Solid-phase microextraction (SPME) followed by gas chromatography/mass spectrometry (GC/MS) was used for the detection of liver cancer volatile biomarkers. Headspace SPME conditions (fiber coating, extraction temperature and extraction time) and desorption conditions were optimized and applied to the determination of volatiles in human blood. Between the liver cancer group (n = 19) and the normal group (n = 18), positive rates of 19 volatile compounds among the total of 47 detected were found to be different with statistical significance (p < 0.05, chi-squared test). We suggested hexanal, 1-octen-3-ol and octane, of the 19 compounds, as biomarkers of liver cancer with clinical diagnostic value. The sensitivity and specificity of 94.7% and 100% for hexanal, 84.2% and 100% for 1-octen-3-ol, and 89.5% and 100% for octane were obtained, respectively, after the cutoff values had been properly established. These results show that SPME-GC/MS is a simple, rapid and sensitive method for the investigation of volatile disease markers in human blood.

Lysophosphatidate (LPA) mediates multiple cellular responses via heterotrimeric G protein coupled LPA-1, LPA-2, and LPA-3 receptors. Many G protein-coupled receptors stimulate ERK following tyrosine phosphorylation of growth factor receptors; however, the mechanism(s) of transactivation of receptor tyrosine kinases are not well defined. Here, we provide evidence for the involvement of phospholipase D (PLD) in LPA-mediated transactivation of platelet-derived growth factor receptor-beta (PDGF-R beta). In primary cultures of human bronchial epithelial cells (HBEpCs), LPA stimulated tyrosine phosphorylation of PDGF-R beta and threonine/tyrosine phosphorylation of ERK1/2. The LPA-mediated activation of ERK and tyrosine phosphorylation of PDGF-R beta was attenuated by tyrphostin AG 1296, an inhibitor of PDGF-R kinase, suggesting transactivation of PDGF-R by LPA. Furthermore, LPA-, but not PDGF beta-chain homodimer-induced tyrosine phosphorylation of PDGF-R beta was partially blocked by pertussis toxin, indicating coupling of LPA-R(s) to Gi. Exposure of HBEpCs to LPA activated PLD. Butan-1-ol, which acts as an acceptor of phosphatidate generated by the PLD pathway, blocked LPA-mediated transactivation of PDGF-R beta. This effect was not seen with butan-3-ol, suggesting PLD involvement. The role of PLD1 and PLD2 in the PDGF-R beta transactivation by LPA was investigated by infection of cells with adenoviral constructs of wild type and catalytically inactive mutants of PLD. LPA activated both PLD1 and PLD2 in HBEpCs; however, infection of cells with cDNA for wild type PLD2, but not PLD1, increased the tyrosine phosphorylation of PDGF-R beta in response to LPA. Also, the LPA-mediated tyrosine phosphorylation of PDGF-R beta was attenuated by the catalytically inactive mutant mPLD2-K758R. Infection of HBEpCs with adenoviral constructs of wild type hPLD1, mPLD2, and the inactive mutants of hPLD1 and mPLD2 resulted in association of PLD2 wild type and inactive mutant proteins with the PDGF-R beta compared with PLD1. These results show for the first time that transactivation of PDGF-R beta by LPA in HBEpCs is regulated by PLD2.

BACKGROUND

Tea is a popular natural non-alcoholic beverage consumed worldwide due to its bioactive ingredients, particularly catechins (flavan-3-ols). Catechins not only contribute to tea quality but also serve important functions in the anti-stress regulation of secondary metabolic pathways. However, the percentages of various catechins are different among tea plant [Camellia sinensis (L.) O. Kuntze] cultivars. This study aimed to elucidate the biosynthetic mechanism of catechins. Transcriptomes from leaf tissues of four tea plant cultivars, 'Yunnanshilixiang', 'Chawansanhao', 'Ruchengmaoyecha', and 'Anjibaicha', were sequenced using the high-throughput sequencing platform Illumina HiSeq™ 2000. De novo assemble were also performed. Catechins contents were measured through reversed-phase high-performance liquid chromatography (RP-HPLC), and the biosynthetic pathway was also surveyed.

RESULTS

We constructed a unified unigene database. A total of 146,342 pairs of putative orthologs from the four tea plant cultivars, 'Yunnanshilixiang', 'Chawansanhao', 'Ruchengmaoyecha', and 'Anjibaicha' were generated. Approximately 68,890 unigenes (47.1%) were aligned to the sequences of seven public databases with a cut-off E-value of 1E-5. A total of 217 differentially expressed genes were found through RPKM values, and 150 unigenes were assigned to the flavonoid biosynthetic pathway using the integrated function annotation. The (-)-EGC and (-)-EC contents were significantly lower and the (+)-GC and (+)-C contents were abnormally higher in 'Ruchengmaoyecha' than in 'Yunnanshilixiang', 'Chawansanhao', and 'Anjibaicha'. The proportion of catechins was confirmed by selecting critical genes (ANS, ANR, and LAR) for qRT-PCR analysis.

CONCLUSIONS

This study provided a global survey of transcriptomes from four tea plant cultivars and serves as an available resource of genetic diversity. The analyses of transcriptome profiles and physiological indicators not only identified the putative genes involved in the flavonoid biosynthetic pathway but also provided some novel insights for the mechanisms of catechins biosynthesis.

Leucyl-tRNA synthetase (LeuRS) specifically links leucine to the 3' end of tRNA(leu) isoacceptors. The overall accuracy of the two-step aminoacylation reaction is enhanced by an editing domain that hydrolyzes mischarged tRNAs, notably ile-tRNA(leu). We present crystal structures of the editing domain from two eukaryotic cytosolic LeuRS: human and fungal pathogen Candida albicans. In comparison with previous structures of the editing domain from bacterial and archeal kingdoms, these structures show that the LeuRS editing domain has a conserved structural core containing the active site for hydrolysis, with distinct bacterial, archeal, or eukaryotic specific peripheral insertions. It was recently shown that the benzoxaborole antifungal compound AN2690 (5-fluoro-1,3-dihydro-1-hydroxy-1,2-benzoxaborole) inhibits LeuRS by forming a covalent adduct with the 3' adenosine of tRNA(leu) at the editing site, thus locking the enzyme in an inactive conformation. To provide a structural basis for enhancing the specificity of these benzoxaborole antifungals, we determined the structure at 2.2 A resolution of the C. albicans editing domain in complex with a related compound, AN3018 (6-(ethylamino)-5-fluorobenzo[c][1,2]oxaborol-1(3H)-ol), using AMP as a surrogate for the 3' adenosine of tRNA(leu). The interactions between the AN3018-AMP adduct and C. albicans LeuRS are similar to those previously observed for bacterial LeuRS with the AN2690 adduct, with an additional hydrogen bond to the extra ethylamine group. However, compared to bacteria, eukaryotic cytosolic LeuRS editing domains contain an extra helix that closes over the active site, largely burying the adduct and providing additional direct and water-mediated contacts. Small differences between the human domain and the fungal domain could be exploited to enhance fungal specificity.

A complex mixture of fatty acid-derived aldehydes, ketones, and alcohols is released upon wounding of the moss Physcomitrella patens. To investigate the formation of these oxylipins at the molecular level we isolated a lipoxygenase from P. patens, which was identified in an EST library by sequence homology to lipoxygenases from plants. Sequence analysis of the cDNA showed that it exhibits a domain structure similar to that of type2 lipoxygenases from plants, harboring an N-terminal import signal for chloroplasts. The recombinant protein was identified as arachidonate 12-lipoxygenase and linoleate 13-lipoxygenase with a preference for arachidonic acid and eicosapentaenoic acid. In contrast to any other lipoxygenase cloned so far, this enzyme exhibited in addition an unusual high hydroperoxidase and also a fatty acid chain-cleaving lyase activity. Because of these unique features the pronounced formation of (2Z)-octen-1-ol, 1-octen-3-ol, the dienal (5Z,8Z,10E)-12-oxo-dodecatrienoic acid and 12-keto eicosatetraenoic acid was observed when arachidonic acid was administered as substrate. 12-Hydroperoxy eicosatetraenoic acid was found to be only a minor product. Moreover, the P. patens LOX has a relaxed substrate tolerance accepting C(18)-C(22) fatty acids giving rise to even more LOX-derived products. In contrast to other lipoxygenases a highly diverse product spectrum is formed by a single enzyme accounting for most of the observed oxylipins produced by the moss. This single enzyme might, in a fast and effective way, be involved in the formation of signal and/or defense molecules thus contributing to the broad resistance of mosses against pathogens.

ABSTRACT A lethal leaf scorch disease of oleander (Nerium oleander) appeared in southern California in 1993. A bacterium, Xylella fastidiosa, was detected by culturing, enzyme-linked immunoassay, and polymerase chain reaction in most symptomatic plants but not in symptomless plants or negative controls. Inoculating oleanders mechanically with X. fastidiosa cultures from diseased oleanders caused oleander leaf scorch (OLS) disease. The bacterium was reisolated from inoculated plants that became diseased. Three species of xylem sap-feeding leafhoppers transmitted the bacterium from oleander to oleander. The bacterium multiplied, moved systemically, and caused wilting in Madagascar periwinkle (Catharanthus rosea) and leaf scorch in periwinkle (Vinca major) in a greenhouse after inoculation with needle puncture. No bacterium was reisolated from grapevine (Vitis vinifera), peach (Prunus persica), olive (Olea europaea), California blackberry (Rubus ursinus), or valley oak (Quercus lobata) mechanically inoculated with OLS strains of X. fastidiosa. A 500-bp sequence of the 16S-23S ribosomal intergenic region of oleander strains showed 99.2% identity with Pierce's disease strains, 98.4% identity with oak leaf scorch strains, and 98.6% identity with phony peach, plum leaf scald, and almond leaf scorch strains.

Niemann-Pick disease type C (NPC) is an autosomal recessive neurovisceral lipid storage disease caused by a loss of NPCI function, which results in perturbation of intracellular cholesterol transport. In BALB/c npc(nih) mice, the murine ortholog of NPCI gene is mutated. In NPC mouse, hypomyelination is conspicuous in the cerebral white matter and corpus callosum in addition to neuronal storage. However, the pathogenesis on hypomyelination is not well elucidated. We hypothesized that the hypomyelination in NPC mice resulted from either defective differentiation of oligodendrocyte lineage cells or a failure of proper axon-glial interaction. Myelin basic protein immunohistochemistry disclosed severe hypomyelination of cerebral cortex as well. NG2- or O4-positive progenitor cells and premyelinating oligodendrocytes (OLs) were abundant. However, pi-glutathione-S-transferase-positive mature OLs were considerably reduced. In hypomyelinated white matter, strong immunoreactivity of polysialylated-neural cell adhesion molecule, a negative regulator of myelination, was observed in axons. Given the fact that neuro-axonal degeneration has been observed in NPC mouse as early as 9 days of age prior to the commencement of myelination in the corpus callosum and that axonal signals are essential for the proper myelination, subtle axonal injury might be contributing to the pathogenesis of disturbed myelination in the NPC mouse.

OBJECTIVE

Previous literature has reported lower morbidity for video-assisted thoracoscopic surgery lobectomy (VL) compared with open lobectomy (OL); however, most comparative studies have been retrospective and have failed to compare well-matched patient groups, therefore allowing selection bias to influence results. Furthermore, oncological adequacy of VL has recently been questioned, particularly with respect to lymphadenectomy. This study aimed to evaluate short- and long-term outcomes of a large cohort of consecutive patients with c-stage I non-small-cell lung cancer (NSCLC) that underwent either VL or OL.

METHODS

Consecutive patients with c-stage I NSCLC who underwent lobectomy without preoperative therapy were reviewed. Univariable, multivariable and propensity-matched analyses were performed. VL patients who underwent conversion to OL were analysed within the VL group.

RESULTS

VL was performed in 307 (32%) patients and OL in 656 (68%). Twenty-two (7%) patients converted from VL to OL. Although there were no differences in overall p-stage grouping, there were fewer patients with pT2 tumours in the VL group (39 vs 48%, P = 0.012) and fewer patients with squamous cell histology (26 vs 18%, P = 0.006). These differences resolved after propensity matching. In unmatched and matched analyses, VL was associated with less overall morbidity, less pulmonary morbidity, fewer atrial arrhythmias, shorter chest tube duration and shorter hospital stay than patients who had OL. Thirty-day in-hospital mortality was 0.3 and 1.4%, for VL and OL groups, respectively (P = NS). In unmatched analysis (log rank), 5-year survival favoured VL (78 vs 68%, P = 0.007); however, after propensity matching there was only a trend towards improved survival with VL (78 vs 73%, P = 0.071). Multivariable Cox regression analysis revealed VL (hazard ratio (HR) 0.64, 95% confidence interval (CI) 0.46-0.92), male sex (HR 1.43, 95% CI 1.10-1.86), Zubrod performance status (HR 3.42, 95% CI 1.26-9.29) and increasing age (HR 1.04, 95% CI 1.03-1.06) to be independent predictors of survival.

CONCLUSIONS

Patients with clinical Stage I NSCLC undergoing VL have less perioperative morbidity compared with matched OL controls. Regional lymphadenectomy, nodal upstaging, overall and disease-free survival were similar between VL and OL groups. In experienced centres, VL is an acceptable operation for patients with c-stage I NSCLC.

Liquid chromatography/ultraviolet (LC/UV) and mass spectrometry/mass spectrometry (MS/MS) libraries containing 39 phenolic compounds were established by coupling a LC and an ion trap MS with an electrospray ionization (ESI) source, operated in negative ion mode. As a result, the deprotonated [M-H]- molecule was observed for all the analyzed compounds. Using MS/MS hydroxybenzoic acid and hydroxycinnamic acids showed a loss of CO2 and production of a [M-H-44]- fragment and as expected, the UV spectra of these two compounds were affected by their chemical structures. For flavonol and flavonol glycosides, the spectra of their glycosides and aglycones produced deprotonated [M-H]- and [A-H]- species, respectively, and their UV spectra each presented two major absorption peaks. The UV spectra and MS/MS data of flavan-3-ols and stilbenes were also investigated. Using the optimized LC/MS/MS analytical conditions, the phenolic extracts from six representative wine samples were analyzed and 31 phenolic compounds were detected, 26 of which were identified by searching the LC/UV and MS/MS libraries. Finally, the presence of phenolic compounds was confirmed in different wine samples using the LC/UV and LC/MS/MS libraries.

CNS damage often results in demyelination of spared axons due to oligodendroglial cell death and dysfunction near the injury site. Although new oligodendroglia are generated following CNS injury and disease, the process of remyelination is typically incomplete resulting in long-term functional deficits. Chondroitin sulfate proteoglycans (CSPGs) are upregulated in CNS grey and white matter following injury and disease and are a major component of the inhibitory scar that suppresses axon regeneration. CSPG inhibition of axonal regeneration is mediated, at least in part, by the protein tyrosine phosphatase sigma (PTPσ) receptor. Recent evidence demonstrates that CSPGs inhibit OL process outgrowth, however, the means by which their effects are mediated remains unclear. Here we investigate the role of PTPσ in CSPG inhibition of OL function. We found that the CSPGs, aggrecan, neurocan and NG2 all imposed an inhibitory effect on OL process outgrowth and myelination. These inhibitory effects were reversed by degradation of CSPGs with Chondroitinase ABC prior to OL exposure. RNAi-mediated down-regulation of PTPσ reversed the inhibitory effect of CSPGs on OL process outgrowth and myelination. Likewise, CSPG inhibition of process outgrowth and myelination was significantly reduced in cultures containing PTPσ(-/-) OLs. Finally, inhibition of Rho-associated kinase (ROCK) increased OL process outgrowth and myelination during exposure to CSPGs. These results suggest that in addition to their inhibitory effects on axon regeneration, CSPGs have multiple inhibitory actions on OLs that result in incomplete remyelination following CNS injury. The identification of PTPσ as a receptor for CSPGs, and the participation of ROCK downstream of CSPG exposure, reveal potential therapeutic targets to enhance white matter repair in the damaged CNS.

The endogenous neurosteroid pregnenolone sulfate (PS) is known to enhance memory and cognitive function at nanomolar concentrations. However, the effect of these low concentrations on synaptic transmission has not been previously studied. The effects of PS on GABAA receptor-mediated inhibitory postsynaptic currents were studied in cultured hippocampal pyramidal neurons. Concentrations of PS similar to those endogenous in the hippocampus (10-30 nM) reduced the frequency of both action potential-dependent (spontaneous inhibitory postsynaptic current) and -independent (miniature inhibitory postsynaptic current; mIPSC) inhibitory postsynaptic currents. This effect of PS was mimicked by the selective sigma1 receptor agonist [2S-(2alpha,6alpha,11R]-1,2,3,4,5,6-hexahydro-6,11-dimethyl-3-(2-propenyl)-2,6-methano-3-benzazocin-8-ol hydrochloride [(+)-SKF 10047] and blocked the specific sigma1 receptor antagonists 1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine dihydrochloride (BD-1063) and haloperidol and by pertussis toxin. The GABAB antagonist baclofen and the metabotropic glutamate receptor antagonist (R,S)-a-cyclopropyl-4-phosphonophenylglycine had no effect on the PS-mediated inhibition of mIPSC frequency. The postsynaptic effects of PS occurred at micromolar concentrations but not at nanomolar concentrations. A comparison of the pre- and postsynaptic effects of PS demonstrated that it was 100-fold more potent in inhibiting presynaptic GABAergic synaptic mechanisms than GABAA receptors. These studies demonstrate that concentrations of PS, similar to those endogenous in the hippocampus, inhibit GABAergic synaptic transmission by a presynaptic effect. PS causes specific activation of G protein-coupled sigma1 receptors, resulting in modulation of both action potential-dependent and -independent IPSCs. These findings improve our understanding of the physiological function of PS.

The mechanism of methanol photorelease from 2-nitrobenzyl methyl ether (1) and 1-(2-nitrophenyl)ethyl methyl ether (2), and of ATP release from adenosine-5'-triphosphate-[P(3)-(1-(2-nitrophenyl)ethyl)] ester ('caged ATP', 3) was studied in various solvents by laser flash photolysis with UV-vis and IR detection. In addition to the well-known primary aci-nitro transients (A, lambda(max) approximately 400 nm), two further intermediates preceding the release of methanol, namely the corresponding 1,3-dihydrobenz[c]isoxazol-1-ol derivatives (B) and 2-nitrosobenzyl hemiacetals (C), were identified. The dependencies of the reaction rates of A-C on pH and buffer concentrations in aqueous solution were studied in detail. Substantial revision of previously proposed reaction mechanisms for substrate release from 2-nitrobenzyl protecting groups is required: (a) A novel reaction pathway of the aci-tautomers A prevailing in buffered aqueous solutions, e.g., phosphate buffer with pH 7, was found. (b) The cyclic intermediates B were identified for the first time as the products formed by the decay of the aci-tautomers A in solution. A recently proposed reaction pathway bypassing intermediates B (Corrie et al. J. Am. Chem. Soc., 2003, 125, 8546-8554) is shown not to be operative. (c) Hemiacetals C limit the release rate of both 1 (pH < 8) and 2 (pH < 10). This observation is in contrast to a recent claim for related 2-nitrobenzyl methyl ethers (Corrie et al.). Our findings are important for potential applications of the 2-nitrobenzyl protecting group in the determination of physiological response times to bioagents ('caged compounds').

BACKGROUND

Olfactorily mediated puberty acceleration in female mice (measured by an increase in uterine weight) has been observed since the 1960s without the active chemosignal being structurally identified. There are many controversies in the literature as to whether this male-originated pheromone is a volatile substance. We investigated the chemical nature of the urinary fractions that are responsible for the characteristic uterine weight increases.

RESULTS

The active pheromone was identified as 5,5-dimethyl-2-ethyltetrahydrofuran-2-ol and/or its open-chain tautomer (6-hydroxy-6-methyl-3-heptanone). A series of cyclic vinyl ethers were isolated from chromatographically active fractions of the urine. Because these compounds did not accelerate puberty, we postulated that these ethers were degradation products of a lactol (5,5-dimethyl-2-ethyltetrahydrofuran-2-ol). The lactol was then detected directly in the mouse urine extract using a silylation agent. Synthetic 6-hydroxy-6-methyl-3-heptanone had strong biological activity, whereas its close structural analogs did not.

CONCLUSIONS

The male house mouse excretes into its urine a large quantity of a volatile substance that has a unique lactol/hydroxyketone structure. This substance is capable of binding to the less volatile urinary constituents, such as proteins or peptides, and is active in puberty-acceleration bioassays. The controversies regarding the volatility of the puberty-accelerating pheromones can now be explained by considering a complex of volatile lactol/hydroxyketone and urinary proteins.

The first oxygenation step in the biosynthesis of the anticancer drug taxol in Taxus species is the cytochrome p450-mediated hydroxylation (with double bond migration) of the diterpene olefin precursor taxa-4(5),11(12)-diene to taxa-4(20),11(12)-dien-5alpha-ol. A homology-based cloning strategy, employing an induced Taxus cell library, yielded a cDNA encoding taxadiene 5alpha-hydroxylase, which was functionally expressed in yeast and insect cells. The recombinant enzyme was characterized and shown to efficiently utilize both taxa-4(5),11(12)-diene and taxa-4(20),11(12)-diene (as an adventitious substrate) to synthesize taxa-4(20),11(12)-dien-5alpha-ol. This hydroxylase resembles, in sequence and properties, other cytochrome p450 oxygenases of taxol biosynthesis. The utilization of both taxadiene isomers in the formation of taxa-4(20),11(12)-dien-5alpha-ol is novel, suggesting a reaction mechanism involving promiscuous radical abstraction with selective oxygen insertion rather than epoxidation of the C4,C5-alkene of the natural substrate and allylic rearrangement of the resulting taxa-11(12)-en-4,5epoxide.

Deficits in the expression of oligodendrocyte (Ol) and myelin genes have been described in numerous brain regions in schizophrenia (SZ) in association with abnormalities of cell cycle markers. We have previously reported a SZ-associated decrease in the expression of genes expressed after, but not prior to, the terminal differentiation of Ols in the posterior limb of the internal capsule (ICp). This pattern of deficits could reflect a failure of Ol precursors to exit the cell cycle and differentiate to meet the demands imposed by the high rate of apoptosis among myelinating Ols. Here we explore this hypothesis using quantitative real time PCR to examine the mRNA expression of additional genes in the ICp of the previously examined sample of 14 subjects with SZ and 15 normal controls (NCs). The genes examined in the present study were chosen because they are associated with particular phases of the cell cycle (CCND1, CCND2, p21(Cip1), p27(Kip1), and p57(Kip2)), with DNA replication and repair (PCNA), apoptosis (CASP3), or the Notch signaling pathway (JAG1, HES1, HES5, andDTX1). The Notch pathway influences whether Ol precursors continue to proliferate or exit the cell cycle. We also determined the densities of Ols in the ICp. Genes associated with maintenance of the cell cycle tended to exhibit increased expression levels in SZ relative to NCs and to be negatively correlated with the expression levels of the previously assessed mature Ol genes. In contrast, genes associated with cell cycle arrest tended to show the opposite pattern (decreased expression in SZ and positive correlations with mature Ol genes). CASP3 and PCNA expression levels were significantly decreased in SZ and positively correlated with mature Ol genes, suggesting that myelinating Ols may turnover more rapidly in normal controls than in subjects with SZ. JAG1 expression was significantly increased in SZ and exhibited positive correlations with mediators of the canonical Notch pathway but negative correlations with mature Ol genes. Ol densities were significantly decreased in SZ. These data are consistent with the hypothesis that Ol and myelin deficits in SZ involve a failure of Ol precursors to appropriately exit the cell cycle in order to differentiate and mature into myelinating Ols.

Pancreatic β-cell apoptosis induced by palmitate requires high glucose concentrations. Ceramides have been suggested to be important mediators of glucolipotoxicity-induced β-cell apoptosis. In INS-1 β-cells, 0.4 mM palmitate with 5 mM glucose increased the levels of dihydrosphingosine and dihydroceramides, two lipid intermediates in the de novo biosynthesis of ceramides, without inducing apoptosis. Increasing glucose concentrations to 30 mM amplified palmitate-induced accumulation of dihydrosphingosine and the formation of (dihydro)ceramides. Of note, glucolipotoxicity specifically induced the formation of C(18:0), C(22:0) and C(24:1) (dihydro)ceramide molecular species, which was associated with the up-regulation of CerS4 (ceramide synthase 4) levels. Fumonisin-B1, a ceramide synthase inhibitor, partially blocked apoptosis induced by glucolipotoxicity. In contrast, apoptosis was potentiated in the presence of D,L-threo-1-phenyl-2-palmitoylamino-3-morpholinopropan-1-ol, an inhibitor of glucosylceramide synthase. Moreover, overexpression of CerS4 amplified ceramide production and apoptosis induced by palmitate with 30 mM glucose, whereas down-regulation of CerS4 by siRNA (short interfering RNA) reduced apoptosis. CerS4 also potentiates ceramide accumulation and apoptosis induced by another saturated fatty acid: stearate. Collectively, our results suggest that glucolipotoxicity induces β-cell apoptosis through a dual mechanism involving de novo ceramide biosynthesis and the formation of ceramides with specific N-acyl chain lengths rather than an overall increase in ceramide content.

Lineage progression and diversification is regulated by the coordinated action of unique sets of transcription factors. Oligodendrocytes (OL) and astrocytes (AS) comprise the glial sub-lineages in the CNS, and the manner in which their associated regulatory factors orchestrate lineage diversification during development and disease remains an open question. Sox10 and NFIA are key transcriptional regulators of gliogenesis associated with OL and AS. We found that NFIA inhibited Sox10 induction of OL differentiation through direct association and antagonism of its function. Conversely, we found that Sox10 antagonized NFIA function and suppressed AS differentiation in mouse and chick systems. Using this developmental paradigm as a model for glioma, we found that this relationship similarly regulated the generation of glioma subtypes. Our results describe the antagonistic relationship between Sox10 and NFIA that regulates the balance of OL and AS fate during development and demonstrate for the first time, to the best of our knowledge, that the transcriptional processes governing glial sub-lineage diversification oversee the generation of glioma subtypes.

The blend of volatile compounds emitted by bean plants (Phaseolus vulgaris) infested with greenhouse whitefly (Trialeurodes vaporariorum) has been studied comparatively with undamaged plants and whiteflies themselves. Collection of the volatiles and analysis by gas chromatography revealed more than 20 compounds produced by plants infested with whitefly. Of these, 4 compounds, (Z)-3-hexen-1-ol, 4,8-dimethyl-1,3,7-nonatriene, 3-octanone, and one unidentified compound were emitted at higher levels than from the undamaged control plants. Synthetic (Z)-3-hexen-1-ol, 4,8-dimethyl-1,3,7-nonatriene, or 3-octanone all elicited a significant increase in oriented flight and landing on the source by the parasitoid, Encarsia formosa, in wind tunnel bioassays. Two-component mixtures of the compounds and the three-component mixture all elicited a similar or, in most cases, a better response by the parasitoid, the most effective being a mixture of (Z)-3-hexen-1-ol and 3-octanone. These results demonstrate that E. formosa uses volatiles from the plant-host complex as olfactory cues for host location.

OBJECTIVE

To compare two validated closed-loop (CL) algorithms versus patient self-control with CSII in terms of glycemic control.

METHODS

This study was a multicenter, randomized, three-way crossover, open-label trial in 48 patients with type 1 diabetes mellitus for at least 6 months, treated with continuous subcutaneous insulin infusion. Blood glucose was controlled for 23 h by the algorithm of the Universities of Pavia and Padova with a Safety Supervision Module developed at the Universities of Virginia and California at Santa Barbara (international artificial pancreas [iAP]), by the algorithm of University of Cambridge (CAM), or by patients themselves in open loop (OL) during three hospital admissions including meals and exercise. The main analysis was on an intention-to-treat basis. Main outcome measures included time spent in target (glucose levels between 3.9 and 8.0 mmol/L or between 3.9 and 10.0 mmol/L after meals).

RESULTS

Time spent in the target range was similar in CL and OL: 62.6% for OL, 59.2% for iAP, and 58.3% for CAM. While mean glucose level was significantly lower in OL (7.19, 8.15, and 8.26 mmol/L, respectively) (overall P = 0.001), percentage of time spent in hypoglycemia (<3.9 mmol/L) was almost threefold reduced during CL (6.4%, 2.1%, and 2.0%) (overall P = 0.001) with less time ≤2.8 mmol/L (overall P = 0.038). There were no significant differences in outcomes between algorithms.

CONCLUSIONS

Both CAM and iAP algorithms provide safe glycemic control.

Streptococcus pneumoniae is a Gram-positive human pathogen with a complex lipoteichoic acid (pnLTA) structure. Because the current structural model for pnLTA shows substantial inconsistencies, we reinvestigated purified and, more importantly, O-deacylated pnLTA, which is most suitable for NMR spectroscopy and electrospray ionization-MS spectrometry. We analyzed pnLTA of nonencapsulated pneumococcal strains D39Δcps and TIGR4Δcps, respectively. The data obtained allowed us to (re)define (i) the position and linkage of the repeating unit, (ii) the putative α-GalpNAc substitution at the ribitiol 5-phosphate (Rib-ol-5-P), and (iii) the length of (i.e. the number of repeating units in) the pnLTA chain. We here also describe for the first time that the terminal sugar residues in the pnLTA (Forssman disaccharide; α-D-GalpNAc-(1→3)-β-D-GalpNAc-(1→)), responsible for the cross-reactivity with anti-Forssman antigen antibodies, can be heterogeneous with respect to its degree of phosphorylcholine substitution in both O-6-positions. To assess the proinflammatory potency of pnLTA, we generated a (lipopeptide-free) Δlgt mutant of strain D39Δcps, isolated its pnLTA, and showed that it is capable of inducing IL-6 release in human mononuclear cells, independent of TLR2 activation. This finding was quite in contrast to LTA of the Staphylococcus aureus SA113Δlgt mutant, which did not activate human mononuclear cells in our experiments. Remarkably, this is also contrary to various other reports showing a proinflammatory potency of S. aureus LTA. Taken together, our study refines the structure of pnLTA and indicates that pneumococcal and S. aureus LTAs differ not only in their structure but also in their bioactivity.

The half-lives (t 1/2) of alcohol sex pheromones, 1-alkanols, acetate sex pheromones, and an epoxide (disparlure) were determined on natural rubber septa. Thet 1/2 values for the homologous alcohols from decanol to heptadecanol increased regularly from 2.2 to 1117 days, but thet 1/2 of octadecanol was 609 days. Thet 1/2 values of (Z)7-, (E)7-, and (Z)9-tetradecen-1-ol acetates were 154, 168, and 199 days, respectively, whereas those of five other tested 14-carbon acetates ranged from 310 to 350 days. The dependence oft 1/2 values on chain length and double-bond position is consistent with the hypothesis that molecular size is an important variable affectingt 1/2 values. Also, in accordance with the hypothesis, when aZ-alkenyl compound has a much shortert 1/2 than the corresponding saturated compound, thet 1/2 values of theZ compound and itsE isomer may be quite different. Thus, (E)-9-tetradecen-l-ol acetate had at 1/2 of 331 days. Thet 1/2 of disparlure was 180 days. The effect of thecis-7,8 epoxide group is apparently similar to that of the olefin group in lowering thet 1/2 below the value that would be expected solely on the basis of chain length.

Desensitization of the micro-opioid receptor (MOR) has been implicated as an important regulatory process in the development of tolerance to opiates. Monitoring the release of intracellular Ca(2+) ([Ca(2+)](i)), we reported that [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO)-induced receptor desensitization requires receptor phosphorylation and recruitment of beta-arrestins (betaArrs), while morphine-induced receptor desensitization does not. In current studies, we established that morphine-induced MOR desensitization is protein kinase C (PKC)-dependent. By using RNA interference techniques and subtype specific inhibitors, PKCepsilon was shown to be the PKC subtype activated by morphine and the subtype responsible for morphine-induced desensitization. In contrast, DAMGO did not increase PKCepsilon activity and DAMGO-induced MOR desensitization was not affected by modulating PKCepsilon activity. Among the various proteins within the receptor signaling complex, Galphai2 was phosphorylated by morphine-activated PKCepsilon. Moreover, mutating three putative PKC phosphorylation sites, Ser(44), Ser(144) and Ser(302) on Galphai2 to Ala attenuated morphine-induced, but not DAMGO-induced desensitization. In addition, pretreatment with morphine desensitized cannabinoid receptor CB1 agonist WIN 55212-2-induced [Ca(2+)](i) release, and this desensitization could be reversed by pretreating the cells with PKCepsilon inhibitor or overexpressing Galphai2 with the putative PKC phosphorylation sites mutated. Thus, depending on the agonist, activation of MOR could lead to heterologous desensitization and probable crosstalk between MOR and other Galphai-coupled receptors, such as the CB1.

Sesquiterpene cyclases (synthases) catalyze the conversion of the isoprenoid intermediate farnesyl diphosphate to various sesquiterpene structural types. In plants, many sesquiterpenes are produced as defensive chemicals (phytoalexins) or mediators of chemical communication (i.e., pollinator attractants). A number of sesquiterpene synthases are present in Artemisia annua L. (annual wormwood). We have isolated a cDNA clone encoding one of these, epi-cedrol synthase. This clone contains a 1641-bp open reading frame coding for 547 amino acids (63.5 kDa), a 38-bp 5'-untranslated end, and a 272-bp 3'-untranslated sequence. The deduced amino acid sequence was 32 to 43% identical with the sequences of other known sesquiterpene cyclases from angiosperms. When expressed in Escherichia coli, the recombinant enzyme catalyzed the formation of both olefinic (3%) and oxygenated (97%) sesquiterpenes from farnesyl diphosphate. GC-MS analysis identified the olefins as alpha-cedrene (57% of the olefins), beta-cedrene (13%), (E)-beta-farnesene (5%), alpha-acoradiene (1%), (E)-alpha-bisabolene (8%), and three unknown olefins (16%) and the oxygenated sesquiterpenes (97% of total sesquiterpene generated, exclusive of farnesol and nerolidol) as cedrol (4%) and epi-cedrol (96%). epi-Cedrol synthase was not active with geranylgeranyl diphosphate as substrate, whereas geranyl diphosphate was converted to monoterpenes by the recombinant enzyme at a rate of about 15% of that observed with farnesyl diphosphate as substrate. The monoterpene olefin products are limonene (45%), terpinolene (42%), gamma-terpinene (8%), myrcene (5%), and alpha-terpinene (2%); a small amount of the monoterpene alcohol terpinen-4-ol is also produced. The pH optimum for the recombinant enzyme is 8.5-9.0 (with farnesyl diphosphate as substrate) and the K(m) values for farnesyl diphosphate are 0.4 and 1.3 microM at pH 7. 0 and 9.0, respectively. The K(m) for Mg(2+) is 80 microM at pH 7.0 and 9.0.

The cytochrome P450 sterol 14alpha-demethylase (CYP51) of Candida albicans is involved in an essential step of ergosterol biosynthesis and is the target for azole antifungal compounds. We have undertaken site-directed mutation of C. albicans CYP51 to produce a recombinant mutant protein with the amino acid substitution R467K corresponding to a mutation observed clinically. This alteration perturbed the heme environment causing an altered reduced-carbon monoxide difference spectrum with a maximum at 452 nm and reduced the affinity of the enzyme for fluconazole, as shown by ligand binding studies. The specific activity of CYP51(R467K) for the release of formic acid from 3beta-[32-(3)H]hydroxylanost-7-en-32-ol was 70 pmol/nmol of P450/min for microsomal protein compared to 240 pmol/nmol of P450/min for microsomal fractions expressing wild-type CYP51. Furthermore, inhibition of activity by fluconazole revealed a 7.5-fold-greater azole resistance of the recombinant protein than that of the wild type. This study demonstrates that resistance observed clinically can result from the altered azole affinity of the fungal CYP51 enzyme.