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Obesity-associated low-grade inflammation in metabolically relevant tissues contributes to insulin resistance. We recently reported monocyte/macrophage infiltration in mouse and human skeletal muscles. However, the molecular triggers of this infiltration are unknown, and the role of muscle cells in this context is poorly understood. Animal studies are not amenable to the specific investigation of this vectorial cellular communication. Using cell cultures, we investigated the crosstalk between myotubes and monocytes exposed to physiological levels of saturated and unsaturated fatty acids. Media from L6 myotubes treated with palmitate—but not palmitoleate—induced THP1 monocyte migration across transwells. Palmitate activated the Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) pathway in myotubes and elevated cytokine expression, but the monocyte chemoattracting agent was not a polypeptide. Instead, nucleotide degradation eliminated the chemoattracting properties of the myotube-conditioned media. Moreover, palmitate-induced expression and activity of pannexin-3 channels in myotubes were mediated by TLR4-NF-κB, and TLR4-NF-κB inhibition or pannexin-3 knockdown prevented monocyte chemoattraction. In mice, the expression of pannexin channels increased in adipose tissue and skeletal muscle in response to high-fat feeding. These findings identify pannexins as new targets of saturated fatty acid–induced inflammation in myotubes, and point to nucleotides as possible mediators of immune cell chemoattraction toward muscle in the context of obesity.

A set of 17,600 samples belonging to our compound collection is selectively examined by liquid chromatography with UV, evaporative light scattering, and mass spectrometric detection methods. At least 70% of this set consists of pure samples with the expected structures. Subsequent studies by flow injection mass spectrometry show that this value is a conservative estimate and that the actual percentage of pure and correct compounds is close to 80%. Because this is the first time that sample quality information becomes available on such a large scale for the compound collection, it offers an opportunity to perform chemi-informatic studies for which structural integrity is essential. Results of these studies can be used to improve the selection of compounds for screening and evaluate the quality of compounds from particular sources.

PNU‐107859, an important representative structure in a novel class of matrix metalloproteinases (MMP) inhibitors known as thiadiazoles, was found to be quickly eliminated from rats. A major metabolite (approximately 10% of total dose) was found to be present in the bile of rats. The metabolite in question was isolated and purified from the bile fluids collected from six cannulated rats. From a total of approximately 75 mg of PNU‐107859 administered to rats, 3.3 mg of the metabolite was recovered. The NMR and mass spectrometry results indicated that the metabolite is a glucuronide conjugate (1‐deoxy‐1& bgr; ‐substituted D ‐glucopyranosiduronic acid) of the intact drug. Furthermore, the UV, MS, and NMR data established that the conjugate is located at the nitrogen α to the thiocarbonyl of the thiadiazole ring.

Lincosamide antibiotics include lincomycin, naturally produced by several actinomycetes, and clindamycin, a semisynthetic derivative obtained by the chlorination of lincomycin. These antibiotics are active against many gram-positive cocci and anaerobes; they inhibit protein synthesis by blocking the peptidyltransferase activity of the 50S subunit of the bacterial ribosome (11). Resistance to lincosamides is usually due to alteration of the ribosome following the N6 dimethylation of a specific adenine in the 23S rRNA, which confers cross-resistance to macrolide, lincosamide, and streptogramin B type antibiotics, i.e., the MLSB phenotype (22, 32). In contrast to this broad-spectrum resistance, resistance specific to lincosamides, gained by bacterial modification of those antibiotics, has been reported. Phosphorylation (1) and nucleotidylation (2, 26) of the hydroxyl group in position 3 of lincosamide molecules (24, 26) have been detected in several species of Streptomyces. Inactivation of lincosamides was also observed in strains of staphylococci, streptococci, enterococci, and lactobacilli of animal origin (10, 12, 13) and in staphylococci isolated from humans (5, 20, 21). Clinical isolates of Staphylococcus haemolyticus BM4610 and Staphylococcus aureus BM4611 are highly resistant to lincomycin (MIC = 64 μg/ml) and are apparently susceptible to clindamycin (MIC = 0.12 μg/ml). In these strains, lincosamide O-nucleotidyltransferases encoded by two closely related genes named linA (lincosamide inactivation nucleotidylation) and linA′, respectively, were characterized (4, 5). These genes encode two 161-amino-acid isoenzymes that differ by 14 amino acids. These enzymes inactivate lincomycin and clindamycin by converting them to lincomycin 3-(5′-adenylate) and clindamycin 4-(5′-adenylate) by using ATP, GTP, CTP, or UTP as a nucleotidyl donor and MgCl2 as a cofactor (5). The distribution of linA and linA′ genes was studied by using DNA-DNA hybridization, and related sequences were found in strains belonging to various species of staphylococci (20). In this paper, we report the nucleotide sequence of a new linB gene that confers resistance to lincosamides on a clinical strain of Enterococcus faecium, HM1025, by inactivating the compounds, and we further report on our study of the biochemical mechanism of the resistance.

SummaryFGF21, a member of the fibroblast growth factor (FGF) superfamily, has recently emerged as a regulator of metabolism and energy utilization. However, the exact mechanism(s) whereby FGF21 mediates its actions have not been elucidated. There is considerable evidence that insulin resistance may arise from aberrant accumulation of intracellular lipids in insulin-responsive tissues due to lipotoxicity. In particular, the sphingolipid ceramide has been implicated in this process. Here, we show that FGF21 rapidly and robustly stimulates adiponectin secretion in rodents while diminishing accumulation of ceramides in obese animals. Importantly, adiponectin-knockout mice are refractory to changes in energy expenditure and ceramide-lowering effects evoked by FGF21 administration. Moreover, FGF21 lowers blood glucose levels and enhances insulin sensitivity in diabetic Lepob/ob mice and diet-induced obese (DIO) mice only when adiponectin is functionally present. Collectively, these data suggest that FGF21 is a potent regulator of adiponectin secretion and that FGF21 critically depends on adiponectin to exert its glycemic and insulin sensitizing effects.

Serine palmitoyltransferase (SPT) is the first and rate-limiting enzyme of the de novo biosynthetic pathway of sphingomyelin (SM). Both SPT and SM have been implicated in the pathogenesis of atherosclerosis, the development of which is driven by macrophages; however, the role of SPT in macrophage-mediated atherogenesis is unknown. To address this issue, we have analyzed macrophage inflammatory responses and reverse cholesterol transport, 2 key mediators of atherogenesis, in SPT subunit 2-haploinsufficient (Sptlc2(+/-)) macrophages. We found that Sptlc2(+/-) macrophages have significantly lower SM levels in plasma membrane and lipid rafts. This reduction not only impaired inflammatory responses triggered by TLR4 and its downstream NF-κB and MAPK pathways, but also enhanced reverse cholesterol transport mediated by ABC transporters. LDL receptor-deficient (Ldlr(-/-)) mice transplanted with Sptlc2(+/-) bone marrow cells exhibited significantly fewer atherosclerotic lesions after high-fat and high-cholesterol diet feeding. Additionally, Ldlr(-/-) mice with myeloid cell-specific Sptlc2 haploinsufficiency exhibited significantly less atherosclerosis than controls. These findings suggest that SPT could be a novel therapeutic target in atherosclerosis.

Two pairs of universal oligonucleotide primers were designed to probe the most conserved regions of all known cryI-type gene sequences so that the amplified PCR fragments of the DNA template from Bacillus thuringiensis strains may contain all possible cryI-type gene sequences. The restriction fragment length polymorphism (RFLP) patterns of the PCR-amplified fragments revealed that 14 distinct cry-type genes have been identified from 20 B. thuringiensis strains. Those cry-type genes included cryIA(a), cryIA(a), cryIA(b), cryIA(b), cryIA(c), cryIB, cryIC, cryIC, cryIC(b), cryID, cryIE, cryIF, cryIF, and cryIII (a dagger at the end of a gene designation indicates a novel cry-type gene determined by restriction mapping or DNA sequences). Among them, the sequences of cryIA(a), cryIA(b), cryIB, cryIC, cryIF, and cryIII were found to be different from the corresponding published cry gene sequences. Interestingly, five cry-type genes [cryIA(a)-, cryIB-, cryIC-, cryIC(b)-, and cryIF-type genes] and seven cry-type genes [cryIA(a)-, cryIA(b)-, cryIB-, cryIC-, cryIC(b)-, cryIF-, and cryIII-type genes] have been detected from B. thuringiensis subsp. morrisoni HD-12 and B. thuringiensis subsp. wuhanensis, respectively. Therefore, the PCR-RFLP typing system is a facile method to detect both known and novel cry genes existing in B. thuringiensis strains.

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first rate-limiting step in converting nicotinamide to NAD(+), essential for cellular metabolism, energy production, and DNA repair. NAMPT has been extensively studied because of its critical role in these cellular processes and the prospect of developing therapeutics against the target, yet how it regulates cellular metabolism is not fully understood. In this study we utilized liquid chromatography-mass spectrometry to examine the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on glycolysis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and serine biosynthesis in cancer cells and tumor xenografts. We show for the first time that NAMPT inhibition leads to the attenuation of glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step due to the reduced availability of NAD(+) for the enzyme. The attenuation of glycolysis results in the accumulation of glycolytic intermediates before and at the glyceraldehyde 3-phosphate dehydrogenase step, promoting carbon overflow into the pentose phosphate pathway as evidenced by the increased intermediate levels. The attenuation of glycolysis also causes decreased glycolytic intermediates after the glyceraldehyde 3-phosphate dehydrogenase step, thereby reducing carbon flow into serine biosynthesis and the TCA cycle. Labeling studies establish that the carbon overflow into the pentose phosphate pathway is mainly through its non-oxidative branch. Together, these studies establish the blockade of glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step as the central metabolic basis of NAMPT inhibition responsible for ATP depletion, metabolic perturbation, and subsequent tumor growth inhibition. These studies also suggest that altered metabolite levels in tumors can be used as robust pharmacodynamic markers for evaluating NAMPT inhibitors in the clinic.

The principle of interference imaging spectrometer is presented. According to the drift of recovery spectral line position, two representative methods of calibrating the laboratory spectral line position are proposed, and the calibration results and their comparative analyses are given. One method of calibration is to correct the principle, which embarks from parameter selection of interference imaging spectrometer and the analysis of the reason why the spectral line position is drifted. Aiming at the problem that the position of spectral line changes with row, the correction scheme is given to improve the accuracy of spectral line position. For four given laser wavelengths, which are 543.5 nm, 594.1 nm, 612 nm, and 632.8 nm, the root-mean-square (RMS) error of spectral line position is reduced from 28.3914 to 5.5371 after calibration. For the interferometer system which has no dispersion, the accuracy of calibration is better than the dispersion system, and can be the same at all detected wavelengths. In this article, the calibration accuracy of long wave is better than that of short wave, which is dependent on the selection of the initial correction wavelength. This method achieves a kind of universality for interference imaging spectrometer and its calibration parameters provide a convenient way to analyze the instrument indexes. Another calibration method is data processing. It makes up the deficiencies of the method mentioned above: a large number of data are needed and the effect of calibration at short wave is not good enough. The RMS error of spectral line position is reduced to 0.9178, which proves that the calibration has a really high precision. This method is simple and can correct all the detected wavelengths and spectral lines by using two united formula. Though this method is not applicable for all the interference imaging spectrometers, the idea that makes hard things simple is deserving of our attention. We can use it in many other fields. The essence of the method is to change a variable quantity into a slowly varying quantity by algorithms, and then establish the relationship between the slowly varying quantity and the standard value. This idea can always make a substantial increase in efficiency of calibration and has a satisfied accuracy. Each of the two methods has advantages and disadvantages: which method we choose to use is dependent on the effect we want to achieve, and it is better to make their combination. This study provides a theoretical and practical guidance for study, design, modulation, experiment and engineering of interference imaging spectrometers.

We have investigated the effect of substrate biasing on the subthreshold characteristics and noise levels of Si/Si/sub 1-x/Ge/sub x/ (x=0,0.15,0.3) heterostructure MOSFETs. A detailed analysis of the dependence of threshold voltage, off-state current, and low-frequency noise level on the substrate-source (V/sub bs/) biasing showed that SiGe heterostructure MOSFETs offer a significant speed advantage, an extended subthreshold operation region, a reduced noise level, and reduced bulk potential sensitivity compared to Si bulk devices. These experimental results demonstrate that SiGe heterostructure MOSFETs render a promising extension to the CMOS technologies at the low-power limit of operation, eventually making the micropower implementation of radio frequency (RF) functions feasible.

The GPR119 receptor plays an important role in the secretion of incretin hormones in response to nutrient consumption. We have studied the ability of an array of naturally occurring endocannabinoid-like lipids to activate GPR119 and have identified several lipid receptor agonists. The most potent receptor agonists identified were three N-acylethanolamines: oleoylethanolamine (OEA), palmitoleoylethanolamine, and linoleylethanolamine (LEA), all of which displayed similar potency in activating GPR119. Another lipid, 2-oleoylglycerol (2-OG), also activated GPR119 receptor but with significantly lower potency. Endogenous levels of endocannabinoid-like lipids were measured in intestine in fasted and refed mice. Of the lipid GPR119 agonists studied, the intestinal levels of only OEA, LEA, and 2-OG increased significantly upon refeeding. Intestinal levels of OEA and LEA in the fasted mice were low. In the fed state, OEA levels only moderately increased, whereas LEA levels rose drastically. 2-OG was the most abundant of the three GPR119 agonists in intestine, and its levels were radically elevated in fed mice. Our data suggest that, in lean mice, 2-OG and LEA may serve as physiologically relevant endogenous GPR119 agonists that mediate receptor activation upon nutrient uptake.

Atypical antipsychotics such as clozapine and olanzapine have been shown to enhance histamine turnover and this effect has been hypothesized to contribute to their improved therapeutic profile compared to typical antipsychotics. In the present study, we examined the effects of antipsychotic drugs on histamine (HA) efflux in the mPFC of the rat by means of in vivo microdialysis and sought to differentiate the receptor mechanisms which underlie such effects. Olanzapine and clozapine increased mPFC HA efflux in a dose related manner. Increased HA efflux was also observed after quetiapine, chlorpromazine and perphenazine treatment. We found no effect of the selective 5-HT2A antagonist MDL100907, 5-HT2c antagonist SB242084 or the 5-HT6 antagonist Ro 04-6790 on mPFC HA efflux. HA efflux was increased following treatment with selective H1 receptor antagonists pyrilamine, diphenhydramine and triprolidine, the H3 receptor antagonist ciproxifan and the mixed 5HT2A/H1 receptor antagonist ketanserin. The potential novel antipsychotic drug FMPD, which has a lower affinity at H1 receptors than olanzapine, did not affect HA efflux. Similarly, other antipsychotics with lower H1 receptor affinity (risperidone, aripiprazole and haloperidol) were also without effect on HA efflux. Perfusion of clozapine and pyrilamine into the TMN, but not the mPFC, increased local HA efflux. Finally, HA efflux after antipsychotic treatment was significantly correlated with affinity at H1 receptors whereas 9 other receptors, including 5-HT2A, were not. These results demonstrate that both typical and atypical antipsychotics increase mPFC histamine efflux and this effect may be mediated via antagonism of histamine H1 receptors.

After de novo biosynthesis phospholipids undergo extensive remodeling by the Lands' cycle. Enzymes involved in phospholipid biosynthesis have been studied extensively but not those involved in reacylation of lysophosphopholipids. One key enzyme in the Lands' cycle is fatty acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT), which utilizes lysophosphatidylcholine (LysoPC) and fatty acyl-CoA to produce various phosphatidylcholine (PC) species. Four isoforms of LPCAT have been identified. In this study we found that LPCAT3 is the major hepatic isoform, and its knockdown significantly reduces hepatic LPCAT activity. Moreover, we report that hepatic LPCAT3 knockdown increases certain species of LysoPCs and decreases certain species of PC. A surprising observation was that LPCAT3 knockdown significantly reduces hepatic triglycerides. Despite this, these mice had higher plasma triglyceride and apoB levels. Lipoprotein production studies indicated that reductions in LPCAT3 enhanced assembly and secretion of triglyceride-rich apoB-containing lipoproteins. Furthermore, these mice had higher microsomal triglyceride transfer protein (MTP) mRNA and protein levels. Mechanistic studies in hepatoma cells revealed that LysoPC enhances secretion of apoB but not apoA-I in a concentration-dependent manner. Moreover, LysoPC increased MTP mRNA, protein, and activity. In short, these results indicate that hepatic LPCAT3 modulates VLDL production by regulating LysoPC levels and MTP expression.

It has been shown that inhibition of de novo sphingolipid synthesis increases insulin sensitivity. For further exploration of the mechanism involved, we utilized two models: heterozygous serine palmitoyltransferase (SPT) subunit 2 (Sptlc2) gene knockout mice and sphingomyelin synthase 2 (Sms2) gene knockout mice. SPT is the key enzyme in sphingolipid biosynthesis, and Sptlc2 is one of its subunits. Homozygous Sptlc2-deficient mice are embryonic lethal. However, heterozygous Sptlc2-deficient mice that were viable and without major developmental defects demonstrated decreased ceramide and sphingomyelin levels in the cell plasma membranes, as well as heightened sensitivity to insulin. Moreover, these mutant mice were protected from high-fat diet-induced obesity and insulin resistance. SMS is the last enzyme for sphingomyelin biosynthesis, and SMS2 is one of its isoforms. Sms2 deficiency increased cell membrane ceramide but decreased SM levels. Sms2 deficiency also increased insulin sensitivity and ameliorated high-fat diet-induced obesity. We have concluded that Sptlc2 heterozygous deficiency- or Sms2 deficiency-mediated reduction of SM in the plasma membranes leads to an improvement in tissue and whole-body insulin sensitivity.

The normalization of excessive glutamatergic neurotransmission through the activation of metabotropic glutamate 2 (mGlu2) receptors may have therapeutic potential in a variety of psychiatric disorders, including anxiety/depression and schizophrenia. Here, we characterize the pharmacological properties of N-(4-((2-(trifluoromethyl)-3-hydroxy-4-(isobutyryl)phenoxy)methyl)benzyl)-1-methyl-1H-imidazole-4-carboxamide (THIIC), a structurally novel, potent, and selective allosteric potentiator of human and rat mGlu2 receptors (EC(50) = 23 and 13 nM, respectively). THIIC produced anxiolytic-like efficacy in the rat stress-induced hyperthermia assay and the mouse stress-induced elevation of cerebellar cGMP and marble-burying assays. THIIC also produced robust activity in three assays that detect antidepressant-like activity, including the mouse forced-swim test, the rat differential reinforcement of low rate 72-s assay, and the rat dominant-submissive test, with a maximal response similar to that of imipramine. Effects of THIIC in the forced-swim test and marble burying were deleted in mGlu2 receptor null mice. Analysis of sleep electroencephalogram (EEG) showed that THIIC had a sleep-promoting profile with increased non-rapid eye movement (REM) and decreased REM sleep. THIIC also decreased the dark phase increase in extracellular histamine in the medial prefrontal cortex and decreased levels of the histamine metabolite tele-methylhistamine (t-MeHA) in rat cerebrospinal fluid. Collectively, these results indicate that the novel mGlu2-positive allosteric modulator THIIC has robust activity in models used to predict anxiolytic/antidepressant efficacy, substantiating, at least with this molecule, differentiation in the biological impact of mGlu2 potentiation versus mGlu2/3 orthosteric agonism. In addition, we provide evidence that sleep EEG and CSF t-MeHA might function as viable biomarker approaches to facilitate the translational development of THIIC and other mGlu2 potentiators.

Phosphatidylcholine (PC) is synthesized from choline via the CDP-choline pathway. Liver cells can also synthesize PC via the sequential methylation of phosphatidylethanolamine, catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). The current study investigates whether or not hepatic PC biosynthesis is linked to diet-induced obesity. Pemt(+/+) mice fed a high fat diet for 10 weeks increased in body mass by 60% and displayed insulin resistance, whereas Pemt(-/-) mice did not. Compared with Pemt(+/+) mice, Pemt(-/-) mice had increased energy expenditure and maintained normal peripheral insulin sensitivity; however, they developed hepatomegaly and steatosis. In contrast, mice with impaired biosynthesis of PC via the CDP-choline pathway in liver became obese when fed a high fat diet. We, therefore, hypothesized that insufficient choline, rather than decreased hepatic phosphatidylcholine, was responsible for the lack of weight gain in Pemt(-/-) mice despite the presence of 1.3 g of choline/kg high fat diet. Supplementation with an additional 2.7 g of choline (but not betaine)/kg of diet normalized energy metabolism, weight gain, and insulin resistance in high fat diet-fed Pemt(-/-) mice. Furthermore, Pemt(+/+) mice that were fed a choline-deficient diet had increased oxygen consumption, had improved glucose tolerance, and gained less weight. Thus, de novo synthesis of choline via PEMT has a previously unappreciated role in regulating whole body energy metabolism.

Herein we demonstrate that a small panel of variants of cytochrome P450 BM3 from Bacillus megaterium covers the breadth of reactivity of human P450s by producing 12 of 13 mammalian metabolites for two marketed drugs, verapamil and astemizole, and one research compound. The most active enzymes support preparation of individual metabolites for preclinical bioactivity and toxicology evaluations. Underscoring their potential utility in drug lead diversification, engineered P450 BM3 variants also produce novel metabolites by catalyzing reactions at carbon centers beyond those targeted by animal and human P450s. Production of a specific metabolite can be improved by directed evolution of the enzyme catalyst. Some variants are more active on the more hydrophobic parent drug than on its metabolites, which limits production of multiply-hydroxylated species, a preference that appears to depend on the evolutionary history of the P450 variant.

Sphingomyelin (SM) is one of the major lipid components of plasma lipoproteins. Serine palmitoyltransferase (SPT) is the key enzyme in SM biosynthesis. Mice totally lacking in SPT are embryonic lethal. The liver is the major site for plasma lipoprotein biosynthesis, secretion, and degradation, and in this study we utilized a liver-specific knock-out approach for evaluating liver SPT activity and also its role in plasma SM and lipoprotein metabolism. We found that a deficiency of liver-specific Sptlc2 (a subunit of SPT) decreased liver SPT protein mass and activity by 95 and 92%, respectively, but had no effect on other tissues. Liver Sptlc2 deficiency decreased plasma SM levels (in both high density lipoprotein and non-high density lipoprotein fractions) by 36 and 35% (p < 0.01), respectively, and increased phosphatidylcholine levels by 19% (p < 0.05), thus increasing the phosphatidylcholine/SM ratio by 77% (p < 0.001), compared with controls. This deficiency also decreased SM levels in the liver by 38% (p < 0.01) and in the hepatocyte plasma membranes (based on a lysenin-mediated cell lysis assay). Liver-specific Sptlc2 deficiency significantly increased hepatocyte apoE secretion and thus increased plasma apoE levels 3.5-fold (p < 0.0001). Furthermore, plasma from Sptlc2 knock-out mice had a significantly stronger potential for promoting cholesterol efflux from macrophages than from wild-type mice (p < 0.01) because of a greater amount of apoE in the circulation. As a result of these findings, we believe that the ability to control liver SPT activity could result in regulation of lipoprotein metabolism and might have an impact on the development of atherosclerosis.

Rationale: Sphingomyelin synthase (SMS)2 contributes to de novo sphingomyelin (SM) biosynthesis and plasma membrane SM levels. SMS2 deficiency in macrophages diminishes nuclear factor κB and mitogen-activated protein kinase activation induced by inflammatory stimuli. Objective: The effects of SMS2 deficiency on the development of atherosclerosis are investigated. Methods and Results: We measured cholesterol efflux from macrophages of wild-type (WT) and SMS2 knockout (KO) mice. We transplanted SMS2 KO mouse bone marrow into low-density lipoprotein (LDL) receptor (LDLr) knockout mice (SMS2 −/− →LDLr −/− ), creating a mouse model of SMS2 deficiency in the macrophages. We found that SMS2 deficiency caused significant induction of cholesterol efflux in vitro and in vivo. Moreover, we found that SMS2 KO mice had less interleukin-6 and tumor necrosis factor α in the circulation before and after endotoxin stimulation, compared with controls. More importantly, after 3 months on a western-type diet, SMS2 −/− →LDLr −/− mice showed decreased atherosclerotic lesions in the aortic arch, root (57%, P P −/− mice. Analysis of plaque morphology revealed that SMS2 −/− →LDLr −/− mice had significantly less necrotic core area (71%, P P P −/− →LDLr −/− mice had significantly lower free cholesterol and cholesteryl ester levels in the brachiocephalic artery than WT→LDLr −/− mice (33 and 52%, P P Conclusions: SMS2 deficiency in the macrophages reduces atherosclerosis in mice. Macrophage SMS2 is thus a potential therapeutic target for treatment of this disease.

Background—NFκB has long been regarded as a proatherogenic factor, mainly because of its regulation of many of the proinflammatory genes linked to atherosclerosis. Metabolism of sphingomyelin (SM) has been suggested to affect NFκB activation, but the mechanism is largely unknown. SMS2 regulates SM levels in cell plasma membrane and lipid rafts and has a potential to regulate NFκB activation.Methods and Results—To investigate the role of SMS2 in NFκB activation we used macrophages from SMS2 knockout (KO) mice and SMS2 siRNA-treated HEK 293 cells. We found that NFκB activation and its target gene expression are attenuated in macrophages from SMS2 KO mice in response to lipopolysaccharide (LPS) stimulation and in SMS2 siRNA- treated HEK 293 cells after tumor necrosis factor (TNF)–α simulation. In line with attenuated NFκB activation, we found that SMS2 deficiency substantially diminished the abundance of toll like receptor 4 (TLR4)-MD2 complex levels on the surface of macrophages after LPS stimulation, and SMS2 siRNA treatment reduced TNF-α-stimulated lipid raft recruitment of TNF receptor-1 (TNFR1) in HEK293 cells. SMS2 deficiency decreased the relative amounts of SM and diacylglycerol (DAG) and increased ceramide, suggesting multiple mechanisms for the decrease in NFκB activation.Conclusions—SMS2 is a modulator of NFκB activation, and thus it could play an important role in NFκB-mediated proatherogenic process.

Stearoyl-CoA desaturase (SCD) catalyzes the formation of monounsaturated fatty acids from saturated fatty acids. It plays a key role in lipid metabolism and energy expenditure in mammals. In mice, four SCD isoforms (SCD1–4) have been identified. Here we report the identification of cDNA sequences corresponding to SCD1, SCD2 and SCD3 of golden hamster. The deduced amino acid sequences of these hamster SCD (hmSCD) isoforms display a high degree of homologies to their mouse counterparts (mouse SCD). Polyclonal antibodies specific to rodent SCDs detected proteins of predicted size in the human embryonic kidney 293 cells transfected with hmSCD cDNAs. Microsome fractions prepared from these cells also displayed increased SCD activity versus cells transfected with vector alone. Real-time reverse transcription-polymerase chain reaction analysis revealed the highest expression of hmSCD1 in liver and adipose tissue, while the highest hmSCD2 expression was detected in the brain. Very low levels of hmSCD3 mRNA can be detected in the tissues tested. This report is the first description of three SCD isoforms in the hamster and will provide useful tools in the further study of fatty acids metabolism in this species.

TE (Thermoelectric) materials have been widely used in clean energy system as low-power generator and Peliter cooler, due to its salient features of being compact, light-weighted, noiseless in operation, highly reliable, and environment friendly. Recently, another application has been explored on TE materials as gas sensors based on Seebeck effect and exothermic reaction of hydrogen oxidation on catalyst. In this paper, a TE hydrogen gas sensor with a simple structure, low energy consumption and a high sensitivity was reported. Bi-Te (bismuth telluride) with a high Seebeck coefficient at room-temperature was deposited onto thin glass substrates by RF magnetron sputtering technology. Four pairs of PN film couples were connected in series to improve the output voltage. Pt/ ACC (Activated Carbon Fiber Cloth) was mounted at the joint of PN couples, acting as catalyst so as to accelerate the oxidation of hydrogen. The influences of reduction temperature and Pt content on the generated temperature difference were investigated. The voltage output and selectivity to combustible gas mixture were measured. Experimental results showed that when exposed to 3vol% H2 / air, as-prepared sensor gave out a high output signal of 33.1mV, and the response time was about 50s with recovery time of 50s.Copyright © 2007 by ASME

Nicotinamide phosphoribosyltransferase (NAMPT) plays an important role in cellular bioenergetics. It is responsible for converting nicotinamide to nicotinamide adenine dinucleotide, an essential molecule in cellular metabolism. NAMPT has been extensively studied over the past decade due to its role as a key regulator of nicotinamide adenine dinucleotide–consuming enzymes. NAMPT is also known as a potential target for therapeutic intervention due to its involvement in disease. In the current study, we used a global mass spectrometry–based metabolomic approach to investigate the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on metabolic perturbations in human cancer cells. We treated A2780 (ovarian cancer) and HCT-116 (colorectal cancer) cell lines with FK866 in the presence and absence of nicotinic acid. Significant changes were observed in the amino acids metabolism and the purine and pyrimidine metabolism. We also observed metabolic alterations in glycolysis, the citric acid cycle (TCA), and the pentose phosphate pathway. To expand the range of the detected polar metabolites and improve data confidence, we applied a global metabolomics profiling platform by using both non-targeted and targeted hydrophilic (HILIC)-LC-MS and GC-MS analysis. We used Ingenuity Knowledge Base to facilitate the projection of metabolomics data onto metabolic pathways. Several metabolic pathways showed differential responses to FK866 based on several matches to the list of annotated metabolites. This study suggests that global metabolomics can be a useful tool in pharmacological studies of the mechanism of action of drugs at a cellular level.

Therapies targeting EGFR often fail after certain period of treatment due to development of resistance. There are many causes of resistance such as genetic mutations of KRAS, BRAF, etc.. Overexpression of other receptor tyrosine kinases can also lead to resistance of EGFR therapies. A therapeutic targeting metabolic phenotypes that can overcome multiple resistance mechanisms is highly desired. In order to identify the Achilles heel of resistant cell metabolism, we investigated the metabolic effects of the current EGFR targeting agents as well as the metabolic differences between sensitive and resistant cancer cells. Citation Format: Yang Zhao, Bo Tan, Ming-Shang Kuo, Ling Liu, Matthew D. Breyer. Metabolomic study of EGFR drug resistance mechanisms. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5439. doi:10.1158/1538-7445.AM2014-5439

This paper describes the structure activity relationships of a new class of cytomegalovirus DNA polymerase inhibitors having two aryl groups joined by an acyloxyamidine linker. Examination of a series of analogues in which the terminal groups are varied revealed a very narrow SAR around the 2,4-dichlorophenyl group of the lead compound, but a variety of replacements for the benzothiazole ring are compatible with activity. The most notable of these is the isoxazole ring of compound 78, which provides a 30-fold enhancement in potency compared to the lead compound. We also describe the design, synthesis and evaluation of 10 analogues in which the acyloxyamidine linker is modified or replaced by an isosteric group. Structure-activity relationship studies identified the linker -NH2 group as a critical pharmacophoric element. Ab initio molecular orbital calculations combined with qualitative estimates of steric interaction energies suggest that the lowest energy conformations of the acyloxyamidine linker are characterized by an extended planar CAr-C=N-O-C arrangement and either a syn-periplanar or anti-periplanar N-O-C-C(Ar') arrangement. Only the anti-periplanar conformation was observed in the crystal structures of three acyloxyamidines. The most active of the linker-modified compounds designed on the basis of these studies is the amidine carbamate 20, which is approximately one-third as potent in the cytomegalovirus DNA polymerase inhibition assay as the comparator acyloxyamidine 53. The activity of 20 suggests that acyloxyamidines may bind to the cytomegalovirus DNA polymerase via an anti-periplanar conformation similar to that observed in the crystal structure of acyloxyamidine 36.

With PCR products as probes, we have cloned two new cry-type genes from Bacillus thuringiensis subsp. wuhanensis. The deduced amino acid sequence of the first clone is 77.3% identical to Cry1Ga1. The deduced protein sequence of the second clone is 69.8–78.7% identical to that of Cry1B group. The nomenclature assignment of these two clones is, therefore, named Cry1Gb1 and Cry1Bd1, respectively. The Cry1Bd1 is toxic to Plutella xylostella larvae, and the Cry1Gb1 is toxic to Pieris rapae larvae.

The rationale of this cell based study was to examine the metabolic effects of a synthetic monocarboxylate transporter 1 (MCT1) inhibitor (AR-C155858) on multiple cellular metabolites in colorectal tumor cells using [1,2,3,4,5,6-13C6]-D-glucose or [1,2,3,4,5-13C5]-D-glutamine as the single precursor metabolic tracer. DiFi colorectal cancer cells were cultured for 24 hrs in the presence of 0.1% DMSO (vehicle control) or 100 nM of MCT-1 inhibitor. Intracellular glycolysis intermediates measured by liquid chromatography/mass spectrometry (LC/MS) included phosphoenolpyruvate (PEP), phosphoglycerate (PG), fructose 1,6 bisphosphate (FBP), Glucose-6-phosphate (G6P), Gluconate-6-phosphate, glyceraldehydes 3-phosphate (G3P), ribose-5-phosphate (R5P), seduheptulose-7-phosphate (S7P), pyruvate, and lactate. TCA cycle intermediates measured included malate, α-ketoglutarate, succinate, fumarate, and citrate. Both glutamine and glutamate were also measured. Increases in PEP, PG3, G6P, Gluconate-6-phosphate, G3P, S7P, pyruvate, and lactate were induced by MCT1 inhibitor when traced with 13C6 glucose. When traced with 13C5 glutamine, PEP, G6P, S7P, pyruvate, and lactate were increased by MCT1 inhibitor. In contrast, decreases in α-ketoglutarate and glutamate were seen with both tracers. Citation Format: Bo Tan, Ming-Shang Kuo, Matthew D. Breyer, Ling Liu, Yang Zhao. Stable isotope resolved metabolomics of colorectal cancer cells treated with inhibitor of monocarboxylate transporter-1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1866. doi:10.1158/1538-7445.AM2013-1866