NF-kappaB and not the MAPK signaling pathway regulates GADD45beta expression during acute inflammation

The GADD45 (growth arrest and DNA damage-inducible) family of genes is involved in the regulation of cell cycle progression and apoptosis. To study signaling pathways affecting GADD45beta expression and to examine systematically in vivo the GADD45beta expression in tissues following various toxic stresses, we created a transgenic mouse by fusing the GADD45beta promoter to firefly luciferase (Gadd45beta-luc). In vivo GADD45beta expression was assessed by measuring the luciferase activity in the Gadd45beta-luc transgenic mouse using a non-invasive imaging system (IVIS Imaging System, Xenogen Corporation). We found that a number of agents that induce oxidative stress, such as sodium arsenite, CCl4, lipopolysaccharide (LPS), or tumor necrosis factor-alpha, are able to induce luciferase expression throughout the entire animal. In liver, spleen, lung, intestine, kidney, and heart, we observed an induction of luciferase activity after LPS treatment, which correlates with an increase of GADD45beta mRNA in these tissues. Processes that induce DNA damage activate the NF-kappaB signaling pathway. Several inhibitors of the NF-kappaB signaling pathway, including dexamethasone, thalidomide, and a proteasome inhibitor, bortezomib, showed inhibitory effects on LPS-induced GADD45beta expression as indicated by a decrease of the luciferase activity. Northern blot analysis confirmed a broad inhibitory effect of bortezomib on LPS-induced GADD45beta mRNA expression in spleen, lung, and intestine. In liver of bortezomib-treated mice, we observed a reverse correlation between the luciferase activity and the GADD45beta mRNA level. We speculate that such a discrepancy could be due to severe liver toxicity caused by bortezomib and LPS co-treatment. MAPK inhibitors had transient and inconsistent effects on LPS-induced luciferase expression. Our data are consistent with the notion that NF-kappaB, but not the MAPK signaling pathways, is involved in the in vivo regulation of GADD45beta expression. Thus, NF-kappaB signaling involves induction of GADD45beta expression, which supports the proposed role of GADD45beta in protecting cells against DNA damaged under various stress conditions.     

Regulatory mechanism of histidine-tagged homocitrate synthase from Saccharomyces cerevisiae. I. Kinetic studies

Homocitrate synthase (HCS) catalyzes one of the regulated steps of the alpha-aminoadipate pathway for lysine biosynthesis in fungi. The kinetic mechanism of regulation of HCS from Saccharomyces cerevisiae by Na+ and the feedback inhibitor lysine was studied by measuring the initial rate in the absence and presence of the effectors. The data suggest that Na+ is an activator at low concentrations and an inhibitor at high concentrations and that these effects occur as a result of the monovalent ion binding to two different sites in the free enzyme. Inhibition and activation by Na+ can occur simultaneously, with the net rate of the enzyme determined by Na+/K(iNa+) and Na+/K(act), where K(iNa+) and K(act) are the inhibition and activation constants, respectively. The inhibition by Na+ was eliminated at high concentrations of acetyl-CoA, the second substrate bound, but the activation remained. Fluorescence binding studies indicated that lysine bound with high affinity to its binding site as an inhibitor. The inhibition by lysine was competitive versus alpha-ketoglutarate and linear in the physiological range of lysine concentrations up to 5 mm. The effects of Na+ and lysine were independent of one another. A model is developed for regulation of HCS that takes into account all of the effects discussed above.     

Dynamic osmotic loading of chondrocytes using a novel microfluidic device

Many cells exhibit disparate responses to a mechanical stimulus depending on whether it is applied dynamically or statically. In this context, few studies have examined how cells respond to dynamic changes of the extracellular osmolality. In this study, we hypothesized that the cell size change response of cultured articular chondrocytes would be dependent on the frequency of applied osmotic loading. To test this hypothesis, we developed a novel microfluidic device, to apply hydrostatic pressure-driven dynamic osmotic loading by applying composition modulated flow, adapted from Tang and co-workers. This microfluidic device was used to study osmotic loads of +/-180 mOsm at a frequency up to 0.1 Hz with a constant minimal fluid-shear stress, and permit real-time monitoring of cell responses. Bovine articular chondrocytes were observed to exhibit increasing changes in cell volume with decreasing osmotic loading frequency. When the cell volume response was modeled by an exponential function, chondrocytes exhibited significantly different volume change responses to dynamic osmotic loading at 0.0125 Hz and static osmotic loading applied for a period of four minutes (Delta = +/-180 mOsm relative to the isotonic 360 mOsm). The intracellular calcium response at 0.0125 Hz was also monitored and compared with the response to static loading. Coupled with phenomenological or constitutive models, this novel approach could yield new information regarding cell material properties in response to dynamic loading that may contribute new insights into mechanisms of cellular homeostasis and mechanotransduction.     

Mouse HFE inhibits Tf-uptake and iron accumulation but induces non-transferrin bound iron (NTBI)-uptake in transformed mouse fibroblasts

Iron-uptake and storage are tightly regulated to guarantee sufficient iron for essential cellular processes and to prevent the production of damaging free radicals. A non-classical class I MHC molecule, the hemochromatosis factor (HFE), has been shown to regulate iron metabolism, potentially via its interaction with the transferrin receptor. Whereas, the effect of human HFE (hHFE) on transferrin/transferrin receptor association, as well as on transferrin receptor recycling and the level of cellular iron pools in various cell lines was analyzed, very little is known about the mouse HFE (mHFE) protein. In the following study, our aim was to analyze in more detail the function of mHFE. Surprisingly, we observed that over-expression of mHFE, but not of hHFE, in a mouse transformed cell line, results in a most significant inhibition of transferrin-uptake which correlated with apoptotic cell death. mHFE inhibited transferrin-uptake immediately following transfection and this inhibition persisted in the surviving stable transfectants. Concomitantly, cellular iron derived from transferrin-iron uptake was dramatically limited. The activation of a non-transferrin bound iron-uptake pathway that functions in the stable mHFE-transfected clones could explain their normal growth curves and survival. The hypothesis that iron starvation can induce iron-uptake by a novel transferrin-independent pathway is discussed.     

Soluble methionine enhances accumulation of a 15 kDa zein, a methionine-rich storage protein, in transgenic alfalfa but not in transgenic tobacco plants

With the general aim of elevating the content of the essential amino acid methionine in vegetative tissues of plants, alfalfa (Medicago sativa L.) and tobacco plants, as well as BY2 tobacco suspension cells, were transformed with a beta-zein::3HA gene under the 35S promoter of cauliflower mosaic virus encoding a rumen-stable methionine-rich storage protein of 15 kDa zein. To examine whether soluble methionine content limited the accumulation of the 15 kDa zein::3HA, methionine was first added to the growth medium of the different transgenic plants and the level of the alien protein was determined. Results demonstrated that the added methionine enhanced the accumulation of the 15 kDa zein::3HA in transgenic alfalfa and tobacco BY2 cells, but not in whole transgenic tobacco plants. Next, the endogenous levels of methionine were elevated in the transgenic tobacco and alfalfa plants by crossing them with plants expressing the Arabidopsis cystathionine gamma-synthase (AtCGS) having significantly higher levels of soluble methionine in their leaves. Compared with plants expressing only the 15 kDa zein::3HA, transgenic alfalfa co-expressing both alien genes showed significantly enhanced levels of this protein concurrently with a reduction in the soluble methionine content, thus implying that soluble methionine was incorporated into the 15 kDa zein::3HA. Similar phenomena also occurred in tobacco, but were considerably less pronounced. The results demonstrate that the accumulation of the 15 kDa zein::3HA is regulated in a species-specific manner and that soluble methionine plays a major role in the accumulation of the 15 kDa zein in some plant species but less so in others.     

Systematic segregation to mutant mitochondrial DNA and accompanying loss of mitochondrial DNA in human NT2 teratocarcinoma Cybrids

In this study a well-characterized pathological mutation at nucleotide position 3243 of human mitochondrial DNA was introduced into human rho(0) teratocarcinoma (NT2) cells. In cloned and mixed populations of NT2 cells heteroplasmic for the mutation, mitotic segregation toward increasing levels of mutant mitochondrial DNA always occurred. Rapid segregation was frequently followed by complete loss of mitochondrial DNA. These findings support the idea that pathological mitochondrial DNA mutations are particularly deleterious in specific cell types, which can explain some of the tissue-specific aspects of mitochondrial DNA diseases. Moreover, these findings suggest that mitochondrial DNA depletion may be an important and widespread feature of mitochondrial DNA disease.     

Inhibitor and ion binding sites on the gastric H,K-ATPase

The gastric H,K-ATPase catalyzes electroneutral exchange of H(+) for K(+) as a function of enzyme phosphorylation and dephosphorylation during transition between E(1)/E(1)-P (ion site in) and E(2)-P/E(2) (ion site out) conformations. Here we present homology modeling of the H,K-ATPase in the E(2)-P conformation as a means of predicting the interaction of the enzyme with two known classes of specific inhibitors. All known proton pump inhibitors, PPIs, form a disulfide bond with cysteine 813 that is accessible from the luminal surface. This allows allocation of the binding site to a luminal vestibule adjacent to Cys813 enclosed by part of TM4 and the loop between TM5 and TM6. K(+) competitive imidazo-1,2alpha-pyridines also bind to the luminal surface of the E(2)-P conformation, and their binding excludes PPI reaction. This overlap of the binding sites of the two classes of inhibitors combined with the results of site-directed mutagenesis and cysteine cross-linking allowed preliminary assignment of a docking mode for these reversible compounds in a position close to Glu795 that accounts for the detailed structure/activity relationships known for these compounds. The new E(2)-P model is able to assign a possible mechanism for acid secretion by this P(2)-type ATPase. Several ion binding side chains identified in the sr Ca-ATPase by crystallography are conserved in the Na,K- and H,K-ATPases. Poised in the middle of these, the H,K-ATPase substitutes lysine in place of a serine implicated in K(+) binding in the Na,K-ATPase. Molecular models for hydronium binding to E(1) versus E(2)-P predict outward displacement of the hydronium bound between Asp824, Glu820, and Glu795 by the R-NH(3)(+) of Lys791 during the conformational transition from E(1)P and E(2)P. The site for luminal K(+) binding at low pH is proposed to be between carbonyl oxygens in the nonhelical part of the fourth membrane span and carboxyl oxygens of Glu795 and Glu820. This site of K(+) binding is predicted to destabilize hydrogen bonds between these carboxylates and the -NH(3)(+) group of Lys791, allowing the Lys791 side chain to return to its E(1) position.     

Kinetic and chemical mechanisms of the fabG-encoded Streptococcus pneumoniae beta-ketoacyl-ACP reductase

Beta-ketoacyl-acyl carrier protein reductase (KACPR) catalyzes the NADPH-dependent reduction of beta-ketoacyl-acyl carrier protein (AcAc-ACP) to generate (3S)-beta-hydroxyacyl-ACP during the chain-elongation reaction of bacterial fatty acid biosynthesis. We report the evaluation of the kinetic and chemical mechanisms of KACPR using acetoacetyl-CoA (AcAc-CoA) as a substrate. Initial velocity, product inhibition, and deuterium kinetic isotope effect studies were consistent with a random bi-bi rapid-equilibrium kinetic mechanism of KACPR with formation of an enzyme-NADP(+)-AcAc-CoA dead-end complex. Plots of log V/K(NADPH) and log V/K(AcAc)(-)(CoA) indicated the presence of a single basic group (pK = 5.0-5.8) and a single acidic group (pK = 8.0-8.8) involved in catalysis, while the plot of log V vs pH indicated that at high pH an unprotonated form of the ternary enzyme complex was able to undergo catalysis. Significant and identical primary deuterium kinetic isotope effects were observed for V (2.6 +/- 0.4), V/K(NADPH) (2.6 +/- 0.1), and V/K(AcAc)(-)(CoA) (2.6 +/- 0.1) at pH 7.6, but all three values attenuated to values of near unity (1.1 +/- 0.03 or 0.91 +/- 0.02) at pH 10. Similarly, the large alpha-secondary deuterium kinetic isotope effect of 1.15 +/- 0.02 observed for [4R-(2)H]NADPH on V/K(AcAc)(-)(CoA) at pH 7.6 was reduced to a value of unity (1.00 +/- 0.04) at high pH. The complete analysis of the pH profiles and the solvent, primary, secondary, and multiple deuterium isotope effects were most consistent with a chemical mechanism of KACPR that is stepwise, wherein the hydride-transfer step is followed by protonation of the enolate intermediate. Estimations of the intrinsic primary and secondary deuterium isotope effects ((D)k = 2.7, (alpha)(-D)k = 1.16) and the correspondingly negligible commitment factors suggest a nearly full expression of the intrinsic isotope effects on (D)V/K and (alpha)(-D)V/K, and are consistent with a late transition state for the hydride transfer step. Conversely, the estimated intrinsic solvent effect ((D)2(O)k) of 5.3 was poorly expressed in the experimentally derived parameters (D)2(O)V/K and (D)2(O)V (both = 1.2 +/- 0.1), in agreement with the estimation that the catalytic commitment factor for proton transfer to the enolate intermediate is large. Such detailed knowledge of the chemical mechanism of KAPCR may now help guide the rational design of, or inform screening assay-design strategies for, potent inhibitors of this and related enzymes of the short chain dehydrogenase enzyme class.     

A four-gene study of evolutionary relationships in Solanum section Acanthophora

The "spiny solanums," Solanum subgenus Leptostemonum (Solanaceae), comprise a large lineage with over 350 species and include the cultivated eggplant, Solanum melongena. Despite the importance of this subgenus, phylogenetic relationships among these taxa are currently unclear. The present research contributes to this understanding while focusing on Solanum section Acanthophora, a group of ca. 19 species defined by the presence of simple hairs, rather than the stellate hairs common across the rest of subgenus Leptostemonum. In this study we inferred phylogenetic relationships among 29 Solanum taxa, including 14 species of section Acanthophora, using DNA sequence data from two nuclear regions (ITS and the granule-bound starch synthase gene [GBSSI or waxy]) and two chloroplast regions (trnT-trnF and trnS-trnG). This combination of gene regions resulted in a well resolved phylogenetic hypothesis, with results strongly suggesting that Solanum sect. Acanthophora is not monophyletic, although the majority of taxa comprise a monophyletic lineage that is sister to Solanum section Lasiocarpa. Of the four gene regions, waxy was especially useful for phylogenetic inference, with both a high percentage of parsimony-informative sites as well as a low level of homoplasy. Further studies in progress will help elucidate relationships of sect. Acanthophora with respect to other members of subgenus Leptostemonum.     

The Y deletion gr/gr and susceptibility to testicular germ cell tumor

Testicular germ cell tumor (TGCT) is the most common cancer in young men. Despite a considerable familial component to TGCT risk, no genetic change that confers increased risk has been substantiated to date. The human Y chromosome carries a number of genes specifically involved in male germ cell development, and deletion of the AZFc region at Yq11 is the most common known genetic cause of infertility. Recently, a 1.6-Mb deletion of the Y chromosome that removes part of the AZFc region—known as the “gr/gr” deletion—has been associated with infertility. In epidemiological studies, male infertility has shown an association with TGCT that is out of proportion with what can be explained by tumor effects. Thus, we hypothesized that the gr/gr deletion may be associated with TGCT. Using logistic modeling, we analyzed this deletion in a large series of TGCT cases with and without a family history of TGCT. The gr/gr deletion was present in 3.0% (13/431) of TGCT cases with a family history, 2% (28/1,376) of TGCT cases without a family history, and 1.3% (33/2,599) of unaffected males. Presence of the gr/gr deletion was associated with a twofold increased risk of TGCT (adjusted odds ratio [aOR] 2.1; 95% confidence interval [CI] 1.3–3.6; P = .005) and a threefold increased risk of TGCT among patients with a positive family history (aOR 3.2; 95% CI 1.5–6.7; P = .0027). The gr/gr deletion was more strongly associated with seminoma (aOR 3.0; 95% CI 1.6–5.4; P = .0004) than with nonseminoma TGCT (aOR 1.5; 95% CI 0.72–3.0; P = .29). These data indicate that the Y microdeletion gr/gr is a rare, low-penetrance allele that confers susceptibility to TGCT.