OBJECTIVE

The main objective of this study is to better understand the associations between forced sex history and history of sexually transmitted disease (STD) infection. Three research questions are investigated. Is history of forced sex associated with risk-taking behaviors? Are these risk-taking behaviors associated with history of STD? Is history of forced sex independently associated with history of STD?

METHODS

Information on the sexual and STD histories is obtained from 3,579 sexually active adolescent girls using data from the National Longitudinal Study of Adolescent Health. Weighted logistic and OLS regressions are employed, using techniques that account for the complex sampling design.

RESULTS

Girls with a history of forced sex are significantly more likely to have a greater number of sexual partners, be younger at first sex, and be more likely to use alcohol or drugs at last sex; there is no difference in condom use at last sex. These factors, in turn, are significantly associated with a positive STD history. Condom use at last sex is negatively associated with ever having had an STD. When all five sexual and protective practices are investigated simultaneously, history of forced sex remains significantly associated with STD history (odds ratio [OR] = 1.39, p =.014); number of sexual partners and early onset of sex remain significant. Condom use and substance use at last sex reduce to marginal significance.

CONCLUSIONS

These results suggest that a history of forced sex is independently associated with a history of STD among sexually active adolescent girls. Programmatic strategies aimed at reducing STDs through encouraging responsible sexual behavior would potentially benefit from also including a component that addresses sexual victimization.

The nucleus accumbens shell region (sNAcc) and the ventral tegmental area (VTA) are two major nodes in the mesolimbic dopamine pathway, which mediates reward for various survival behaviors, including feeding. Opioids increase and maintain food intake when injected peripherally and centrally. Opioids in the VTA cause increased release of dopamine in the sNAcc, and when injected into either site, cause an increase in food intake. Animals in this study were double cannulated in the VTA and in the sNAcc and injected with various combinations of naltrexone (NTX) (2.5, 5, and 25 microg/side) and Tyr-d-Ala-Gly-(Me)Phe-Gly-ol (DAMGO) (0.1, 0.3, 1, 3, and 5 nmol/side) in both sites. DAMGO was found to dose dependently increase intake to an equal extent when injected into either site. DAMGO-induced increases in food intake when injected into the VTA were blocked to control levels with the highest dose of NTX injected bilaterally into the sNAcc; however, increases in intake when injected into the sNAcc were blocked only partially by the highest dose of NTX injected bilaterally into the VTA. These results indicate opioid-opioid communication between the two sites; however, the communication may be quite indirect, requiring other sites and transmitters to elicit a change in behavior.

The influence of aging on serum LH and testosterone (T) pulse frequency and gonadotroph sensitivity to androgen and estrogen feedback was studied in young (less than 55 yr old) and elderly (greater than 65 yr) Trappist monks. LH pulse frequency (sampling interval, 20 min) was significantly lower [0.25 +/- 0.03 (+/- SEM) vs. 0.38 +/- 0.02 pulses/h; P less than 0.01] in elderly (n = 21) than in young monks (n = 27); the pulse amplitudes were similar. Similarly, T pulse frequency was lower in the elderly than in the young monks (0.13 +/- 0.04 vs. 0.23 +/- 0.02 pulses/h; P less than 0.01). In elderly men, the hypothalamo-pituitary complex was more sensitive to 5 alpha-androstan-17 beta-ol-3-one feedback, as determined by the decrease in serum LH and T levels. Moreover, during 5 alpha-androstan-17 beta-ol-3-one (125 mg/day, percutaneously, for 10 days) administration, the LH response to LHRH (100 micrograms, iv) was significantly higher in the elderly men compared to the pretreatment response. During estradiol (1.5 mg/day, percutaneously for 10 days) administration, the LH response to LHRH was decreased in the elderly men, but unchanged in the young men, suggesting greater responsiveness to estradiol in the elderly men. We conclude that in aged men, decreased testicular androgen secretion is not exclusively the consequence of a primary testicular alteration, but that important changes occur in hypothalamo-pituitary function, specifically decreased LH pulse frequency and increased LH responsiveness to sex hormone feedback.

The causes of irritable bowel syndrome remain elusive and there are few effective treatments for pain in this syndrome. Electroacupunture (EA) is used extensively for treatment of various painful conditions including chronic visceral hyperalgesia (CVH). However, mechanism of its analgesic effect remains unknown. This study was designed to investigate effect of EA on colon specific dorsal root ganglion (DRG) neurons in rats with CVH. CVH was induced by intracolonic injection of acetic acid (AA) in 10-day-old rats. Electromyography and patch clamp recordings were performed at age of 8-10 weeks. Colon DRG neurons were labelled by injection of DiI into the colon wall. EA was given at ST36 in both hindlimbs. As adults, neonatal AA-injected rats displayed an increased sensitivity to colorectal distension (CRD) and an enhanced excitability of colon DRG neurons. EA treatment for 40 min significantly attenuated the nociceptive responses to CRD in these rats; this attenuation was reversed by pretreatment with naloxone. EA treatment for 40 min per day for 5 days produced a prolonged analgesic effect and normalized the enhanced excitability of colon DRG neurons. Furthermore, in vitro application of [D-Ala(2), N-MePhe(4), Gly(5)-Ol] enkephalin (DAMGO) suppressed the enhanced excitability of colon neurons from rats with CVH. These findings suggest that EA produced-visceral analgesia, which might be mediated in a large part by endogenous opioids pathways, is associated with reversal of the enhanced excitability of colon DRG neurons in rats with CVH.

The synthesis and evaluation of a refined series of alpha-ketoheterocycles based on the oxazole 2 (OL-135) incorporating systematic changes in the central heterocycle bearing a key set of added substituents are described. The nature of the central heterocycle, even within the systematic and minor perturbations explored herein, significantly influenced the inhibitor activity: 1,3,4-oxadiazoles and 1,2,4-oxadiazoles 9>> tetrazoles, the isomeric 1,2,4-oxadiazoles 10, 1,3,4-thiadiazoles>> oxazoles including 2>> 1,2-diazines>> thiazoles>> 1,3,4-triazoles. Most evident in these trends is the observation that introduction of an additional heteroatom at position 4 (oxazole numbering, N>> O>> CH) substantially increases activity that may be attributed to a reduced destabilizing steric interaction at the FAAH active site. Added heterocycle substituents displaying well-defined trends may be utilized to enhance the inhibitor potency and, more significantly, to enhance the inhibitor selectivity. These trends, exemplified herein, emerge from both enhancements in the FAAH activity and simultaneous disruption of binding affinity for competitive off-target enzymes.

Systemic morphine (1.0 mg/kg i.v. or s.c.) consistently inhibited spontaneous urinary bladder contractions recorded isometrically in the anesthetized rat. This effect was reversed by intrathecal (i.t.) naloxone (1-4 micrograms) at doses which were ineffective systemically. Intrathecal morphine was also inhibited bladder activity when administered into cervical, thoracic or lumbar regions but was faster acting and more effective when injected i.t. directly in the vicinity of the lumbo-sacral cord. The effect of morphine was dose-related and appeared to be a stereospecific opiate effect being observed with i.t. levorphanol but not with dextrorphan. Bladder inhibition by i.t. morphine at low doses could be overcome by increasing the intravesicular pressure. Intrathecal morphine was also reversed by i.t. (1-2 micrograms) or s.c. (0.5 mg/kg) naloxone but not by i.c.v. naloxone (1-2 micrograms). Intrathecal naloxone in naive animals increased bladder contraction frequency and intravesicular pressure only at high doses (10-20 micrograms i.t.). Bladder activity was consistently inhibited by [D-Ala2,MePhe4,Gly-(ol)5] enkephalin (mu agonist) and [D-Ala2-D-Leu5]enkephalin (delta agonist) but was unaffected by U-50, 488H i.t., (kappa agonist). The observations support the hypothesis that spinal opioid mechanisms are involved in the neurogenic control of bladder function and that systemically or i.t. administered opioid drugs mediate inhibition of bladder motility by spinal mechanisms involving mu and delta opioid receptors.

A reconstituted monooxygenase system containing a form of cytochrome P-450, termed P-450(14)DM, and NADPH-cytochrome P-450 reductase, both purified from yeast microsomes, catalyzed the conversion of lanosterol (4,4,14 alpha-trimethyl-5 alpha-cholesta-8,24-dien-3 beta-01) to a sterol metabolite in the presence of NADPH and molecular oxygen. This conversion did not occur anaerobically or when either P-450(14)DM, the reductase, or NADPH was omitted from the system. In both free and trimethylsilylated forms, this metabolite showed a relative retention time (relative to lanosterol) of 1.10 in gas chromatography on OV-17 columns. Comparison of its mass spectrum and retention time with those of lanosterol and 4,4-dimethylzymosterol (4,4-dimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol) indicated that the metabolite was 4,4-dimethyl-5 alpha-cholesta-8,14,24-trien-3 beta-ol. Upon aerobic incubation of microsomes from semianaerobically grown yeast cells in the presence of NADPH and cyanide, endogenous lanosterol was converted to 4,4-dimethylzymosterol. This metabolism was inhibited by CO, metyrapone, SKF-525A, and antibodies to P-450(14)DM. It is concluded that in yeast microsomes lanosterol is 14 alpha-demethylated by a P-450(14)DM-containing monooxygenase system to give rise to 4,4-dimethyl-5 alpha-cholesta-8,14,24-trien-3 beta-ol, which is then reduced to 4,4-dimethylzymosterol by an NADPH-linked reductase.

Dietary saturated fatty acids containing 12- to 18-carbon atoms satisfy growth requirements of Neurospora crassa mutant cel (previously named ol; Perkins et al., reference 11); unsaturated fatty acids are synthesized by direct desaturation when an appropriate saturate is available. Odd-chain saturates, 15 carbons and 17 carbons long, satisfy the requirement, and elaidic acid (18:1 Delta(9)trans) results in slow growth. Oleic acid and other cis-unsaturated fatty acids do not satisfy growth requirements; however, oleic acid plus elaidic acid result in growth at a faster rate than elaidate alone. The use of a spin-label fatty acid reveals that hyphae produced by cel during a slow basal level of growth have lipids that reflect a relatively rigid state of viscosity compared to wild type. cel Supplemented with fatty acids and wild type supplemented in the same way have lipids of the same viscosities as reflected by electron spin resonance.

We have previously described the expression of a functional full-length trkC transcript for neurotrophin-3 (NT-3) receptor in oligodendroglia (OL) cells (Kumar and de Vellis, 1996). To date, the role of NT-3 and its signal transduction cascade in OL remains poorly defined. We report that the NT-3 responsive population of cells in the OL lineage are the progenitor cells and that the addition of NT-3 results in the autophosphorylation of p145TrkC. Furthermore, NT-3-mediated activation of p21ras and mitogen-activated protein kinase (MAPK), extracellular signal-regulated protein kinase2 (ERK2), were also observed in the progenitor OL cells. These protein tyrosine kinase (PTK)-induced responses were sensitive to the presence of K252a, an inhibitor for tyrosine kinase. We have determined that NT-3 promotes progenitor OL cell commitment to enter into S-phase of cell cycle to initiate DNA synthesis, in a manner similar to platelet-derived growth factor-AA (PDGF-AA). NT-3 thus plays a role in cell proliferation when present alone, while augmenting the proliferation capacity of PDGF-AA as indicated by the nuclear binding activity of the transcription factor, E2F-1. Both the initiation and progression of mitotic events were confirmed by the expression of c-myc and cdc2 in the presence of NT-3, PDGF-AA or NT-3 plus PDGF-AA. A cell survival assay examining interleukin 1-beta-converting enzyme (ICE)-like protease-mediated cleavage of poly (ADP-ribose) polymerase (PARP) revealed an increase in OL progenitor cell death in the absence of NT-3 or PDGF-AA. In corroboration with our in vitro studies, in vivo results show an increased expression of the progenitor OL cell marker, glycerol phosphate dehydrogenase (GPDH) within 48 hr following an intracranial injection of NT-3, PDGF-AA, or NT-3 plus PDGF-AA in PN4-5 rats. These novel findings suggest that PDGF-AA potentiates the OL progenitor cell's ability to enter into the S-phase of the cell cycle and that NT-3 can augment this activity. Furthermore, PDGF-AA and NT-3 can block ICE-like protease-mediated PARP fragmentation in progenitor OL cells. These results provide important information which further delineates the signal transduction cascades and the role of NT-3 and PDGF-AA on OL progenitor cells.

Sphingosine 1-phosphate (S1P), a metabolite of sphingomyelin degradation, stimulates interleukin-8 (IL-8) secretion in human bronchial epithelial (Beas-2B) cells. The molecular mechanisms regulating S1P-mediated IL-8 secretion are yet to be completely defined. Here we provide evidence that activation of phospholipases D1 and D2 (PLD1 and PLD2) by S1P regulates the phosphorylation of extracellular-signal-regulated kinase (ERK) and IL-8 secretion in Beas-2B cells. S1P, in a time- and dose-dependent manner, enhanced the threonine/tyrosine phosphorylation of ERK. The inhibition of S1P-induced ERK phosphorylation by pertussis toxin and PD 98059 indicated coupling of S1P receptors to G(i) and the ERK signalling cascade respectively. Treatment of Beas-2B cells with butan-1-ol, but not butan-3-ol, abrogated the S1P-induced phosphorylation of Raf-1 and ERK, suggesting that PLD is involved in this activation. The roles of PLD1 and PLD2 in ERK activation and IL-8 secretion activated by S1P were investigated by infecting cells with adenoviral constructs of wild-type and catalytically inactive mutants of PLD1 and PLD2. Infection of Beas-2B cells with the wild-type constructs resulted in the activation of PLD1 and PLD2 by S1P and PMA. Also, the enhanced production of [(32)P]phosphatidic acid and [(32)P]phosphatidylbutanol in the presence of butan-1-ol and the increased phosphorylation of ERK by S1P were blocked by the catalytically inactive mutants hPLD1-K898R and mPLD2-K758R. Transient transfection of Beas-2B cells with human PLD1 and mouse PLD2 cDNAs potentiated S1P-mediated IL-8 secretion compared with vector controls. In addition, PD 98059 attenuated IL-8 secretion induced by S1P in a dose-dependent fashion. These results demonstrate that both PLD1 and PLD2 participate in S1P stimulation of ERK phosphorylation and IL-8 secretion in bronchial epithelial cells.

Sixty-one sterols and pentacyclic triterpenes have been isolated and characterized by chromatographic and spectral methods from Zea mays (corn). Several plant parts were examined; seed, pollen, cultured hypocotyl cells, roots, coleoptiles (sheaths), and blades. By studying reaction pathways and mechanisms on plants fed radiotracers ([2-(14)C]mevalonic acid, [2-(14)C]acetate, and [2-(3)H]acetate), and stable isotopes (D2O), we discovered that hydroxymethylgutaryl CoA reductase is not "the" rate-limiting enzyme of sitosterol production. Additionally, we observed an ontogenetic shift and kinetic isotope effect in sterol biosynthesis that was associated with the C-24 alkylation of the sterol side chain. Blades synthesized mainly 24 alpha-ethyl-sterols, sheaths synthesized mainly 24-methyl-sterols, pollen possessed an interrupted sterol pathway, accumulating 24(28)-methylene-sterols, and germinating seeds were found to lack an active de novo pathway. Shoots, normally synthesizing (Z)-24(28)-ethylidine-cholesterol, after incubation with deuterated water, synthesized the rearranged double-bond isomer, stigmasta-5,23-dien-3 beta-ol. Examination of the mass spectrum and 1H nuclear magnetic resonance spectrum of the deuterated 24-ethyl-sterol indicated the Bloch-Cornforth route originating with acetyl-CoA and passing through mevalonic acid to sterol was not operative at this stage of development. An alternate pathway giving rise to sterols is proposed.

A detailed analysis of the endogenous sterols present in the clinically relevant intracellular (amastigote) stages of Trypanosoma cruzi, is presented. The parasites were grown in cultured Vero cells in the absence or presence of different sterol biosynthesis inhibitors, including the C14alpha demethylase inhibitor ketoconazole and two inhibitors of delta24(25)-sterol methyl transferase, 20 piperidin-2-yl-5alpha-pregnan-3beta-20-R-diol (22,26-azasterol) and 24-(R,S),25-epiminolanosterol. Amastigotes were isolated and purified from their host cells and neutral lipids were extracted, separated and analyzed by chromatographic and mass spectrometric methods. Control (untreated) amastigotes contained as main endogenous sterols 24-methyl-cholesta-7-en-3beta-ol (ergosta-7-en-3beta-ol) and its 24-ethyl analog, plus smaller amounts of their precursor, ergosta-7,24(28)dien-3beta-ol; these cells also contained cholesterol (up to 80% by weight of total sterols), probably derived from host cells. Amastigotes that proliferated in the presence of 10 nM ketoconazole (minimal inhibitory concentration, MIC) for 24 h had a sharply reduced content of endogenous 4-desmethyl sterols with a concomitant accumulation of 24-methyl-dihydrolanosterol and 24-methylene-dihydrolanosterol. On the other hand, amastigotes incubated during the same period of time with the two inhibitors of 24(25)-SMT at their respective MICs (100-300 nM) accumulated large amounts of C27 sterols whose structure suggested, in the case of 22,26-azasterol, that delta14 sterol reductase was also inhibited. Ketoconazole produced a dose-dependent reduction in the incorporation of [2-(14)C]-acetate into the parasite's endogenous C4-desmethyl sterols with an IC50 of 50 nM, indistinguishable from the value reported previously for the extracellular epimastigote form. Taken together, the results showed that amastigotes have a simpler sterol biosynthetic pathway than that previously described for epimastigotes, lacking both delta5 and delta22 reductases. They also suggest that the 100-fold higher potency of antifungal azoles as antiproliferative agents against amastigotes, when compared with epimastigotes, is most probably due to a smaller pool of endogenous sterols in the intracellular parasites.

BACKGROUND

Two of the most widely used mouse strains for studying the behavioral effects of ethanol are C57BL/6J (B6) and DBA/2J (D2) mice. These strains exhibit marked differences in behavioral and physiological responses to ethanol. The subjective discriminative stimulus effects of ethanol may play a role in ethanol abuse, but the discriminative stimulus profile of ethanol has not been compared in B6 and D2 mice. Examination of the discriminative stimulus effects of ethanol in B6 and D2 mouse strains may enhance our understanding of the relationship between the subjective effects of ethanol and other ethanol-induced behavioral effects.

METHODS

Twelve adult male C57BL/6J mice and 12 male DBA/2J mice were trained to discriminate 1.5 g/kg ethanol from saline in daily 15 min, milk-reinforced operant sessions. After training, ethanol substitution and response-rate suppression dose response curves were determined for ethanol, midazolam, diazepam, pentobarbital, pregnanolone, 4,5,6,7-Tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), dizocilpine, and morphine.

RESULTS

D2 mice learned the ethanol discrimination significantly more quickly than did B6 mice. Ethanol, midazolam, pregnanolone, and dizocilpine fully substituted for ethanol in both strains. Pentobarbital was more potent in producing ethanol-like discriminative stimulus effects in D2 than B6 mice. Midazolam and diazepam were significantly more potent in suppressing response rates in D2 than B6 mice. Morphine failed to substitute for ethanol in either strain, but the ED50 for morphine suppression of responding was significantly lower in B6 than D2 mice.

CONCLUSIONS

The initial stimulus effects of 1.5 g/kg ethanol may be more salient in D2 than B6 mice. This does not appear to result from differences in the neurotransmitter systems that mediate ethanol's discriminative stimulus effects. In both strains, gamma-aminobutyric acid-positive modulators and a noncompetitive NMDA antagonist substituted for ethanol. However, strain differences did exist in the potency of gamma-aminobutyric acid-positive modulators and morphine for suppressing operant responding.

There are several currently employed classification systems for diffuse gliomas that sort tumors based on histological features. Contemporary molecular techniques, however, offer the promise of improved tumor classification and resultant patient stratification for treatment and prognosis. In particular, gene expression profiling has shown exceptional promise for providing an alternative and more objective molecular approach to glioma classification. In this study, we used cDNA array technology to profile the gene expression of 30 primary human glioma tissue samples comprising 4 different glioma subtypes as defined by current World Health Organization (WHO 2000) criteria: glioblastoma (GM, WHO grade IV), anaplastic astrocytoma (AA, WHO grade III), anaplastic oligodendroglioma (AO, WHO grade III), and oligodendroglioma (OL, WHO grade II). Gene expression data alone were used to group the tumors using multidimensional scaling, which is an unsupervised statistical method. Results show that impressive separation of the 4 glioma subtypes can be achieved solely on the basis of molecular data. In addition, a subcluster of 3 glioblastomas was identified as distinct from other GMs and from the oligodendroglial tumors. These 3 patients have shown extended survival compared to other GMs in the study. Survival analysis of the full data set revealed a good correlation with the molecular classification. Results of this proof-of-principle study demonstrate that molecular profiling alone can recapitulate conventional histologic classification and grading with high fidelity. In addition, results show that the molecular approach to tumor classification can generate clinically meaningful patient stratification, and, more importantly, is an efficient class-discovery tool for human gliomas, permitting the identification of previously unrecognized, clinically relevant tumor subsets.

The density of bark-beetle colonization of a tree could be regulated by a quantitative effect of the pheromone signal from beetles in the tree (cessation of release of attractive pheromone) or by a qualitative effect (production of pheromone components inhibiting attraction). The quantitative hypothesis was tested onIps typographus by varying the release rate of the two known attractive compounds, 2-methyl-3-buten-2-ol (MB) and (4S)-cis-verbenol (cV). The highest number of beetles were captured at traps with the highest release rates. The catch was nearly proportional to the release of MB and cV at a distance between traps of 12 m or more. At 6-, 3-, and 1.5-m distances between traps deployed in a triangular arrangement there was still a good discrimination between release rates, but relatively more beetles, especially males, were caught on the blank. The lower release rates caught an equal sex ratio while the highest release rate caught only about 30% males. The qualitative hypothesis was tested by releasing the suspected inhibitors ipsdienol (Id) and ipsenol (Ie), from traps in the same amounts as cV. Only small effects were noted forI. typographus. However, the competitorI. duplicatus was attracted to Id and inhibited by Ie, while the predatorThanasimus formicarius was attracted to both compounds. On the other hand, when the ratio of Id or Ie to cV was 10∶1 or 0.1∶1 rather than 1∶1, they affected the numbers ofI. typographus attracted. A small amount of Id combined with the attractants increased trap catch, while large amounts of Id or Ie decreased attraction, especially when combined. Attack density regulation is modeled as an effect of both quantitative and qualitative mechanisms acting in sequence.

Cognitive decline in Alzheimer's disease (AD) stems from the progressive dysfunction of synaptic connections within cortical neuronal microcircuits. Recently, soluble amyloid beta protein oligomers (Abeta(ol)s) have been identified as critical triggers for early synaptic disorganization. However, it remains unknown whether a deficit of Hebbian-related synaptic plasticity occurs during the early phase of AD. Therefore, we studied whether age-dependent Abeta accumulation affects the induction of spike-timing-dependent synaptic potentiation at excitatory synapses on neocortical layer 2/3 (L2/3) pyramidal cells in the APPswe/PS1dE9 transgenic mouse model of AD. Synaptic potentiation at excitatory synapses onto L2/3 pyramidal cells was significantly reduced at the onset of Abeta pathology and was virtually absent in mice with advanced Abeta burden. A decreased alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/N-methyl-D-aspartate (NMDA) receptor-mediated current ratio implicated postsynaptic mechanisms underlying Abeta synaptotoxicity. The integral role of Abeta(ol)s in these processes was verified by showing that pretreatment of cortical slices with Abeta((25-35)ol)s disrupted spike-timing-dependent synaptic potentiation at unitary connections between L2/3 pyramidal cells, and reduced the amplitude of miniature excitatory postsynaptic currents therein. A robust decrement of AMPA, but not NMDA, receptor-mediated currents in nucleated patches from L2/3 pyramidal cells confirmed that Abeta(ol)s perturb basal glutamatergic synaptic transmission by affecting postsynaptic AMPA receptors. Inhibition of AMPA receptor desensitization by cyclothiazide significantly increased the amplitude of excitatory postsynaptic potentials evoked by afferent stimulation, and rescued synaptic plasticity even in mice with pronounced Abeta pathology. We propose that soluble Abeta(ol)s trigger the diminution of synaptic plasticity in neocortical pyramidal cell networks during early stages of AD pathogenesis by preferentially targeting postsynaptic AMPA receptors.

Epidermal slices from pig, human, and rat skin were treated with dilute buffered trypsin solution (0.005%, w/v), and suspensions of mixed basal and spinous cells were obtained in good yield. Total lipids accounted for approximately 8% of the pig, 10% of the human, and 20% of the rat epidermal cell (dry weight). Phospholipids in pig, human, and rat cells accounted for, respectively, 62%, 53%, and 35% of the total lipids. Phosphatidylcholine (34-38%), phosphatidylethanolamine (18-23%), and sphingomyelin (17-21%) were major compounds in all species. The major neutral lipids were sterols (mostly cholesterol) and triglycerides. Free fatty acids were a major lipid class in pig and human cells, whereas wax esters were a major component in rat epidermal cells. Nearly half (45%) of the sterols in rat cells but less than 10% of those in pig and human cells were esterified. Cholest-7-ene-3beta-ol accounted for 20% of the total sterols in rat cells. Cholesteryl sulfate and ceramide were minor lipids in the three species. The predominant glycosphingolipid (greater than 99%) was glucosylceramide, which accounted for 7% and 9%, respectively, of the total lipids in pig and human cells. A significant proportion (pig, 17%; human, 11%) of the fatty acids in the glucosylceramides were C26:0 and C28:0.

GABA transmission in the ventral tegmental area (VTA) is critical for fine tuning the activity of dopamine neurons in response to opioids. However, the precise mechanism by which GABA input shapes opioid reward is poorly understood. We observed a reduction of conditioned place preference for low doses of the opioid [d-Ala2, N-MePhe4, Gly5-ol]-enkephalin (DAMGO) and a switch in the functional effects of μ-opioid receptor modulation of GABA postsynaptic currents in the mouse VTA 1 d after chronic morphine treatment. Specifically, whereas in naive mice DAMGO inhibits GABA postsynaptic currents, GABAergic currents are potentiated by DAMGO after chronic morphine treatment. Importantly, pretreatment with the cAMP signaling inhibitor (R)-adenosine, cyclic 3',5'-(hydrogenphosphorothioate) triethylammonium both restored DAMGO reward and reversed the DAMGO-mediated potentiation, thereby reestablishing the inhibitory effects of opioids on GABA currents. Thus, a paradoxical bidirectionality in μ-receptor-mediated control of GABA transmission following chronic morphine treatment is a critical mechanism that determines the expression of opioid reward in the VTA.

Very few antagonists have been identified for the human pregnane X receptor (PXR). These molecules may be of use for modulating the effects of therapeutic drugs, which are potent agonists for this receptor (e.g., some anticancer compounds and macrolide antibiotics), with subsequent effects on transcriptional regulation of xenobiotic metabolism and transporter genes. A recent novel pharmacophore for PXR antagonists was developed using three azoles and consisted of two hydrogen bond acceptor regions and two hydrophobic features. This pharmacophore also suggested an overall small binding site that was identified on the outer surface of the receptor at the AF-2 site and validated by docking studies. Using computational approaches to search libraries of known drugs or commercially available molecules is preferred over random screening. We have now described several new smaller antagonists of PXR discovered with the antagonist pharmacophore with in vitro activity in the low micromolar range [S-p-tolyl 3',5-dimethyl-3,5'-biisoxazole-4'-carbothioate (SPB03255) (IC(50), 6.3 microM) and 4-(3-chlorophenyl)-5-(2,4-dichlorobenzylthio)-4H-1,2,4-triazol-3-ol (SPB00574) (IC(50), 24.8 microM)]. We have also used our computational pharmacophore and docking tools to suggest that most of the known PXR antagonists, such as coumestrol and sulforaphane, could also interact on the outer surface of PXR at the AF-2 domain. The involvement of this domain was also suggested by further site-directed mutagenesis work. We have additionally described an FDA approved prodrug, leflunomide (IC(50), 6.8 microM), that seems to be a PXR antagonist in vitro. These observations are important for predicting whether further molecules may interact with PXR as antagonists in vivo with potential therapeutic applications.

Two thirds of breast cancers express estrogen receptors (ER). ER alpha (ERα) mediates breast cancer cell proliferation, and expression of ERα is the standard choice to indicate adjuvant endocrine therapy. ERbeta (ERβ) inhibits growth in vitro; its effects in vivo have been incompletely investigated and its role in breast cancer and potential as alternative target in endocrine therapy needs further study. In this work, mammary epithelial (EpH4 and HC11) and breast cancer (MC4-L2) cells with endogenous ERα and ERβ expression and T47-D human breast cancer cells with recombinant ERβ (T47-DERβ) were used to explore effects exerted in vitro and in vivo by the ERβ agonists 2,3-bis (4-hydroxy-phenyl)-propionitrile (DPN) and 7-bromo-2-(4-hydroxyphenyl)-1,3-benzoxazol-5-ol (WAY). In vivo, ERβ agonists induced mammary gland hyperplasia and MC4-L2 tumour growth to a similar extent as the ERα agonist 4,4',4''-(4-propyl-(1H)-pyrazole-1,3,5-triyl) trisphenol (PPT) or 17β-estradiol (E2) and correlated with higher number of mitotic and lower number of apoptotic features. In vitro, in MC4-L2, EpH4 or HC11 cells incubated under basal conditions, ERβ agonists induced apoptosis measured as upregulation of p53 and apoptosis-inducible factor protein levels and increased caspase 3 activity, whereas PPT and E2 stimulated proliferation. However, when extracellular signal-regulated kinase 1 and 2 (ERK ½) were activated by co-incubation with basement membrane extract or epidermal growth factor, induction of apoptosis by ERβ agonists was repressed and DPN induced proliferation in a similar way as E2 or PPT. In a context of active ERK ½, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/RAC-alpha serine/threonine-protein kinase (AKT) signalling was necessary to allow proliferation stimulated by ER agonists. Inhibition of MEK ½ with UO126 completely restored ERβ growth-inhibitory effects, whereas inhibition of PI3K by LY294002 inhibited ERβ-induced proliferation. These results show that the cellular context modulates ERβ growth-inhibitory effects and should be taken into consideration upon assessment of ERβ as target for endocrine treatment.

A series of substituted 4-(4-fluoro-1H-indol-5-yloxy)pyrrolo[2,1-f][1,2,4]triazine-based inhibitors of vascular endothelial growth factor receptor-2 kinase is reported. Structure-activity relationship studies revealed that a methyl group at the 5-position and a substituted alkoxy group at the 6-position of the pyrrolo[2,1-f][1,2,4]triazine core gave potent compounds. Biochemical potency, kinase selectivity, and pharmacokinetics of the series were optimized and in vitro safety liabilities were minimized to afford BMS-540215 (12), which demonstrated robust preclinical in vivo activity in human tumor xenograft models. The l-alanine prodrug of 12, BMS-582664 (21), is currently under evaluation in clinical trials for the treatment of solid tumors.

Menstrual irregularity is a common complaint at presentation in women with Cushing's syndrome, although the etiology has been little studied. We have assessed 45 female patients (median age, 32 yr; range, 16-41 yr) with newly diagnosed pituitary-dependent Cushing's syndrome. Patients were subdivided into 4 groups according to the duration of their menstrual cycle: normal cycles (NC; 26-30 days), oligomenorrhea (OL; 31-120 days), amenorrhea (AM;>> 120 days), and polymenorrhea (PM; < 26 days). Blood was taken at 0900 h for measurement of LH, FSH, PRL, testosterone, androstenedione, dehydroepiandrosterone sulfate, estradiol (E2), sex hormone-binding globulin (SHBG), and ACTH; cortisol was sampled at 0900, 1800, and 2400 h. The LH and FSH responses to 100 micrograms GnRH were analyzed in 23 patients. Statistical analysis was performed using the nonparametric Mann-Whitney U and Spearman tests. Only 9 patients had NC (20%), 14 had OL (31.1%), 15 had AM (33.3%), and 4 had PM (8.8%), whereas 3 had variable cycles (6.7%). By group, AM patients had lower serum E2 levels (median, 110 pmol/L) than OL patients (225 pmol/L; P < 0.05) or NC patients (279 pmol/L; P < 0.05), and higher serum cortisol levels at 0900 h (800 vs. 602 and 580 nmol/L, respectively; P < 0.05) and 1800 h (816 vs. 557 and 523 nmol/L, respectively; P < 0.05) and higher mean values from 6 samples obtained through the day (753 vs. 491 and 459 nmol/L, respectively; P < 0.05). For the whole group of patients there was a negative correlation between serum E2 and cortisol at 0900 h (r = -0.50; P < 0.01) and 1800 h (r = -0.56; P < 0.01) and with mean cortisol (r = -0.46; P < 0.05). No significant correlation was found between any serum androgen and E2 or cortisol. The LH response to GnRH was normal in 43.5% of the patients, exaggerated in 52.1%, and decreased in 4.4%, but there were no significant differences among the menstrual groups. No differences were found in any other parameter. In summary, in our study 80% of patients with Cushing's syndrome had menstrual irregularity, and this was most closely related to serum cortisol rather than to circulating androgens. Patients with AM had higher levels of cortisol and lower levels of E2, while the GnRH response was either normal or exaggerated. Our data suggest that the menstrual irregularity in Cushing's disease appears to be the result of hypercortisolemic inhibition of gonadotropin release acting at a hypothalamic level, rather than raised circulating androgen levels.