Neuroprotection by valproic acid in an intrastriatal rotenone model of Parkinson's disease.

Rotenone, which is used as a pesticide and insecticide, has been shown to cause systemic inhibition of mitochondrial complex I activity, with consequent degeneration of dopaminergic neurons within the substantia nigra and striatum, as observed in Parkinson's disease. A novel intrastriatal rotenone model of Parkinson's disease was used to examine the neuroprotective effects of valproic acid (VPA), which is known to upregulate neurotrophic factors and other protective proteins in the brain. Sham or lesioned rats were treated with either vehicle or VPA at a dose of 4mg/mL in drinking water. The right striatum was lesioned by infusion of rotenone at three sites (2µg/site) along its rostro-caudal axis. A forelimb asymmetry (cylinder) test indicated a significant (p<0.01) decrease in use of the contralateral forelimb in rotenone-lesioned animals, in the third week post-lesioning, which was abolished by VPA treatment. Similarly, a significant (p<0.01) and persistent increase in use of the ipsilateral forelimb in lesioned animals over the 4weeks of testing, was not seen in animals treated with VPA. Results of the asymmetry test illustrate that intrastriatal infusion of rotenone causes contralateral motor dysfunction, which is blocked by VPA. The significant increase in ipsilateral forelimb use has not been documented previously, and presumably represents a compensatory response in lesioned animals. Six weeks post-surgery, animals were sacrificed by transcardial perfusion. Subsequent immunohistochemical examination revealed a decrease in tyrosine hydroxylase immunoreactivity within the striatum and substantia nigra of rotenone-lesioned animals. VPA treatment attenuated the decrease in tyrosine hydroxylase in the striatum and abolished it in the substantia nigra. Stereological cell counting indicated a significant (p<0.05) decrease in tyrosine hydroxylase-positive dopamine neurons in the substantia nigra of rotenone-lesioned animals, which was confirmed by Nissl staining. Importantly, this loss of dopamine neurons in rotenone-lesioned animals, was blocked by chronic VPA treatment. These findings strongly support the therapeutic potential of VPA in Parkinson's disease.

Detection of avian influenza viruses from shorebirds: evaluation of surveillance and testing approaches.

Although influenza A viruses have been isolated from numerous shorebird species (Family: Scolopacidae) worldwide, our understanding of natural history of these viruses in this diverse group is incomplete. Gaining this information can be complicated by sampling difficulties related to live capture, the need for large sample sizes related to a potentially low prevalence of infection, and the need to maintain flexibility in diagnostic approaches related to varied capabilities and resources. To provide information relevant to improving sampling and testing of shorebirds for influenza A viruses, we retrospectively evaluated a combined data set from Delaware Bay, USA, collected from 2000 to 2009. Our results indicate that prevalence trends and subtype diversity can be effectively determined by either direct sampling of birds or indirect sampling of feces; however, the extent of detected subtype diversity is a function of the number of viruses recovered during that year. Even in cases where a large number of viruses are identified, an underestimate of true subtype diversity is likely. Influenza A virus isolation from Ruddy Turnstones can be enhanced by testing both cloacal and tracheal samples, and matrix real-time PCR can be used as an effective screening tool. Serologic testing to target species of interest also has application to shorebird surveillance. Overall, all of the sampling and diagnostic approaches have utility as applied to shorebird surveillance, but all are associated with inherent biases that need to be considered when comparing results from independent studies.

Is the occurrence of avian influenza virus in Charadriiformes species and location dependent?

Birds in the order Charadriiformes were sampled at multiple sites in the eastern half of the continental USA, as well as at Argentina, Chile, and Bermuda, during 1999-2005, and tested for avian influenza virus (AIV). Of more than 9,400 birds sampled, AIV virus was isolated from 290 birds. Although Ruddy Turnstones (Arenaria interpres) comprised just 25% of birds sampled, they accounted for 87% of isolates. Only eight AIV isolations were made from birds at four locations outside of the Delaware Bay, USA, region; six of these were from gulls (Laridae). At Delaware Bay, AIV isolations were predominated by hemagglutinin (HA) subtype H10, but subtype diversity varied each year. These results suggest that AIV infection among shorebirds (Scolopacidae) may be localized, species specific, and highly variable in relation to AIV subtype diversity.

A randomized, placebo-controlled study of rofecoxib with paracetamol in early post-tonsillectomy pain in adults.

BACKGROUND AND OBJECTIVE: Effective and early treatment of postoperative pain and nausea have become pivotal for the early discharge of patients after tonsillectomy. Opioid-based analgesia is standard practice but the use of non-steroidal anti-inflammatory drugs is discouraged due to their platelet inhibiting properties. The cyclo-oxygenase-2 selective non-steroidal anti-inflammatory drugs are effective analgesics and do not affect platelet function. We hypothesized that premedication with cyclo-oxygenase-2 selective non-steroidal anti-inflammatory drug in addition to paracetamol would provide effective analgesia and decrease opioid consumption during early recovery from tonsillectomy. METHODS: In a randomized, placebo-controlled study of adult tonsillectomy patients (n=40) one group (R-group; n=20) was premedicated with paracetamol 1.5 g and rofecoxib 50 mg and a control group (P-group; n=20) was premedicated with paracetamol 1.5 g and placebo. Morphine was used as rescue medication. Postoperative (24 h) pain scores (0--10), morphine consumption as well as intraoperative blood loss were recorded. RESULTS: We found no overall difference in pain scores between the groups but significantly more patients in the placebo group had pain scores >5 within the first 8 h. The rofecoxib group consumed less morphine during the first 12 h. A lower intraoperative blood loss was observed in the rofecoxib group. CONCLUSION: Our results suggest an early although clinically minor analgesic benefit of the addition of a cyclo-oxygenase-2 selective non-steroidal anti-inflammatory drug to paracetamol as premedication for adult tonsillectomy.

Changes in contaminant levels in New Jersey osprey eggs and prey, 1989 to 1998.

Ospreys are good indicators of the health of estuarine areas because they feed almost exclusively on fish with the balance on other aquatic biota. Through the 1980s, ospreys nesting on Delaware Bay in New Jersey had reduced reproductive success relative to those nesting on the Atlantic coast and the Maurice River, a tributary of Delaware Bay. Earlier research suggested that elevated levels of DDT and polychlorinated biphenyl (PCB) contaminants identified in addled osprey eggs contributed to this reduced productivity. We repeated egg and prey sampling initially conducted in 1989 to evaluate the trends of contaminants in the last decade. Most organochlorine contaminants declined in osprey eggs in 1998 relative to 1989. Across three study areas, PCBs decreased from 4.1-7.7 ppm in 1989 to 1.8-3.2 ppm in 1998; DDE decreased from 1.2-3.2 ppm in 1989 to 0.7-1.2 ppm in 1998. Lead in eggs increased from an average of 0.01 to 0.30 ppm wet weight, and mercury averaged 0.12 ppm and increased only in Atlantic coast eggs. Most of these contaminant changes were also found in typical prey fish: PCBs decreased from 0.18-1.2 ppm in 1989 to 0.06-0.43 ppm in 1998; DDE decreased from 0.05-0.69 ppm in 1989 to 0.03-0.13 ppm in 1998. Lead and mercury increased in most fish samples. The improvement in most organochlorine contaminants in osprey eggs and prey reflected improved nest success in the Delaware Bay study area, and the nesting populations in the Atlantic and Maurice River study areas increased approximately 200% since 1989. PCBs and DDE in osprey eggs were below levels considered to be toxic to egg development. This study documents significant improvements in organochlorine contaminants in southern New Jersey ospreys, but justifies continued monitoring of heavy metals, such as lead and mercury, in aquatic ecosystems.

Melatonin receptor mRNA expression in human granulosa cells.

We have shown that the melatonin receptor agonist, 2-[125I] iodomelatonin, binds to high-affinity guanine nucleotide-sensitive sites on human granulosa (HG) cell membranes. In order to confirm the presence of melatonin receptors in HG cells, we have now used a reverse transcriptase-polymerase chain reaction (RT-PCR) procedure to examine receptor subtype expression. RT-PCR studies revealed the presence of the mt1 (Mel1alpha) melatonin receptor subtype in ten single or pooled HG cell samples which were obtained from 14 patients. In contrast, expression of MT2 ( Mel1b) mRNA was observed in only two of these HG samples. DNA sequencing of the mt1 PCR product confirmed its identity with the reported human mt1 melatonin receptor. The expression of mt1 and MT2 receptor mRNA in HG cells and the reported presence of melatonin in human follicular fluid indicate a potentially important role for this hormone in regulating human ovarian and reproductive function.

Diazepam inhibits forskolin-stimulated adenylyl cyclase activity in human tumour cells.

Previous studies have shown that the benzodiazepine agonist, diazepam, suppresses adenylyl cyclase activity in rat brain, via a G protein-coupled benzodiazepine receptor. Since diazepam binding sites are also present in diverse non-neuronal tissues including tumour cells, its effects on adenylyl cyclase activity were examined in membranes from human MCF-7 (breast cancer) and M-6 (melanoma) cells. Diazepam caused a biphasic and concentration-dependent inhibition of forskolin-stimulated adenylyl cyclase activity in MCF-7 membranes. The first phase of inhibition, at picomolar to nanomolar drug concentrations (EC50=5.7 x 10(-12)M), is similar to the receptor mediated phase observed in the rat brain. At micromolar concentrations of diazepam (EC50= 1.8 x 10(-4)M), the steep decrease in adenylyl cyclase activity may involve a direct action on the enzyme itself, as detected previously in rat brain membranes. Diazepam-induced suppression of adenylyl cyclase activity was also detected in M-6 membranes. However, in contrast to MCF-7 findings, only micromolar concentrations of diazepam (EC50=5.2 x 10(-4)M) inhibited enzyme activity in M-6 membranes. These findings suggest that G protein-coupled benzodiazepine receptors, which mediate inhibition of the adenylyl cyclase-cAMP pathway in the brain, are also expressed in MCF-7 cells.

Modulation of c-fos expression in the rat striatum by diazepam.

The benzodiazepine agonist, diazepam, inhibits cAMP production in the rat brain. Since cAMP influences c-fos activity, we examined the effects of diazepam on expression of this immediate early gene, as indicated by Western blot analysis. Intraperitoneal administration of diazepam increased Fos protein levels in the striatum, but not in the hippocampus. In contrast, pretreatment with diazepam blocked the potent inducing effect of amphetamine, in both brain regions. Similar induction and blockade effects were also observed for a 90 kDa Fos related antigen (Fra), in the striatum and hippocampus. The possible mechanisms underlying the modulatory effects of diazepam on c-fos expression in the brain are discussed.

A marked diurnal rhythm of melatonin ML1A receptor mRNA expression in the suprachiasmatic nucleus.

Melatonin (ML1A) receptor binding in the suprachiasmatic nucleus (SCN) exhibits a diurnal rhythm, with significant increases during the daytime. In order to determine whether the diurnal variation in binding is reflected by a cycling of melatonin ML1A receptor mRNA levels, we utilized a semiquantitative reverse transcriptase-polymerase chain reaction procedure to examine receptor expression throughout the light-dark cycle. Here we report the first evidence of a significant diurnal variation in ML1A receptor mRNA levels within the SCN. Furthermore, this mRNA expression occurs approximately 3 h prior to, but is tightly correlated to the diurnal rhythm in high-affinity melatonin binding in the SCN.

The antidopaminergic action of S-20098 is mediated by benzodiazepine/GABA(A) receptors in the striatum.

The naphthalenic compound S-20098, which is a melatonergic agonist, inhibits [3H]diazepam binding in striatal membranes. S-20098 also inhibits apomorphine-induced turning in 6-hydroxydopamine lesioned rats. This antidopaminergic effect is blocked by either intraperitoneal injection of the central-type benzodiazepine (BZ) antagonist, flumazenil, or intrastriatal injection of the GABA(A) antagonist, bicuculline. These findings indicate that S-20098 can activate central-type BZ receptors, and its antidopaminergic action, like that of melatonin, involves a GABAergic mechanism in the striatum.

Mechanisms underlying the antidopaminergic effect of clonazepam and melatonin in striatum.

Intrastriatal injection of the GABA(A) antagonist, bicuculline, caused about a 75% decrease in the inhibitory effect of the central-type benzodiazepine (BZ) agonist, clonazepam or the indoleamine hormone, melatonin, on apomorphine-induced rotation in a 6-hydroxydopamine model of dopaminergic supersensitivity. Pretreatment with the peripheral-type BZ antagonist, PK 11195 (intrastriatally or intraperitoneally), also attenuated the antidopaminergic effect of these drugs but with much less potency than bicuculline. However, the combination of both bicuculline and PK 11195, injected directly into the striatum, completely blocked the antidopaminergic action of clonazepam or melatonin. These results indicate that the antidopaminergic action of clonazepam and melatonin in the striatum involves two distinct mechanisms: (1) a predominant GABAergic activation via the BZ/GABA(A) receptor complex, and (2) a secondary mechanism linked to a PK 11195-sensitive BZ receptor pathway. Recent studies indicate that PK 11195 blocks BZ-induced inhibition of the adenylyl cyclase-cyclic AMP pathway in the striatum. Since cyclic AMP has been implicated in the rotational behaviour of 6-hydroxydopamine-lesioned animals, it is possible that the antidopaminergic action of clonazepam and melatonin also involves suppression of this second messenger.

Sensitization of G protein-coupled benzodiazepine receptors in the striatum of 6-hydroxydopamine-lesioned rats.

The nonselective benzodiazepine (BZ) agonist diazepam is a potent inhibitor of adenylyl cyclase (AC) activity in the rat striatum. To examine this inhibitory action of diazepam further, its effects were examined in 6-hydroxydopamine-lesioned animals, which reportedly exhibit sensitization of the striatal AC pathway. As previously observed, inhibition of AC activity by diazepam was biphasic, with the first phase being receptor-mediated, whereas the second phase involves a direct action on the enzyme itself. In the presence of NaCl (120 mM), a marked sensitization to the receptor-mediated inhibitory effect of diazepam on AC activity was observed in striatal membranes of lesioned animals. EC50 values were 10.4 +/- 1.1 and 4.8 +/- 0.9 nM (p < 0.05) for intact and lesioned striata, respectively. An examination of [3H]diazepam binding revealed a significant increase in the density of binding sites in denervated striata, with no change in affinity. A time-dependent increase in [alpha-32P]GTP labeling of two distinct striatal proteins with apparent molecular masses of 40 and 45 kDa, suggestive of the alpha subunits of Gi and Gs, respectively, was observed. There was a significant increase in basal [alpha-32P]GTP binding to both proteins in lesioned striata. In addition, diazepam stimulated [alpha-32P]GTP binding to the 40-kDa protein, especially in lesioned striata. These data indicate that the sensitization of the receptor-mediated inhibitory effect of diazepam on AC activity in denervated striata may involve up-regulation of BZ receptors as well as enhanced functional coupling of these receptors to inhibitory G proteins.

PK 11195 blockade of benzodiazepine-induced inhibition of forskolin-stimulated adenylate cyclase activity in the striatum.

1. The effects of benzodiazepine receptor antagonists on the inhibition of forskolin-stimulated adenylate cyclase (AC) activity by various benzodiazepine (BZ) and indoleamine agonists in the rat striatum were investigated. 2. A biphasic inhibition of forskolin-stimulated AC activity by the peripheral-type agonist, Ro5-4864, and a multiphasic inhibition by the non-selective BZ, diazepam, was observed. One phase of AC inhibition is consistent with a Gi-coupled receptor-mediated action, whereas the other phases appear to involve a direct effect on the enzyme itself. 3. While the central-type antagonist, flumazenil, had no effect on the ability of Ro5-4864 to inhibit AC activity, the peripheral-type receptor ligand, PK 11195, abolished the first phase of inhibition. 4. PK 11195 and pertussis toxin were found to block the inhibitory effect of various BZs and the indoleamines, melatonin and 2-iodomelatonin, on induced AC activity. 5. Saturation binding studies, conducted at 30 degrees C with [3H]-diazepam revealed a single binding site in the rat striatum (KD = 19.3 +/- 0.80 nM) which significantly decreased in affinity in the presence of GTP (KD = 30.5 +/- 2.6 nM; P < 0.05). No significant change in Bmax was observed. 6. These findings indicate the presence of Gi-coupled BZ receptors in the rat striatum. Thus, suppression of cyclic AMP production may contribute to the diverse neuropharmacological effects of BZs, melatonin and related drugs.

Benzodiazepine-induced inhibition of human malignant melanoma (M-6) cell growth.

Since benzodiazepines (BZs) have been shown to inhibit the growth of some cell lines, the effects of these drugs on human melanoma (M-6) cell growth were examined. Cell growth was measured by the tetrazolium salt (MTT) assay or the Hoechst 33258 DNA assay. Diazepam, a non-selective BZ agonist, and Ro5-4864, a peripheral-type agonist, inhibited M-6 cell proliferation by 36% and 55% with EC50s of 139 microM and 107 microM respectively, after four days of treatment in culture. The central-type agonists, clonazepam and flunitrazepam, were ineffective. The antiproliferative effect of diazepam was partially reversed by treatment with phorbol 12-myristate 13-acetate (PMA). Neither PK 11195, a peripheral-type BZ receptor antagonist, nor flumazenil a central-type antagonist, blocked the effect of diazepam, indicating that these BZ receptors are not involved. The effect of PMA suggests that the antiproliferative effect of the BZs may involve inhibition of a calcium/protein kinase C-related pathway in M-6 cells.

Central-type benzodiazepine receptors mediate the antidopaminergic effect of clonazepam and melatonin in 6-hydroxydopamine lesioned rats: involvement of a GABAergic mechanism.

In this study, we examined the effect of the central-type benzodiazepine agonist, clonazepam, and the indoleamine hormone, melatonin, on central dopaminergic function using the 6-hydroxydopamine model of dopamine receptor supersensitivity. Unilateral lesioning of the nigrostriatal pathway with 6-hydroxydopamine was carried out in Sprague-Dawley rats. Two weeks after surgery, the animals were examined for the presence of dopaminergic supersensitivity by their response to the dopamine receptor agonist, apomorphine. Clonazepam, melatonin and its analogs, 6-chloromelatonin and 2-iodomelatonin, significantly inhibited apomorphine-induced turning behavior (P < .01). Pretreatment with a central-type benzodiazepine antagonist, flumazenil, significantly reduced the effect of melatonin and clonazepam (P < .01). The peripheral-type benzodiazepine antagonist, PK 11195, caused some attenuation of melatonin's effect (P < .05), but it was significantly less potent than flumazenil. Bicuculline, a GABAA receptor antagonist, was also found to reduce the inhibitory effect of melatonin on the induced rotational response (P < .01). These results indicate that the antidopaminergic effect of clonazepam and melatonin is mediated predominantly by central-type benzodiazepine receptors in the central nervous system, via a GABAergic mechanism.

Melatonin receptors on human granulosa cell membranes.

Putative melatonin binding sites were detected in the membrane fraction of gonadotropin-stimulated human granulosa cells using the melatonin analogue 2-[125I]-iodomelatonin (125I-IML). Saturation studies and Scatchard analysis revealed the presence of a major binding site with a Kd of 99 pM. Guanosine triphosphate shifted the receptor affinity to 380 pM. In competition studies, the rank order of potency of indoles for inhibition of 125I-IML binding at these sites was typical of melatonin receptors: 2-iodomelatonin > melatonin > N-acetylserotonin > 5-methoxytryptamine > serotonin. Culture of cells for 7 days in vitro increased receptor density but not the affinity. These findings strongly suggest that melatonin found in follicular fluid may have a physiological role.

Heparin inhibits melatonin binding and signal transduction in chick brain.

The high-affinity receptor for melatonin is coupled to a pertussis toxin-sensitive, inhibitory guanine nucleotide regulatory protein, Gi, which mediates inhibition of adenylate cyclase activity in the chick and other species. Heparin has been found to uncouple alpha 2-adrenoceptors from a similar Gi; therefore it was of interest to examine the effect of this agent on melatonin binding and signal transduction in chick brain. In competition studies, melatonin inhibited the binding of 2-[125I]iodo-melatonin with high- and low-affinities of KH = 20 pM and KL = 15 nM, respectively. In the presence of heparin (100 units/ml), a single site with an affinity of about 6 nM was detected. The monovalent cations, Na+ and Li+, produced a greater rightward shift of the agonist competition curve than heparin and converted all high-affinity sites to a low affinity of approximately 15-18 nM. In saturation studies, heparin reduced the affinity of high-affinity sites and caused a significant decrease in the density of low-affinity sites. In keeping with its ability to reduce high-affinity binding, heparin blocked melatonin's inhibitory effect on forskolin-stimulated adenylate cyclase activity in chick brain synaptosomal membranes. These findings indicate that heparin impairs the coupling between the melatonin receptor and Gi, with a consequent decrease in binding affinity and a loss of receptor-mediated signalling.

Physiological regulation of melatonin receptors in rat suprachiasmatic nuclei: diurnal rhythmicity and effects of stress.

A marked diurnal variation in high-affinity binding of 2-[125I]iodomelatonin ([125I]MEL) in rat brain sections containing the suprachiasmatic nuclei (SCN) was observed. Binding was highest late in the light phase and lowest during darkness, in inverse correlation to the serum melatonin rhythm. Interestingly, only high-affinity sites were present during most of the light phase while both high- and low-affinity sites were detected just before and during darkness. Guanosine triphosphate (GTP) in combination with sodium converted all high affinity sites to a low affinity state suggesting that the two sites observed during darkness represent the two states of the melatonin receptor. Acute swim-stress caused a significant elevation of serum melatonin, together with a decrease in the density of [125I]MEL binding in the SCN. The inverse relationship between circulating melatonin levels and binding, under two different physiological conditions, indicates that this hormone is involved in regulating its own receptors in the SCN.