Lamin B receptor (LBR), a nuclear protein of avian and mammalian cells, contains an hydrophobic domain that shares extensive structural similarities with the members of the sterol reductase family. To test if the sterol-reductase-like domain of LBR could be enzymatically competent, several sterol reductase-defective strains of Saccharomyces cerevisiae were transformed with a human-LBR expressing vector. LBR production did not change the ergosterol biosynthesis defect in an erg4 mutant impaired in sterol C24(28) reductase. In contrast, the sterol C14 reduction step and ergosterol prototrophy were restored in LBR-producing erg24 transformants which lack endogenous sterol C14 reductase. To test the effects of C14 reductase inhibitors on LBR activity, we constructed EMY54, an ergosterol-requiring strain that is devoid of both sterol C8-C7 isomerase and sterol C14 reductase activities. EMY54 cells recovered the capability of synthesizing ergost-8-en-3beta-ol upon transformation with a vector that expressed either yeast sterol C14 reductase or hLBR. In addition, growth in sterol-free medium was restored in these transformants. Sterol biosynthesis and proliferation of LBR-producing cells were found to be highly susceptible to fenpropimorph and tridemorph, but only moderately susceptible to SR 31747. Our results strongly suggest that hLBR is a sterol C14 reductase.

Isoprenoids are a class of natural products with more than 55,000 members. All isoprenoids are constructed from two precursors, isopentenyl diphosphate and its isomer dimethylallyl diphosphate. Two of the most important discoveries in isoprenoid biosynthetic studies in recent years are the elucidation of a second isoprenoid biosynthetic pathway [the methylerythritol phosphate (MEP) pathway] and a modified mevalonic acid (MVA) pathway. In this review, we summarize mechanistic insights on the MEP pathway enzymes. Because many isoprenoids have important biological activities, the need to produce them in sufficient quantities for downstream research efforts or commercial application is apparent. Recent advances in both MVA and MEP pathway-based synthetic biology are also illustrated by reviewing the landmark work of artemisinic acid and taxadien-5α-ol production through microbial fermentations.

The socio-demographic factors have a substantial impact on the overall casualties caused by the Coronavirus (COVID-19). In this study, the global and local spatial association between the key socio-demographic variables and COVID-19 cases and deaths in the European regions were analyzed using the spatial regression models. A total of 31 European countries were selected for modelling and subsequent analysis. From the initial 28 socio-demographic variables, a total of 2 (for COVID-19 cases) and 3 (for COVID-19 deaths) key variables were filtered out for the regression modelling. The spatially explicit regression modelling and mapping were done using four spatial regression models such as Geographically Weighted Regression (GWR), Spatial Error Model (SEM), Spatial Lag Model (SLM), and Ordinary Least Square (OLS). Additionally, Partial Least Square (PLS) and Principal Component Regression (PCR) was performed to estimate the overall explanatory power of the regression models. For the COVID cases, the local R² values, which suggesting the influences of the selected socio-demographic variables on COVID cases and death, were found highest in Germany, Austria, Slovenia, Switzerland, Italy. The moderate local R² was observed for Luxembourg, Poland, Denmark, Croatia, Belgium, Slovakia. The lowest local R² value for COVID-19 cases was accounted for Ireland, Portugal, United Kingdom, Spain, Cyprus, Romania. Among the 2 variables, the highest local R² was calculated for income (R² = 0.71), followed by poverty (R² = 0.45). For the COVID deaths, the highest association was found in Italy, Croatia, Slovenia, Austria. The moderate association was documented for Hungary, Greece, Switzerland, Slovakia, and the lower association was found in the United Kingdom, Ireland, Netherlands, Cyprus. This suggests that the selected demographic and socio-economic components, including total population, poverty, income, are the key factors in regulating overall casualties of COVID-19 in the European region. In this study, the influence of the other controlling factors, such as environmental conditions, socio-ecological status, climatic extremity, etc. have not been considered. This could be the scope for future research.

Keywords: COVID-19; Demography; Outbreak; Pandemic; Spatial regression; Virus.

We describe a selection procedure which utilizes the vapor from an unsaturated alcohol, 1-pentene-3-ol, for the detection and isolation of mutant flies with little or no alcohol dehydrogenase activity. ADH-negative flies are unaffected by exposure to the unsaturated alcohol, but ADH positives (wild-types) die after short exposure. The technique can be used to select rare ADH-negative individuals from large populations of wild-type flies.

Quality-adjusted life years (QALYs) are well recognized as a valid measure for outcomes in cost-effectiveness analyses. A summary health utility score is necessary to evaluate QALYs. The objective of this study was to predict a summary utility score (represented by the Health Utility Index [HUI2]) from scores on the SF-36.

METHODS

A structural equation framework was applied to longitudinal data collected from 1992 to 1995 on a sample of patients insured by Southem California Kaiser Permanente (N = 6921). An ordinary least squares (OLS) method was used to estimate the HUI2.

RESULTS

The OLS model on cross-sectional data predicted 50.5% of the observed variance in HUI2 scores. Parameter estimates of all SF-36 components showed statistical significance at the P < 0.05 level.

CONCLUSIONS

Results of this study provide a quantitative link between two important measures of health status. The present model can be used to estimate health utility summary scores in studies that have collected SF-36 data.

The actions of various opioids were examined on calcium action potentials in the cell somata of guinea pig myenteric neurones and on the release of acetylcholine at synapses onto these cells. The opioids morphine, normorphine, and [D-Ala2, MePhe4, Met5(O)]enkephalin-ol caused membrane hyperpolarizations resulting from an increase in potassium conductance; opioids that are more selective agonists for the kappa receptor subtype (dynorphin, tifluadom, U50488H) did not. Conversely, calcium action potentials were depressed or abolished by the kappa opioids but were not affected by morphine and [D-Ala2, MePhe4, Met(O)5]enkephalin-ol. Both groups of opioids caused presynaptic inhibition of acetylcholine release in the myenteric plexus, depressing the amplitude of the fast excitatory postsynaptic potential. The presynaptic inhibition caused by [D-Ala2, MePhe4, Met(O)5]enkephalin-ol, morphine, and normorphine, but not that caused by the kappa opioids, was prevented by pretreatment with the selective mu site-directed irreversible antagonist beta-funaltrexamine. Furthermore, the presynaptic inhibitory action of morphine and [D-Ala2, MePhe4, Met(O)5]enkephalin-ol, but not that of the kappa-receptor agonists, was reversibly blocked by barium. The results suggest that presynaptic inhibition caused by mu receptor activation probably results from an increase in potassium conductance, whereas kappa-receptor agonists may depress the release of acetylcholine by directly reducing calcium entry into the nerve terminals.

Fourteen hormone-producing gastrointestinal tract tumors were tested for their content of somatostatin (SRIH) receptors, using receptor autoradiography and in vitro binding assay with tumor homogenates. All four gastrinomas tested had high levels of SRIH receptors, as did two of five insulinomas and four of five vasoactive intestinal peptide-producing tumors. Receptor visualization was obtained with two different radioligands, either a SRIH-28 analog, [125I]-[Leu8,D-Trp22,Tyr25]SRIH-28, or a SRIH octapeptide, the [125I]Tyr3 derivative of SMS 201-995 [H-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr(ol)], [125I]204-090. In both cases receptors were localized over the tumor cell area only. Biochemical and pharmacological analyses of one insulinoma and two vipomas revealed saturable, high affinity binding sites with pharmacological specificity for SRIH. However, differences in receptor affinity of selected SRIH analogs, in particular SRIH-28 and SRIH octapeptides, were found between the insulinomas and the two other tumor types, vipoma and gastrinoma. The presence of SRIH receptors on various hormone-producing gastrointestinal tumors suggests that at least part of the beneficial effects of chronic therapy with SRIH analogs may be mediated through such membrane-bound receptors located on the tumor itself. SRIH receptor measurement may be of prognostic value in assessment of the therapeutic efficacy of SRIH analogs. They may also be of diagnostic value, if used as in vivo markers for the localization of small hormone-producing gastrointestinal tumors or their metastases.

The prion, the transmissible agent that causes spongiform encephalopathies such as scrapie, bovine spongiform encephalopathy (BSE) and Creutzfeldt-Jakob disease, is believed to be devoid of nucleic acid and identical with PrPSc, a modified form of the normal host protein PrPC which is encoded by the single copy gene Prnp. The 'protein only' hypothesis proposes that PrPSc, when introduced into a normal host, causes the conversion of PrPC into PrPSc; it therefore predicts that an animal devoid of PrPC should be resistant to prion diseases. We generated homozygous Prnp(olo) ('PrP knockout') mice and showed that, after inoculation with prions, they remained free of scrapie for at least 2 years while wild-type controls all died within 6 months. There was no propagation of prions in the Prnp(olo) animals. Surprisingly, heterozygous Prnp(ol+) mice, which express PrPC at about half the normal level, also showed enhanced resistance to scrapie disease despite high levels of infectious agent and PrPSc in the brain early on. After introduction of murine PrP transgenes Prnp(olo) mice became highly susceptible to mouse but not to hamster prions, while the insertion of Syrian hamster PrP transgenes rendered them susceptible to hamster but to a much lesser extent to mouse prions. These complementation experiments paved the way to the application of reverse genetics. We have prepared animals transgenic for genes encoding PrP with amino terminal deletions of various lengths and have found that PrP lacking 48 amino proximal amino acids, which comprise four of the five octa repeats of PrP, is still biologically active.

Estrogens are classically viewed as hormones that bind to intracellular receptors, which then act as transcription factors to modulate gene expression; however, they also affect many aspects of neuronal functioning by rapid nongenomic actions. Brain estrogen production can be regulated within minutes by changes in aromatase (estrogen synthase) activity as a result of calcium-dependent phosphorylations of the enzyme. To determine the effects of rapid changes in estrogen availability on male copulatory behavior, we mimicked in male mice the rapid upregulation and downregulation of brain estrogen concentration that should occur after inactivation or activation of aromatase activity. A single injection of different aromatase inhibitors [Vorozole, 1,4,6-androstatrien-3,17-dione (ATD), or its metabolite 17-OH-ATD (1,4,6-androstatrien-17beta-ol-3-one)] almost completely suppressed male sexual behavior (mounts and intromissions) expressed 10-20 min later by C57BL/6J mice but did not affect behavior in aromatase knock-out (ArKO) mice, activated by daily injections of estradiol benzoate, thereby confirming the specificity of the behavioral inhibition observed in wild-type mice. The rapid ATD-induced inhibition was reversed by the simultaneous injection of a large dose of estradiol. A single injection of estradiol to ArKO mice also activated male sexual behavior within 15 min. Thus, rapid increases or decreases in brain estrogen concentrations are followed within minutes by corresponding changes in male sexual behavior. Sexual behavior can thus be used to monitor changes in local estrogen concentrations and analyze the mechanisms mediating the rapid decline in estrogen signaling that takes place after inhibition of estrogen synthesis.

Following p.o. administration of 14C-labelled rac.-1-(e)-(m-methoxyphenyl)-2-(e)-dimethylaminomethyl-cyclohexan-1-(a)-ol hydrochloride (tramadol hydrochloride, CG 315, Tramal) to mice, hamsters, rats, guinea pigs, rabbits, dogs and man the metabolic pathways were investigated and the results compared. After synthesis of the reference substances the metabolites were identified by co-chromatography using both TLC (thin-layer chromatography) and HPLC (high-performance liquid chromatography) methods, by co-crystallization and by gas chromatography-mass spectrometry. In all species the main metabolic pathways are N- and O-demethylation (phase I reactions) and conjugation of O-demethylated compounds (phase II reactions). 11 metabolites are known, 5 arising by phase I reactions (M1 to M5) and 6 by phase II reactions (glucuronides and sulfates of M1, M4 and M5). The 5 phase I metabolites are mono-O-demethyl-tramadol (M1), mono-N-demethyl-tramadol (M2), di-N-demethyl-tramadol (M3), tri-N,O-demethyl-tramadol (M4) and di-N,O-demethyl-tramadol (M5). The biotransformation scheme of tramadol is qualitatively identical in man, dog, rabbit, guinea pig, rat, hamster and mouse. In all species M1 and M1-conjugates, M5 and M5-conjugates and M2 are the main metabolites, whereas M3, M4 and M4-conjugates were only formed in minor quantities. Following p.o. administration to man and animals 14C-tramadol are rapidly and almost completely absorbed. The unchanged drug and metabolites are mainly excreted via kidneys. The cumulative renal excretion of total radioactivity accounts for approximately 90% in man and varies from 86 to 100% in mouse, hamster, rat, guinea pig, rabbit and dog; the residual of the applied radioactivity appears in the feces. Apparently tramadol is metabolized much more rapidly in animals than in man. For that reason there are appreciable differences between man and animals in the amount of tramadol excreted unchanged in the urine (about 30% and 1% of the p.o. dose, respectively). After incubation with beta-glucuronidase and arylsulfatase at least 81% of the excreted radioactivity could be extracted from the urine of man animals (with the exception of the guinea pig and the rabbit). In man all extractable metabolites were identified.

BACKGROUND

Health-related quality of Life in patients with chronic urticaria is evaluated by mean of generic instruments or questionnaire designed for skin diseases. No disease-specific tool is now available for the assessment of chronic urticaria impact from patients' viewpoint.

OBJECTIVE

The aim of our study is to develop and validate a new questionnaire specifically designed for the assessment of quality of life in chronic urticaria (Chronic Urticaria Quality of Life Questionnaire -- CU-Q(2)oL).

METHODS

In the development phase of CU-Q(2)oL an initial list of items of 37 items was compiled and given to 80 patients with chronic urticaria; the 23 most significant items were selected and converted into questions evaluating the answers on a Likert scale of five steps. The validation procedure involved 125 patients (86 F and 39 M) (age 42.17 +/- 9.24 years).

RESULTS

Following a statistical analysis, CU-Q(2)oL showed a six-dimensional structure and good levels of internal consistency for the extracted factors: Pruritus (0.79), Swelling (0.65), Impact on life activities (0.83), Sleep problems (0.77), Looks (0.83) and Limits (0.74). In stable conditions CU-Q(2)oL showed a good reliability, ranged between 0.64 and 0.92. Responsiveness to clinical changes was accomplished.

CONCLUSIONS

These results provide evidence that CU-Q(2)oL has specificity enough for being a valid tool for detecting the relative burden of CU on subjective wellbeing, and for obtaining a global evaluation both of CU impact and of treatments, taking into account the patient's point of view. The CU-Q(2)oL was easily and quickly filled up and well accepted by the patients.

The three dominant oxidative biotransformations of estradiol were examined in 10 normal women and 33 females with breast cancer by using a recently devised radiometric method. Estradiol tracers, labeled with 3H specifically in the 17 alpha, C-2, or 16 alpha position, were used to measure both the rate and extent of 17 beta-ol oxidation (the initial metabolic step) and the subsequent 2- and 16 alpha-oxidative reactions. The mean +/- SEM values for the extent of extradiol metabolism at these three specific sites for the extent of estradiol metabolism at these three specific sites were 76.9 +/- 5.3%, 31.1 +/- 4.0%, and 9.3 +/- 0.8%, respectively in normal subjects. Corresponding data in patients with breast cancer--i.e., 73.0 +/- 4.2%, 32.7 +/- 2.7%, and 14.9 +/- 1.5%--revealed a significantly greater extent of 16 alpha-hydroxylation in the latter population. Because the 16 alpha-hydroxylated compounds (including estriol) are themselves potent estrogens, these changes may have important hyperestrogenic consequences that could have a bearing on the etiology of the disease.

Adverse early-life experiences, such as poor maternal care, program an abnormal stress response that may involve an altered balance between excitatory and inhibitory signals. Here, we explored how early-life stress (ELS) affects excitatory and inhibitory transmission in corticotrophin-releasing factor (CRF)-expressing dorsal-medial (mpd) neurons of the neonatal mouse hypothalamus. We report that ELS associates with enhanced excitatory glutamatergic transmission that is manifested as an increased frequency of synaptic events and increased extrasynaptic conductance, with the latter associated with dysfunctional astrocytic regulation of glutamate levels. The neurosteroid 5α-pregnan-3α-ol-20-one (5α3α-THPROG) is an endogenous, positive modulator of GABAA receptors (GABAARs) that is abundant during brain development and rises rapidly during acute stress, thereby enhancing inhibition to curtail stress-induced activation of the hypothalamic-pituitary-adrenocortical axis. In control mpd neurons, 5α3α-THPROG potently suppressed neuronal discharge, but this action was greatly compromised by prior ELS exposure. This neurosteroid insensitivity did not primarily result from perturbations of GABAergic inhibition, but rather arose functionally from the increased excitatory drive onto mpd neurons. Previous reports indicated that mice (dams) lacking the GABAAR δ subunit (δ(0/0)) exhibit altered maternal behavior. Intriguingly, δ(0/0) offspring showed some hallmarks of abnormal maternal care that were further exacerbated by ELS. Moreover, in common with ELS, mpd neurons of δ(0/0) pups exhibited increased synaptic and extrasynaptic glutamatergic transmission and consequently a blunted neurosteroid suppression of neuronal firing. This study reveals that increased synaptic and tonic glutamatergic transmission may be a common maladaptation to ELS, leading to enhanced excitation of CRF-releasing neurons, and identifies neurosteroids as putative early regulators of the stress neurocircuitry.

The myelin sheath forms by the spiral wrapping of a glial membrane around the axon. The mechanisms responsible for this process are unknown but are likely to involve coordinated changes in the glial cell cytoskeleton. We have found that inhibition of myosin II, a key regulator of actin cytoskeleton dynamics, has remarkably opposite effects on myelin formation by Schwann cells (SC) and oligodendrocytes (OL). Myosin II is necessary for initial interactions between SC and axons, and its inhibition or down-regulation impairs their ability to segregate axons and elongate along them, preventing the formation of a 1:1 relationship, which is critical for peripheral nervous system myelination. In contrast, OL branching, differentiation, and myelin formation are potentiated by inhibition of myosin II. Thus, by controlling the spatial and localized activation of actin polymerization, myosin II regulates SC polarization and OL branching, and by extension their ability to form myelin. Our data indicate that the mechanisms regulating myelination in the peripheral and central nervous systems are distinct.

Insect chemosensory proteins (CSPs) have been proposed to capture and transport hydrophobic chemicals from air to olfactory receptors in the lymph of antennal chemosensilla. They may represent a new class of soluble carrier protein involved in insect chemoreception. However, their specific functional roles in insect chemoreception have not been fully elucidated. In this study, we report for the first time three novel CSP genes (AlinCSP1-3) of the alfalfa plant bug Adelphocoris lineolatus (Goeze) by screening the antennal cDNA library. The qRT-PCR examinations of the transcript levels revealed that all three genes (AlinCSP1-3) are mainly expressed in the antennae. Interestingly, these CSP genes AlinCSP1-3 are also highly expressed in the 5(th) instar nymphs, suggesting a proposed function of these CSP proteins (AlinCSP1-3) in the olfactory reception and in maintaining particular life activities into the adult stage. Using bacterial expression system, the three CSP proteins were expressed and purified. For the first time we characterized the types of sensilla in the antennae of the plant bug using scanning electron microscopy (SEM). Immunocytochemistry analysis indicated that the CSP proteins were expressed in the pheromone-sensitive sensilla trichodea and general odorant-sensitive sensilla basiconica, providing further evidence of their involvement in chemoreception. The antennal activity of 55 host-related semiochemicals and sex pheromone compounds in the host location and mate selection behavior of A. lineolatus was investigated using electroantennogram (EAG), and the binding affinities of these chemicals to the three CSPs (AlinCSP1-3) were measured using fluorescent binding assays. The results showed several host-related semiochemicals, (Z)-3-hexen-1-ol, (E)-2-hexen-1-al and valeraldehyde, have a high binding affinity with AlinCSP1-3 and can elicit significant high EAG responses of A. lineolatus antennae. Our studies indicate the three antennae-biased CSPs may mediate host recognition in the alfalfa plant bug A. lineolatus.

Ligands of structurally diverse natures are able to bind at the CB(1) cannabinoid receptor, suggesting the existence of multiple binding sites on the receptor. Modeling studies have implicated Ser2.60(173) and Ser7.39(383) as possible interaction site(s) for CB(1) agonists. To test the importance of these residues for receptor recognition, recombinant human CB(1) receptors, stably expressed in human embryonic kidney 293 cells, were used to investigate the consequences of mutating Ser2.60 (to S2.60A) or Ser7.39 (to S7.39A) in radioligand binding and guanosine 5'-3-O-(thio)triphosphate functional assays. The S7.39A mutant resulted in a total ablation of [(3)H](-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl] cyclohexan-1-ol (CP55,940) high-affinity binding. However, [(3)H](R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]-pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN55,212-2) binding properties at S7.39A were comparable with those of the wild-type (WT) receptor. The binding affinity of (-)-11beta-hydroxy-3-(1',1'-dimethylheptyl)hexahydrocannabinol (AM4056) and (-)-11-hydroxydimethylheptyl-Delta(8)-tetrahydrocannabinol (HU210) were drastically reduced (50- to 100-fold) at the S7.39A mutant. Likewise, the EC(50) for HU210 and AM4056-mediated activation of the S7.39A receptor was increased by >200-fold. In contrast, the binding affinity and potency of WIN55,212-2, CP55,940, HU210, and AM4056 were unaltered at the S2.60A mutant compared with WT human CB(1) receptors. These results clearly suggest that Ser7.39, but not Ser2.60, plays a crucial role in mediating ligand specific interactions for CP55,940, HU210, and AM4056 at the human CB(1) receptor. Our modeling studies predict that Ser7.39 in a g-chi1 conformation may induce a helix bend in TMH7 that provides docking space for CP55,940 binding; the S7.39A mutation may alter this binding space, precluding CP55,940 binding.

The alpha(2A)-adrenergic receptor (AR) subtype mediates antinociception induced by the alpha(2)AR agonists clonidine, dexmedetomidine, norepinephrine, and 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK-14,304) as well as antinociceptive synergy of UK-14,304 with opioid agonists [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin and deltorphin II. Differential localization of alpha(2)-adrenergic (alpha(2A)-, alpha(2B)-(,) alpha(2C)-) and opioid (mu-, delta-, kappa-) subtypes suggests differential involvement of subtype pairs in opioid-adrenergic analgesic synergy. The present study applies a novel imidazoline(1)/alpha(2)-adrenergic receptor analgesic, moxonidine, to test for involvement of alpha(2B)- and alpha(2C)ARs in antinociception and antinociceptive synergy, because spinal antinociceptive activity of moxonidine shows minimal dependence on alpha(2A)AR. Intrathecal administration of moxonidine produced similar (2-3-fold) decreases in both mutant mice with a functional knockout of alpha(2A)AR (D79N-alpha(2A)AR) and alpha(2C)AR knockout (KO) mice. The potency of moxonidine was not altered in alpha(2B)KO mice, indicating that this subtype does not participate in moxonidine-induced spinal antinociception. Moxonidine-mediated antinociception was dose dependently inhibited by the selective alpha(2)-receptor antagonist SK&F 86466 in both D79N-alpha(2A) mice and alpha(2C)KO mice, indicating that alpha(2)AR activation is required in the absence of either alpha(2A)- or alpha(2C)AR. Spinal administration of antisense oligodeoxynucleotides directed against the alpha(2C)AR decreased both alpha(2C)AR immunoreactivity and the antinociceptive potency of moxonidine. Isobolographic analysis demonstrates that moxonidine-deltorphin antinociceptive synergy is present in the D79N-alpha(2A) mice but not in the alpha(2C)AR-KO mice. These results confirm that the alpha(2C)AR subtype contributes to spinal antinociception and synergy with opioids.

Insulin-like growth factor-1 (IGF-1) interacts with the Type I receptor to activate two main signaling pathways, the mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) and the phosphatidylinositol 3-kinase (PI3K)-Akt cascades, which mediate proliferation or survival of oligodendrocyte (OL) progenitors (OLPs). In other cellular systems, mammalian target of rapamycin (mTOR) and the p70 S6 kinase are downstream effectors that phosphorylate translation initiation factors (e.g. eIF-4E), their regulators (e.g. 4E-binding protein 1, 4E-BP1) and ribosomal protein S6 (S6). The aim of this study was to determine whether these pathways are involved in IGF-1-stimulated protein synthesis, important for growth and differentiation of OLs. Rat cultured OLPs were treated with IGF-1 with or without inhibitors of PI3K (LY294002 or Wortmannin), mTOR (rapamycin), MEK (PD98059), and Akt (III or IV), as well as an adenovirus encoding a dominant negative form of Akt. Protein synthesis, as assessed by [(35)S]-methionine incorporation, was stimulated by IGF-1 and required the upstream activation of PI3K, Akt, mTOR and MEK/ERK. Concordant with the experiments using protein kinase inhibitors, western blotting revealed that IGF-1 stimulates phosphorylation of Akt, mTOR, ERK, S6 and 4E-BP1. Activation of S6 and inactivation of 4E-BP1, necessary for protein synthesis to take place, were dependent on the upstream activation of PI3K and mTOR. Finally, IGF-1 consistently stimulated protein synthesis through mTOR in differentiating OLPs but mRNA transcription was not required at day 4, indicating a differential role of IGF-1 throughout OL development.

This paper offers a review of current scientific research regarding the potential cardiovascular health benefits of flavonoids found in cocoa and chocolate. Recent reports indicate that the main flavonoids found in cocoa, flavan-3-ols and their oligomeric derivatives, procyanidins, have a variety of beneficial actions, including antioxidant protection and modulation of vascular homeostasis. These findings are supported by similar research on other flavonoid-rich foods. Other constituents in cocoa and chocolate that may also influence cardiovascular health are briefly reviewed. The lipid content of chocolate is relatively high; however, one third of the lipid in cocoa butter is composed of the fat stearic acid, which exerts a neutral cholesterolemic response in humans. Cocoa and chocolate contribute to trace mineral intake, which is necessary for optimum functioning of all biologic systems and for vascular tone. Thus, multiple components in chocolate, particularly flavonoids, can contribute to the complex interplay of nutrition and health. Applications of this knowledge include recommendations by health professionals to encourage individuals to consume a wide range of phytochemical-rich foods, which can include dark chocolate in moderate amounts.

Plant-derived polyphenols are increasingly receiving attention as dietary supplements for the homeostatic management of inflammation, to support detoxication, and for anticancer, weight loss, and other benefits. Their pro-homeostatic effects on genes, transcription factors, enzymes, and cell signaling pathways are being intensively explored, but the poor bioavailability of some polyphenols likely contributes to poor clinical trial outcomes. This review covers four polyphenol preparations with poor bioavailability and their complexation into phytosomes to bypass this problem. Silybin and the other silymarin flavonolignans from milk thistle conserve tissue glutathione, are liver-protective, and have anticancer potential. Curcumin and its related diphenolic curcuminoids have potent antioxidant, anti-inflammatory, and anti-carcinogenic properties. The green tea flavan-3-ol catechins have antioxidant, anti-inflammatory, cardio- and neuro-protective effects, and anti-carcinogenic benefits, with fat oxidation effects coupled to weight loss. The complex grape seed proanthocyanidin mix (including catechin and epicatechin monomers and oligomers) counters oxidative stress and protects the circulatory system. For each of these preparations, conversion into phytosomes has improved efficacy without compromising safety. The phytosome technology creates intermolecular bonding between individual polyphenol molecules and one or more molecules of the phospholipid, phosphatidylcholine (PC). Molecular imaging suggests that PC molecule(s) enwrap each polyphenol; upon oral intake the amphipathic PC molecules likely usher the polyphenol through the intestinal epithelial cell outer membrane, subsequently accessing the bloodstream. PC itself has proven clinical efficacy that contributes to phytosome in vivo actions. As a molecular delivery vehicle, phytosome technology substantially improves the clinical applicabilities of polyphenols and other poorly absorbed plant medicinals.

A central feature in the biosynthesis of Taxol is oxygenation at multiple positions of the taxane core structure, reactions that are considered to be mediated by cytochrome P450-dependent monooxygenases. A PCR-based differential display-cloning approach, using Taxus (yew) cells induced for Taxol production, yielded a family of related cytochrome P450 genes, one of which was assigned as a taxane 10 beta-hydroxylase by functional expression in yeast. The acquired clones that did not function in yeast were heterologously expressed by using the Spodoptera fugiperda-baculovirus-based system and were screened for catalytic capability by using taxa-4(20),11(12)-dien-5 alpha-ol and its acetate ester as test substrates. This approach allowed identification of one of the cytochrome P450 clones (which bore 63% deduced sequence identity to the aforementioned taxane 10 beta-hydroxylase) as a taxane 13 alpha-hydroxylase by chromatographic and spectrometric characterization of the corresponding recombinant enzyme product. The demonstration of a second relevant hydroxylase from the induced family of cytochrome P450 genes validates this strategy for elucidating the oxygenation steps of taxane diterpenoid (taxoid) metabolism. Additionally, substrate specificity studies with the available cytochrome P450 hydroxylases now indicate that there is likely more than one biosynthetic route to Taxol in yew species.

BACKGROUND

D-Serine, an endogenous agonist of the N-methyl-D-aspartate (NMDA) receptors, is effective in the treatment of schizophrenia. However, orally administered D-serine is metabolized substantially by D-amino acid oxidase (DAAO), diminishing its oral bioavailability. In this study, we examined the effects of oral D-serine administration with or without a DAAO inhibitor, 5-chloro-benzo[d]isoxazol-3-ol (CBIO), on the prepulse inhibition (PPI) deficits after administration of the NMDA receptor antagonist dizocilpine.

METHODS

Vehicle or D-serine (30, 300, or 900 mg/kg) with or without CBIO (30 mg/kg) was orally administered to mice 1 hour before administration of dizocilpine (.1 mg/kg), and then the PPI of the acoustic startle response was measured. We measured the extracellular levels of D-serine in the frontal cortex after oral administration of D-serine with or without CBIO.

RESULTS

Coadministration of CBIO with D-serine (30 mg/kg), but not D-serine (30 mg/kg) alone, significantly attenuated dizocilpine-induced PPI deficits. Furthermore, coadministration of CBIO significantly increased the extracellular levels of D-serine in the frontal cortex after administration of D-serine.

CONCLUSIONS

These findings suggest that coadministration of CBIO significantly enhanced the efficacy of D-serine in attenuating PPI deficits by administration of dizocilpine. Therefore, coadministration of D-serine and a DAAO inhibitor has therapeutic potential for the treatment of schizophrenia.

Twenty-six Streptomyces spp. were screened for their volatile production capacity on yeast starch agar. The volatile organic compounds (VOCs) were concentrated on a porous polymer throughout an 8-day growth period. VOCs were analyzed by gas chromatography with flame ionization detection and identified or characterized by gas chromatography-mass spectrometry. A total of 120 VOCs were characterized by retention index and mass spectra. Fifty-three compounds were characterized as terpenoid compounds, among which 18 could be identified. Among the VOCs were alkanes, alkenes, alcohols, esters, ketones, sulfur compounds, and isoprenoid compounds. Among the most frequently produced compounds were isoprene, acetone, 1-butanol, 2-methyl-1-propanol, 3-methyl-3-buten-1-ol, 3-methyl-1-butanol, 2-methyl-1-butanol, cyclopentanone, dimethyl disulfide, dimethyl trisulfide, 2-phenylethanol, and geosmin. The relationship between the excretion of geosmin and the production of spores was examined for one isolate. A good correlation between headspace geosmin and the number of spores was observed, suggesting that VOCs could be used to indicate the activity of these microorganisms in heterogeneous substrates.

OBJECTIVE

The molecular basis of agonist-selective signalling at the µ-opioid receptor is poorly understood. We have recently shown that full agonists such as [D-Ala(2)-MePhe(4)-Gly-ol]enkephalin (DAMGO) stimulate the phosphorylation of a number of carboxyl-terminal phosphate acceptor sites including threonine 370 (Thr(370)) and serine 375 (Ser(375)), and that is followed by a robust receptor internalization. In contrast, morphine promotes a selective phosphorylation of Ser(375) without causing rapid receptor internalization.

METHODS

Here, we identify kinases and phosphatases that mediate agonist-dependent phosphorylation and dephosphorylation of the µ-opioid receptor using a combination of phosphosite-specific antibodies and siRNA knock-down screening in HEK293 cells.

RESULTS

We found that DAMGO-driven phosphorylation of Thr(370) and Ser(375) was preferentially catalysed by G-protein-coupled receptor kinases (GRKs) 2 and 3, whereas morphine-driven Ser(375) phosphorylation was preferentially catalysed by GRK5. On the functional level, inhibition of GRK expression resulted in enhanced µ-opioid receptor signalling and reduced receptor internalization. Analysis of GRK5-deficient mice revealed that GRK5 selectively contributes to morphine-induced Ser(375) phosphorylation in brain tissue. We also identified protein phosphatase 1γ as a µ-opioid receptor phosphatase that catalysed Thr(370) and Ser(375) dephosphorylation at or near the plasma membrane within minutes after agonist removal, which in turn facilitates receptor recycling.

CONCLUSIONS

Together, the morphine-activated µ-opioid receptor is a good substrate for phosphorylation by GRK5 but a poor substrate for GRK2/3. GRK5 phosphorylates µ-opioid receptors selectively on Ser(375), which is not sufficient to drive significant receptor internalization.