Spectral observation of an acyl-enzyme intermediate of lipoprotein lipase

There have been several studies indicating that hydrolysis reactions of fatty acid esters catalyzed by lipases proceed through an acyl-enzyme intermediate typical of serine proteases. In particular, one careful kinetic study with the physiologically important enzyme lipoprotein lipase (LPL) is consistent with rate-limiting deacylation of such an intermediate. To observe the spectrum of acyl-enzyme and study the mechanism of LPL-catalyzed hydrolysis of substrate, we have used a variety of furylacryloyl substrates including 1,2-dipalmitoyl-3-[(beta-2-furylacryloyl)triacyl]glyceride (DPFATG) to study the intermediates formed during the hydrolysis reaction catalyzed by the enzyme. After isolation and characterization of the molecular weight of adipose LPL, we determined its extinction coefficient at 280 nm to quantitate the formation of any acyl-enzyme intermediate formed during substrate hydrolysis. We observed an intermediate at low pH during the enzyme-catalyzed hydrolysis of (furylacryloyl)imidazole. This intermediate builds early in the reaction when a substantial amount of substrate has hydrolyzed but no product, furylacrylate, has been formed. The acyl-enzyme has a lambda max = 305 nm and a molar extinction coefficient of 22,600 M-1 cm-1; these parameters are similar to those for furylacryloyl esters including the serine ester. These data provide the first spectral evidence for a serine acyl-enzyme in lipase-catalyzed reactions. The LPL hydrolysis reaction is base catalyzed, exhibiting two pKa values; the more acidic of these is 6.5, consistent with base catalysis by histidine. The biphasic rates for substrate disappearance or product appearance and the absence of leaving group effect indicate that deacylation of intermediate is rate limiting.     

Loss of protein targeting to glycogen sensitizes human hepatocellular carcinoma cells towards glucose deprivation mediated oxidative stress and cell death

Protein targeting to glycogen (PTG) is a ubiquitously expressed scaffolding protein that critically regulates glycogen levels in many tissues, including the liver, muscle and brain. However, its importance in transformed cells has yet to be explored in detail. Since recent studies have demonstrated an important role for glycogen metabolism in cancer cells, we decided to assess the effect of PTG levels on the ability of human hepatocellular carcinoma (HepG2) cells to respond to metabolic stress. Although PTG expression did not significantly affect the proliferation of HepG2 cells under normal culture conditions, we determined that PTG plays an important role during glucose deprivation. Overexpression of PTG protected cells from cell death in the absence of glucose, whereas knocking down PTG further promoted cytotoxicity, as measured by the release of lactate dehydrogenase (LDH) into the media. Additionally, we demonstrated that PTG attenuates glucose deprivation induced haeme oxygenase-1 (HO-1) expression, suggesting that PTG protects against glucose deprivation-induced oxidative stress. Indeed, treating cells with the antioxidant N-acetyl cysteine (NAC) rescued cells from cytotoxicity caused by glucose deprivation. Finally, we showed that loss of PTG resulted in enhanced autophagy. In control cells, glucose deprivation suppressed autophagy as determined by the increase in the levels of p62, an autophagy substrate. However, in knockdown cells, this suppression was relieved. Blockade of autophagy also attenuated cytotoxicity from glucose deprivation in PTG knockdown cells. Taken together, our findings identify a novel role for PTG in protecting hepatocellular carcinoma cells from metabolic stress, in part by regulating oxidative stress and autophagy.     

Kinetic modeling of <Emphasis Type="Italic">Moorella thermoacetica</Emphasis> growth on single and dual-substrate systems

Acetic acid is an important chemical raw material that can be produced directly from sugars in lignocellulosic biomass. Development of kinetic models that capture the bioconversion dynamics of multiple sugar systems will be critical to optimization and process control in future lignocellulosic biorefinery processes. In this work, a kinetic model was developed for the single- and dual-substrate conversion of xylose and glucose to acetic acid using the acetogen Moorella thermoacetica. Batch fermentations were performed experimentally at 20 g L^?1 total sugar concentration using synthetic glucose, xylose, and a mixture of glucose and xylose at a 1:1 ratio. The product yield, calculated as total product formed divided by total sugars consumed, was 79.2, 69.9, and 69.7 % for conversion of glucose, xylose, and a mixture of glucose and xylose (1:1 ratio), respectively. During dual-substrate fermentation, M. thermoacetica demonstrated diauxic growth where xylose (the preferred substrate) was almost entirely consumed before consumption of glucose began. Kinetic parameters were similar for the single-substrate fermentations, and a strong linear correlation was determined between the maximum specific growth rate μ _max and substrate inhibition constant, K _s . Parameters estimated for the dual-substrate system demonstrated changes in the specific growth rate of both xylose and glucose consumption. In particular, the maximum growth rate related to glucose tripled compared to the single-substrate system. Kinetic growth is affected when multiple substrates are present in a fermentation system, and models should be developed to reflect these features.     

Integrating ecophysiology and forest landscape models to improve projections of drought effects under climate change

Fundamental drivers of ecosystem processes such as temperature and precipitation are rapidly changing and creating novel environmental conditions. Forest landscape models (FLM) are used by managers and policy‐makers to make projections of future ecosystem dynamics under alternative management or policy options, but the links between the fundamental drivers and projected responses are weak and indirect, limiting their reliability for projecting the impacts of climate change. We developed and tested a relatively mechanistic method to simulate the effects of changing precipitation on species competition within the LANDIS‐II FLM. Using data from a field precipitation manipulation experiment in a piñon pine (Pinus edulis) and juniper (Juniperus monosperma) ecosystem in New Mexico (USA), we calibrated our model to measurements from ambient control plots and tested predictions under the drought and irrigation treatments against empirical measurements. The model successfully predicted behavior of physiological variables under the treatments. Discrepancies between model output and empirical data occurred when the monthly time step of the model failed to capture the short‐term dynamics of the ecosystem as recorded by instantaneous field measurements. We applied the model to heuristically assess the effect of alternative climate scenarios on the piñon–juniper ecosystem and found that warmer and drier climate reduced productivity and increased the risk of drought‐induced mortality, especially for piñon. We concluded that the direct links between fundamental drivers and growth rates in our model hold great promise to improve our understanding of ecosystem processes under climate change and improve management decisions because of its greater reliance on first principles.     

Expressed sequence tag-based gene expression analysis under aluminum stress in rye

To understand the mechanisms responsible for aluminum (Al) toxicity and tolerance in plants, an expressed sequence tag (EST) approach was used to analyze changes in gene expression in roots of rye (Secale cereale L. cv Blanco) under Al stress. Two cDNA libraries were constructed (Al stressed and unstressed), and a total of 1,194 and 774 ESTs were generated, respectively. The putative proteins encoded by these cDNAs were uncovered by Basic Local Alignment Search Tool searches, and those ESTs showing similarity to proteins of known function were classified according to 13 different functional categories. A total of 671 known function genes were used to analyze the gene expression patterns in rye cv Blanco root tips under Al stress. Many of the previously identified Al-responsive genes showed expression differences between the libraries within 6 h of Al stress. Certain genes were selected, and their expression profiles were studied during a 48-h period using northern analysis. A total of 13 novel genes involved in cell elongation and division (tonoplast aquaporin and ubiquitin-like protein SMT3), oxidative stress (glutathione peroxidase, glucose-6-phosphate-dehydrogenase, and ascorbate peroxidase), iron metabolism (iron deficiency-specific proteins IDS3a, IDS3b, and IDS1; S-adenosyl methionine synthase; and methionine synthase), and other cellular mechanisms (pathogenesis-related protein 1.2, heme oxygenase, and epoxide hydrolase) were demonstrated to be regulated by Al stress. These genes provide new insights about the response of Al-tolerant plants to toxic levels of Al.     

High Local Concentrations and Effects on Differentiation Implicate Interleukin‐6 as a Paracrine Regulator

Objective: To examine the possibility that interleukin‐6 (IL‐6) can act as a paracrine regulator in adipose tissue by examining effects on adipogenic genes and measuring interstitial IL‐6 concentrations in situ. Research Methods and Procedures: Circulating and interstitial IL‐6 concentrations in abdominal and femoral adipose tissue were measured using the calibrated microdialysis technique in 20 healthy male subjects. The effects of adipose cell enlargement on gene expression and IL‐6 secretion were examined, as well as the effect of IL‐6 in vitro on gene expression of adiponectin and other markers of adipocyte differentiation. Results: The IL‐6 concentration in the interstitial fluid was ～100‐fold higher than that in plasma, suggesting that IL‐6 may be a paracrine regulator of adipose tissue. This was further supported by the finding that adding IL‐6 in vitro at similar concentrations down‐regulated the expression of adiponectin, aP2, and PPARγ‐2 in cultured human adipose tissue. In addition, gene expression and release of IL‐6, both in vivo and in vitro, correlated with adipose cell size. Discussion: These data suggest that IL‐6 may be a paracrine regulator of adipose tissue. Furthermore, increased adipose tissue production of IL‐6 after hypertrophic enlargement of the adipose cells may detrimentally affect systemic insulin action by inducing adipose tissue dysfunction with impaired differentiation of the pre‐adipocytes and/or adipocytes and lower adiponectin.     

Transmission of chromosomes through the eggs and pollen of triticale × wheat F1 hybrids

Summary The substitution patterns of rye chromosomes in hexaploid triticale × wheat F₂ hybrids, along with the transmission patterns of rye chromosomes through egg cells and pollen when several of the F₁ hybrids were test crossed to triticale and wheat were investigated. The data indicated that the rye chromosome transmission through both the egg and pollen was random in number and in composition. The test crosses suggested that it was best to use wheat pollen for the transmission of rye chromosomes through the egg cells of the F₁ hybrids and triticale egg cells for the transmission of rye chromosomes through F₁ hybrid pollen. A deviation from random segregation in the F₂ and the transmission rate was observed for rye chromosomes 1R, 4R/7R, and 6R. The transmission rates of 1R and 6R varied depending on the direction in which the cross was made. The results also indicated that there was little or no compensation between the R- and D-genomes and that the chromosomes of these two genomes appeared to be transmitted independently of each other.     

Overexpression of protein kinase C betaII induces colonic hyperproliferation and increased sensitivity to colon carcinogenesis.

Protein kinase C betaII (PKC betaII) has been implicated in proliferation of the intestinal epithelium. To investigate PKC betaII function in vivo, we generated transgenic mice that overexpress PKC betaII in the intestinal epithelium. Transgenic PKC betaII mice exhibit hyperproliferation of the colonic epithelium and an increased susceptibility to azoxymethane-induced aberrant crypt foci, preneoplastic lesions in the colon. Furthermore, transgenic PKC betaII mice exhibit elevated colonic beta-catenin levels and decreased glycogen synthase kinase 3beta activity, indicating that PKC betaII stimulates the Wnt/adenomatous polyposis coli (APC)/beta-catenin proliferative signaling pathway in vivo. These data demonstrate a direct role for PKC betaII in colonic epithelial cell proliferation and colon carcinogenesis, possibly through activation of the APC/beta-catenin signaling pathway.     

Proprotein convertase subtilisin/kexin type 9 (PCSK9) affects gene expression pathways beyond cholesterol metabolism in liver cells

Proprotein convertase subtilisin/kexin type 9 (PCSK9) induces degradation of low‐density lipoprotein receptor (LDLR) in the liver. It is being pursued as a therapeutic target for LDL‐cholesterol reduction. Earlier genome‐wide gene expression studies showed that PCSK9 over‐expression in HepG2 cells resulted in up‐regulation of genes in cholesterol biosynthesis and down‐regulation of genes in stress response pathways; however, it was not known whether these changes were directly regulated by PCSK9 or were secondary to PCSK9‐induced changes to the intracellular environment. In order to further understand the biological function of PCSK9 we treated HepG2 cells with purified recombinant wild type (WT) and D374Y gain‐of‐function PCSK9 proteins for 8, 24, and 48 h, and used microarray analysis to identify genome‐wide expression changes and pathways. These results were compared to the changes induced by culturing HepG2 cells in cholesterol‐free medium, mimicking the intracellular environment of cholesterol starvation. We determined that PCSK9‐induced up‐regulation of cholesterol biosynthesis genes resulted from intracellular cholesterol starvation. In addition, we identified novel pathways that are presumably regulated by PCSK9 and are independent of its effects on cholesterol uptake. These pathways included “protein ubiquitination,” “xenobiotic metabolism,” “cell cycle,” and “inflammation and stress response.” Our results indicate that PCSK9 affects metabolic pathways beyond cholesterol metabolism in HepG2 cells. J. Cell. Physiol. 224:273–281, 2010 © 2010 Wiley‐Liss, Inc.