Homeostasis of cytoplasmic crowding by cell wall fluidization and ribosomal counterions.

In bacteria, algae, fungi, and plant cells, the wall must expand in concert with cytoplasmic biomass production, otherwise cells would experience toxic molecular crowding1,2 or lyse. But how cells achieve expansion of this complex biomaterial in coordination with biosynthesis of macromolecules in the cytoplasm remains unexplained3, although recent works have revealed that these processes are indeed coupled4,5. Here, we report a striking increase of turgor pressure with growth rate in E. coli, suggesting that the speed of cell wall expansion is controlled via turgor. Remarkably, despite this increase in turgor pressure, cellular biomass density remains constant across a wide range of growth rates. By contrast, perturbations of turgor pressure that deviate from this scaling directly alter biomass density. A mathematical model based on cell wall fluidization by cell wall endopeptidases not only explains these apparently confounding observations but makes surprising quantitative predictions that we validated experimentally. The picture that emerges is that turgor pressure is directly controlled via counterions of ribosomal RNA. Elegantly, the coupling between rRNA and turgor pressure simultaneously coordinates cell wall expansion across a wide range of growth rates and exerts homeostatic feedback control on biomass density. This mechanism may regulate cell wall biosynthesis from microbes to plants and has important implications for the mechanism of action of antibiotics6.

Plasticity of growth laws tunes resource allocation strategies in bacteria.

Bacteria like E. coli grow at vastly different rates on different substrates, however, the precise reason for this variability is poorly understood. Different growth rates have been attributed to 'nutrient quality', a key parameter in bacterial growth laws. However, it remains unclear to what extent nutrient quality is rooted in fundamental biochemical constraints like the energy content of nutrients, the protein cost required for their uptake and catabolism, or the capacity of the plasma membrane for nutrient transporters. Here, we show that while nutrient quality is indeed reflected in protein investment in substrate-specific transporters and enzymes, this is not a fundamental limitation on growth rate, at least for certain 'poor' substrates. We show that it is possible to turn mannose, one of the 'poorest' substrates of E. coli, into one of the 'best' substrates by reengineering chromosomal promoters of the mannose transporter and metabolic enzymes required for mannose degradation. This result falls in line with previous observations of more subtle growth rate improvement for many other carbon sources. However, we show that this faster growth rate comes at the cost of diverse cellular capabilities, reflected in longer lag phases, worse starvation survival and lower motility. We show that addition of cAMP to the medium can rescue these phenotypes but imposes a corresponding growth cost. Based on these data, we propose that nutrient quality is largely a self-determined, plastic property that can be modulated by the fraction of proteomic resources devoted to a specific substrate in the much larger proteome sector of catabolically activated genes. Rather than a fundamental biochemical limitation, nutrient quality reflects resource allocation decisions that are shaped by evolution in specific ecological niches and can be quickly adapted if necessary.

A universal mechanism of biomass density homeostasis via ribosomal counterions.

In all growing cells, the cell envelope must expand in concert with cytoplasmic biomass to prevent lysis or molecular crowding. The complex cell wall of microbes and plants makes this challenge especially daunting and it unclear how cells achieve this coordination. Here, we uncover a striking linear increase of cytoplasmic pressure with growth rate in E. coli. Remarkably, despite this increase in turgor pressure with growth rate, cellular biomass density was constant across a wide range of growth rates. In contrast, perturbing pressure away from this scaling directly affected biomass density. A mathematical model, in which endopeptidase-mediated cell wall fluidization enables turgor pressure to set the pace of cellular volume expansion, not only explains these confounding observations, but makes several surprising quantitative predictions that we validated experimentally. The picture that emerges is that changes in turgor pressure across growth rates are mediated by counterions of ribosomal RNA. Profoundly, the coupling between rRNA and cytoplasmic pressure simultaneously coordinates cell wall expansion across growth rates and exerts homeostatic feedback control on biomass density. Because ribosome content universally scales with growth rate in fast growing cells, this universal mechanism may control cell wall biosynthesis in microbes and plants and drive the expansion of ribosome-addicted tumors that can exert substantial mechanical forces on their environment.

Focusing of surface plasmon polaritons propagating at the SiO2/Ag interface with 2-level and 4-level Fresnel phase zone pad structures.

We propose and demonstrate dielectric Fresnel phase zone pad (FPZP) structures for focusing surface plasmon polaritons (SPPs) propagating at the SiO2/Ag interfaces. We exploited up-conversion fluorescence microscopy to characterize the SPP focusing. We first report on the SPP focusing with 2-level FPZP structures that introduced a π-phase shift in the SPP wavefront between adjacent zones. We optimized the SPP focusing by fine-tuning the longitudinal width of the FPZP structure. This led to the enhancement of the peak intensity of the SPP focal spot and the reduction of the focal spot size in both the longitudinal and transverse directions. Such focusing was also demonstrated with different focal lengths. To further improve the SPP focusing, we developed a 4-level FPZP structure, which introduced a π/2-phase shift in the SPP wavefront between adjacent zones. With the optimized 4-level FPZP structure, the SPP focal spot peak intensity is further improved, and the spot size is reduced. To assist the design of the FPZP structures, we carried out theoretical analysis and numerical calculations to determine the SPP wavelengths at various oxide/Ag interfaces. We also carried out finite difference time domain (FDTD) calculations to simulate the SPP focusing with the FPZP structures. The results of the FDTD simulation agree with the experimental results qualitatively.

Long-term history dependence of growth rates of E. coli after nutrient shifts.

According to a widely accepted paradigm of microbiology, steady-state growth rates are determined solely by current growth conditions1-3 and adaptations between growth states are rapid, as recently recapitulated by simple resource allocation models4. However, even in microbes overlapping regulatory networks can yield multi-stability or long-term cellular memory. Species like Listeria monocytogenes5 and Bacillus subtilis "distinguish" distinct histories for the commitment to sporulation6, but it is unclear if these states can persist over many generations. Remarkably, studying carbon co-utilization of Escherichia coli, we found that growth rates on combinations of carbon sources can depend critically on the previous growth condition. Growing in identical conditions, we observed differences in growth rates of up to 25% and we did not observe convergence of growth rates over 15 generations. We observed this phenomenon occurs across combinations of different phosphotransferase (PTS) substrates with various gluconeogenic carbon sources and found it to depend on the transcription factor Mlc.

Plasticity of growth laws tunes resource allocation strategies in bacteria.

Bacteria like E. coli grow at vastly different rates on different substrates, however, the precise reason for this variability is poorly understood. Different growth rates have been attributed to 'nutrient quality', a key parameter in bacterial growth laws. However, it remains unclear to what extent nutrient quality is rooted in fundamental biochemical constraints like the energy content of nutrients, the protein cost required for their uptake and catabolism, or the capacity of the plasma membrane for nutrient transporters. Here, we show that while nutrient quality is indeed reflected in protein investment in substrate-specific transporters and enzymes, this is not a fundamental limitation on growth rate. We show that it is possible to turn mannose, one of the 'poorest' substrates of E. coli, into one of the 'best' substrates by reengineering chromosomal promoters of the mannose transporter and metabolic enzymes required for mannose degradation. However, we show that this faster growth rate comes at the cost of diverse cellular capabilities, reflected in longer lag phases, worse starvation survival and lower motility. We show that addition of cAMP to the medium can rescue these phenotypes but imposes a corresponding growth cost. Based on these data, we propose that nutrient quality is largely a self-determined, plastic property that can be modulated by the fraction of proteomic resources devoted to a specific substrate in the much larger proteome sector of catabolically activated genes. Rather than a fundamental biochemical limitation, nutrient quality reflects resource allocation decisions that are shaped by evolution in specific ecological niches and can be quickly adapted if necessary.

Analysis of proteome adaptation reveals a key role of the bacterial envelope in starvation survival.

Bacteria reorganize their physiology upon entry to stationary phase. What part of this reorganization improves starvation survival is a difficult question because the change in physiology includes a global reorganization of the proteome, envelope, and metabolism of the cell. In this work, we used several trade-offs between fast growth and long survival to statistically score over 2,000 Escherichia coli proteins for their global correlation with death rate. The combined ranking allowed us to narrow down the set of proteins that positively correlate with survival and validate the causal role of a subset of proteins. Remarkably, we found that important survival genes are related to the cell envelope, i.e., periplasm and outer membrane, because the maintenance of envelope integrity of E. coli plays a crucial role during starvation. Our results uncover a new protective feature of the outer membrane that adds to the growing evidence that the outer membrane is not only a barrier that prevents abiotic substances from reaching the cytoplasm but also essential for bacterial proliferation and survival.

MetA is a "thermal fuse" that inhibits growth and protects Escherichia coli at elevated temperatures.

Adaptive stress resistance in microbes is mostly attributed to the expression of stress response genes, including heat-shock proteins. Here, we report a response of E. coli to heat stress caused by degradation of an enzyme in the methionine biosynthesis pathway (MetA). While MetA degradation can inhibit growth, which by itself is detrimental for fitness, we show that it directly benefits survival at temperatures exceeding 50°C, increasing survival chances by more than 1,000-fold. Using both experiments and mathematical modeling, we show quantitatively how protein expression, degradation rates, and environmental stressors cause long-term growth inhibition in otherwise habitable conditions. Because growth inhibition can be abolished with simple mutations, namely point mutations of MetA and protease knockouts, we interpret the breakdown of methionine synthesis as a system that has evolved to halt growth at high temperatures, analogous to "thermal fuses" in engineering that shut off electricity to prevent overheating.

Phosphatidic acid species 34:1 mediates expression of the myo-inositol 3-phosphate synthase gene INO1 for lipid synthesis in yeast.

Depletion of exogenous inositol in yeast results in rising levels of phosphatidic acid (PA) and is correlated with increased expression of genes containing the inositol-dependent upstream activating sequence promoter element (UASINO). INO1, encoding myo-inositol 3-phosphate synthase, is the most highly regulated of the inositol-dependent upstream activating sequence-containing genes, but its mechanism of regulation is not clear. In the current study, we determined the relative timing and kinetics of appearance of individual molecular species of PA following removal of exogenous inositol in actively growing wild type, pah1Δ, and ole1ts strains. We report that the pah1Δ strain, lacking the PA phosphatase, exhibits a delay of about 60 min in comparison to wildtype before initiating derepression of INO1 expression. The ole1ts mutant on the other hand, defective in fatty acid desaturation, when grown at a semirestrictive temperature, exhibited reduced synthesis of PA species 34:1 and elevated synthesis of PA species 32:1. Importantly, we found these changes in the fatty acid composition in the PA pool of the ole1ts strain were associated with reduced expression of INO1, indicating that synthesis of PA 34:1 is involved in optimal expression of INO1 in the absence of inositol. Using deuterium-labeled glycerol in short-duration labeling assays, we found that changes associated with PA species 34:1 were uniquely correlated with increased expression of INO1 in all three strains. These data indicate that the signal for activation of INO1 transcription is not necessarily the overall level of PA but rather levels of a specific species of newly synthesized PA 34:1.

Subwavelength-resolution imaging of surface plasmon polaritons with up-conversion fluorescence microscopy.

Imaging and characterization of surface plasmon polaritons (SPPs) are crucial for the research and development of the plasmonic devices and circuits. Here, we report on direct imaging of SPPs propagation on SiO2/metal interface with subwavelength spatial resolution using up-conversion fluorescence microscopy, that exploits rare-earth ions, such as Er3+, Yb3+, and Nd3+, doped nanoparticles as the fluorophores. We demonstrated that by further taking the intensity ratio of the image obtained with fluorescent emission at different wavelengths, we are able to substantially enhance the features associated to the SPP wavefronts in the image for quantitative analysis, such as the wavevector and propagation direction of the SPPs. Our results agree with the theoretic prediction of the SPP wavelengths quantitatively. We further demonstrate the evolution of the SPP wavefronts due to refraction SPPs, and reproduced the experiment with finite difference time domain (FDTD) method simulations. The relative refractive index of SPP estimated from the experiment also agrees quantitatively with those extracted from the theory and the simulation.

From DNA Damage to Cancer Progression: Potential Effects of Cytolethal Distending Toxin.

Cytolethal distending toxin (CDT), one of the most important genotoxins, is produced by several gram-negative bacteria and is involved in bacterial pathogenesis. Recent studies have shown that bacteria producing this peculiar genotoxin target host DNA, which potentially contributes to development of cancer. In this review, we highlighted the recent studies focusing on the idea that CDT leads to DNA damage, and the cells with inappropriately repaired DNA continue cycling, resulting in cancer development. Understanding the detailed mechanisms of genotoxins that cause DNA damage might be useful for targeting potential markers that drive cancer progression and help to discover new therapeutic strategies to prevent diseases caused by pathogens.

Bacterial Genotoxin-Coated Nanoparticles for Radiotherapy Sensitization in Prostate Cancer.

Prostate cancer (PCa) is one of the most commonly diagnosed cancers in men and usually becomes refractory because of recurrence and metastasis. CD44, a transmembrane glycoprotein, serves as a receptor for hyaluronic acid (HA). It has been found to be abundantly expressed in cancer stem cells (CSCs) that often exhibit a radioresistant phenotype. Cytolethal distending toxin (CDT), produced by Campylobacter jejuni, is a tripartite genotoxin composed of CdtA, CdtB, and CdtC subunits. Among the three, CdtB acts as a type I deoxyribonuclease (DNase I), which creates DNA double-strand breaks (DSBs). Nanoparticles loaded with antitumor drugs and specific ligands that recognize cancerous cell receptors are promising methods to overcome the therapeutic challenges. In this study, HA-decorated nanoparticle-encapsulated CdtB (HA-CdtB-NPs) were prepared and their targeted therapeutic activity in radioresistant PCa cells was evaluated. Our results showed that HA-CdtB-NPs sensitized radioresistant PCa cells by enhancing DSB and causing G2/M cell-cycle arrest, without affecting the normal prostate epithelial cells. HA-CdtB-NPs possess maximum target specificity and delivery efficiency of CdtB into the nucleus and enhance the effect of radiation in radioresistant PCa cells. These findings demonstrate that HA-CdtB-NPs exert target specificity accompanied with radiomimetic activity and can be developed as an effective strategy against radioresistant PCa.

A universal trade-off between growth and lag in fluctuating environments.

The rate of cell growth is crucial for bacterial fitness and drives the allocation of bacterial resources, affecting, for example, the expression levels of proteins dedicated to metabolism and biosynthesis1,2. It is unclear, however, what ultimately determines growth rates in different environmental conditions. Moreover, increasing evidence suggests that other objectives are also important3-7, such as the rate of physiological adaptation to changing environments8,9. A common challenge for cells is that these objectives cannot be independently optimized, and maximizing one often reduces another. Many such trade-offs have indeed been hypothesized on the basis of qualitative correlative studies8-11. Here we report a trade-off between steady-state growth rate and physiological adaptability in Escherichia coli, observed when a growing culture is abruptly shifted from a preferred carbon source such as glucose to fermentation products such as acetate. These metabolic transitions, common for enteric bacteria, are often accompanied by multi-hour lags before growth resumes. Metabolomic analysis reveals that long lags result from the depletion of key metabolites that follows the sudden reversal in the central carbon flux owing to the imposed nutrient shifts. A model of sequential flux limitation not only explains the observed trade-off between growth and adaptability, but also allows quantitative predictions regarding the universal occurrence of such tradeoffs, based on the opposing enzyme requirements of glycolysis versus gluconeogenesis. We validate these predictions experimentally for many different nutrient shifts in E. coli, as well as for other respiro-fermentative microorganisms, including Bacillus subtilis and Saccharomyces cerevisiae.

Effect of Ruellia tuberosa L. on aorta endothelial damage-associated factors in high-fat diet and streptozotocin-induced type 2 diabetic rats.

Hyperglycemia plays crucial roles in vascular disease development, including macrovascular and microvascular diseases from diabetes mellitus (DM). Our previous study demonstrated that Ruellia tuberosa L. (RTL) aqueous and ethanol extracts alleviate hyperglycemia and inhibit insulin resistance in diabetic rats. This study investigated the protective effect of RTL ethanol extract against aorta dysfunction in high-fat diet (HFD) and streptozotocin (STZ)-induced type 2 DM (T2DM) rats. Results showed that RTL ethanol extract (100 and 400 mg/kg BW/day) ameliorated serum lipid profiles, including triglyceride, free fatty acid, low-density lipoprotein cholesterol, very low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol levels. It also significantly reduced the level of serum cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 in T2DM rats. Additionally, RTL extract decreased endothelin-1 and endothelial nitric oxide contents, reduced the level of cell adhesion factors, including monocyte chemoattractant protein-1 and cell adhesion factor vascular cell adhesion molecule-1, while decreasing content of damage factors, namely tissue factor and von Willebrand factor in aortic tissues of diabetic rats. Equally noteworthy is that RTL extract enhanced the activity of aorta antioxidative enzymes, including superoxidase dismutase and catalase in diabetic rats, suggesting that RTL ethanol extract may ameliorate aorta dysfunction via enhancing aortic antioxidative enzyme activity, which subsequently suppresses aorta endothelial damage-associated factors in HFD with STZ-induced T2DM rats.

Ameliorative effect of Ruellia tuberosa L. on hyperglycemia in type 2 diabetes mellitus and glucose uptake in mouse C2C12 myoblasts.

Ruellia tuberosa L. (RTL) exhibits a wide range of phytochemical activities, for example, on treatment of diabetes mellitus (DM), in Orient. There is, however, few study regarding the effect of RTL on glycemic-related homeostasis in type 2 DM (T2DM). We investigated the effect of RTL aqueous and ethanolic extracts on hypoglycemia in high-fat diet (HFD)-fed plus streptozotocin (STZ)-induced T2DM rats, and examined the effect of RTL on glucose uptake in tumor necrosis factor-α-induced insulin-resistant mouse C2C12 myoblasts, a mouse skeletal muscle cell line. The administration of 100 or 400 mg kg BW-1 day-1 of RTL aqueous or ethanolic extracts once a day for 4 weeks significantly ameliorated hyperglycemia, hyperinsulinemia, and the insulin resistance (IR) index in diabetic rats. RTL either aqueous or ethanolic extract at a concentration of 25-800 µg/ml significantly improved glucose uptake in insulin-resistant mouse C2C12 myoblasts, indicating inhibiting the IR in skeletal muscles. These evidences suggest that RTL ameliorates hyperglycemia in HFD/STZ-induced T2DM rats may be attributed to the alleviation of IR in skeletal muscles.

Protective effect of Ruellia tuberosa L. extracts against abnormal expression of hepatic detoxification enzymes in diabetic rats.

Ruellia tuberosa L. (RTL) has been used as a folk medicine for curing diabetes mellitus in East Asia decades. This study investigated the effect of RTL on hepatic detoxification enzyme expression in diabetic rats. Male Wistar rats were fed a high fat diet (HFD) and intraperitoneally injected with streptozotocin (STZ) to induce diabetes. Subsequently, rats received oral administrations of 100 or 400 mg kg-1 body weight RTL extract, in either water (RTLW) or ethanol (RTLE), once a day for 4 weeks. The real-time PCR analyses showed that abnormality of hepatic phase I and II detoxification enzyme expression was observed in diabetic rats. However, both RTLW and RTLE significantly normalized the expression of hepatic phase I detoxification enzymes such as CYP 2E1, and expression of phase II detoxification enzymes such as UGT 1A7 and GST M1 in diabetic rats. Furthermore, we found that fasting serum glucose, hemoglobin A1C (HbA1C) and the area under the curve of oral glucose tolerance test (AUCOGTT) levels were significantly reduced in both RTLW and RTLE treated diabetic rats. Moreover, both RTLW and RTLE significantly increased the activity of hepatic anti-oxidative enzymes such as superoxide dismutase (SOD) in diabetic rats. The present study suggests that RTL may ameliorate abnormal hepatic detoxification function via alleviating hyperglycemia and enhancing hepatic antioxidant capacity in HFD/STZ-induced diabetic rats.

Interaction between repressor Opi1p and ER membrane protein Scs2p facilitates transit of phosphatidic acid from the ER to mitochondria and is essential for INO1 gene expression in the presence of choline.

In the yeast Saccharomyces cerevisiae, the Opi1p repressor controls the expression of INO1 via the Opi1p/Ino2p-Ino4p regulatory circuit. Inositol depletion favors Opi1p interaction with both Scs2p and phosphatidic acid at the endoplasmic reticulum (ER) membrane. Inositol supplementation, however, favors the translocation of Opi1p from the ER into the nucleus, where it interacts with the Ino2p-Ino4p complex, attenuating transcription of INO1 A strain devoid of Scs2p (scs2Δ) and a mutant, OPI1FFAT, lacking the ability to interact with Scs2p were utilized to examine the specific role(s) of the Opi1p-Scs2p interaction in the regulation of INO1 expression and overall lipid metabolism. Loss of the Opi1p-Scs2p interaction reduced INO1 expression and conferred inositol auxotrophy. Moreover, inositol depletion in strains lacking this interaction resulted in Opi1p being localized to sites of lipid droplet formation, coincident with increased synthesis of triacylglycerol. Supplementation of choline to inositol-depleted growth medium led to decreased TAG synthesis in all three strains. However, in strains lacking the Opi1p-Scs2p interaction, Opi1p remained in the nucleus, preventing expression of INO1 These data support the conclusion that a specific pool of phosphatidic acid, associated with lipid droplet formation in the perinuclear ER, is responsible for the initial rapid exit of Opi1p from the nucleus to the ER and is required for INO1 expression in the presence of choline. Moreover, the mitochondria-specific phospholipid, cardiolipin, was significantly reduced in both strains compromised for Opi1p-Scs2p interaction, indicating that this interaction is required for the transfer of phosphatidic acid from the ER to the mitochondria for cardiolipin synthesis.

Dissecting seipin function: the localized accumulation of phosphatidic acid at ER/LD junctions in the absence of seipin is suppressed by Sei1p(ΔNterm) only in combination with Ldb16p.

BACKGROUND: Seipin is required for the correct assembly of cytoplasmic lipid droplets. In the absence of the yeast seipin homolog Sei1p (formerly Fld1p), droplets are slow to bud from the endoplasmic reticulum, lack the normal component of proteins on their surface, are highly heterogeneous in size and shape, often bud into the nucleus, and promote local proliferation of the endoplasmic reticulum in which they become tangled. But the mechanism by which seipin catalyzes lipid droplet formation is still uncertain. RESULTS: Seipin prevents a localized accumulation of phosphatidic acid (PA puncta) at ER-droplet junctions. PA puncta were detected with three different probes: Opi1p, Spo20p(51-91) and Pah1p. A system of droplet induction was used to show that PA puncta were not present until droplets were formed; the puncta appeared regardless of whether droplets consisted of triacylglycerol or steryl ester. Deletion strains were used to demonstrate that a single phosphatidic acid-producing enzyme is not responsible for the generation of the puncta, and the puncta remain resistant to overexpression of enzymes that metabolize phosphatidic acid, suggesting that this lipid is trapped in a latent compartment. Suppression of PA puncta requires the first 14 amino acids of Sei1p (Nterm), a domain that is also important for initiation of droplet assembly. Consistent with recent evidence that Ldb16p and Sei1p form a functional unit, the PA puncta phenotype in the ldb16Δ sei1Δ strain was rescued by human seipin. Moreover, PA puncta in the sei1Δ strain expressing Sei1p(ΔNterm) was suppressed by overexpression of Ldb16p, suggesting a functional interaction of Nterm with this protein. Overexpression of both Sei1p and Ldb16p, but not Sei1p alone, is sufficient to cause a large increase in droplet number. However, Ldb16p alone increases triacylglycerol accumulation in the ldb16Δ sei1Δ background. CONCLUSION: We hypothesize that seipin prevents formation of membranes with extreme curvature at endoplasmic reticulum/droplet junctions that would attract phosphatidic acid. While Ldb16p alone can affect triacylglycerol accumulation, proper droplet formation requires the collaboration of Sei1p and Ldb16.

CGI-58/ABHD5 is phosphorylated on Ser239 by protein kinase A: control of subcellular localization.

CGI-58/ABHD5 coactivates adipose triglyceride lipase (ATGL). In adipocytes, CGI-58 binds to perilipin 1A on lipid droplets under basal conditions, preventing interaction with ATGL. Upon activation of protein kinase A (PKA), perilipin 1A is phosphorylated and CGI-58 rapidly disperses into the cytoplasm, enabling lipase coactivation. Because the amino acid sequence of murine CGI-58 has a predicted PKA consensus sequence of RKYS(239)S(240), we hypothesized that phosphorylation of CGI-58 is involved in this process. We show that Ser239 of murine CGI-58 is a substrate for PKA using phosphoamino acid analysis, MS, and immuno-blotting approaches to study phosphorylation of recombinant CGI-58 and endogenous CGI-58 of adipose tissue. Phosphorylation of CGI-58 neither increased nor impaired coactivation of ATGL in vitro. Moreover, Ser239 was not required for CGI-58 function to increase triacylglycerol turnover in human neutral lipid storage disorder fibroblasts that lack endogenous CGI-58. Both CGI-58 and S239A/S240A-mutated CGI-58 localized to perilipin 1A-coated lipid droplets in cells. When PKA was activated, WT CGI-58 dispersed into the cytoplasm, whereas substantial S239A/S240A-mutated CGI-58 remained on lipid droplets. Perilipin phosphorylation also contributed to CGI-58 dispersion. PKA-mediated phosphorylation of CGI-58 is required for dispersion of CGI-58 from perilipin 1A-coated lipid droplets, thereby increasing CGI-58 availability for ATGL coactivation.

Synthesis and anti-osteoporotic evaluation of certain 3-amino-2-hydroxypropoxyisoflavone derivatives.

We report herein the synthesis and anti-osteoporotic evaluation of certain 3-amino-2-hydroxypropoxyisoflavone derivatives. The results indicated that 3-(3,4-dimethoxyphenyl)-7-(oxiran-2-ylmethoxy)-4H-chromen-4-one (4) and 3-{4-[3-(cyclohexylamino)-2-hydroxypropoxy]phenyl}-7-methoxy-4H-chromen-4-one (5a) exhibited significant inhibitory effects on osteoclast activity (TRAP activity in RAW 264.7 with an ED(50) of 0.56 and 2.28 microM respectively). Both compounds have also exhibited very strong osteogenic effects, approximately a 10-fold effect of Ipriflavone on mineralization of osteoblasts (MC3T3E1 cells, a preosteoblast cell line derived from calvaria of C57BL/6 mice). Results indicated the potency on enhancing mineralization in D1 cells (a bone marrow mesenchymal cell line derived from BALB/c mice) decreased in an order 4>Ipriflavone>5a. However, the potency on enhancing mineralization in human adipose tissue derived stem cells (hADSCs) decreased in an order 5a>4>Raloxifene>Ipriflavone. Compound 5a has been found to be non-cytotoxic and especially active in the enhancement of mineralization in human adipose tissue derived stem cells. Therefore, 5a was selected as a potential lead for further structural optimization.