content, the immediate biosynthetic precursor of cardiolipin, was observed without alterations in the content of other brain phospholipids. Consistent with these changes, more ordered lipid head group and acyl chain packing with enhanced rotational motion of diphenylhexatriene (DPH) about its long axis were demonstrated in time-resolved DPH fluorescence lifetime experiments. These abnormalities in mitochondrial membrane properties were associated with a 15% reduction in linked complex I/III activity of the electron transport chain, without reductions in mitochondrial number, complex II/III activity, or individual complex I, II, III, or IV activity. Reduced complex I activity is thought to be a critical factor in the development of PD. Thus, altered membrane composition and structure and impaired complex I/III function in Snca-/- brain suggest a relationship between alpha-synuclein's role in brain lipid metabolism, mitochondrial function, and PD.     

Akt-dependent cell size regulation by the adhesion molecule on glia occurs independently of phosphatidylinositol 3-kinase and Rheb signaling

The role of cell adhesion molecules in mediating interactions with neighboring cells and the extracellular matrix has long been appreciated. More recently, these molecules have been shown to modulate intracellular signal transduction cascades critical for cell growth and proliferation. Expression of adhesion molecule on glia (AMOG) is downregulated in human and mouse gliomas, suggesting that AMOG may be important for growth regulation in the brain. In this report, we examined the role of AMOG expression on cell growth and intracellular signal transduction. We show that AMOG does not negatively regulate cell growth in vitro or in vivo. Instead, expression of AMOG in AMOG-deficient cells results in a dramatic increase in cell size associated with protein kinase B/Akt hyperactivation, which occurs independent of phosphatidylinositol 3-kinase activation. AMOG-mediated Akt phosphorylation specifically activates the mTOR/p70S6 kinase pathway previously implicated in cell size regulation, but it does not depend on tuberous sclerosis complex/Ras homolog enriched in brain (Rheb) signaling. These data support a novel role for a glial adhesion molecule in cell size regulation through selective activation of the Akt/mTOR/S6K signal transduction pathway.     

Tetracycline-inducible gene regulation in mycobacteria

A system for the tetracycline-inducible regulation of gene expression in mycobacteria has been developed. We have sub-cloned the tetRO region from the Corynebacterium glutamicum TetZ locus into a mycobacterial shuttle plasmid, making expression of genes cloned downstream of tetRO responsive to tetracycline. Using the luxAB-encoded luciferase from Vibrio harveyi as a reporter (pMind-Lx), we observed a 40-fold increase in light output from Mycobacterium smegmatis cultures 2 h after adding 20 ng ml⁻¹ of tetracycline. Similarly, exposure to the drug resulted in up to 20-fold increase in relative light units from M.bovis BCG carrying the reporter construct, and a 10-fold increase for M.tuberculosis. Tetracycline induction was demonstrated in log and stationary phase cultures. To evaluate whether this system is amenable to use in vivo, J774 macrophages were infected with M.bovis BCG[pMind-Lx], treated with amikacin to kill extracellular bacteria, and then incubated with tetracycline. A 10-fold increase in light output was measured after 24 h, indicating that intracellular bacteria are accessible and responsive to exogenously added tetracycline. To test the use of the tetracycline-inducible system for conditional gene silencing, mycobacteria were transformed with a pMind construct with tetRO driving expression of antisense RNA for the ftsZ gene. Bacterial cells containing the antisense construct formed filaments after 24 h exposure to tetracycline. These results demonstrate the potential of this tetracycline-regulated system for the manipulation of mycobacterial gene expression inside and outside cells.     

Protein kinase C and preconditioning: role of the sarcoplasmic reticulum

Activation of protein kinase C (PKC) is cardioprotective, but the mechanism(s) by which PKC mediates protection is not fully understood. Inasmuch as PKC has been well documented to modulate sarcoplasmic reticulum (SR) Ca2+ and because altered SR Ca2+ handling during ischemia is involved in cardioprotection, we examined the role of PKC-mediated alterations of SR Ca2+ in cardioprotection. Using isolated adult rat ventricular myocytes, we found that addition of 1,2-dioctanoyl-sn-glycerol (DOG), to activate PKC under conditions that reduced myocyte death associated with simulated ischemia and reperfusion, also reduced SR Ca2+. Cell death was 57.9 +/- 2.9% and 47.3 +/- 1.8% in untreated and DOG-treated myocytes, respectively (P < 0.05). Using fura 2 fluorescence to monitor Ca2+ transients and caffeine-releasable SR Ca2+, we examined the effect of DOG on SR Ca2+. Caffeine-releasable SR Ca2+ was significantly reduced (by approximately 65%) after 10 min of DOG treatment compared with untreated myocytes (P < 0.05). From our examination of the mechanism by which PKC alters SR Ca2+, we present the novel finding that DOG treatment reduced the phosphorylation of phospholamban (PLB) at Ser16. This effect is mediated by PKC-epsilon, because a PKC-epsilon-selective inhibitory peptide blocked the DOG-mediated decrease in phosphorylation of PLB and abolished the DOG-induced reduction in caffeine-releasable SR Ca2+. Using immunoprecipitation, we further demonstrated that DOG increased the association between protein phosphatase 1 and PLB. These data suggest that activated PKC-epsilon reduces SR Ca2+ content through PLB dephosphorylation and that reduced SR Ca2+ may be important in cardioprotection.     

Chronic xanthine oxidase inhibition prevents myofibrillar protein oxidation and preserves cardiac function in a transgenic mouse model of cardiomyopathy

Heart failure is a clinical syndrome associated with elevated levels of oxygen-derived free radicals. Xanthine oxidase activity is believed to be one source of reactive oxygen species in the failing heart. Interventions designed to reduce oxidative stress are believed to have significant therapeutic potential in heart failure. This study tested the hypothesis that xanthine oxidase activity would be elevated in a mouse model of dilated cardiomyopathy and evaluated the effect of chronic oral allopurinol, an inhibitor of xanthine oxidase, on contractility and progressive ventricular dilation in these mice. Nontransgenic and transgenic mice containing a troponin I truncation were treated with oral allopurinol from 2-4 mo of age. Myocardial xanthine oxidase activity was threefold higher in untreated transgenic mice compared with nontransgenic mice. Analyses of myofilament proteins for modification of carbonyl groups demonstrated myofibrillar protein damage in untreated transgenic mice. Treatment with allopurinol for 2 mo suppressed xanthine oxidase activity and myofibrillar protein oxidation. Allopurinol treatment also alleviated ventricular dilation and preserved shortening fraction in the transgenic animals. In addition, cardiac muscle twitch tension was preserved to 70% of nontransgenic levels in allopurinol-treated transgenic mice, a significant improvement over untreated transgenic mice. These findings indicate that chronic inhibition of xanthine oxidase can alter the progression of heart failure in dilated cardiomyopathy.     

Estrogen receptor-beta mediates male-female differences in the development of pressure overload hypertrophy

The goal of this study was to determine the role of estrogen receptor subtypes in the development of pressure overload hypertrophy in mice. Epidemiological studies have suggested gender differences in the development of hypertrophy and heart disease, but the mechanism and the role of estrogen receptor subtypes are not established. We performed transverse aortic constriction (TAC) and sham operations in male and female wild-type (WT) mice and mice lacking functional estrogen receptor-alpha [alpha-estrogen receptor knockout (alpha-ERKO)] and mice lacking estrogen receptor-beta (beta-ERKO). Body, heart, and lung weights were measured 2 wk postsurgery. WT male mice subjected to TAC showed a 64% increase in the heart weight-to-body weight ratio (HW/BW) compared with sham, and WT males have increased lung weight at 2 wk. WT female mice subjected to TAC showed a 31% increase in HW/BW compared with sham, which was significantly less than their male counterparts and with no evidence of heart failure. alpha-ERKO females developed HW/BW nearly identical to that seen in WT littermate females in response to TAC, indicating that estrogen receptor-alpha is not essential for the attenuation of hypertrophy observed in WT females. In contrast, beta-ERKO females responded to TAC with a significantly greater increase in HW/BW than WT littermate females. beta-ERKO females have lower expression of lipoprotein lipase at baseline than WT or alpha-ERKO females. These data suggest an important role for estrogen receptor-beta in attenuating the hypertrophic response to pressure overload in females.     

Quantification of supplemental enzymes in animal feedingstuffs by radial enzyme diffusion

Methods are described which facilitate quantification of supplemental cellulase, protease and -amylase when added to animal feedingstuffs at normal industrial inclusion levels. The methods entail extraction of the enzymes from the feedingstuffs by agitation in buffer followed by quantification of extract activity using radial diffusion techniques. A linear relationship between the diameter of the zone of hydrolyzed substrate and the log of the enzyme activity applied is observed over a broad activity range. Assay of a feedingstuff supplemented with 1 kg t^–1 cellulase, protease and -amylase yielded net supplemental activity recoveries of 104±11.7%, 91.3±6.74% and 126±29.5%, respectively. A similar assay method did not prove sufficiently sensitive to facilitate detection of xylanase at typical in-feed inclusion levels. The levels of endogenous cellulase, protease and -amylase activity detected in the unsupplemented feedingstuffs were equivalent to 6.4±0.47%, 6.6±0.82% and 29.0±14.1%, respectively, of a 1 kg t^–1 supplement. The methods are technically straightforward and will facilitate determination of enzyme stabilities during processes such as high-temperature pelleting of feedingstuffs, as well as allowing more rigorous quality control related to enzyme-supplemented animal feedingstuffs.