Design, synthesis and biological evaluation of novel (E)-2-benzylidene-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)hydrazine-1-carboxamide derivatives as α-glucosidase inhibitors.

In this present study, a series of novel (E)-2-benzylidene-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)hydrazine-1-carboxamide derivatives against α-glucosidase were designed and synthesized, and their biological activities were evaluated in vitro and in vivo. Most of the designed analogues exhibited better inhibitory activity than the marketed acarbose, especially the most potent compound 7 with an IC50 value of 9.26 ± 1.84 µM. The direct binding of 7 and 8 with α-glucosidase was confirmed by fluorescence quenching experiments, and the kinetic and molecular docking studies revealed that 7 and 8 inhibited α-glucosidase in a non-competitive manner. Cytotoxicity bioassay indicated compounds 7 and 8 were non-toxic towards LO2 and HepG2 at 100 µM. Furthermore, both compounds were demonstrated to have in vivo hypoglycemic activity by reducing the blood glucose levels in sucrose-treated rats.

Cress seed gum improves rheological, textural and physicochemical properties of native wheat starch-sucrose mixture.

In this paper, the impact of different substitution levels of cress seed gum (CSG, 0, 5, 10, and 15%) and sucrose (SUC, 0, 5, and 10%) on the rheological properties, textural attributes, syneresis, FTIR and microstructure of native wheat starch (NWS, 4%) gel was investigated. According to the rheological tests, the NWS-CSG and NWS-CSG-SUC gels showed thixotropic behavior and all the samples exhibited shear-thinning flow behavior. Increasing the CSG substitution level up to 15% elevated the apparent viscosity, consistency coefficient whereas the SUC substitution with NWS reduced these values. The higher apparent viscosity, consistency coefficient, and stronger pseudoplastic behavior were obtained for NWS-CSG-SUC gel than NWS gel. The addition of CSG greatly decreased hardness and consistency from 140 to 55.5 g and from 6.9 to 3.0 mJ, respectively during storage at 4 °C for 14 days; while in the presence of SUC these values slightly decreased. After storage, syneresis of NWS and NWS-10%SUC gels increased by 46.78% and 32.11%, respectively; whereas it decreased 19.88% for NWS-15%CSG gel. The SEM images showed that the mixed gels had a denser structure with a smaller pore size. The results indicated that CSG had positive effect in modifying the properties of NWS-SUC mixed gels.

Ces1d deficiency protects against high-sucrose diet-induced hepatic triacylglycerol accumulation.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Triacylglycerol accumulation in the liver is a hallmark of NAFLD. Metabolic studies have confirmed that increased hepatic de novo lipogenesis (DNL) in humans contributes to fat accumulation in the liver and to NAFLD progression. Mice deficient in carboxylesterase (Ces)1d expression are protected from high-fat diet-induced hepatic steatosis. To investigate whether loss of Ces1d can also mitigate steatosis induced by over-activated DNL, WT and Ces1d-deficient mice were fed a lipogenic high-sucrose diet (HSD). We found that Ces1d-deficient mice were protected from HSD-induced hepatic lipid accumulation. Mechanistically, Ces1d deficiency leads to activation of AMP-activated protein kinase and inhibitory phosphorylation of acetyl-CoA carboxylase. Together with our previous demonstration that Ces1d deficiency attenuated high-fat diet-induced steatosis, this study suggests that inhibition of CES1 (the human ortholog of Ces1d) might represent a novel pharmacological target for prevention and treatment of NAFLD.

Dopamine D3R antagonist VK4-116 attenuates oxycodone self-administration and reinstatement without compromising its antinociceptive effects.

Prescription opioids such as oxycodone are highly effective analgesics for clinical pain management, but their misuse and abuse have led to the current opioid epidemic in the United States. In order to ameliorate this public health crisis, the development of effective pharmacotherapies for the prevention and treatment of opioid abuse and addiction is essential and urgently required. In this study, we evaluated-in laboratory rats-the potential utility of VK4-116, a novel and highly selective dopamine D3 receptor (D3R) antagonist, for the prevention and treatment of prescription opioid use disorders. Pretreatment with VK4-116 (5-25 mg/kg, i.p.) dose-dependently inhibited the acquisition and maintenance of oxycodone self-administration. VK4-116 also lowered the break-point (BP) for oxycodone self-administration under a progressive-ratio schedule of reinforcement, shifted the oxycodone dose-response curve downward, and inhibited oxycodone extinction responding and reinstatement of oxycodone-seeking behavior. In addition, VK4-116 pretreatment dose-dependently enhanced the antinociceptive effects of oxycodone and reduced naloxone-precipitated conditioned place aversion in rats chronically treated with oxycodone. In contrast, VK4-116 had little effect on oral sucrose self-administration. Taken together, these findings indicate a central role for D3Rs in opioid reward and support further development of VK4-116 as an effective agent for mitigating the development of opioid addiction, reducing the severity of withdrawal and preventing relapse.

Detection and modulation of capsaicin perception in the human oral cavity.

Capsaicin causes a burning or spicy sensation when this vanilloid compound comes in contact with trigeminal neurons of the tongue. This compound has low solubility in water, which presents difficulties in examining the psychophysical properties of capsaicin by standard aqueous chemosensory tests. This report describes a new approach that utilizes edible strips for delivering precise amounts of capsaicin to the human oral cavity for examining threshold and suprathreshold amounts of this irritant. When incorporated into pullulan-based edible strips, recognition thresholds for capsaicin occurred over a narrow range, with a mean value near 1 nmol. When incorporated into edible strips at suprathreshold amounts, capsaicin yielded robust intensity values that were readily measured in our subject population. Maximal capsaicin intensity was observed 20 s after strips dissolved on the tongue surface, and then decreased in intensity. Suprathreshold studies showed that complete blockage of nasal airflow diminished capsaicin perception in the oral cavity. Oral rinses with vanillin-linoleic acid emulsions decreased mean intensity values for capsaicin by approximately 75%, but only modestly affected recognition threshold values. Also, oral rinses with isointense amounts of aqueous sucrose and sucralose solutions decreased mean intensity values for capsaicin by approximately 50%. In addition, this decrease in capsaicin intensity following an oral rinse with sucrose was partially reversed by the sweet taste inhibitor lactisole. These results suggest that blockage of nasal airflow, vanillin, sucrose, and sucralose modulate capsaicin perception in the human oral cavity. The results further suggest a chemosensory link between receptor cells that detect sweet taste stimuli and trigeminal neurons that detect capsaicin.

Muscarinic M5 receptors modulate ethanol seeking in rats.

Despite the cost to both individual and society, alcohol use disorders (AUDs) remain a major health risk within society, and both relapse and heavy drinking are still poorly controlled with current medications. Here we demonstrate for the first time that a centrally active and selective negative allosteric modulator for the rat M5 muscarinic acetylcholine receptor (mAChR), ML375, decreases ethanol self-administration and attenuates cue-induced reinstatement of ethanol seeking in ethanol-preferring (iP) rats. Importantly, ML375 did not affect sucrose self-administration or general locomotor activity indicative of a selective effect on ethanol seeking. Based on the expression profile of M5 mAChRs in the brain and the distinct roles different aspects of the dorsal striatum have on long-term and short-term ethanol use, we studied whether intra-striatal microinjection of ML375 modulated ethanol intake in rats. We show in iP rats with an extensive history of ethanol intake that intra-dorsolateral (DL), but not intra-dorsomedial, striatal injections of ML375 reduced ethanol self-administration to a similar extent as the nicotinic acetylcholine receptor ligand varenicline, which has preclinical and clinical efficacy in reducing the reinforcing effects of ethanol. These data implicate the DL striatum as a locus for the effects of cholinergic-acting drugs on ethanol seeking in rats with a history of long-term ethanol use. Accordingly, we demonstrate in rats that selectively targeting the M5 mAChR can modulate both voluntary ethanol intake and cue-induced ethanol seeking and thereby provide direct evidence that the M5 mAChR is a potential novel target for pharmacotherapies aimed at treating AUDs.

Use of D-glucose-fenpiclonil conjugate as a potent and specific inhibitor of sucrose carriers.

Until now, specific inhibitors of sucrose carriers were not available. This led us to study the properties of the recently synthesized D-glucose-fenpiclonil conjugate (D-GFC). This large amphiphilic glucoside exhibited an extremely low phloem systemicity in contrast to L-amino acid-fenpiclonil conjugates. Using Ricinus seedlings, the effect of D-GFC on 0.5 mM [14C]sucrose (Suc), 3-O-[3H]methylglucose, and [3H]glutamine uptake by cotyledon tissues was compared with that of p-chloromercuribenzenesulfonic acid (PCMBS). D-GFC dramatically inhibited H+-Suc symport at the same concentrations as PCMBS (0.5 and 1 mM), but in contrast to the thiol reagent, it did not affect 3-O-methylglucose and glutamine transport, nor the acidification of the incubation medium by cotyledon tissues. Similarly, 0.5 mM D-GFC inhibited active Suc uptake by Vicia faba leaf tissues and by Saccharomyces cerevisiae cells transformed with AtSUC2, a gene involved in Suc phloem loading in Arabidopsis, by approximately 80%. The data indicated that D-GFC was a potent inhibitor of Suc uptake from the endosperm and of Suc phloem loading. It is the first chemical known to exhibit such specificity, at least in Ricinus, and this property permitted the quantification of the two routes involved in phloem loading of endogenous sugars after endosperm removal.

Modulation of sweet taste by umami compounds via sweet taste receptor subunit hT1R2.

Although the five basic taste qualities-sweet, sour, bitter, salty and umami-can be recognized by the respective gustatory system, interactions between these taste qualities are often experienced when food is consumed. Specifically, the umami taste has been investigated in terms of whether it enhances or reduces the other taste modalities. These studies, however, are based on individual perception and not on a molecular level. In this study we investigated umami-sweet taste interactions using umami compounds including monosodium glutamate (MSG), 5'-mononucleotides and glutamyl-dipeptides, glutamate-glutamate (Glu-Glu) and glutamate-aspartic acid (Glu-Asp), in human sweet taste receptor hT1R2/hT1R3-expressing cells. The sensitivity of sucrose to hT1R2/hT1R3 was significantly attenuated by MSG and umami active peptides but not by umami active nucleotides. Inhibition of sweet receptor activation by MSG and glutamyl peptides is obvious when sweet receptors are activated by sweeteners that target the extracellular domain (ECD) of T1R2, such as sucrose and acesulfame K, but not by cyclamate, which interact with the T1R3 transmembrane domain (TMD). Application of umami compounds with lactisole, inhibitory drugs that target T1R3, exerted a more severe inhibitory effect. The inhibition was also observed with F778A sweet receptor mutant, which have the defect in function of T1R3 TMD. These results suggest that umami peptides affect sweet taste receptors and this interaction prevents sweet receptor agonists from binding to the T1R2 ECD in an allosteric manner, not to the T1R3. This is the first report to define the interaction between umami and sweet taste receptors.

Binge-like consumption of ethanol and other salient reinforcers is blocked by orexin-1 receptor inhibition and leads to a reduction of hypothalamic orexin immunoreactivity.

BACKGROUND: Orexin (OX) neurons originating in the lateral hypothalamus (LH) are ideally positioned to modulate reward processing as they form connections with several key brain regions known to be involved in the reward pathway. Consistent with these findings, a growing number of studies have implicated the OX system in modulating the rewarding properties of several drugs of abuse, including ethanol (EtOH). However, the role of the OX system in excessive binge-like EtOH intake remains relatively unexplored. Here, we assessed changes in OX immunoreactivity (IR) in the hypothalamus following repeated cycles of binge-like EtOH drinking and assessed the participation of the OX-1 receptor (OX1R) in binge-like EtOH consumption. METHODS: The drinking-in-the-dark (DID) paradigm was used to model binge-like EtOH drinking in male C57BL/6J mice. In the first experiment, mice experienced 1 or 3 cycles of binge-like EtOH or sucrose drinking with DID procedures to assess changes in OX IR in distinct subregions of the hypothalamus. Subsequent experiments examined binge-like EtOH and saccharin drinking following peripheral injections of 0.0, 5.0, or 10.0 mg/kg SB-334867 (SB), a selective OX1R antagonist. Finally, mice were given peripheral injections of SB and open-field locomotor activity was measured. RESULTS: Relative to water drinking controls, binge-like consumption of EtOH and sucrose resulted in a marked reduction in OX IR in the LH. Inhibition of the OX1R via SB blunted EtOH and saccharin drinking, but did not alter open-field locomotor activity. CONCLUSIONS: Our observed reduction in OX IR in the LH indicates that the OX system in engaged during binge-like consumption of EtOH and sucrose. The observation that inhibition of the OX1R signaling blunted binge-like EtOH, and saccharin drinking suggests that reward-related OX circuits originating in the LH participate in the consumption of salient reinforcers regardless of calories.

Immune tolerance induction in haemophilia A patients with inhibitors by treatment with recombinant factor VIII: a retrospective non-interventional study.

Immune tolerance induction (ITI) can overcome inhibitory factor VIII (FVIII) antibodies in haemophilia A patients receiving FVIII replacement therapy. The objective was to evaluate the use of sucrose-formulated, full-length recombinant FVIII (rFVIII-FS) for ITI therapy. Patients (<8 years at ITI start) with severe haemophilia A and a peak inhibitor titre >5 Bethesda units (BU) who underwent ITI with any rFVIII-FS dose for ≥ 9 months (or until success) were eligible for this retrospective study. Efficacy analyses included descriptions of ITI treatment regimens and outcomes; ITI success was determined solely at the discretion of the investigator. Safety analyses included assessment of adverse events. Of 51 enrolled patients, 32 [high dose (≥ 85 IU kg(-1) day(-1)), n = 21; low dose, n = 11] were eligible for analysis. ITI was successful in 69% (22/32) of patients (high dose, 66.7%; low dose, 72.7%) after a median of 1.4 years (range, 0.1-3.6 years). Influencing factors for ITI success were start of ITI <1 year after inhibitor detection and an inhibitor titre <10 BU at ITI start. All patients successfully tolerized with ITI continued to receive rFVIII-FS prophylaxis as maintenance therapy, with no inhibitor recurrence from the end of ITI until study enrolment. Use of rFVIII-FS for ITI was effective and well tolerated; success rates were similar to those in published studies.

Four alleles of AtCESA3 form an allelic series with respect to root phenotype in Arabidopsis thaliana.

Plant cell shape is determined by the orientation of cellulose microfibrils in the primary cell wall. Consequently, mutations that affect genes encoding the enzymes responsible for the synthesis of cellulose, namely, the cellulose synthase catalytic subunits, can alter cell shape substantially, particularly in the roots of affected plants. The multiple response expansion1 (mre1) mutant of Arabidopsis thaliana results from a point mutation in the AtCESA3 gene, which encodes one of the three isoforms of the cellulose synthase catalytic subunit required for synthesis of cellulose in the primary cell wall. Phenotypic comparison of the mre1 mutant with three other alleles (ectopic lignification1-1, ectopic lignification1-2 and constitutive expression of vsp1) showed that these four alleles form an allelic series with respect to their root phenotypes, with mre1 being the weakest allele identified to date. These analyses demonstrated that sucrose affects a significant alteration of cell shape in the roots of these mutants and likely suppresses root cell division in them as well, and that the chemical aminoisobutyric acid can suppress these effects of sucrose. Interestingly, the cell walls in the roots of these four AtCESA3 alleles contain different percentages of cellulose, and these percentages correlate with the lengths of the roots and cortex cells in these roots when grown on media containing high levels of sucrose.

Evaluation of the 'liking' and 'wanting' properties of umami compound in rats.

Food reward is neurologically and psychologically divided into at least two properties; 'liking' and 'wanting'. Although umami taste enhances food palatability, the liking and wanting properties of umami taste, and the underlying neural mechanisms for these properties are not clear. Here, we compared sucrose (0, 10, 30, 120 and 480 mM) and monosodium l-glutamate (MSG; 0, 10, 30, 60 and 120 mM) solutions using a taste reactivity test to evaluate liking, and fixed/progressive-ratio operant licking tasks to evaluate wanting. To determine the underlying neural mechanisms, we also conducted systemic blockade of opioid receptors in both tests. In the taste reactivity test, the hedonic reactions to 30, 60 and 120 mM MSG were greater than those to water (0mM) but lower than those to 480 mM sucrose. In the operant task, the intake, number of licks, and breakpoint to MSG reached peaks at around 60mM but they were lower than those to 30-480 mM sucrose. The systemic naloxone treatment decreased the hedonic responses to MSG and sucrose, and reduced the incentive salience of MSG but not sucrose. These findings indicate that the hedonic response and incentive salience of MSG is lower than those of sucrose when compared at the maximum response and that the incentive salience of MSG is lower than sucrose even where the hedonic response is similar. The present study also suggest that the hedonic response and incentive salience of umami compound is modulated by brain opioid signaling.

Nonselective suppression of operant ethanol and sucrose self-administration by the mGluR7 positive allosteric modulator AMN082.

Emerging evidence indicates that specific metabotropic glutamate receptors (mGluRs) modulate ethanol self-administration. In general, inhibition of glutamate transmission through blockade of postsynaptic mGluRs, or activation of presynaptic mGluRs, inhibits ethanol self-administration. The goal of this preclinical study was to further characterize mGluR regulation of ethanol self-administration by examining effects of AMN082, an allosteric positive modulator of presynaptic mGluR7 activity. Separate groups of C57BL/6J male mice were trained to self-administer ethanol or sucrose on a fixed-ratio 4 schedule of reinforcement during 1 h sessions. On test days, mice were pretreated with AMN082 (0, 1.0, 3.0, 5.6, or 10 mg/kg) 30 min prior to self-administration sessions. Functional specificity and activity was examined by testing the effects of AMN082 (0-10 mg/kg) on open-field locomotor activity and HPA axis function as measured by plasma corticosterone levels. AMN082 (10 mg/kg) produced a significant reduction in ethanol and sucrose reinforced responding, and inhibited locomotor activity. Plasma corticosterone levels were significantly increased following AMN082 (5.6 and 10 mg/kg) suggesting a dose-dependent dissociation between the behavioral and hormonal effects of the compound. These data suggest that activation of mGluR7 by AMNO82 produces nonspecific reductions in motivated behavior that are associated with negative effects on motor activity.

Sugarcane ShSUT1: analysis of sucrose transport activity and inhibition by sucralose.

Plant sucrose transporters (SUTs) are members of the glycoside-pentoside-hexuronide (GPH) cation symporter family (TC2.A.2) that is part of the major facilitator superfamily (MFS). All plant SUTs characterized to date function as proton-coupled symporters and catalyze the cellular uptake of sucrose. SUTs are involved in loading sucrose into the phloem and sink tissues, such as seeds, roots and flowers. Because monocots are agriculturally important, SUTs from cereals have been the focus of recent research. Here we present a functional analysis of the SUT ShSUT1 from sugarcane, an important crop species grown for its ability to accumulate high amounts of sucrose in the stem. ShSUT1 was previously shown to be expressed in maturing stems and plays an important role in the accumulation of sucrose in this tissue. Using two-electrode voltage clamping in Xenopus oocytes expressing ShSUT1, we found that ShSUT1 is highly selective for sucrose, but has a relatively low affinity for sucrose (K(0.5) = 8.26 mM at pH 5.6 and a membrane potential of -137 mV). We also found that the sucrose analog sucralose (4,1',6'-trichloro-4,1',6'-trideoxy-galacto-sucrose) is a competitive inhibitor of ShSUT1 with an inhibition coefficient (K(i)) of 16.5 mM. The presented data contribute to our understanding of sucrose transport in plants in general and in monocots in particular.

The liaison of sweet and savory.

The sense of taste provides humans with necessary information about the composition and quality of food. For humans, five basic tastes are readily distinguishable and include sweet, bitter, salty, sour, and savory (or umami). Although each of these qualities has individualized transduction pathways, sweet and umami tastes are believed to share a common receptor element, the T1R3 receptor subunit. The two G-protein-coupled heteromer receptors that comprise an umami stimulus receptor (T1R1-T1R3) and a sweetener receptor (T1R2-T1R3) constitute a potential link between these two qualities of perception. While the role of the individual monomers in each human heteromer has been examined in vitro, very little is known of the implication of this research for human perception, or specifically, how sweet and savory taste perceptions may be connected. Using a psychophysical approach, we demonstrate that lactisole, a potent sweetness inhibitor that binds in vitro to hT1R3, also inhibits a significant portion of the perception of umami taste from monosodium glutamate. Following the molecular logic put forward by Xu et al. (2004, Proc. Natl Acad. Sci. USA, 101, 14258-14263), our psychophysical data support the in vitro hypothesis that the shared T1R3 monomer moderates the activation of both T1R2 and T1R1 in humans and impairs suprathreshold perception, respectively, of sweetness and, to a lesser degree, umaminess in the presence of lactisole.

Activation of the immune system in rats with lipopolysaccharide reduces voluntary sucrose intake but not intraoral intake.

Traditional intake measures of voluntary consumption of food or fluid from a specific location involve both appetitive and consummatory behaviors. Appetitive behaviors are food finding behaviors displayed by an animal prior to the consumption of the food, whereas consummatory behaviors are the behaviors involved in the actual consumption of the food. Intraoral intake of a fluid can be measured by directly infusing it into the oral cavity of an animal and quantifying the consummatory behaviors. The present study compared the effects of immune activation (lipopolysaccharide, LPS) and toxin (lithium chloride, LiCl)-induced changes on both a traditional intake measure (bottle drinking) and an intraoral intake measure. In Experiment 1, rats were injected intraperitoneally with LPS (200 microg/kg), LiCl (0.15 M, 20 ml/kg) or NaCl vehicle, and voluntary sucrose (0.3 M) intake was monitored for 1 h from a graduated drinking tube. Voluntary intake was again assessed on a second test day, 72 h later under the same conditions. In Experiment 2, a continuous intraoral infusion of sucrose (0.3 M) was given via intraoral cannulae following systemic injections of LPS, LiCl or NaCl vehicle on two different test days, 72 h apart. Rats injected with LiCl displayed reduced sucrose intake on both the voluntary intake measure and the intraoral intake measure relative to controls (P's<.05). The reduced intake observed was of greater magnitude on the second test day of both experiments, consistent with conditioning effects. In contrast, LPS reduced sucrose intake only when assessed with the traditional intake measure. Intraoral sucrose intake remained unchanged relative to controls. The present results provide further evidence that activation of the immune system has adverse effects on the appetitive phase of ingestion, whereas the consummatory aspects are unaffected.

Naloxone nonselective suppression of drinking of ethanol, sucrose, saccharin, and water by rats.

Naloxone, a nonselective opioid antagonist, has been demonstrated to reduce oral self-administration of ethanol (EtOH) in rats. Conflicting conclusions have been drawn about the effects of naloxone on consumption of non-EtOH control liquids. A preliminary meta-analysis found large and homogeneous effects of naloxone on EtOH consumption and heterogeneous effects on the consumption of control liquids. Although many of the authors concluded that their control liquid results were "not significant," when they were combined using meta-analytic techniques, it was apparent that there were some strong, but widely divergent, effect sizes. In the first experiment in the current study, 60 male Sprague-Dawley rats were trained to drink 10% EtOH in tap water over 3 weeks of limited-access sessions. Then, their limited-access consumption was measured in single-bottle tests of four liquids (water, 10% EtOH in water, an isocaloric sucrose solution, and an "equally sweet" saccharin solution) 15 min following an intraperitoneal injection of either saline or 1.0 mg/kg naloxone. Every animal was tested 36 times in a counterbalanced order: three times for each liquid following an injection of naloxone and six times for each liquid following an injection of saline. There were distinct differences in the quantity of each liquid consumed in the saline trials. However, the suppression percentages for each liquid in the naloxone trials were identical ( approximately 50%). There were significant correlations, in the range of.23-.42, between the mean amount of each liquid consumed during saline trials for each animal and the suppression percentage during naloxone trials for the same animal and liquid. When the animals were divided into high, low, and medium drinkers for each liquid, the low drinkers demonstrated a much lower suppression after naloxone treatment than did the other two groups. The data confirm that blockade of opioid receptors suppresses consumption of both EtOH and non-EtOH liquids to a degree that is related to the amount of voluntary, untreated consumption of the liquids.

A comparison of the effects of 6-beta naltrexol and naltrexone on the consumption of ethanol or sucrose using a limited-access procedure in rats.

We recently reported that 6-beta naltrexol, the major metabolite of naltrexone in humans, reduced ethanol consumption in rats. Two new experiments were designed to compare 6-beta naltrexol and naltrexone across three dose levels on an ethanol or sucrose baseline using a limited-access procedure in Wistar rats. The results of Experiment 1 showed that both 6-beta naltrexol and naltrexone reduced ethanol consumption across a range of doses. An in vivo assay showed that naltrexone was approximately 25 times more potent than 6-beta naltrexol at comparable ED50 doses. In addition, there was no indication of systematic development of tolerance to the effect of either drug across the 4 days of drug administration. In Experiment 2, both 6-beta naltrexol and naltrexone reduced the consumption of a sucrose solution using a limited-access procedure. The implications of these data for the development of pharmacotherapeutic agents capable of reducing drinking in recovering alcoholics are discussed.

A florigenic effect of sucrose in Fuchsia hybrida is blocked by gibberellin-induced assimilate competition.

The use of gas chromatography-mass spectrometry-selected ion monitoring along with a (13)C internal standard has allowed sensitive measurements of the sucrose (Suc) content of individual shoot apices of Fuchsia hybrida. With intact plants, as the photosynthetic irradiance increased, so did shoot apex Suc content, reaching saturation at about 500 micromol m(-2) s(-1). These same plants flowered at the higher irradiances, remaining vegetative in 10-h short days at an irradiance of 230 micromol m(-2) s(-1). The strong correlation (r = 0.93) in these studies between flowering and shoot apex Suc content indicates a role for Suc as a stimulus to flowering in this species. However, Suc is not the long-day (LD) "florigen" of F. hybrida because 2 to 4 LD given as a 14-h low-irradiance photoperiod extension (10-15 micromol m(-2) s(-1)) induced flowering but without increase in shoot apex Suc content. Flowering induced by either pathway, the LD- or the Suc-mediated one, was inhibited by applying gibberellin (GA) to the shoot tip. Such inhibition of flowering by GA, at least for the LD pathway, was associated with a reduced apex Suc content, enhanced elongation of subapical stem tissue, and a reduced import into the shoot apex of leaf-sourced assimilate. Thus, our findings show how GA inhibits flowering of F. hybrida and confirm the importance of nutrient diversion in regulating flowering.

The sucrose permease of Escherichia coli: functional significance of cysteine residues and properties of a cysteine-less transporter.

The sucrose (CscB) permease belongs to the oligosaccharide:H(+) symporter family of the Major Facilitator Superfamily and is homologous to the lactose permease from Escherichia coli. Sucrose transport in cells expressing sucrose permease is completely inhibited by N-ethylmaleimide (NEM), suggesting that one or more of the seven native Cys residues may be important for transport. In this paper, each Cys residue was individually replaced with Ser, and transport activity, membrane expression, and NEM sensitivity are documented. All seven single Cys-->Ser mutants are expressed normally in the membrane and catalyze sucrose transport with activities ranging from 80% to 180% of wild type. Six of the seven Ser mutants are completely inactivated by NEM, while Cys122-->Ser permease is insensitive to the sulfhydryl reagent, indicating that NEM inhibition results from alkylation of Cys122. Subsequently, a sucrose permease devoid of Cys residues (Cys-less) was constructed in which all Cys residues were replaced with Ser simultaneously by using a series of overlap-extension PCRs. Membrane expression and kinetic parameters for Cys-less [K(m) 4.8 mM, V(max) 192 nmol min(-1) (mg of protein)(-1)] are essentially identical to those of wild type [K(m) 5.4 mM, V(max) 196 nmol min(-1) (mg of protein)(-1)]. However, Cys-less permease catalyzes sucrose accumulation to steady-state levels that are approximately 2-fold higher than those of wild type. As anticipated, Cys-less permease is completely resistant to NEM inhibition. The observations demonstrate that Cys residues play no functional role in sucrose permease. Furthermore, the approach described to create the Cys-less transporter is generally applicable to other proteins. An application of Cys-less permease in the study of the substrate binding site is presented in the accompanying paper.