The Arabidopsis thaliana transparent testa10 (tt10) mutant exhibits a delay in developmentally determined browning of the seed coat, also called the testa. Seed coat browning is caused by the oxidation of flavonoids, particularly proanthocyanidins, which are polymers of flavan-3-ol subunits such as epicatechin and catechin. The tt10 mutant seeds accumulate more epicatechin monomers and more soluble proanthocyanidins than wild-type seeds. Moreover, intact testa cells of tt10 cannot trigger H2O2-independent browning in the presence of epicatechin and catechin, in contrast with wild-type cells. UV-visible light detection and mass spectrometry revealed that the major oxidation products obtained with epicatechin alone are yellow dimers called dehydrodiepicatechin A. These products differ from proanthocyanidins in the nature and position of their interflavan linkages. Flavonol composition was also affected in tt10 seeds, which exhibited a higher ratio of quercetin rhamnoside monomers versus dimers than wild-type seeds. We identified the TT10 gene by a candidate gene approach. TT10 encodes a protein with strong similarity to laccase-like polyphenol oxidases. It is expressed essentially in developing testa, where it colocalizes with the flavonoid end products proanthocyanidins and flavonols. Together, these data establish that TT10 is involved in the oxidative polymerization of flavonoids and functions as a laccase-type flavonoid oxidase.

BACKGROUND

Dietary flavonoids have beneficial effects on blood pressure in intervention settings, but there is limited information on habitual intake and risk of hypertension in population-based studies.

OBJECTIVE

We examined the association between habitual flavonoid intake and incident hypertension in a prospective study in men and women.

METHODS

A total of 87,242 women from the Nurses' Health Study (NHS) II, 46,672 women from the NHS I, and 23,043 men from the Health Professionals Follow-Up Study (HPFS) participated in the study. Total flavonoid and subclass intakes were calculated from semiquantitative food-frequency questionnaires collected every 4 y by using an updated and extended US Department of Agriculture database.

RESULTS

During 14 y of follow-up, 29,018 cases of hypertension in women and 5629 cases of hypertension in men were reported. In pooled multivariate-adjusted analyses, participants in the highest quintile of anthocyanin intake (predominantly from blueberries and strawberries) had an 8% reduction in risk of hypertension [relative risk (RR): 0.92; 95% CI: 0.86, 0.98; P < 0.03] compared with that for participants in the lowest quintile of anthocyanin intake; the risk reduction was 12% (RR: 0.88; 95% CI: 0.84, 0.93; P < 0.001) in participants ≤60 y of age and 0.96 (0.91, 1.02) in participants >60 y of age (P for age interaction = 0.02). Although intakes of other subclasses were not associated with hypertension, pooled analyses for individual compounds suggested a 5% (95% CI: 0.91, 0.99; P = 0.005) reduction in risk for the highest compared with the lowest quintiles of intake of the flavone apigenin. In participants ≤60 y of age, a 6% (95% CI: 0.88, 0.97; P = 0.002) reduction in risk was observed for the flavan-3-ol catechin when the highest and the lowest quintiles were compared.

CONCLUSIONS

Anthocyanins and some flavone and flavan-3-ol compounds may contribute to the prevention of hypertension. These vasodilatory properties may result from specific structural similarities (including the B-ring hydroxylation and methyoxylation pattern).

In order to investigate external factors that may influence the magnitude of placebo analgesia as well as psychological factors that mediate placebo analgesia, 13 irritable bowl syndrome (IBS) patients rated evoked rectal distension and cutaneous heat pain under the conditions of natural history (NH), rectal placebo (RP), rectal nocebo (RN), rectal lidocaine (RL) and oral lidocaine (OL). Patients were given verbal suggestions for pain relief and rated expected pain levels and desire for pain relief for both evoked visceral and cutaneous pain, respectively. Large reductions in pain intensity and pain unpleasantness ratings were found in the RP, RL and OL condition as compared to the natural history condition, whereas no significant difference in pain reduction between the three treatment conditions was found. Ratings during RN and NH were not statistically different. Compared to a previous study, which shows that rectal lidocaine reverses visceral and cutaneous hyperalgesia, these results suggest that adding a verbal suggestion for pain relief can increase the magnitude of placebo analgesia to that of an active agent. Since IBS patients rate these stimuli as much higher than do normal control subjects and since placebo effects were very large, they probably reflect anti-hyperalgesic mechanisms to a major extent. Expected pain levels and desire for pain relief accounted for large amounts of the variance in visceral pain intensity in the RP, RL, and OL condition (up to 81%), and for lower amounts of the variance in cutaneous pain intensity. Hence, the combination of expected pain levels and desire for pain relief may offer an alternative means of assessing the contribution of placebo factors during analgesia.

The overriding of satiety and homeostatic control mechanisms by cognitive, rewarding, and emotional aspects of palatable foods may contribute to the evolving obesity crisis, but little is known about neural pathways and mechanisms responsible for crosstalk between the "cognitive" and "metabolic" brain in the control of appetite. Here we show that neural connections between the nucleus accumbens and hypothalamus might be part of this link. Using the well known model of selective stimulation of high-fat intake induced by intra-accumbens injection of the mu-opioid receptor agonist D-Ala2-N-Me-Phe4-gly5-ol-enkephalin (DAMGO), we demonstrate that orexin signaling in the ventral tegmental area is important for this reward-driven appetite to override metabolic repletion signals in presatiated rats. We further show that accumbens DAMGO in the absence of food selectively increases the proportion of orexin neurons expressing c-Fos in parts of the perifornical hypothalamus and that neural projections originating in DAMGO-responsive sites of the nucleus accumbens make close anatomical contacts with hypothalamic orexin neurons. These findings suggest that direct accumbens-hypothalamic projections can stimulate hypothalamic orexin neurons, which in turn through orexin-1 receptor signaling in the ventral tegmental area and possibly other sites interfaces with the motivational and motor systems to increase intake of palatable food.

Learning and memory processes may be influenced by fluctuations in steroid hormones, such as estrogens and progestins. In this study, we have used an animal model to investigate the effects of endogenous fluctuations in ovarian steroids in intact female rats and effects of administration of ovarian steroids to ovariectomized rats for non-spatial, working memory using the object recognition task. Performance in this task relies on cortical and hippocampal function. As such, serum, cortical, and hippocampal concentrations of estradiol (E2), progesterone (P4), and P4's metabolite, 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP), were measured by radioimmunoassay. Experiment 1: Rats in behavioral estrus, compared to those in diestrus or estrus, spent a greater percentage of time exploring a novel object concomitant with increases in serum E2, P4, and 3alpha,5alpha-THP levels. Regression analyses revealed that there was a significant positive relationship between E2 levels in the hippocampus and 3alpha,5alpha-THP levels in the hippocampus and cortex and performance in this task. Experiment 2: Administration of E2 and/or P4 immediately post-training increased the percentage of time spent exploring the novel object and produced levels of E2, P4, and 3alpha,5alpha-THP akin to that of rats in behavioral estrus. Experiment 3: Post-training administration of selective estrogen receptor modulators, including 17beta-E2, propyl pyrazole triol, and diarylpropionitrile increased the percentage of time spent exploring the novel object compared to vehicle-administration. Experiment 4: Post-training P4 or 3alpha,5alpha-THP administration, compared to vehicle, increased the percentage of time spent exploring the novel object and produced P4 and/or 3alpha,5alpha-THP levels within the physiological range typically observed for rats in behavioral estrus. Experiment 5: If post-training administration of E2 and/or P4 was delayed one hour, no enhancement in object recognition was observed. Together, these results suggest that E2 and progestins can have mnemonic effects through actions in the cortex and/or hippocampus.

1. The rat hippocampal slice preparation has been used in conjunction with intracellular recording and ionophoresis to study the action of gamma-aminobutyric acid (GABA) on CA1 pyramidal cells.2. GABA elicits a hyperpolarizing (h.) response at the soma. The reversal potential of this h. response is the same as for inhibitory post-synaptic potentials (i.p.s.p.s) evoked by stimulating pyramidal cell axons.3. GABA elicits primarily depolarizing (d.) responses when applied to the apical dendrites, but h. responses can also be found.4. The GABA antagonists bicuculline methiodide, picrotoxin, penicillin, and pentylenetetrazole are all ten to one hundred times more potent on the d. response than on the h. response. Hyperpolarizing responses are uncovered in the dendrites when intermediate doses of these drugs block the d. response.5. The GABA analogue, 4,5,6,7-tetrahydroisoxazolo [5,4-c]pyridine-3-ol (THIP), which has been proposed to activate synaptic receptors preferentially in other systems, elicits h. responses in the dendrites. It is one seventh as potent as GABA in eliciting d. responses.6. Pentobarbitone enhances d. responses to a much greater extent than h. responses, while diazepam enhances h. responses to a greater extent.7. Nipecotic acid, low temperature, and low sodium media all increase the size of d. responses to ionophoretically applied GABA indicating that an active uptake process limits their size.8. We conclude that h. responses reflect the activation of synaptic receptors which are highly concentrated on the pyramidal cell soma-initial segment, but are also present on the dendrites. Depolarizing responses, which are evoked in the dendrites, reflect the activation of extrasynaptic receptors.9. We propose that an ordinarily undetectable amount of synaptically released GABA can ;spill' over onto extrasynaptic (d.) receptors. Depolarizing receptor activation can be detected in the presence of pentobarbitone. Spillover is markedly enhanced at subphysiological temperatures presumably due to enhanced release of GABA and impairment of the GABA uptake system.

The utility of morphine for the treatment of chronic pain is hindered by the development of tolerance to the analgesic effects of the drug. Morphine is unique among opiates in its ability to activate the mu opioid receptor (MOR) without promoting its desensitization and endocytosis. Here we demonstrate that [D-Ala(2)-MePhe(4)-Gly(5)-ol] enkephalin (DAMGO) can facilitate the ability of morphine to stimulate MOR endocytosis. As a consequence, rats treated chronically with both drugs show reduced analgesic tolerance compared to rats treated with morphine alone. These results demonstrate that endocytosis of the MOR can reduce the development of tolerance, and hence suggest an approach for the development of opiate analogs with enhanced efficacy for the treatment of chronic pain.

The essential oil of Melaleuca alternifolia (tea tree) has broad-spectrum antimicrobial activity. The mechanisms of action of tea tree oil and three of its components, 1,8-cineole, terpinen-4-ol, and alpha-terpineol, against Staphylococcus aureus ATCC 9144 were investigated. Treatment with these agents at their MICs and two times their MICs, particularly treatment with terpinen-4-ol and alpha-terpineol, reduced the viability of S. aureus. None of the agents caused lysis, as determined by measurement of the optical density at 620 nm, although cells became disproportionately sensitive to subsequent autolysis. Loss of 260-nm-absorbing material occurred after treatment with concentrations equivalent to the MIC, particularly after treatment with 1,8-cineole and alpha-terpineol. S. aureus organisms treated with tea tree oil or its components at the MIC or two times the MIC showed a significant loss of tolerance to NaCl. When the agents were tested at one-half the MIC, only 1,8-cineole significantly reduced the tolerance of S. aureus to NaCl. Electron microscopy of terpinen-4-ol-treated cells showed the formation of mesosomes and the loss of cytoplasmic contents. The predisposition to lysis, the loss of 260-nm-absorbing material, the loss of tolerance to NaCl, and the altered morphology seen by electron microscopy all suggest that tea tree oil and its components compromise the cytoplasmic membrane.

Mitochondrial oxidative damage contributes significantly to a range of human disorders, including neurodegenerative diseases, ischaemia-reperfusion injury and ageing-associated dysfunction. To prevent this damage we have delivered a molecule containing the active antioxidant moiety of vitamin E to mitochondria. This was carried out by covalently coupling the antioxidant moiety to a lipophilic triphenylphosphonium cation. This mitochondrially targeted antioxidant, 2-[2-(triphenylphosphonio)ethyl]-3,4-dihydro-2, 5,7,8-tetramethyl-2H-1-benzopyran-6-ol bromide (TPPB), accumulated several-hundred fold within the mitochondrial matrix, driven by the organelle's large membrane potential. When cells were incubated with micromolar concentrations of TPPB, they accumulated millimolar concentrations within their mitochondria. The amount of TPPB taken up by mitochondria was approximately 80-fold greater than endogenous levels of vitamin E. Consequently the targeted derivative of vitamin E protected mitochondrial function from oxidative damage far more effectively than vitamin E itself. The mitochondrially targeted antioxidant TPPB has potential as an antioxidant therapy for disorders involving mitochondrial oxidative damage. It also suggests a new family of mitochondrially targeted antioxidants, redox-active and pharmacologically active molecules designed to prevent damage or manipulate mitochondrial function.

BACKGROUND

Acquired immunodeficiency syndrome-related opportunistic illnesses (Ols) continue to occur after initiation of potent antiretroviral therapy in patients with human immunodeficiency virus (HIV) infection. Risk factors for clinical progression to Ols during potent therapy are not well defined.

OBJECTIVE

To examine the incidence of and risk factors for Ols among patients treated with potent antiretroviral therapy in a population-based study.

METHODS

The Swiss HIV Cohort Study, a prospective cohort study of adult HIV-infected persons.

METHODS

Seven study centers throughout Switzerland.

METHODS

A total of 2410 cohort study participants with a potential follow-up of at least 15 months after starting potent therapy between September 1995 and December 1997.

METHODS

Disease-specific incidence of Ols during the 6 months preceding potent antiretroviral therapy and at 3 intervals after initiating therapy; risk factors for development of Ols during therapy.

RESULTS

Of the 2410 participants, 143 developed 186 Ols after initiation of potent antiretroviral therapy. Incidence of any OI decreased from 15.1 per 100 person-years in the 6 months before therapy to 7.7 in the first 3 months after starting treatment, 2.6 in the following 6 months, and 2.2 per 100 person-years between 9 and 15 months. Reductions in incidence ranged from 38% per month for Kaposi sarcoma (P<.001) to 5% per month for non-Hodgkin lymphoma (P = .31). Baseline CD4 cell count continued to predict the risk of disease progression after initiating potent therapy. Compared with CD4 cell counts above 200 x 10(6)/L, the hazard ratio for developing Ols was 2.5 (95% confidence interval [CI], 1.4-4.5) for counts between 51 and 200 x 10(6)/L and 5.8 (95% CI, 3.2-10.5) for counts below 51 x 10(6)/L at baseline. Independent of baseline CD4 cell count, a rise in CD4 cell count by 50 x 10(6)/L or more and undetectable HIV-1 RNA in plasma (<400 copies/mL) by 6 months reduced risk of subsequent events, with hazard ratios of 0.32 (95% CI, 0.20-0.52) and 0.39 (0.24-0.65), respectively.

CONCLUSIONS

Our data indicate that the risk of developing an OI for a person receiving potent antiretroviral therapy is highest during the initial months of therapy. Baseline CD4 cell count and immunologic and virologic response to treatment were strong predictors of disease progression in patients receiving potent therapy. Individuals with CD4 cell counts of 50 x 10(6)/L or below may need close clinical surveillance after initiation of potent therapy.

Steroid modulation of cognitive function has focused on estrogen (E(2)), but progestins naturally co-vary with E(2) and may also influence cognitive performance. Spatial performance in the object placement task over endogenous hormonal states in which E(2) and progestins vary, and when E(2) and/or progestins were administered, was examined. Experiment 1: Rats in proestrus or estrus had significantly better performance in the object placement task than did diestrus rats. Experiment 2: Rats in the third trimester, post-partum, or lactation exhibited significantly better performance in the object placement task than did rats in the first trimester. Experiment 3: Ovariectomized (ovx) rats administered 17beta-estradiol (0.9 mg/kg), subcutaneously (sc), progesterone (P; 4 mg/kg, sc), or E(2) and P, immediately after training in the object placement task, performed significantly better when tested 4h later, than did control rats administered vehicle (sesame oil 0.2 cc). Experiment 4: ovx rats administered E(2) or P with a 1.5h delay after training in the object placement task, did not perform differently than vehicle-administered controls. Experiment 5: ovx rats administered post-training E(2), which has a high affinity for both E(2) receptor (ER)alpha and beta isoforms, or propyl pyrazole triol (PPT; 0.9 mg/kg, sc), which is more selective for ERalpha than ERbeta, had significantly better performance in the object placement task than did rats administered vehicle or diarylpropionitrile (DPN; 0.9 mg/kg, sc), an ERbeta selective ligand. Experiment 6: ovx rats administered P, or its metabolite, 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP; 4 mg/kg, sc), immediately post-training performed significantly better in the object placement task than did vehicle control rats. Thus, performance in the object placement task is better when E(2) and/or P are naturally elevated or when E(2), the ERalpha selective ER modulator PPT, P, or its metabolite, 3alpha,5alpha-THP, are administered post-training.

In a previous publication, we described the use of biradicals, in that case two TEMPO molecules tethered by an ethylene glycol chain of variable length, as polarizing agents for microwave driven dynamic nuclear polarization (DNP) experiments. The use of biradicals in place of monomeric paramagnetic centers such as TEMPO yields enhancements that are a factor of approximately 4 larger (epsilon approximately 175 at 5 T and 90 K) and concurrently the concentration of the polarizing agent is a factor of 4 smaller (10 mM electron spins), reducing the residual electron nuclear dipole broadening. In this paper we describe the synthesis and characterization by EPR and DNP/NMR of an improved polarizing agent 1-(TEMPO-4-oxy)-3-(TEMPO-4-amino)propan-2-ol (TOTAPOL). Under the same experimental conditions and using 2.5 mm magic angle rotors, this new biradical yields larger enhancements (epsilon approximately 290) at lower concentrations (6 mM electron spins) and has the additional important property that it is compatible with experiments in aqueous media, including salt solutions commonly used in the study of proteins and nucleic acids.

Fifty-kHz ultrasonic vocalizations have been proposed to reflect a positive appetitive affective state in rats, being consistently linked to the positive appetitive behavior. In the first study, we examined the brain substrates of 50-kHz ultrasonic vocalizations (USVs) by using localized electrical stimulation of the brain (ESB) at various sites that are known to mediate reward. We found that the brain areas that produced ESB-induced 50-kHz calls are the areas that have previously been shown to support the most vigorous self-stimulation behavior (prefrontal cortex, nucleus accumbens, ventral pallidum, lateral preoptic area, lateral hypothalamus, ventral tegmental area, and raphe). Importantly, all animals that showed repeatable ESB-induced 50-kHz USVs demonstrated self-stimulation behavior. In the second study, conditioned place preference was assessed following microinjection of the mu-opiate agonist Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol (DAMGO) directly into the ventral tegmental area (VTA) at a dose previously found to be rewarding. Animals that showed more 50-kHz USVs in response to drug injections compared to vehicle injections showed significant place preferences, whereas animals that did not show elevated vocalization to DAMGO did not show place preference. In experiment 3, we examined the effect of VTA electrolytic lesions, 6-OHDA lesions, and the effect of the D1/D2 dopamine antagonist flupenthixol (0 and 0.8 mg/kg, i.p.) on 50-kHz ultrasonic vocalizations. We found that these manipulations all selectively reduced 50-kHz ultrasonic vocalizations, and that these effects could be disassociated from any side effects. These data are consistent with the proposition that 50-kHz calls are tightly linked to reward in rats and that the neural circuit of 50-kHz calls closely overlaps that of ESB self-stimulation reward, drug reward, and the mesolimbic dopamine system.

Effective repression of cI transcription from PRM by the bacteriophage lambda CI repressor requires binding sites (<em>OL</em>) located 2.4 kb from the promoter. A CI tetramer bound to <em>OL</em>1.<em>OL</em>2 interacts with a tetramer bound near PRM (OR1.OR2), looping the intervening DNA. We previously proposed that in this CI octamer:DNA complex, the distant <em>OL</em>3 operator and the weak OR3 operator overlapping PRM are juxtaposed so that a CI dimer at <em>OL</em>3 can cooperate with a CI dimer binding to OR3. Here we show that <em>OL</em>3 is necessary for effective repression of PRM and that the repressor at <em>OL</em>3 appears to interact specifically with the repressor at OR3. The <em>OL</em>3-CI-OR3 interaction involves the same CI interface used for short-range dimer-dimer interactions and does not occur without the other four operators. The long-range interactions were incorporated into a physicochemical model, allowing estimation of the long-range interaction energies and showing the lysogenic state to be ideally poised for CI negative autoregulation. The results establish the lambda system as a powerful tool for examining long-range gene regulatory interactions in vivo.

Certain neurosteroids rapidly alter the excitability of neurons, in part by potentiating gamma-aminobutyric acid (GABA)-evoked chloride currents, and, like other GABA potentiating drugs, may have anticonvulsant activity. We compared the abilities of a series of isomeric metabolites of progesterone and deoxycorticosterone (3-hydroxy pregnane-20-ones and 3-hydroxy pregnane-21-ol-20-ones) to enhance GABA-evoked chloride currents in cultured hippocampal neurons with their abilities to protect against pentylenetetrazol (PTZ)-induced seizures in mice. Metabolites with 3-hydroxy in the alpha-position and 5-H in the alpha- or beta-configuration were highly effective at potentiating GABA-evoked chloride current and also showed potent anticonvulsant activity in the PTZ seizure test. The corresponding metabolites with hydroxyl groups in the 3 beta-position were considerably less potent in enhancing GABA responses and were inactive in the PTZ test. All of the neurosteroids failed to protect against tonic hindlimb extension in the maximal electroshock seizure test. 5 alpha-Pregnane-3 alpha,11 beta,21-triol-20-one, a corticosterone metabolite reported to block voltage-dependent Ca++ channels, was inactive in either of the anticonvulsant tests. At higher doses, neurosteroids effective in the PTZ test also produced motor impairment. Relative motor toxicity was lower (higher protective index) for compounds with the 5 alpha-configuration than for their corresponding 5 beta-epimers. The anticonvulsant profile of the neurosteroids resembled that of the benzodiazepine clonazepam. Although the anticonvulsant steroids had greater in vitro potencies than clonazepam, they were less potent in vivo, and they had lower protective indices.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood pressure varies in relation to factors such as physical activity, body position, ambient temperature, and autonomic nervous system activity. Therefore, we have developed a portable multibiomedical (PMB) recorder that monitors five parameters: indirect blood pressure, physical activity, body position, ambient temperature, and RR interval of the electrocardiogram. In the present study, we applied the PMB recorder over a 24-hour period to study the effect of insufficient sleep on blood pressure in subjects doing extensive overtime work. The parameters listed above were measured by the PMB recorder throughout a normal workday (mean period of sleep, 8 hours) and throughout a day with insufficient sleep (mean period of sleep, 3.6 hours) in 18 male technical workers aged 23 to 48 years old. Blood pressure (mean systolic/diastolic pressure +/- SD) significantly increased the day after a sleep-insufficient night (129 +/- 8/79 +/- 6 mm Hg) compared with the day after a normal night (123 +/- 8/76 +/- 7 mm Hg, P<.05). However, ambient temperature, mean number of steps per minute, and percentage of time spent in a standing position showed no significant difference between these days. Spectral analysis of RR intervals showed that the ratio of the low-frequency component on the RR power spectrum (0.05 to 0.15 Hz) to the high-frequency component (0.15 to 0.40 Hz) was higher on the sleep-insufficient day (2.17 +/- 0.37 versus 1.81 +/- 0.37), as was the urinary excretion of norepinephrine (P<.05). Heart rate was significantly higher on the sleep-insufficient day (81 +/- ll versus 76 +/- 8 beats per minute), after the data of two subjects with abnormal levels of physical activity were excluded (P<.Ol). These data suggest that lack of sleep may increase sympathetic nervous system activity on the following day, leading to increased blood pressure. The PMB recorder was useful for precisely evaluating the relationship between blood pressure and environmental factors.

Alcohol withdrawal syndrome (AWS) symptoms include hyperexcitability, anxiety, and sleep disorders. Chronic intermittent ethanol (CIE) treatment of rats with subsequent withdrawal of ethanol (EtOH) reproduced AWS symptoms in behavioral assays, which included tolerance to the sleep-inducing effect of acute EtOH and its maintained anxiolytic effect. Electrophysiological assays demonstrated a CIE-induced long-term loss of extrasynaptic GABAA receptor (GABAAR) responsiveness and a gain of synaptic GABAAR responsiveness of CA1 pyramidal and dentate granule neurons to EtOH that we were able to relate to behavioral effects. After CIE treatment, the alpha4 subunit-preferring GABAAR ligands 4,5,6,7 tetrahydroisoxazolo[5,4-c]pyridin-3-ol, La3+, and Ro15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5alpha][1,4]benzodiazepine-3-carboxylate) exerted decreased effects on extrasynaptic currents but had increased effects on synaptic currents. Electron microscopy revealed an increase in central synaptic localization of alpha4 but not delta subunits within GABAergic synapses on the dentate granule cells of CIE rats. Recordings in dentate granule cells from delta subunit-deficient mice revealed that this subunit is not required for synaptic GABAAR sensitivity to low [EtOH]. The profound alterations in EtOH sensitivity and alpha4 subunit localization at hippocampal GABAARs of CIE rats suggest that such changes in these and other relevant brain circuits may contribute to the development of tolerance to the sleep-inducing effects and long-term dependence on alcohol.

Agonists stimulate guanylyl 5'-[gamma-[35S]thio]-triphosphate (GTP[gamma-35S]) binding to receptor-coupled guanine nucleotide binding protein (G proteins) in cell membranes as revealed in the presence of excess GDP. We now report that this reaction can be used to neuroanatomically localize receptor-activated G proteins in brain sections by in vitro autoradiography of GTP[gamma-35S] binding. Using the mu opioid-selective peptide [D-Ala2,N-MePhe4,Gly5-ol]enkephalin (DAMGO) as an agonist in rat brain sections and isolated thalamic membranes, agonist stimulation of GTP[gamma-35S] binding required the presence of excess GDP (1-2 mM GDP in sections vs. 10-30 microM GDP in membranes) to decrease basal G-protein activity and reveal agonist-stimulated GTP[gamma-35S] binding. Similar concentrations of DAMGO were required to stimulate GTP[gamma-35S] binding in sections and membranes. To demonstrate the general applicability of the technique, agonist-stimulated GTP[gamma-35S] binding in tissue sections was assessed with agonists for the mu opioid (DAMGO), cannabinoid (WIN 55212-2), and gamma-aminobutyric acid type B (baclofen) receptors. For opioid and cannabinoid receptors, agonist stimulation of GTP[gamma-35S] binding was blocked by incubation with agonists in the presence of the appropriate antagonists (naloxone for mu opioid and SR-141716A for cannabinoid), thus demonstrating that the effect was specifically receptor mediated. The anatomical distribution of agonist-stimulated GTP[gamma-35S] binding qualitatively paralleled receptor distribution as determined by receptor binding autoradiography. However, quantitative differences suggest that variations in coupling efficiency may exist between different receptors in various brain regions. This technique provides a method of functional neuroanatomy that identifies changes in the activation of G proteins by specific receptors.

OBJECTIVE

We sought to determine the optimal dose of the selective endothelin A (ET(A)) receptor antagonist sitaxsentan for the treatment of pulmonary arterial hypertension (PAH); for observation only, an open-label (OL) bosentan arm was included.

BACKGROUND

Endothelin is a mediator of PAH. In a preliminary PAH study, the selective ET(A) receptor antagonist sitaxsentan improved six-min walk (6MW) distance, World Health Organization (WHO) functional class (FC), and hemodynamics.

METHODS

In this double-blind, placebo-controlled 18-week study, 247 PAH patients (idiopathic, or associated with connective tissue disease or congenital heart disease) were randomized; 245 patients were treated: placebo (n = 62), sitaxsentan 50 mg (n = 62) or 100 mg (n = 61), or OL (6MW tests, Borg dyspnea scores, and WHO FC assessments third-party blind) bosentan (n = 60). The primary end point was change in 6MW distance from baseline to week 18. Secondary end points included change in WHO FC, time to clinical worsening, and change in Borg dyspnea score.

RESULTS

At week 18, patients treated with sitaxsentan 100 mg had an increased 6MW distance compared with the placebo group (31.4 m, p = 0.03), and an improved WHO FC (p = 0.04). The placebo-subtracted treatment effect for sitaxsentan 50 mg was 24.2 m (p = 0.07) and for OL bosentan, 29.5 m (p = 0.05). The incidence of elevated hepatic transaminases (>3x the upper limit of normal) was 6% for placebo, 5% for sitaxsentan 50 mg, 3% for sitaxsentan 100 mg, and 11% for bosentan.

CONCLUSIONS

Treatment with the selective ET(A) receptor antagonist sitaxsentan, orally once daily at a dose of 100 mg, improves exercise capacity and WHO FC in PAH patients, with a low incidence of hepatic toxicity.

The retinoic acid-related orphan receptor alpha (RORalpha) is an orphan member of the subfamily 1 of nuclear hormone receptors. No X-ray structure of RORalpha has been described so far, and no ligand has been identified. We describe the first crystal structure of the ligand binding domain (LBD) of RORalpha, at 1.63 A resolution. This structure revealed a ligand present in the ligand binding pocket (LBP), which was identified by X-ray crystallography as cholest-5-en-3beta-ol (cholesterol). Moreover, RORalpha transcriptional activity could be modulated by changes in intracellular cholesterol level or mutation of residues involved in cholesterol binding. These findings suggest that RORalpha could play a key role in the regulation of cholesterol homeostasis and thus represents an important drug target in cholesterol-related diseases.

BACKGROUND

Flavonoids are plant-based phytochemicals with cardiovascular protective properties. Few studies have comprehensively examined flavonoid classes in relation to cardiovascular disease mortality.

OBJECTIVE

We examined the association between flavonoid intake and cardiovascular disease (CVD) mortality among participants in a large, prospective US cohort.

METHODS

In 1999, a total of 38,180 men and 60,289 women in the Cancer Prevention Study II Nutrition Cohort with a mean age of 70 and 69 y, respectively, completed questionnaires on medical history and lifestyle behaviors, including a 152-item food-frequency questionnaire. Cox proportional hazards modeling was used to calculate multivariate-adjusted hazard RRs and 95% CIs for associations between total flavonoids, 7 flavonoid classes, and CVD mortality.

RESULTS

During 7 y of follow-up, 1589 CVD deaths in men and 1182 CVD deaths in women occurred. Men and women with total flavonoid intakes in the top (compared with the bottom) quintile had a lower risk of fatal CVD (RR: 0.82; 95% CI: 0.73, 0.92; P-trend = 0.01). Five flavonoid classes-anthocyanidins, flavan-3-ols, flavones, flavonols, and proanthocyanidins-were individually associated with lower risk of fatal CVD (all P-trend < 0.05). In men, total flavonoid intakes were more strongly associated with stroke mortality (RR: 0.63; 95% CI: 0.44, 0.89; P-trend = 0.04) than with ischemic heart disease (RR: 0.90; 95% CI: 0.72, 1.13). Many associations appeared to be nonlinear, with lower risk at intakes above the referent category.

CONCLUSIONS

Flavonoid consumption was associated with lower risk of death from CVD. Most inverse associations appeared with intermediate intakes, suggesting that even relatively small amounts of flavonoid-rich foods may be beneficial.

c-erbB receptors are usually located in cell membranes and are activated by extracellular binding of EGF-like growth factors. Unexpectedly, using immunofluorescence we found high levels of c-erbB-3 within the nuclei of MTSV1-7 immortalized nonmalignant human mammary epithelial cells. Nuclear localization was mediated by the COOH terminus of c-erbB-3, and a nuclear localization signal was identified by site-directed mutagenesis and by transfer of the signal to chicken pyruvate kinase. A nuclear export inhibitor caused accumulation of c-erbB-3 in the nuclei of other mammary epithelial cell lines as demonstrated by immunofluorescence and biochemical cell fractionation, suggesting that c-erbB-3 shuttles between nuclear and nonnuclear compartments in these cells. Growth of MTSV1-7 on permeable filters induced epithelial polarity and concentration of c-erbB-3 within the nucleoli. However, the c-erbB-3 ligand heregulin beta1 shifted c-erbB-3 from the nucleolus into the nucleoplasm and then into the cytoplasm. The subcellular localization of c-erbB-3 obviously depends on exogenous stimuli and on the stage of epithelial polarity and challenges the specific function of c-erbB-3 as a transmembrane receptor protein arguing for additional, as yet unidentified, roles of c-erbB-3 within the nucle(ol)us of mammary epithelial cells.

The development of a rodent model in the perinatal rat or mouse that reproduces the principal features of human perinatal white matter injury (periventricular leukomalacia) has been hampered by uncertainty about the developmental window in the rodent that coincides temporally with cerebral white matter development in the premature infant. We recently determined oligodendrocyte (OL) lineage progression in human cerebral white matter and found that the late OL progenitor (preOL) predominates throughout the high-risk period for periventricular leukomalacia [J. Neurosci. 21(2001), 1302-1312]. Here, we determined in the perinatal rat and mouse when each species displays a distribution of OL stages that is similar to the premature human cerebral white matter. PreOLs are abundant in the rat and mouse at P2. By P7, extensive OL maturation occurs in both species and coincides with the onset of early myelination. PreOLs and immature OLs mature in the P2 white matter along a medial to lateral gradient. This may provide an explanation for regional variation in the susceptibility of perinatal white matter to injury. We propose that the sequence of OL lineage progression is a useful means to estimate developmental windows of white matter maturation in perinatal rodents that coincide with those of developing human cerebral white matter. These studies support that the vulnerable period for white matter injury in the rodent is centered around P2 and should decline thereafter, coincident with the onset of myelination.

Retrospective studies suggest that online hemodiafiltration (OL-HDF) may reduce the risk of mortality compared with standard hemodialysis in patients with ESRD. We conducted a multicenter, open-label, randomized controlled trial in which we assigned 906 chronic hemodialysis patients either to continue hemodialysis (n=450) or to switch to high-efficiency postdilution OL-HDF (n=456). The primary outcome was all-cause mortality, and secondary outcomes included cardiovascular mortality, all-cause hospitalization, treatment tolerability, and laboratory data. Compared with patients who continued on hemodialysis, those assigned to OL-HDF had a 30% lower risk of all-cause mortality (hazard ratio [HR], 0.70; 95% confidence interval [95% CI], 0.53-0.92; P=0.01), a 33% lower risk of cardiovascular mortality (HR, 0.67; 95% CI, 0.44-1.02; P=0.06), and a 55% lower risk of infection-related mortality (HR, 0.45; 95% CI, 0.21-0.96; P=0.03). The estimated number needed to treat suggested that switching eight patients from hemodialysis to OL-HDF may prevent one annual death. The incidence rates of dialysis sessions complicated by hypotension and of all-cause hospitalization were lower in patients assigned to OL-HDF. In conclusion, high-efficiency postdilution OL-HDF reduces all-cause mortality compared with conventional hemodialysis.