OBJECTIVE

Our study has two goals: to evaluate variation in symptoms of substance abuse/dependence by family structure and to examine several potential explanations for this association, including differences in socio-economic status, social support, social stress and perceived approval and use of substances by family and friends.

METHODS

Ordinary least squares (OLS) regression is used to examine the association between family type and problematic substance use and to assess the hypothesized mediators.

METHODS

Data were collected between 1998 and 2000 as part of a study of the prevalence and social distributions of psychiatric and substance use disorders. The study involved face-to-face interviews with a representative sample of young adults in a South Florida community.

METHODS

Respondents (n = 1760) were between 18 and 23 years of age. Approximately 25% were of Cuban origin, 25% other Caribbean basin Hispanic, 25% African American and 25% non-Hispanic white.

METHODS

Four family types are examined: mother-father families, single-parent families, single-parent families that include other adult relative(s) and stepfamilies. Problematic substance use is measured by a set of 22 substance abuse/dependence symptoms.

RESULTS

Controlling for race-ethnicity and gender, respondents from single-parent families report a significantly higher level of problematic substance use than those from mother-father families. Although nearly all explanations receive support, we find the strongest evidence for differential association with deviant peers and exposure to stress.

CONCLUSIONS

Our findings suggest that--rather than representing a unique and independent predictor of substance use problems--family structure can be viewed as a marker of the unequal distribution of factors influencing the risk of problematic substance use.

While the EuroQOL instrument (EQ-5D) is being considered and used in clinical trials in the United States and Canada, and in large international multicentre studies, population weights for the instrument have never been established in North America. The primary purpose of this investigation was to derive a set of US-based population weights for the standard set of health states described in the EQ-5D health questionnaire. Valuations for EQ-5D health states were obtained via a postal survey using the visual analogue scale (VAS) format. A sample of 3,500 adults from the continental US were surveyed. A response rate of 25.8% was obtained. Mean and median valuations for 45 distinct health states, including dead and unconscious, were calculated. An ordinary least squares (OLS) regression-based weighting scheme was constructed to impute scores for states not directly valued in the questionnaire formats. Valuations for the standard EQ-5D health states displayed similar characteristics to VAS valuations obtained in previous investigations in European countries. The OLS model fit the observed data relatively well, achieving an adjusted R2 of 0.42. However, the diagnostic testing indicated that the initial model was misspecified. Subsequent alternative models alleviated some, but not all, of the problems of misspecification. The EQ-5D valuations from adult Americans in this sample appeared to behave in much the same fashion as in previous valuation studies. However, the generalisability of results to the entire adult American population may be limited. Violations of assumptions of the OLS regression model indicate the need for further investigation into the modelling technique used in deriving a single index score.

1. Single-channel kinetics of steroid enhancement of single gamma-aminobutyric acidA (GABA) receptor currents obtained from somata of mouse spinal cord neurones in culture were investigated using the excised outside-out patch-clamp recording technique. GABA (2 microM) and GABA (2 microM) plus androsterone (5 alpha-androstan-3 alpha-ol-17-one, AND, 10 nM-10 microM) or pregnanolone (5 beta-pregnan-3 alpha-ol-20-one, PRE, 100 nM-10 microM) applied by pressure ejection from micropipettes evoked inward currents when patches were voltage clamped at -75 mV in symmetrical chloride solutions. Averaged GABA receptor currents were increased in the presence of the steroids. 2. GABA receptor currents were recorded with at least two conductance levels, a predominant or main-conductance level of about 28 pS (which contributed 96% of the current evoked) and a minor or sub-conductance level of about 20 pS. The current amplitudes of the two conductance levels were unchanged by the steroids. The gating (opening and closing) kinetics of both of the conductance levels were analysed. Findings for the main-conductance level are summarized below. 3. Both steroids increased the average GABA receptor channel open duration. Consistent with the increased GABA receptor channel average open duration, the steroids shifted frequency histograms of GABA receptor channel open durations to longer durations. Three exponential functions were required to fit best the frequency histograms of GABA open durations, consistent with at least three kinetic open states of the main-conductance level. Time constants obtained from the GABA receptor channel open-duration frequency histograms were unchanged in the presence of the steroids. The basis for the increased average GABA receptor channel open durations by the steroids was due to an increased relative proportion of the two longer open-duration time constants. The GABA receptor channel average open durations were increased by AND and PRE in a concentration-dependent manner by shifting the proportion of openings to the longer open time constants. At a concentration of 10 microM, the prolongation of the average open duration was decreased, suggesting that the GABA receptor channel was blocked by these steroids. 4. GABA receptor channel opening frequency was increased and average channel-closed duration was decreased by AND or PRE. Consistent with this, areas of the frequency histograms of channel closed durations were shifted to shorter durations. Closed frequency distributions were fitted best with five to six exponential functions, suggesting that the channel had multiple kinetic closed states. The three briefest time constants were not greatly altered by the steroids.(ABSTRACT TRUNCATED AT 400 WORDS)

Canine distemper virus (CDV) and measles virus (MV) cause severe illnesses in their respective hosts. The viruses display a characteristic cytopathic effect by forming syncytia in susceptible cells. For CDV, the proficiency of syncytium formation varies among different strains and correlates with the degree of viral attenuation. In this study, we examined the determinants for the differential fusogenicity of the wild-type CDV isolate 5804Han89 (CDV(5804)), the small- and large-plaque-forming variants of the CDV vaccine strain Onderstepoort (CDV(OS) and CDV(OL), respectively), and the MV vaccine strain Edmonston B (MV(Edm)). The cotransfection of different combinations of fusion (F) and hemagglutinin (H) genes in Vero cells indicated that the H protein is the main determinant of fusion efficiency. To verify the significance of this observation in the viral context, a reverse genetic system to generate recombinant CDVs was established. This system is based on a plasmid containing the full-length antigenomic sequence of CDV(OS). The coding regions of the H proteins of all CDV strains and MV(Edm) were introduced into the CDV and MV genetic backgrounds, and recombinant viruses rCDV-H(5804), rCDV-H(OL), rCDV-H(Edm), rMV-H(5804), rMV-H(OL), and rMV-H(OS) were recovered. Thus, the H proteins of the two morbilliviruses are interchangeable and fully functional in a heterologous complex. This is in contrast with the glycoproteins of other members of the family Paramyxoviridae, which do not function efficiently with heterologous partners. The fusogenicity, growth characteristics, and tropism of the recombinant viruses were examined and compared with those of the parental strains. All these characteristics were found to be predominantly mediated by the H protein regardless of the viral backbone used.

The opioid receptors involved in the mediation of thermal analgesia (55 degrees C hot-plate) and inhibition of gastrointestinal transit at the spinal and supraspinal levels were studied in unanesthetized mice. Five receptor-selective compounds were evaluated for effectiveness in eliciting analgesia and inhibiting transit after both i.c.v. and intrathecal administration; these included the proposed mu agonist, [D-Ala2, N-methyl-Phe4, Gly5-ol]enkephalin (DAGO), the proposed delta agonists, [D-Pen2, L-Pen5]enkephalin (DPLPE), [D-Pen2, D-Pen5]enkephalin (DPDPE) (conformationally constrained delta selective enkephalin analogs) and [D-Thr2, Thr6, Leu5]enkephalin (DTTLE), and the proposed kappa agonist, trans-3,4-dichloro-N-methyl-N-[2-(1-pyrolidinyl)-cyclohexyl]- benzeneacetamide methanesulfonate (U-50,488H), as well as the nonselective mu-acting agonist, morphine. All compounds were found to produce analgesia after i.c.v. administration; the rank order of potency by the i.c.v. route was DAGO greater than DTTLE greater than morphine greater than DPLPE greater than DPDPE greater than U-50,488H. The analgesic effectiveness of most of these agonists given i.c.v. was evident for up to 40 min, with only DTTLE and U-50,488H having briefer time courses. Similarly, all the compounds produced analgesic responses after intrathecal administration, with the rank order of potency by this route being DTTLE greater than morphine greater than DAGO greater than DPLPE greater than DPDPE greater than U-50,488H, and all compounds (except U-50,488H) had durations of action of up to 20 to 40 min. These agonists also inhibited gastrointestinal transit after intrathecal administration, with a rank order of potency of DAGO greater than DTTLE greater than DPLPE greater than morphine greater than DPDPE greater than U-50,488H.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurosteroids typified by 5alpha-pregnan-3alpha-ol-20-one (5alpha3alpha) have emerged as the most potent endogenous positive modulators of the GABAA receptor, the principal mediator of fast inhibitory transmission within the CNS. Neurosteroids can be synthesized de novo in the brain in levels sufficient to modulate GABA(A) receptor function and, thus, might play an important physiological-pathophysiological role. Indirect support for this proposal comes from the observation that neurosteroid action is region and neuron selective. However, the mechanism(s) that imparts specificity of action remains primarily elusive. Although neurosteroids are relatively promiscuous toward different GABA(A) receptor isoforms, the contribution of local neurosteroid metabolism has been relatively unexplored. Here, we investigate the role of neurosteroid metabolism by using electrophysiological techniques to compare the actions of 5alpha3alpha and its metabolically stable synthetic analog ganaxolone on inhibitory neurotransmission in CA1 and dentate gyrus neurons. Furthermore, we evaluate the contribution of a key enzyme in neurosteroid metabolism [i.e., 3alpha-hydroxysteroidoxidoreductase (3alpha-HSOR)] to the inactivation of endogenous, or exogenously applied 5alpha3alpha. We show that low concentrations of ganaxolone, but not of 5alpha3alpha, enhance inhibitory transmission in dentate gyrus, whereas both steroids are similarly effective in CA1 neurons. Furthermore, inhibition of 3alpha-HSOR by the contraceptive agent Provera results in enhanced synaptic and extrasynaptic GABA(A) receptor-mediated inhibition in the dentate gyrus but not in the CA1 region. Collectively, these findings advocate a crucial role for local steroid metabolism in shaping GABA(A) receptor-mediated inhibition in a regionally dependent manner and suggest a novel action by the contraceptive agent on inhibitory centers in the CNS.

Damage to oligodendrocyte (OL) progenitor cells (OPCs) and hypomyelination are two hallmark features of periventricular leukomalacia (PVL), the most common form of brain damage in premature infants. Clinical and animal studies have linked the incidence of PVL to maternal infection/inflammation, and activated microglia have been proposed to play a central role. However, the precise mechanism of how activated microglia adversely affects the survival and development of OPCs is still not clear. Here we demonstrate that lipopolysaccharide (LPS)-activated microglia are deleterious to OPCs, that is, impeding OL lineage progression, reducing the production of myelin basic protein (MBP), and mediating OPC death. We further demonstrate that LPS-activated microglia mediate OPC death by two distinct mechanisms in a time-dependent manner. The early phase of cell damage occurs within 24 h after LPS treatment, which is mediated by nitric oxide (NO)-dependent oxidative damage and is prevented by N(G)-nitro-l-arginine methyl ester (l-NAME), a general inhibitor of nitric oxide synthase. The delayed cell death is evident at 48 h after LPS treatment, is mediated by cytokines, and is prevented by blocking the activity of tumor necrosis factor-alpha (TNF-alpha) and pro-nerve growth factor (proNGF), but not by l-NAME. Furthermore, microglia-derived insulin-like growth factor-1 (IGF-1) and ciliary neurotrophic factor (CNTF) were significantly suppressed by LPS, and exogenous IGF-1 and CNTF synergistically protected OLs from death induced by LPS-treated microglia conditioned medium, indicating that a deficiency in trophic support may also be involved in OL death. Our finding that LPS-activated microglia not only induce two waves of cell death but also greatly impair OL development may shed some light on the mechanisms underlying selective white matter damage and hypomyelination in PVL.

Observational studies have suggested that the intake of flavonoids is associated with a decreased risk of CVD. However, the results of these studies remain controversial. The aim of the present study was to evaluate the association between dietary flavonoid intake and CVD risk by conducting a systematic review of prospective cohort studies. Electronic reference databases were searched to identify studies that met the pre-stated inclusion criteria. The studies were assessed for eligibility and data were extracted by two authors independently. For each study, relative risks (RR) and 95 % CI were extracted and pooled using either a fixed-effects or a random-effects model. Generalised least-squares trend estimation analysis was used to evaluate dose-response relationships. The inclusion criteria were met by fourteen prospective cohort studies. The intakes of anthocyanidins (RR 0·89, 95 % CI 0·83, 0·96), proanthocyanidins (RR 0·90, 95 % CI 0·82, 0·98), flavones (RR 0·88, 95 % CI 0·82, 0·96), flavanones (RR 0·88, 95 % CI 0·82, 0·96) and flavan-3-ols (RR 0·87, 95 % CI 0·80, 0·95) were inversely associated with the risk of CVD when comparing the highest and lowest categories of intake. A similar association was observed for flavonol intake and CVD risk. Sensitivity and subgroup analyses further supported this association. The summary RR for CVD for every 10 mg/d increment in flavonol intake was 0·95 (95 % CI 0·91, 0·99). The present systematic review suggests that the dietary intakes of six classes of flavonoids, namely flavonols, anthocyanidins, proanthocyanidins, flavones, flavanones and flavan-3-ols, significantly decrease the risk of CVD.

Oligodendrocyte progenitor cells (OPCs) in the postnatal mouse corpus callosum (CC) and motor cortex (Ctx) reportedly generate only oligodendrocytes (OLs), whereas those in the piriform cortex may also generate neurons. OPCs have also been subdivided based on their expression of voltage-gated ion channels, ability to respond to neuronal activity, and proliferative state. To determine whether OPCs in the piriform cortex have inherently different physiological properties from those in the CC and Ctx, we studied acute brain slices from postnatal transgenic mice in which GFP expression identifies OL lineage cells. We whole-cell patch clamped GFP-expressing (GFP(+)) cells within the CC, Ctx, and anterior piriform cortex (aPC) and used prelabeling with 5-ethynyl-2'-deoxyuridine (EdU) to assess cell proliferation. After recording, slices were immunolabeled and OPCs were defined by strong expression of NG2. NG2(+) OPCs in the white and gray matter proliferated and coexpressed PDGFRα and voltage-gated Na(+) channels (I(Na)). Approximately 70% of OPCs were capable of generating regenerative depolarizations. In addition to OLIG2(+) NG2(+) I(Na)(+) OPCs and OLIG2(+) NG2(neg) I(Na)(neg) OLs, we identified cells with low levels of NG2 limited to the soma or the base of some processes. These cells had a significantly reduced I(Na) and a reduced ability to incorporate EdU when compared with OPCs and probably correspond to early differentiating OLs. By combining EdU labeling and lineage tracing using Pdgfrα-CreER(T2) : R26R-YFP transgenic mice, we double labeled OPCs and traced their fate in the postnatal brain. These OPCs generated OLs but did not generate neurons in the aPC or elsewhere at any time that we examined.

Using systematic combination of alpha 1, alpha 3, and alpha 5 with beta 1, beta 2, and beta 3, together with gamma 1, gamma 2, and gamma 3, we have investigated the contributions of the various alpha, beta, and gamma subunits to the pharmacology of gamma-aminobutyric acid (GABA)A agonists. We have characterized GABA, (RS)-dihydromuscimol, piperidine-4-sulfonic acid, and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol with recombinant human GABAA receptors expressed in Xenopus oocytes. Our observations indicate that the alpha subunit is the major determinant of efficacy for partial GABAA agonists. When alpha 1 and alpha 3 or alpha 1 and alpha 5 are coexpressed, the alpha 1 subunit determines the maximum efficacy, whereas the affinity is determined by the entire combination of subunits. Thus, the results of the present study demonstrate that the pharmacology of GABAA agonists is dependent on the subunit composition of the GABAA receptor complex. Functional GABAA receptors containing two different alpha subunits show pharmacological profiles distinctly different from those of receptors containing a single alpha subtype, indicating that two different alpha subunits can be coexpressed in one functional GABAA receptor complex.

An enkephalin analogue [D-Ala2, MePhe4, Met(o)-ol] enkephalin (DAMME), given intravenously to normal subjects raised serum prolactin and growth-hormone levels but lowered serum levels of luteinising hormone, follicle-stimulating hormone, cortisol, and corticotrophin. There was also a small fall in total glucagon and gastric inhibitory peptide (G.I.P.) and a rise in thyrotrophin. beta-Lipotrophin, motilin, vasoactive intestinal peptide, insulin, gastrin, and pancreatic glucagon were unchanged. Blood-glycerol increased, and blood lactate, alanine, and glucose fell. Prior administration of the opiate antagonist, naloxone, attenuated the hormonal responses to DAMME. This enkephalin analogue produces endocrine and metabolic changes in man which may be mediated through opiate-binding receptors both within and outside the brain. The enkephalins and related substances may provide an important link between perception, behaviour, and neuroendocrine regulation of hormone secretion and metabolism.

Human sweat samples were chemically fractionated into acid and non-acid components. The most abundant volatile compounds present in the fractions were identified by linked gas chromatography mass spectrometry. The acid fractions were found to be composed of a range of twenty aliphatic and three aromatic carboxylic acids ranging, on average, from 0.02 to 20 micrograms per ml of sweat sampled. Non-acid fractions were found to contain: 6-methyl-5-hepten-2-one, 1-octen-3-ol, decanal, benzyl alcohol, dimethylsulphone, phenylethanol, phenol and 4-methylphenol, collectively amounting to 0.1 and 3 micrograms per ml of sweat. The major component of sweat was found to be L-lactic acid which constituted from 1 to 5 mg/ml. Using the intact antennae of the anthropophilic malaria vector mosquito Anopheles gambiae Giles, the peripheral olfactory activities of compounds identified in the sweat fractions were investigated by electroantennography (EAG). Short-chain saturated carboxylic acids, methanoic, ethanoic, propanoic, butanoic, pentanoic and hexanoic acids were found to elicit significantly larger EAG responses than longer chain saturated carboxylic acids from female An.gambiae. For a given dose the largest amplitude EAG response was elicited by methanoic acid. Pentanoic acid elicited larger EAG responses than either butanoic or hexanoic acids. Two non-acidic compounds, 1-octen-3-ol and 4-methylphenol, were found to elicit significant dose-dependent EAG responses from female An.gambiae. 1-Octen-3-ol elicited larger EAG responses than 4-methylphenol for a given dose, but both compounds elicited smaller EAG responses than the same dose of C1-C6 straight-chain aliphatic carboxylic acids. The possible behavioural significance of the EAG-active compounds identified in human sweat samples is discussed.

Wine contains many phenolic substances, most of which originate in the grape berry. The phenolics have a number of important functions in wine, affecting the tastes of bitterness and astringency, especially in red wine. Second, the color of red wine is caused by phenolics. Third, the phenolics are the key wine preservative and the basis of long aging. Lastly, since phenolics oxidize readily, they are the component that suffers owing to oxidation and the substance that turns brown in wine (and other foods) when exposed to air. Wine phenolics include the non-flavonoids: hydroxycinnamates, hydroxybenzoates and the stilbenes; plus the flavonoids: flavan-3-ols, the flavonols, and the anthocyanins. While polymeric condensed tannins and pigmented tannins constitute the majority of wine phenolics, their large size precludes absorption and thus they are not likely to have many health effects (except, perhaps, in the gut). The total amount of phenols found in a glass of red wine is on the order of 200 mg versus about 40 mg in a glass of white wine.

The dietary flavan-3-ol (-)-epicatechin improves the bioactivity of nitric oxide in arterial vessels in vivo. Moreover, it effectively protects cultured vascular endothelial cells from signs of oxidative stress and elevates intracellular nitric oxide in vitro. We addressed the effects of (-)-epicatechin, its metabolic conversion products and structurally related compounds on NADPH oxidase activity in intact human umbilical vein endothelial cells (HUVEC) and in cell lysates. (-)-Epicatechin proved to be an O2*(-)-scavenger but did not inhibit NADPH oxidase activity, whereas the converse pattern was observed for the metabolites 3'- and 4'-O-methyl epicatechin. The dimer procyanidin B2 and (-)-epicatechin glucuronide were O2*(-)-scavengers and inhibited NADPH oxidase. Analysis of structure-activity relations with 45 compounds suggests an apocynin-like mode of NADPH oxidase inhibition. Notably, HUVEC converted (-)-epicatechin to NADPH oxidase-inhibitory methyl ethers. These data identify endothelial NADPH oxidase as candidate target of dietary flavonoids and particularly of their metabolites.

Plant flavonoid polyphenols continue to find increasing pharmaceutical and nutraceutical applications; however their isolation, especially of pure compounds, from plant material remains an underlying challenge. In the past Escherichia coli, one of the most well-characterized microorganisms, has been utilized as a recombinant host for protein expression and heterologous biosynthesis of small molecules. However, in many cases the expressed protein activities and biosynthetic efficiency are greatly limited by the host cellular properties, such as precursor and cofactor availability and protein or product tolerance. In the present work, we developed E. coli strains capable of high-level flavonoid synthesis through traditional metabolic engineering techniques. In addition to grafting the plant biosynthetic pathways, the methods included engineering of an alternative carbon assimilation pathway and the inhibition of competitive reaction pathways in order to increase intracellular flavonoid backbone precursors and cofactors. With this strategy, we report the production of plant-specific flavanones up to 700 mg/L and anthocyanins up to 113 mg/L from phenylpropanoic acid and flavan-3-ol precursors, respectively. These results demonstrated the efficient and scalable production of plant flavonoids from E. coli for pharmaceutical and nutraceutical applications.

Irradiation of rat skeletal muscles before increased loading has been shown to prevent compensatory hypertrophy for periods of up to 4 wk, possibly by preventing satellite cells from proliferating and providing new myonuclei. Recent work suggested that stem cell populations exist that might allow irradiated muscles to eventually hypertrophy over time. We report that irradiation essentially prevented hypertrophy in rat muscles subjected to 3 mo of functional overload (OL-Ir). The time course and magnitude of changes in cellular and molecular markers of anabolic and myogenic responses were similar in the OL-Ir and the contralateral nonirradiated, overloaded (OL) muscles for the first 3-7 days. These markers then returned to control levels in OL-Ir muscles while remaining elevated in OL muscles. The number of myonuclei and amount of DNA were increased markedly in OL but not OL-Ir muscles. Thus it appears that stem cells were not added to the irradiated muscles in this time period. These data are consistent with the theory that the addition of new myonuclei may be required for compensatory hypertrophy in the rat.

Oligodendrocyte precursor cells (OPCs) originate in the ventricular zones (VZs) of the brain and spinal cord and migrate throughout the developing central nervous system (CNS) before differentiating into myelinating oligodendrocytes (OLs). It is not known whether OPCs or OLs from different parts of the VZ are functionally distinct. OPCs persist in the postnatal CNS, where they continue to divide and generate myelinating OLs at a decreasing rate throughout adult life in rodents. Adult OPCs respond to injury or disease by accelerating their cell cycle and increasing production of OLs to replace lost myelin. They also form synapses with unmyelinated axons and respond to electrical activity in those axons by generating more OLs and myelin locally. This experience-dependent "adaptive" myelination is important in some forms of plasticity and learning, for example, motor learning. We review the control of OL lineage development, including OL population dynamics and adaptive myelination in the adult CNS.

BACKGROUND

Delayed onset of lactogenesis (OL) is most common in primiparas and increases the risk of excess neonatal weight loss, formula supplementation, and early weaning.

OBJECTIVE

We examined variables associated with delayed OL among first-time mothers who delivered at term and initiated breastfeeding (n = 431).

METHODS

We conducted in-person interviews during pregnancy and at days 0, 3, and 7 postpartum and extracted obstetric and newborn information from medical records. We defined OL as delayed if it occurred after 72 h and used chi-square analysis to examine its association with potential risk factors across 6 dimensions: 1) prenatal characteristics, 2) maternal anthropometric characteristics, 3) labor and delivery experience, 4) newborn characteristics, 5) maternal postpartum factors, and 6) infant feeding variables. We examined independent associations by using multivariable logistic regression analysis.

RESULTS

Median OL was 68.9 h postpartum; 44% of mothers experienced delayed OL. We observed significant bivariate associations between delayed OL and variables in all 6 dimensions (P < 0.05). In a multivariate model adjusted for prenatal feeding intentions, independent risk factors for delayed OL were maternal age>> or =30 y, body mass index in the overweight or obese range, birth weight >3600 g, absence of nipple discomfort between 0-3 d postpartum, and infant failing to "breastfeed well">> or =2 times in the first 24 h. Postpartum edema was significant in an alternate model excluding body mass index (P < 0.05).

CONCLUSIONS

The risk factors for delayed OL are multidimensional. Public health and obstetric and maternity care interventions are needed to address what has become an alarmingly common problem among primiparas.

We have developed a highly effective method for in vivo gene silencing in the spinal cord and dorsal root ganglia (DRG) by a cationic lipid facilitated delivery of synthetic, small interfering RNA (siRNA). A siRNA to the delta opioid receptor (DOR), or a mismatch RNA, was mixed with the transfection reagent, i-Fect (vehicle), and delivered as repeated daily bolus doses (0.5 microg to 4 microg) via implanted intrathecal catheter to the lumbar spinal cord of rats. Twenty-four hours after the last injection, rats were tested for antinociception by the DOR selective agonist, [D-Ala(2), Glu(4)]deltorphin II (DELT), or the mu opioid receptor (MOR) selective agonist, [D-Ala(2), N-Me-Phe(4), Gly-ol(5)]enkephalin (DAMGO). Pretreatment with the siRNA, but not the mismatch RNA or vehicle alone, blocked DELT antinociception dose-dependently. The latter was concomitant with a reduction in the spinal immunoreactivity and receptor density of DOR, and in DOR transcripts in the lumbar DRG and spinal dorsal horn. Neither siRNA nor mismatch RNA pretreatment altered spinal immunoreactivity of MOR or antinociception by spinal DAMGO, and had no effect on the baseline thermal nociceptive threshold. The inhibition of function and expression of DOR by siRNA was reversed by 72 hr after the last RNA injection. The uptake of fluorescence-tagged siRNA was detected in both DRG and spinal cord. The low effective dose of siRNA/i-Fect complex reflects an efficient delivery of the siRNA to peripheral and spinal neurons, produced no behavioral signs of toxicity. This delivery method may be optimized for other gene targets.

The importance of progesterone's (P(4)) metabolism by the 5 alpha-reductase type I enzyme was examined in homozygous and heterozygous 5 alpha-reductase type I knockout mice and their wild-type siblings. P(4) (1.0 mg) or vehicle was administered and effects on motor, anxiety, nociceptive, and depression behavior were observed. After testing, whole-brain progesterone and 5 alpha-pregnan-3 alpha-ol-20-one (3 alpha,5 alpha-THP) levels were determined by radioimmunoassay. Motor behavior in the horizontal crossing and open field tasks of 5 alpha-reductase-deficient mice administered P(4) was similar to vehicle control mice and significantly reduced compared to wild-type mice administered P(4). In the open field, 5 alpha-reductase-deficient mice administered P(4) had a similar number of central entries as did vehicle control mice, both were lower than central entries of P(4)-administered wild-type mice. However, in the plus maze, P(4) to 5 alpha-reductase-deficient or wild-type mice significantly increased open arm activity compared to vehicle-administered control mice. P(4) to wild-type, but not 5 alpha-reductase-deficient mice, significantly increased latencies to lick front and back paws in response to radiant heat stimuli compared to vehicle administration to control mice. In the forced swim test, 5 alpha-reductase-deficient mice administered P(4) were similar to vehicle control mice and the latency to immobility was significantly decreased, and the duration of immobility was significantly increased, compared to wild-type mice administered P(4). Thus, these data suggest metabolism by the 5 alpha-reductase type I enzyme may mitigate P(4)'s effects on some tasks of motor, anxiety, nociception, and depression behavior.

OBJECTIVE

Morphine activates the µ-opioid receptor without causing its rapid endocytosis. In contrast, full agonists such as [d-Ala(2) -MePhe(4) -Gly-ol]enkephalin (DAMGO) or etonitazene stimulate a rapid and profound internalization. However, the detailed molecular events underlying the differential regulation of receptor trafficking by distinct opioid agonists remain incompletely understood.

METHODS

Here, we have generated phosphosite-specific antibodies for the carboxyl-terminal residues serine 363 (Ser363), threonine 370 (Thr370) and serine 375 (Ser375), which enabled us to selectively detect either the Ser363-, Thr370- or Ser375-phosphorylated form of the receptor.

RESULTS

We showed that agonist-induced phosphorylation occurs at Thr370 and Ser375, whereas Ser363 is constitutively phosphorylated in the absence of agonist. We further demonstated that DAMGO and etonitazene stimulated the phosphorylation of both Thr370 and Ser375. In contrast, morphine promoted the phosphorylation of Ser375, but failed to stimulate Thr370 phosphorylation. In the presence of DAMGO, Ser375 phosphorylation occurred at a faster rate than phosphorylation of Thr370, indicating that Ser375 is the primary site of agonist-dependent phosphorylation. Activation of PKC by phorbol 12-myristate 13-acetate increased receptor phosphorylation only on Thr370, but not on Ser375, indicating that Thr370 can also undergo heterologous PKC-mediated phosphorylation. We also showed that µ receptor dephosphorylation can occur within minutes at or near the plasma membrane, and that agonist removal is a major prerequisite for Thr370 and Ser375 dephosphorylation.

CONCLUSIONS

Together, we showed for the first time that distinct agonists stimulate site-specific patterns of phosphorylation, which are intimately related to their ability to elicit µ-opioid receptor sequestration.

BACKGROUND

This article is commented on by Kelly, pp. 294-297 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01387.x.

Developing oligodendrocytes (OLs) are highly vulnerable to excitotoxicity and oxidative stress, both of which are important in the pathogenesis of many brain disorders. OL excitotoxicity is mediated by ionotropic glutamate receptors (iGluRs) of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate type on these cells. Here we report that metabotropic GluRs (mGluRs) are highly expressed in OL precursors but are down-regulated in mature OLs. Activation of group 1 mGluRs attenuates OL excitotoxicity by controlling downstream oxidative stress after iGluR overactivation and also prevents nonexcitotoxic forms of oxidative stress by inhibiting reactive oxygen species accumulation and intracellular glutathione loss. The modulating effect of group 1 mGluRs on hypoxic-ischemic OL injury is not due to iGluR endocytosis that occurs in neurons in response to mGluR activation but requires activation of PKC alpha after G protein coupling to phospholipase C. Our results reveal a previously undescribed role for mGluRs in limiting OL injury and suggest that targeting group 1 mGluRs may be a useful therapeutic strategy for treating disorders that involve excitotoxic injury and/or oxidative stress to OLs.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors of the nuclear hormone receptor superfamily that have been described as master genes that switch cells from an undifferentiated phenotype to a differentiated phenotype. In the present investigation, we examined the possibility that ligands for PPARs are potent activators of oligodendrocyte (OL) differentiation and/or proliferation. Primary glial cultures and enriched OL cultures of neonatal mouse cerebra were treated with three different PPAR agonists: a PPAR gamma-selective agonist, a PPAR delta-selective agonist, and a pan agonist selective for both PPAR gamma and delta. Treatment with PPAR gamma agonist does not have an effect on the differentiation of OLs; however, PPAR delta agonist and the pan agonist treatment accelerates the differentiation of OLs within 24 h of application in mixed glial cultures. The number of OLs with processes and huge membrane sheets increases two- to threefold in both groups. The increase in the size of the sheets is also mirrored by changes in the intensity and distribution of myelin basic protein (MBP) and proteolipid protein (PLP) mRNAs. As compared to controls, the PPAR delta agonist-treated groups contain more OLs that have MBP and PLP mRNA extending into distal processes. These results indicate that PPAR delta plays a significant role in the maturation of OLs and regulates the size of OL sheets. BrdU immunostaining reveals that these agonists do not significantly stimulate proliferation of OLs expressing glycolipids. The studies in enriched OL cultures reproduce the effects of the PPAR agonists seen in the mixed glial cultures, indicating that the effect of the PPAR agonists is directly on the OLs and not via astrocytes. In the enriched cultures, the total number of OLs increases significantly in the PPAR delta agonist-treated groups, but BrdU immunostaining does not show an increased proliferation of cells. These findings suggest that PPAR delta increases the survival of cells and/or prevents cell death in enriched cultures. Although PPAR delta is expressed in various cell types, its role as a factor in the transcriptional regulation of OL differentiation has not been explored. We show for the first time that a ligand that serves as an agonist for PPAR delta activates the program of OL differentiation in primary and enriched OL cultures.