Portacaval anastomosis (PCA) in the rat results in a broad spectrum of neurological and neurobehavioral changes, including alterations of circadian rhythms, impaired locomotor activity, and reflexes, as well as decreased threshold to noxious stimuli. In addition, following portacaval shunting, rats drink significantly more ethanol in a free-choice drinking paradigm. Available evidence suggests that many of these behavioral changes may be modulated by the endogenous opioid system of the brain. To evaluate this possibility, the effects of PCA on circulating beta-endorphin (beta-EP), as well as beta-EP content in the pituitary and specific brain nuclei, was evaluated using a sensitive radioimmunoassay. Furthermore, the characteristics and regional densities of mu and delta opioid receptors in the brains of PCA and sham-operated control rats were studied using an in vitro technique, as well as quantitative receptor autoradiography and the specific ligands 125I [D-Ala2, MePhe4, Met(o)ol5]enkephalin (FK 33-824) and 125I [2-D-penicillamine, 5-D-penicillamine]-enkephalin (DPDPE) for micro and delta sites, respectively. PCA resulted in region-selective modifications of beta-EP in brain, but not in pituitary or blood. Autoradiographic studies revealed a generalized decrease in mu binding sites (up to 70% decreases compared with sham-operated controls) and region-selective alterations of delta receptor densities following PCA. Portacaval-shunted rats drank significantly more ethanol in a free-choice drinking paradigm, an effect that was significantly attenuated by the administration of the opiate antagonist naloxone. Increased ethanol preference thus appeared to result from modifications of the endogenous opioid system in nucleus accumbens of rats following PCA.

The African trypanosome, Trypanosoma brucei, can gauge its environment by sensing nutrient availability. For example, procyclic form (PF) trypanosomes monitor changes in glucose levels to regulate surface molecule expression, which is important for survival in the tsetse fly vector. The molecular connection between glycolysis and surface molecule expression is unknown. Here we partially characterize T. brucei homologs of the beta and gamma subunits of the AMP-activated protein kinase (AMPK), and determine their roles in regulating surface molecule expression. Using flow cytometry and mass spectrometry, we found that TbAMPKbeta or TbAMPKgamma-deficient parasites express both of the major surface molecules, EP- and GPEET-procyclin, with the latter being a form that is expressed when glucose is low such as in the tsetse fly. Last, we have found that the putative scaffold component of the complex, TbAMPKbeta, fractionates with organellar components and colocalizes in part with a glycosomal marker as well as the flagellum of PF parasites.

OBJECTIVE

To explore the long-term effects and pain relief mechanism of acupotomy by observing changes in nitric oxide synthase (NOS) and beta-endorphin (beta-EP) in the hypothalamus, spinal cord, and peripheral blood of rats with third lumbar vertebrae (L3) transverse process syndrome.

METHODS

Twenty-eight SD rats were randomly assigned to normal, model, electroacupuncture (EA), and acupotomy group. The last three groups were put through an operation to emulate L3 transverse process syndrome. Fourteen days after the simulation operation, EA and acupotomy treatments were applied to the respective groups. Fifty-six days after the simulation operation, biochemistry tests and enzyme-linked immunosorbent assay were used to measure NOS and beta-EP in the hypothalamus, spinal cord, and peripheral blood.

RESULTS

Rats with the simulation operation showed significantly higher levels of NOS and beta-EP in the hypothalamus, spinal cord, and peripheral blood than those in the normal group. The EA and acupotomy groups had significantly lower levels of NOS and beta-EP than those in the model group. There was no statistical difference between the EA and acupotomy groups.

CONCLUSIONS

EA and acupotomy treatments significantly lowered NOS and beta-EP levels in the hypothalamus, spinal cord, and peripheral blood and alleviated L3 transverse process syndrome.

Placental site trophoblastic tumour (PSTT) is a very rare and unique form of gestational trophoblastic disease (GTD). This tumour represents a neoplastic transformation of intermediate trophoblastic cells that normally play a critical role in implantation. PSTT can occur after a normal pregnancy, abortion, term delivery, ectopic pregnancy or molar pregnancy. It displays a wide clinical spectrum, and when metastatic, can be difficult to control even with surgery and chemotherapy. Unlike other forms of GTD, PSTT is characterized by low beta-hCG levels because it is a neoplastic proliferation of intermediate trophoblastic cells. Expression, however, of human placental lactogen (hPL) is increased on histologic section as well as in the serum. The most common presenting symptoms of PSTT are vaginal bleeding and amenorrhoea. Diagnosis is confirmed by dilatation and curettage (D and E) and hysterectomy but meticulous evaluation of metastasis is mandatory. Most cases are confined to the uterus but pelvic involvement, lung and other organ metastasis has been reported. Unlike other forms of GTD, the WHO prognostic score is of little help. For the PSTT patient, surgery is the primary treatment of choice. For patients desiring future childbearing, D and C and adjuvant chemotherapy is an option. Because these tumours tend to be less sensitive than other types of GTD to chemotherapy, the most successful regimen to date has been with EMA/CO or EMA/EP. Good prognosis is anticipated in cases localized to the uterus, and when the interval between antecedent pregnancy and treatment is less than 2 years. In cases with distant metastasis or delayed treatment, the outcome is dismal. Advances in chemotherapeutic regimens have improved clinical reponse in metastatic disease.

<A<em>b</em>stractText>This study aims to explore the feasi<em>b</em>ility of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and <em>b</em>lood oxygen level-d<em>ep</em>endent magnetic resonance imaging (<em>B</em>OLD-MRI) in assessing vessel function and tumour aggressiveness during anti-angiogenesis treatment.</A<em>b</em>stractText><A<em>b</em>stractText>A colon cancer xenograft model was esta<em>b</em>lished in <em>B</em>AL<em>B</em>/C nude mice with the HCT116 cell line. Sixteen mice were randomly divided into Group A and Group <em>B</em>, which were treated with saline or <em>b</em>evacizuma<em>b</em> <em>b</em>y intraperitoneal injection on the 1st, 4th, 7th, 10th and 13th days and underwent DCE-MRI and <em>B</em>OLD-MRI examinations <em>b</em>efore and on the 3rd, 6th, 9th, 12th and 15th days after treatment. Group C was treated with oxaliplatin monotherapy, and Group D was treated with <em>b</em>evacizuma<em>b</em> and oxaliplatin as a point of comparison for therapeutic effects. The pathological examinations included HE, HIF-1α, fi<em>b</em>ronectin and TUNEL staining, as well as α-SMA and CD31 dou<em>b</em>le staining. One-way analysis of variance and correlation analysis were the main methods used for statistical analysis.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>Group D manifested the highest tumour inhi<em>b</em>ition rate and smallest tumour volume on day 15, followed <em>b</em>y Group C, Group <em>B</em> and Group A. K<su<em>b</em>)trans</su<em>b</em>) (F = 81.386, P < 0.001), K<su<em>b</em>)<em>ep</em></su<em>b</em>) (F = 45.901, P < 0.001), V<su<em>b</em>)e</su<em>b</em>) (F = 384.290, P < 0.001) and R2* values (F = 89.323, P < 0.001) showed meaningful trends with time in Group <em>B</em> <em>b</em>ut not Group A. The K<su<em>b</em>)trans</su<em>b</em>) values and tumour vessel maturity index (VMI) were higher than <em>b</em>aseline values 3-12 days after <em>b</em>evacizuma<em>b</em> treatment. The CD31 positive staining rate and VMI had the strongest correlations with K<su<em>b</em>)trans</su<em>b</em>) values, followed <em>b</em>y AUC<su<em>b</em>)180</su<em>b</em>), V<su<em>b</em>)e</su<em>b</em>) and K<su<em>b</em>)<em>ep</em></su<em>b</em>) values. The R2* value positively correlated with the positive staining rates of HIF-1α and fi<em>b</em>ronectin.</p><A<em>b</em>stractText>Intermittent application of low-dose anti-angiogenic inhi<em>b</em>itor treatment may help improve the effect of chemotherapy <em>b</em>y reducing hypoxia-related treatment resistance and improving drug delivery. DCE-MRI is useful for evaluating vessel maturity and vascular normalization, while <em>B</em>OLD-MRI may help to predict tumour hypoxia and metastatic potential after anti-vascular treatment.</A<em>b</em>stractText>

Several lines of data recently pointed out a role of the serine proteinase thrombin in liver fibrogenesis, but its mechanism of action is unknown. The aim of this study was to evaluate the effect of thrombin on the migration of human liver myofibroblasts. We show here that thrombin inhibits both basal migration and platelet-derived growth factor (PDGF)-BB-induced migration of myofibroblasts. By using a thrombin antagonist, a protease-activated receptor (PAR)-1 mimetic peptide, and a PAR-1 antibody, we show that this effect is dependent on the catalytic activity of thrombin and on PAR-1 activation. Thrombin's effect on basal migration was dependent on cyclooxygenase 2 (COX-2) activation because it was blocked by the COX-2 inhibitors NS-398 and nimesulide, and pharmacological studies showed that it was relayed through prostaglandin E(2) and its EP(2) receptor. On the other hand, thrombin-induced inhibition of PDGF-BB-induced migration was not dependent on COX-2. We show that thrombin inhibits PDGF-induced Akt-1 phosphorylation. This effect was consecutive to inhibition of PDGF-beta receptor activation through active dephosphorylation. Thus thrombin, through two distinct mechanisms, inhibits both basal- and PDGF-BB-induced migration of human hepatic liver myofibroblasts. The fine tuning of myofibroblast migration may be one of the mechanisms used by thrombin to regulate liver fibrogenesis.

The distribution of alpha- and beta-subunits of G-proteins was analyzed in membranes of three cell clones which are derived from the human neuroblastoma cell line SK-N-SH. The neuroblast-like clone SH-SY5Y shows a pattern of G-proteins very similar to that of human brain cortex with high levels of Gi alpha and Go alpha but low levels of G40 alpha. The intermediate clone SH-IN contains high levels of Go alpha and Gi alpha and moderate levels of G40 alpha. The non-neuronal clone SH-EP shows high levels of G40 alpha but lacks Go alpha. Differentiation of the neuroblast-like clone SH-SY5Y by retinoic acid or nerve growth factor does not change the amount of Gi alpha or Go alpha in the membrane.

An improved radio-immunoassay using an antiserum directed towards the N-terminal part of the endogenous opioid peptide beta-endorphin 1-31 (beta-EP) was validated and applied to a study of beta-EP in plasma during ischaemic pain. Experimental ischaemic pain induced in seven healthy volunteers by the submaximal effort tourniquet test did not change plasma beta-EP or adrenocorticotrophin. Plasma beta-EP was determined in 21 patients with acute myocardial infarction (AMI) and in seven patients with unstable angina pectoris. Plasma beta-EP was 4.9 fmol/ml with 95% confidence limits, 3.2-7.8 fmol/ml in AMI patients at admittance, and 2.9 (2.0-3.4) fmol/ml one week later in stable and pain-free condition (p less than 0.05). The level in 49 healthy persons was 2.8 (2.4-2.9) fmol/ml. Elevated beta-EP levels were found in five AMI patients with cardiogenic shock and in four AMI patients dying within 24 h after admittance compared to the rest of AMI patients (p less than 0.02). beta-EP was not elevated during unstable angina pectoris, although pain scores were similar to AMI. The AMI group revealed a significant, although weak, positive correlation between plasma beta-EP and pain score (Spearman r = 0.49, p less than 0.05), while there was no correlation during unstable angina pectoris. beta-EP was not correlated to the amount of morphine required within the 48 h after admittance of AMI patients. We conclude that the increase of beta-EP in plasma during AMI may be due to stressful factors other than ischaemic pain and that it is questionable whether beta-EP in plasma is related to antinociception.

The action of prostacyclin, prostaglandin E1 (PGE1), and their mimetics on myocardial function includes changes in contractility, electrophysiological properties, and protection from injury caused by transient myocardial ischemia. This study was undertaken to investigate the basic properties of myocardial E-type prostaglandin (<em>EP</em>) receptors. Ligand binding studies using an enriched preparation of sarcolemmal membranes prepared from pig hearts revealed a single class of binding sites for [3H]PGE1, with a Kd of 3.7 nmol/L and a <em>B</em>max of 92 fmol/mg protein. Competition experiments indicated highest affinity for <em>EPs</em>, suggesting an <em>EP</em> receptor. In addition, the <em>EP</em> receptor subtype-selective agonists sulprostone (<em>EP</em>1 and <em>EP</em>3) and M&<em>B</em> 28.767 (<em>EP</em>3) were active, suggesting the presence of an <em>EP</em>3 receptor subtype. PGE1 stimulated sarcolemmal GTPase and inhibited sarcolemmal adenylyl cyclase activity, indicating <em>EP</em>3 receptor coupling to an inhibitory G protein (Gi). Additional in vivo experiments showed that intracoronary infusion of PGE1 (1 nmol/min) decreased isoprenaline-stimulated left ventricular contractile activity without altering systemic vascular resistance. This inhibition of <em>beta</em>-adrenergic effects is compatible with the known myocardial anti-ischemic action of prostaglandins. Further experiments examined <em>EP</em>3 receptor density and G-protein coupling in sarcolemma from ischemic and reperfused ischemic myocardium. In anesthetized open-chest minipigs, occlusion of the left anterior descending coronary artery for 60 minutes increased <em>EP</em>3 receptor density by 50%, whereas receptor affinity was unchanged. This upregulation was prevented by pretreatment with colchicine (2 mg/kg i.v.), indicating microtubule-dependent receptor externalization. Northern hybridization showed comparable <em>EP</em>3 receptor mRNA expression in control and ischemic myocardium. The increase of receptor protein was reversed during 60 minutes of reperfusion. G-protein coupling proved to be intact in ischemic and reperfused ischemic myocardial tissue, as shown by preserved GTP-gamma-S-induced decrease of [3H]PGE1 binding. These data demonstrate for the first time that myocardial receptors for PGE1 belong to the <em>EP</em>3 subtype. The properties of this receptor include inhibition of adenylyl cyclase and upregulation during regional myocardial ischemia, suggesting an involvement in the anti-ischemic activity of E- and I-type prostaglandins.

Regulatory mechanisms of the expression of interleukin-10 (IL-10) in brain inflammatory conditions remain elusive. To address this issue, we used multiple primary brain cell cultures to study the expression of IL-10 in lipopolysaccharide (LPS)-elicited inflammatory conditions. In neuron-glia cultures, LPS triggered well-orchestrated expression of various immune factors in the following order: tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and lastly IL-10, and these inflammatory mediators were mainly produced from microglia. While exogenous application of individual earlier-released pro-inflammatory factors (e.g., TNF-α, IL-1β, or PGE2) failed to induce IL-10 expression, removal of LPS from the cultures showed the requirement of continuing presence of LPS for IL-10 expression. Interestingly, genetic disruption of tnf-α, its receptors tnf-r1/r2, and cox-2 and pharmacological inhibition of COX-2 activity enhanced LPS-induced IL-10 production in microglia, which suggests negative regulation of IL-10 induction by the earlier-released TNF-α and PGE2. Further studies showed that negative regulation of IL-10 production by TNF-α is mediated by PGE2. Mechanistic studies indicated that PGE2-elicited suppression of IL-10 induction was eliminated by genetic disruption of the PGE2 receptor EPEPEP receptors. Inhibition of cAMP-dependent signal transduction failed to affect PGE2-mediated inhibition of IL-10 production, suggesting that a G protein-independent pathway was involved. Indeed, deficiency in β-arrestin-1 or β-arrestin-2 abolished PGE2-elicited suppression of IL-10 production. In conclusion, we have demonstrated that COX-2-derived PGE2 inhibits IL-10 expression in brain microglia through a novel EPβ-arrestin-dependent signaling pathway.

The licensed vaccine against hepatitis B virus (HBV) is an effective means to prevent infection, but is not an effective therapeutic strategy to treat established chronic infections when used alone. In an animal model of chronic HBV infection (the woodchuck experimentally infected with woodchuck hepatitis virus (WHV)), the combination of conventional vaccine and potent antiviral drugs has shown promise as a potential therapeutic intervention. This approach might be improved further through the application of newer vaccine technologies. In the present study, we evaluated electroporation (EP)-based intramuscular (i.m.) delivery of a codon-optimized DNA vaccine for the WHV surface antigen (WHsAg) in mice and rabbits. In mice, this immunization procedure compared favorably to vaccination by i.m. injection of the DNA vaccine or i.m. administration of a recombinant WHsAg-alum vaccine, exhibiting characteristics expected to be beneficial for a therapeutic vaccine strategy. These included dose efficiency, consistency, vigorous induction of antibody responses to WHsAg, as well as a Th1 bias. Following scale-up to rabbits, a species that approximates the size of the woodchuck, the EP dosing regimen was markedly more effective than conventional i.m. injection of the DNA vaccine. Taken together, these results provide the foundation for studies of EP-based DNA immunization in the woodchuck in order to further assess its potential as an immunotherapeutic approach for treatment of chronic HBV infection in humans.

An important driving force behind the sequence diversity of hepatitis B virus (HBV) is viral adaptation to host immune responses. To gain an insight into the impact of host immunity on genetic diversification and properties of HBV, we characterized HBV of genotype D from treatment-naive hepatitis B e antigen-positive (EP) and hepatitis B e antigen-negative (EN) patients with chronic hepatitis B (CHB), where HBV is under stronger immune pressure, with that of HBV derived from human immunodeficiency virus (HIV)/HBV-coinfected individuals, where HIV infection has significantly weakened the immune system. Full-length sequence analysis showed that HBV heterogeneity was most extensive in EN-CHB followed by EP-CHB and HIV/HBV coinfection. The relative magnitude of non-synonymous changes within B-cell epitopes was greater than that in T-cell epitopes of HBV open reading frames (ORFs) in both EP-CHB and EN-CHB. Nine amino acid substitutions were identified in B-cell epitopes and one in a T-cell epitope of HBV in EN-CHB, most of which resulted in altered hydrophobicities, as determined using the Kyte and Doolittle method, relative to wild-type residues found in HBV from the HIV-positive group. Additionally, 19 substitutions occurred at significantly higher frequencies in non-epitope regions of HBV ORF-P in EN-CHB than HIV/HBV-coinfected patients. In vitro replication assay demonstrated that the substitutions, particularly in reverse transcriptase and RNaseH domains of ORF-P, resulted in a decline in replication capacity of HBV. Hence, our results indicate that HBV adapts to increasing immune pressure through preferential mutations in B-cell epitopes and by replicative attenuation. The viral epitopes linked to immune response identified in this study bear important implications for future HBV vaccine studies.

OBJECTIVE

To examine the hypothesis that chronic alcohol use causes accelerated aging of the brain.

METHODS

The auditory evoked potentials (EPs) were compared in three groups of 10 subjects each: (a) middle-aged individuals meeting DSM-IV criteria for alcohol dependence, (b) age- and gender-matched group of healthy individuals, and (c) an older (>65 years) group of gender-matched healthy individuals. Multiple levels of cortical information processing were examined using EPs. Early stages of information processing, related to sensory gating and stimulus classification (P50, N100/P200), were studied using a paired-click paradigm. Later stages of information processing associated with memory upgrading and identification of novel stimuli (P300) were studied using an oddball paradigm.

RESULTS

The amplitude and latency of the P300 of the alcoholic patients and the older healthy subjects differed significantly from those of the younger healthy group. Both groups showed changes that have been reported in association with aging. A tendency towards decreased sensory gating in later stages of information processing was noted in the aged healthy individuals.

CONCLUSIONS

These data suggest that alcohol dependence may accelerate the aging process. The tendency towards a sensory gating deficit during the attentive phase of information processing in older healthy subjects requires further investigation because it may be a marker for an increased proneness to developing psychotic symptoms in that group.

Distensibility of the descending aorta was evaluated during routine transesophageal echocardiography (TEE) in 50 subjects (16 to 80 years, average age 53). M-mode measurements of aortic systolic (SD) and diastolic diameter (DD) were taken distal to the left subclavian artery. Simultaneously, cuff brachial artery systolic (SBP) and diastolic (DBP) pressures were measured. Aortic pressure strain modulus (Ep), calculated as brachial artery pulse pressure/aortic strain, averaged 1.19 +/- 0.95 10(6) dynes/cm2. Elasticity index beta, defined as 1n (SBP/DBP)/aortic strain, averaged 3.77 +/- 2.12. Both Ep and beta were correlated with age (r = 0.65, p less than 0.001; and r = 0.70, p less than 0.0001). In 20 subjects aortic pulse wave velocity was assessed at the same time using simultaneous high fidelity recordings of carotid and femoral artery pressure waveforms. Aortic pulse wave velocity averaged 818 +/- 231 cm/sec and was correlated with Ep (r = 0.60, p less than 0.01) and with age (r = 0.55, p less than 0.05). Intraobserver and interobserver variability for aortic diameter measurement ranged from 0.2 to 0.5 mm.

Previous studies have provided evidence of an increased sensitivity to pain, a decreased hypothalamic opioid tone, and decreased cerebrospinal fluid (CSF) beta-endorphin (beta-EP) concentration in patients with primary chronic headache. We applied separate specific radioimmunoassays for beta-EP in CSF and plasma on samples from age-matched controls and a group of 50 patients with chronic tension-type headache (CTH) fulfilling the diagnostic criteria set by the International Headache Society. Median CSF beta-EP concentrations (95% confidence limits) were 12.8 pmol/l (11.0-14.5) in CTH patients and 11.9 pmol/l (10.9-14.2) in the control group, which is not significantly different (P = 0.28). Plasma beta-EP concentrations did not differ either, being 3.1 pmol/l (2.4-3.7) and 3.3 pmol/l (1.8-4.0) in the patients with CTH and in controls, respectively (P = 0.88). Plasma and CSF beta-EP concentrations did not correlate. Reversed-phase high performance liquid chromatography (HPLC) of CSF pools from the headache patients and controls revealed similar profiles of beta-EP-immunoreactivity both when C-terminally and N-terminally directed antisera were used, suggesting a normal post-translational processing of the pro-opiomelanocortin gene in patients with CTH. beta-EP is not involved in the pathogenesis of CTH, or such a role is not reflected in CSF or plasma concentrations of the neuropeptide.

Nineteen patients with acute psychoses, the majority schizophrenics, were studied in the course of chlorpromazine (CPZ) treatment. Plasma levels of the drug, plasma prolactin (PRL), extrapyramidal side-effects (EPS) and changes in mental state were monitored weekly, as in our earlier study. The results confirm some of our previous findings: (a) plasma CPZ levels vary widely among patients and correlate poorly with daily doses of CPZ; (b) increased plasma PRL is associated with higher plasma CPZ levels and is more common among the patients who develop EPS; and (c) none of these three variables differ between groups of patients with good and poor treatment outcome. However we did not confirm our previous finding of a significant association between EPS and higher plasma CPZ, nor did we find that the ratio of CPZ-sulphoxide to CPZ differed between the improved patients and the rest.

This paper describes the structure of neutral exopolysaccharide (EPS) produced by Lactobacillus johnsonii 142, strain of the lactic acid bacteria isolated from the intestine of mice with experimentally induced inflammatory bowel disease (IBD). Sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including two-dimensional (1)H,(1)H COSY, TOCSY, NOESY, and (1)H,(13)C HSQC experiments revealed that the repeating unit of the EPS is a pentasaccharide: -->3)-alpha-D-Galp-(1-->3)-beta-d-Glcp-(1-->5)-beta-D-Galf-(1-->3)-alpha-D-Galp-(1-->3)-alpha-D-Galp-(1->> The rabbit antiserum raised against whole cells of L. johnsonii 142 reacted with homologous EPS, and cross-reacted with exopolysaccharide from Lactobacillus animalis/murinus 148 isolated also from mice with IBD, but not reacted with EPS of L. johnsonii 151 from healthy mice.

Opiates have been reported to suppress POMC in the medial basal hypothalamus (MBH) but studies have been complicated by the fact that acutely, in the rat, opiates stimulate corticosterone and inhibit gonadal steroid release, which could both affect POMC in brain. We have therefore examined POMC gene expression and peptide levels in the MBH of castrated rats after 10 days of treatment with subcutaneous morphine or placebo pellets and after pellet removal. POMC mRNA was measured by solution hybridization assay and beta-endorphin (beta-EP) and alpha-MSH were measured by RIA. In castrated male rats, the mean POMC mRNA concentration in the MBH was 1.67 +/- 0.11 pg/microgram RNA in the control animals and decreased to 1.17 +/- 0.11 pg/microgram RNA in the morphine-treated animals (P < 0.01). Similarly in castrated, estradiol replaced female rats, the mean POMC mRNA level in the MBH was 1.36 +/- 0.19 pg/microgram RNA and decreased to 0.82 +/- 0.08 pg/microgram RNA after morphine treatment (P < 0.05). beta-EP levels were not significantly different in either study. When castrated male rats were similarly morphine pelleted and killed either on day 10 or 2 days later after pellet removal, the mean POMC mRNA level again fell from 1.83 +/- 0.21 in the controls to 1.28 +/- 0.20 pg/microgram RNA after 10 days of morphine; 2 days after pellet removal levels remained suppressed at 0.80 +/- 0.08 pg/microgram RNA (P < 0.01). In this study the concentrations of beta-EP and alpha-MSH were both noted to decline in the MBH after morphine treatment (P < 0.05). When the forms of beta-EP in the MBH were characterized by HPLC, a decrease in the concentration of beta-EP was again seen after morphine but no significant differences in the pattern of beta-EP processing or in the relative amounts of beta-EPbeta-EPbeta-EPbeta-EP or gamma 3-MSH release from the perifused rat hypothalamus in vitro. We conclude that morphine suppresses POMC gene expression in the hypothalamus of chronically treated male and female rats. Persistent changes were also noted during morphine withdrawal. In some cases this was accompanied by a fall in beta-EP peptide content. These effects were seen in castrated animals with and without sex steroid replacement and are thus independent of the effects of morphine on the pituitary-gonadal axis. These results show that opiate drugs modify endogenous opioid systems in the brain and provide further support for the hypothesis that such changes may contribute to mechanisms of opiate dependence and withdrawal.

Low density lipoprotein (LDL) cholesterol is known to be oxidized both in vitro and in vivo giving rise to oxygenated sterols. Conflicting results, however, have been reported concerning both the nature and the relative concentrations of these compounds in oxidized human LDL. We examined the extracts obtained from Cu(2+)-oxidized LDL. Thin layer chromatography analysis showed that the sterol mixture became more complex with reaction time. Analysis of the components by thin layer chromatography and mass spectrometry allowed to establish that 7 alpha- and 7 beta-hydroperoxycholest-5-en-3 beta-ol (7 alpha OOH and beta OOH) are largely prevalent among the oxysterols at early times of oxidation. These hydroperoxy derivatives have not been previously identified in oxidized LDL. The concentration of 7-hydroperoxycholest-5-en-3 beta-ol decreased with oxidation time with a concomitant increase of cholest-5-en-3 beta, 7 alpha-diol (7 alpha OH), cholest-5-en-3 beta, 7 beta-diol (7 beta OH), cholesta-3,5-dien-7-one (CD) and cholest-5-en-3 beta-ol-7-one (7CO). After 24 h of oxidation a minor component of the LDL sterols was cholestan-3 beta-ol-5,6-oxide (EP).

The protective effect of progesterone against cell death of mouse uterine epithelium was evaluated by examining the retention of 5'-[125I]iodo-2'-deoxyuridine [( 125I]IdUrd) incorporated into the whole uterus and the apoptotic index (percentage of apoptotic cells in total cells), which is a good index of physiological cell death. Castrated adult female mice were given a daily injection of oestradiol-17 beta for 3 days, and then an injection of [125I]IdUrd. They were then divided into 4 groups, which received a daily injection of vehicle only, oestradiol-17 beta (E), progesterone (P), or both oestradiol-17 beta and progesterone (EP), and were killed at intervals during these treatments for determination of 125I radioactivity retained in the whole uterus. On treatment with vehicle only, the 125I radioactivity retained in the uterus decreased rapidly, but treatment with E, P or EP reduced the loss of 125I radioactivity significantly. Progesterone did not antagonize the effect of oestradiol-17 beta on the 125I radioactivity retained in the uterus. The apoptotic index of uterine cells was examined by a similar experimental protocol, but without injection of [125I]IdUrd. In the group treated with vehicle only, the apoptotic indices of both luminal and glandular epithelia increased markedly, but the injection of E, P or EP suppressed these increases significantly. Progesterone did not antagonize the effect of oestradiol-17 beta on the apoptotic index. The apoptotic index of stroma was not affected by the injection of E, P or EP. On the other hand, progesterone completely inhibited the increase in the mitotic index of uterine epithelia induced by oestradiol-17 beta. These results show that progesterone alone or in combination with oestrogen reduced cell death in mouse uterine epithelium and that the effects of oestrogen and progesterone on uterine cell death were independent of their actions on cell division.

The aims of this study were: to quantify immunoreactive tumour necrosis factor alpha (TNF-alpha) concentrations in maternal plasma and amniotic fluid obtained from women during pregnancy and labour, both at term and preterm; and to establish the effects of bacterial endotoxin and cytokines on the in vitro release of TNF-alpha from intrauterine tissues. Maternal plasma TNF-alpha concentrations did not change during pregnancy (457.2 +/- 102.9 ng/l, mean +/- SEM, N = 52) or at the time of labour (543.5 +/- 138.6 ng/l, N = 43). In contrast, amniotic fluid TNF-alpha concentrations increased significantly (p < 0.05) during pregnancy (early pregnancy, EP, 93.0 +/- 24.8 ng/l, N = 7; preterm not-in-labour, PNIL, 186.8 +/- 42.9 ng/l, N = 16; term not-in-labour. TNIL, 499.7 +/- 150.9 ng/l, N = 13) and in association with preterm labour (preterm in-labour, PIL, 958.7 +/- 575.6 ng/l, N = 5 vs PNIL, 186.8 +/- 42.9 ng/l, N = 16). Choriodecidual and placental explants (N = 3) maintained in in vitro culture released TNF-alpha. Furthermore, the release of TNF-alpha was increased significantly (p < 0.05) by bacterial endotoxin (lipopolysaccharide, 10 ng/l-10 mg/l) but was not affected by the following cytokines at the indicated doses: interleukin-1 alpha (0.28 nmol/l), interleukin-6 (12.5 nmol/l), granulocyte colony-stimulating factor (2.5 nmol/l), granulocyte-macrophage colony-stimulating factor (35 nmol/l), macrophage colony-stimulating factor (1.2 nmol/l), leukaemia inhibitory factor (0.45 nmol/l) and transforming growth factor-beta (0.4 nmol/l).(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE

To prospectively determine whether a relationship exists between magnetic resonance (MR) imaging abnormalities of the right ventricle (RV) and intracardiac electrophysiologic (EP) test results in patients with myotonic dystrophy.

METHODS

Conventional T1-weighted single-shot black-blood fast spin-echo and gradient-echo MR imaging of the heart was prospectively performed in 32 patients with myotonic dystrophy who required EP testing. Patients were divided into two groups according to EP test results: (a) inducible (n = 15), indicating inducible ventricular tachyarrhythmias, and (b) noninducible (n = 17). Morphologic and functional MR data were analyzed by two independent investigators. Nonparametric statistical methods and kappa statistics were used.

RESULTS

No morphologic or functional abnormalities of the RV wall were observed in noninducible patients. Increased signal intensity of the RV wall, indicative of fatty replacement, was identified in 13 inducible patients. Myocardial thinning of the RV was observed in six inducible patients. An overlap of morphologically abnormal areas and areas of hypo- or dyskinesis were present in 11 inducible patients. RV outflow tract diameter was larger and RV ejection fraction was smaller in inducible patients than in noninducible patients, although differences were not significant. Interobserver agreement for MR findings was good (increased signal intensity: kappa = 0.87, P>>.30 [pairwise Wilcoxon signed rank test]; myocardial thinning: kappa = 0.87, P>>.30; hypo- or dyskinesis: kappa = 1.00, P>>.99). There was a strong relationship between MR abnormalities and inducibility during EP testing (increased signal intensity, P <.001; myocardial thinning, P <.01; hypo- or dyskinesis, P <.01).

CONCLUSIONS

The relationship between MR morphologic and functional RV abnormalities and EP testing suggests potential for the use of MR imaging as a noninvasive method to estimate the individual risk of arrhythmia in patients with myotonic dystrophy.

In the present experiment, we examined the effect of 8-iso-prostaglandin E(2) and 8-iso-prostaglandin F(2 alpha) on the release of noradrenaline from the isolated rat stomach. The postganglionic sympathetic nerves were electrically stimulated twice at 1 Hz for 1 min and test reagents were added during the second stimulation. 8-Iso-prostaglandin E(2) (10(-8)-10(-6) M) and 8-iso-prostaglandin F(2 alpha) (10(-7)-10(-5) M) dose-dependently reduced the evoked noradrenaline release, and these inhibitory potencies were as follows: 8-iso-prostaglandin E(2)>8-iso-prostaglandin F(2 alpha). The inhibitory effect of 8-iso-prostaglandin F(2 alpha), but not 8-iso-prostaglandin E(2), was abolished by 10(-6) M SQ-29548 ([1S-[1 alpha,2 alpha(Z),3 alpha,4 alpha]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino] methyl]-7-oxabicyclo[2,2,1]hept-2-yl]-5-heptenoic acid) (a prostanoid TP receptor antagonist). On the other hand, the inhibitory effect of 8-iso-prostaglandin E(2) was abolished by 10(-5) M AH-6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid) (a prostanoid EP receptor antagonist), which also attenuated the inhibitory effects of ONO-AE-248 (16S-9-deoxy-9 beta-chloro-15-deoxy-16-hydroxy-17,17-trimethylene 19, 20-didehydro prostaglandin F(2)) (a selective EP(3) receptor agonist) on the evoked release of noradrenaline. The inhibitory effect of 8-iso-prostaglandin F(2 alpha), but not 8-iso-prostaglandin E(2), was abolished by pertussis toxin. These results suggest that 8-iso-prostaglandin F(2 alpha) inhibits noradrenaline release through TP receptors, whereas 8-iso-prostaglandin E(2) seems to inhibit noradrenaline release through EP(3) receptors, located on the gastric sympathetic nerve terminals in rats.

A melatonin-induced supersensitivity of the gonadotropin-secretory system to the negative feedback action of sex steroids is thought to be important to the timing of seasonal reproduction. However, little is known concerning this action of melatonin. In the present study the antigonadal action of melatonin in the anterior hypothalamus (AH) of the white-footed mouse, Peromyscus leucopus, was used to examine the neuroendocrine mechanism whereby melatonin enhances the sensitivity to sex steroid negative feedback. Mice received a melatonin-containing pellet in the AH for 14 weeks, at which time they were castrated and treated sc with a Silastic testosterone (T) capsule for 3 weeks. At the time of castration, weight of the testes and the concentration of T in the blood of mice with a melatonin pellet were greatly reduced compared to mice with a blank (melatonin-free) implant in the AH (P less than 0.01). In mice treated with melatonin the physiological dose of T significantly reduced the concentrations of LH in blood and pituitary (P less than 0.05). This dose of T, however, had little effect on LH in mice with a blank pellet in the AH. Melatonin in the AH markedly increased the content of gonadotropin-releasing hormone (GnRH) in the mediobasal hypothalamus (P less than 0.05) in mice treated with T; however, there was little effect of melatonin and/or T in any other region examined. Melatonin and T had little effect on the contents of immunoreactive beta-endorphin (B-EP) in the hypothalamus, but T alone increased the content of B-EP in the preoptic area. These results are evidence that melatonin and T act in concert to induce the reproductively-quiescent state by suppressing secretion of GnRH from the hypothalamus.