A mammalian PAR-3-PAR-6 complex implicated in Cdc42/Rac1 and aPKC signalling and cell polarity.

Cellular asymmetry is critical for the development of multicellular organisms. Here we show that homologues of proteins necessary for asymmetric cell division in Caenorhabditis elegans associate with each other in mammalian cells and tissues. mPAR-3 and mPAR-6 exhibit similar expression patterns and subcellular distributions in the CNS and associate through their PDZ (PSD-95/Dlg/ZO-1) domains. mPAR-6 binds to Cdc42/Rac1 GTPases, and mPAR-3 and mPAR-6 bind independently to atypical protein kinase C (aPKC) isoforms. In vitro, mPAR-3 acts as a substrate and an inhibitor of aPKC. We conclude that mPAR-3 and mPAR-6 have a scaffolding function, coordinating the activities of several signalling proteins that are implicated in mammalian cell polarity.

Expression, activity and regulation of CYP3A in human and rodent brain.

The importance of cytochrome P450 (CYP) 3A enzymes in human drug metabolism is well established. The function of these enzymes has been characterized extensively in liver and intestinal tissues but much less is known about their expression, regulation and functional activity in the brain. Several lines of evidence point to the presence and function of multiple forms of CYP enzymes, including CYP3A, in both human and rodent brain. Expression studies suggest that CYP3A enzymes show regional differences in their distribution in the brain, where they may play a role in steroid metabolism. They also metabolize many psychoactive drugs and may have a profound effect on their efficacy and safety. This review explores the tissue, cellular, and subcellular expression of CYP3A isoforms in human and rodent brain and provides insight into their functional roles and regulation.

Bioavailability and hypoglycemic activity of the semisynthetic bile acid salt, sodium 3alpha,7alpha-dihydroxy-12-oxo-5beta-cholanate, in healthy and diabetic rats.

Previous studies in our laboratory have shown that the semisynthetic bile acid derivative, sodium 3alpha,7alpha-dihydroxy-12-oxo-5beta-cholanate (MKC), has hypoglycemic activity. The aim of this study was to investigate the relationship between the pharmacokinetics and hypoglycemic activity of MKC in healthy and diabetic rats. Groups of healthy and alloxan-induced diabetic rats were dosed intravenously (i.v.) and orally with MKC (4 mg/kg). Blood samples were taken before administration of the dose and at 20, 40, 60, 80, 120, 150, 180, 210 and 240 minutes post-dose. MKC serum concentration was measured by HPLC, and pharmacokinetic parameters determined using the WinNonlin program. The absolute bioavailability of MKC was found to be low in healthy and diabetic rats (29 and 23% respectively) and was not significantly different between the two groups. Mean residence time (MRT), volume of distribution (Vd) and half-life (t1/2) of MKC after oral administration were significantly lower in diabetic than in healthy rats (21, 31 and 29% respectively). After the i.v. dose, the change in blood glucose concentration was not significant in either healthy or diabetic rats. After the oral dose, the decrease in blood glucose concentration was significant, reaching a maximum decrease from baseline of 24% in healthy rats and 15% in diabetic rats. The results suggest that a first-pass effect is crucial for the hypoglycemic activity of MKC, indicating that a metabolite of MKC and/or interference with metabolism and glucose transport is responsible.

Pharmacology of bile acids and their derivatives: absorption promoters and therapeutic agents.

The role of bile acids in pharmacotherapy is reviewed in this article. The therapeutic use of bile has been recognized since ancient times. Previously bile acids were the standard treatment for gallstones where chenodeoxycholic acid and ursodeoxycholic acid were effective in promoting the dissolution of cholesterol gallstones. Today their therapeutic role looks set to expand enormously. Bile acids as absorption promoters have the potential to aid intestinal, buccal, transdermal, ocular, nasal, rectal and pulmonary absorption of various drugs at concentrations that are non-toxic. Keto derivatives of cholic acid, such as 3a,7a,dihydroxy-12-keto-5alpha-cholic acid (sodium salt and methyl ester) are potential modifiers of blood-brain barrier transport and have been shown to promote quinine uptake, enhance the analgesic effect of morphine and prolong the sleeping time induced by pentobarbital. They have also been shown to be hypoglycaemic. Bile acids as therapeutic agents have the potential to produce beneficial effects in sexually transmitted diseases, primary biliary cirrhosis, primary sclerosing cholangitis, gallstones, digestive tract diseases, cystic fibrosis, cancer and diabetes.

Structure and origin of bile acids: an overview.

An overview of the structure and the origin of naturally occurring bile acids is given. Most naturally occurring bile acids belong to the 5beta-series, with hydroxyl groups in the A, B, and C ring of the steroid system. Hydroxyl groups are mostly found at the C3, C6, C7, C12 and C23 positions and are a- rather than beta-oriented. In most bile acids, the A/B ring junction is cis (5beta-series). However, the A ring can be usually present in the more stable (chair) or less stable (boat) conformation. Both B/C and C/D ring junction are trans. With respect to the angular C19-methyl group, the hydrogen atoms at C5 and C8 are cis-oriented whereas those at C9 and C14 are trans-oriented. The archetypal bile acid is 5beta-cholanic acid (3) from which all other C24 bile acids can be derived. In addition to the bile acids with 24 carbons, some naturally occurring C27 bile acids have been identified including di-, tri- and tetra-hydroxy derivatives of coprostanic acid isolated from bile of several reptile species. The most dominant bile acids and their natural sources are given and a selection of naturally occurring bile acids with unusual structures which have been mostly isolated from the bile of reptiles and amphibians is described.

Biosynthesis of bile acids in mammalian liver.

The biosynthesis of bile acids in mammalian liver and its regulation, together with the physiological role of bile acids, are reviewed in this article. Bile acids are biosynthesized from cholesterol in hepatocytes. Several steps are involved including epimerisation of the 3beta-hydroxyl group, reduction of the delta4 double bond to the 5beta-H structural arrangement, introduction of alpha-hydroxyl groups at C7 or C7 and C12 and, finally, oxidative degradation of the side chain by three carbon atoms. This gives the primary bile acids, cholic and chenodeoxycholic acids. Cholesterol-7alpha-hydroxylation is the rate determining step in the biosynthesis of cholic and chenodeoxycholic acids. Feedback regulation of cholesterol biosynthesis occurs by various mechanisms including termination of the synthesis of specific cytochromes P-450, modulation of specific cytosol proteins, short-term changes in the process of phosphorylation-dephosphorylation and changes in the capacity of the cholesterol pool as a substrate. Prior to being exported from the liver, bile acids are conjugated with glycine and taurine to produce the bile salts. After excretion into the intestinal tract, primary bile acids are partly converted to secondary bile acids, deoxycholic and lithocholic acids, by intestinal microorganisms. The majority of bile acids is absorbed from the intestinal tract and returned to the liver via the portal blood, so that only a small fraction is excreted in the feces. Bile acids returned to the liver can be reconjugated and reexcreted into the bile in the process of enterohepatic recycling. In addition to the physiological function of emulsifying lipids in the intestinal tract, bile acids are particularly important in respect of their ability to dissolve and transport cholesterol in the bile.

Chemical and metabolic transformations of selected bile acids.

This article surveys chemical transformations of selected bile acids. Chemical transformations were initially carried out with the aim of determining the structure of bile acids. More recently they have been concerned with bile acid interconversions as well as with the synthesis of steroid hormones, vitamins and therapeutc agents. Studies of similarities and differences in the biosynthesis of bile acids from cholesterol have occupied many researches. However, this article reviews only papers dealing with the synthesis of potential intermediates in the biosynthesis of bile acids. Steroid hormones such as pregnenolone, progesterone and testosterone are synthesized from methyl thiodeoxycholate whereas cortisone is synthesized from methyl deoxycholiate. Numerous papers and patents devoted to the synthesis of ursodeoxycholic acid from cholic or chenodeoxycholic acid testify to its effectiveness in the treatment of cholelithiasis. Chenodeoxycholic acid appears to be an excellent precursor in the synthesis of steroid plant growth regulators, as well as in the synthesis of metabolites and vitamin D analogues. Chirality of bile acids has been exploited in the synthesis of cyclic and acyclic receptors and solvents. Cholic and deoxycholic acids have been used to create new macrocyclic structures which show different capacities to bind and transport other compounds. Another important trend in the chemistry of bile acids is their application in combinatorial chemistry.

Isolation and determination of bile acids.

In this article, the methods of isolation and determination of bile acids are reviewed. Methods for separation of bile acids from cattle and pig bile are given in detail. Isolation of a mixture of cholic acid and deoxycholic acids from cattle bile and their subsequent purification are described. The isolation and purification of hyodeoxycholic acid and other components of pig bile are also included. Methods for the determination of bile acids in various biological samples are reviewed, including enzyme assays, radioimmunoassay, enzyme immunoassay and chromatographic methods. Among chromatographic methods, separation and determination of bile acids by thin-layer chromatography, gas chromatography and high performance liquid chromatography are reviewed. Particular attention is given to the use of high performance liquid chromatography since this has recently been the most commonly applied method for the separation and determination of bile acids.

Data on the catalytic mechanism of thiol peroxidase mimics.

We have recently reported SAR data describing the pharmacological activity of a series of phenyl alkyl selenides and tellurides which catalyse the oxidation of thiols by hydrogen peroxide (H2O2), "The design of redox active thiol peroxidase mimics: dihydrolipoic acid recognition correlates with cytotoxicity and prooxidant action" B. Zadehvakili, S.M. McNeill, J.P. Fawcett, G.I. Giles (2016) [1]. This thiol peroxidase (TPx) activity is potentially useful for a number of therapeutic applications, as it can alter the outcome of oxidative stress related pathologies and modify redox signalling. This article presents data describing the molecular changes that occur to a TPx mimic upon exposure to H2O2, and then the thiol mercaptoethanol, as characterised by UV-vis spectroscopy and HPLC retention time.

The design of redox active thiol peroxidase mimics: Dihydrolipoic acid recognition correlates with cytotoxicity and prooxidant action.

Redox active molecules containing organoselenium or organotellurium groups catalyse the oxidation of cellular thiols by hydrogen peroxide and are currently being developed as therapeutic agents. Potentially these synthetic thiol peroxidase (TPx) mimics can protect cells from oxidative stress by catalysing the reduction of reactive oxygen species by the cellular thiol glutathione, an activity which mimics the function of the antioxidant enzyme glutathione peroxidase. Alternatively they can act as prooxidants by catalysing the oxidation of essential thiol species within the cell. However the structure-activity relationships which determine the choice of thiol substrate, and hence the overall antioxidant or prooxidant outcome of drug administration, remain unknown. We report the first study that relates the pharmacological properties of TPx mimics with their solubility and catalytic activity using different thiol substrates. We used a series of structurally related compounds PhMCnH2n+1 (M=Se, Te; n=4-7) and investigated their ability to catalyse the oxidation of the cellular thiols glutathione and dihydrolipoic acid by hydrogen peroxide. The resulting rate constants (kobs) were then related to compound cytotoxicity and antioxidant versus prooxidant action in A549 cancer cells. The results show that the dihydrolipoic acid kobs values correlate with both cytotoxicity and prooxidant function. This enabled us to define a relationship, IC50=10+280e(-5(DHLA) (kobs)), which allows the prediction of TPx mimic cytotoxicity. In contrast, hydrophobicity and glutathione kobs were unrelated to the compounds' redox pharmacology.

Evidence that brain-derived neurotrophic factor from presynaptic nerve terminals regulates the phenotype of calbindin-containing neurons in the lateral septum.

Brain-derived neurotrophic factor (BDNF) is transported anterogradely in neurons of the CNS and can be released by activity-dependent mechanisms to regulate synaptic plasticity. However, few neural networks have been identified in which the production, transport, and effects of BDNF on postsynaptic neurons can be analyzed in detail. In this study, we have identified such a network. BDNF has been colocalized by immunocytochemistry with tyrosine hydroxylase (TH) in nerve fibers and nerve terminals within the lateral septum of rats. BDNF-containing nerve fibers terminate on a population of calbindin-containing neurons in lateral septum that contain TrkB, the high-affinity receptor for BDNF. Overexpression of BDNF in noradrenergic neurons increased levels of calbindin in septum, as well as in whole-brain lysates. Septal levels of calbindin and BDNF partially decreased after unilateral lesions of the medial forebrain bundle (MFB), induced with 6-hydroxydopamine, a treatment that abolished TH staining. These data suggest that BDNF is anterogradely transported within the MFB in catecholaminergic neurons arising from brainstem nuclei. To determine whether BDNF affects the production of calbindin in lateral septal neurons directly, we tested the effects of BDNF on cultures of septal neurons from embryonic day 16-17 rats. BDNF promoted the expression of calbindin, as well as the arborization of calbindin-containing neurons, but BDNF had no effect on cell division or survival. Together, these results suggest that BDNF, anterogradely transported in catecholaminergic neurons, regulates calbindin expression within the lateral septum.

Whole blood serotonin relates to violence in an epidemiological study.

BACKGROUND: Clinical and animal studies suggest that brain serotonergic systems may regulate aggressive behavior; however, the serotonin/violence hypothesis has not been assessed at the epidemiological level. For study of an epidemiological sample we examined blood serotonin, because certain physiological and behavioral findings suggested that it might serve as an analog marker for serotonergic function. METHODS: Whole blood serotonin was measured in a representative birth cohort of 781 21-year-old women (47%) and men (53%). Violence was measured using cumulative court conviction records and participants' self-reports. Potential intervening factors addressed were: gender, age, diurnal variation, diet, psychiatric medications, illicit drug history, season of phlebotomy, plasma tryptophan, platelet count, body mass, suicide attempts, psychiatric diagnoses, alcohol, tobacco, socioeconomic status, IQ, and overall criminal offending. RESULTS: Whole blood serotonin related to violence among men but not women. Violent men's mean blood serotonin level was 0.48 SD above the male population norm and 0.56 SD above the mean of nonviolent men. The finding was specific to violence, as opposed to general crime, and it was robust across two different methods of measuring violence. Together, the intervening variables accounted for 25% of the relation between blood serotonin and violence. CONCLUSIONS: To our knowledge, this is the first demonstration that an index of serotonergic function is related to violence in the general population.

Pharmacokinetics and pharmacodynamics of single oral doses of albuterol and its enantiomers in humans.

Albuterol (INN, salbutamol) is a beta 2-adrenergic receptor agonist widely used in the treatment of asthma. It is administered clinically as a racemic mixture, but only one enantiomer is active (eutomer) while the other (distomer) has been implicated in causing toxicity. This study used a chiral assay to compare the pharmacokinetics and pharmacodynamics of racemic albuterol with its two enantiomers in a three-way crossover of single oral doses in 12 healthy males. The bioavailability of the eutomer was less than that of the distomer after administration of pure enantiomers and racemate. Apart from causing a small increase in plasma potassium, the distomer had no effect on any pharmacodynamic parameter. The eutomer administered alone was significantly more potent than an equivalent dose given as racemate with regard to its effects on heart rate, QTc interval, plasma potassium levels, and plasma glucose levels. Despite this higher potency, the area under the plasma concentration versus time curve for eutomer after administration of pure eutomer was significantly lower than after administration of the racemate. This difference is probably the result of the more efficient metabolism of the eutomer in the absence of the distomer.

Characterization of dopaminergic midbrain neurons in a DBH:BDNF transgenic mouse.

The neurotrophin brain-derived neurotrophic factor (BDNF) has been implicated in the survival and differentiation of central nervous system neurons, including dopaminergic cells in culture. To determine whether BDNF might play a role in the development of dopaminergic neurons in vivo, we used a previously characterized transgenic mouse (DBH:BDNF) that overexpresses BDNF in adrenergic and noradrenergic neurons as a result of fusion of the BDNF gene to the dopamine beta-hydroxylase (DBH) gene promoter. We quantified dopaminergic neuronal profiles at four midbrain coronal levels and compared DBH:BDNF transgenic animals with wild-type mice of the same genetic background. Analysis of sections immunostained with tyrosine hydroxylase (TH) showed that the mean number of dopaminergic neurons in the four selected midbrain sections was 52% greater (one-way analysis of variance, P < 0.0005) in transgenic mice (2,165 +/- 55 S. E.M., n = 4) than in control mice (1,428 +/- 71 S.E.M., n = 4). The increase in dopaminergic neuron profile count in DBH:BDNF transgenic animals was confirmed by analysis of the pars compacta of the substantia nigra on Nissl-stained sections. Surface area of the reference region of interest containing TH-immunoreactive neurons was similar in transgenic and control mice. Regional analysis of different midbrain areas containing dopaminergic neurons suggested that the increase in cell profile count occurs in a relatively homogeneous manner. Comparison of TH-immunoreactive cell size showed a tendency for smaller neurons in transgenic animals, but the difference was not statistically significant. We conclude that DBH:BDNF transgenic mice show increased number of TH-immunoreactive cells in the midbrain. We propose that BDNF rescues dopaminergic neurons from the perinatal period of developmental cell death as a consequence of increased anterograde transport of the neurotrophin via the coeruleonigral projection.

Antidepressant treatment and chemical sympathectomy fail to modulate alpha 1-adrenoceptor sensitivity in mouse eye.

The mydriatic response to alpha 1-adrenergic agonists was used as a functional index of postsynaptic alpha 1-adrenoceptors in mouse iris dilator muscle. Topical ocular application of methoxamine or phenylephrine caused dose-related mydriasis which was inhibited by pretreatment with prazosin or phentolamine. Chemical sympathectomy with topical 6-hydroxydopamine (6-OHDA) produced supersensitivity to phenylephrine but not methoxamine. Daily antidepressant treatment for 14 days with desipramine (10 mg/kg, i.p.), amitriptyline (10 mg/kg, i.p.), fluoxetine (2 mg/kg, i.p.), or moclobemide (40 mg/kg, i.p.) did not alter the response to methoxamine. Central alpha 1-adrenoceptors labelled with [3H]prazosin were similarly unaffected except for a modest downregulation produced by fluoxetine. These results demonstrate that postsynaptic alpha 1-adrenoceptors in mouse CNS and iris dilator muscle are refractory to manipulations known to alter their sensitivity in other tissues.

The pharmacokinetics of levosalbutamol: what are the clinical implications?

Salbutamol (albuterol) is a beta2-adrenoceptor agonist used as a bronchodilator for the treatment of asthma and as a uterine relaxant for the suspension of premature labour. Salbutamol has been marketed as a racemic mixture, although beta2-agonist activity resides almost exclusively in the (R)-enantiomer. The enantioselective disposition of salbutamol and the possibility that (S)-salbutamol has adverse effects have led to the development of an enantiomerically pure (R)-salbutamol formulation known as levosalbutamol (levalbuterol). Salbutamol is metabolised almost exclusively by sulphotransferase (SULT) 1A3 to an inactive metabolite. (R)-Salbutamol is metabolised up to 12 times faster than (S)-salbutamol. This leads to relatively higher plasma concentrations of (S)- salbutamol following all routes of administration, but particularly following oral administration because of extensive metabolism by the intestine. Enantiomer concentrations are similar for the first hour following an inhaled dose, reflecting the fact that salbutamol in the lung probably undergoes little metabolism. Subsequently, (S)-salbutamol predominates due to absorption and metabolism of the swallowed portion of the inhaled dose. Following oral or inhaled administration of enantiomerically pure salbutamol, a small amount (6%) is converted to the other enantiomer, probably by acid-catalysed racemisation in the stomach. Tissue binding of salbutamol is not enantioselective and plasma protein binding is relatively low. Both enantiomers are actively excreted into the urine. Compared with healthy individuals, patients with asthma do not have substantially different pharmacokinetics of the salbutamol enantiomers, but they do appear to have less drug delivered to the lung following inhaled administration because of their narrowed airways. Levosalbutamol elicits an equal or slightly larger response than an equivalent dose of the racemic mixture. This is probably due to competitive inhibition between the enantiomers at beta-adrenoceptors. Pharmacokinetic-pharmacodynamic relationships for levosalbutamol show relatively large interindividual variations. Functionally significant genetic polymorphisms have been identified for beta2-adrenoceptors, SULT1A3 and organic action transporters, all of which affect the disposition or action of levosalbutamol. Animal, in vitro and some clinical studies have reported deleterious effects of (S)-salbutamol on smooth muscle contractility or lung function. However, well-designed clinical studies in patients with asthma have failed to find evidence of significant toxicity associated with (S)-salbutamol. The clinical consequences of relatively higher plasma concentrations of (S)-salbutamol following administration of racemate remain unclear, but in the absence of clear evidence of toxicity the clinical superiority of levosalbutamol over racemic salbutamol appears to be small.

Too easily lead? Health effects of gasoline additives.

Octane-enhancing constituents of gasoline pose a number of health hazards. This paper considers the relative risks of metallic (lead, manganese), aromatic (e.g., benzene), and oxygenated additives in both industrialized and developing countries. Technological advances, particularly in industrialized countries, have allowed the progressive removal of lead from gasoline and the increased control of exhaust emissions. The developing world, by contrast, has relatively lax environmental standards and faces serious public health problems from vehicle exhaust and the rapid increase in automobile use. Financial obstacles to the modernization of refineries and vehicle fleets compound this problem and the developing world continues to import large quantities of lead additives and other hazardous materials. Progress in decreasing environmental health problems depends both on the adoption of international public health standards as well as efforts to decrease dependence on the private automobile for urban transport.

Distribution of blood lead levels in a birth cohort of New Zealanders at age 21.

Little is known about lead exposure in the general population of young adults. In this study, whole blood lead concentration (PbB) was determined in a sample of the Dunedin Multidisciplinary Health and Development Study, a well-documented birth cohort of New Zealanders aged 21 years in 1993-1994. PbB in those who consented to venipuncture at 21 years of age (n = 779; 411 males, 368 females) was compared to PbB for the same cohort at age 11 years. The PbB at age 21 ranged from 0.4 to 56 micrograms/dl with a geometric mean of 4.5 micrograms/dl (95% CI, 4.3-4.7 micrograms/dl). Only three individuals had a PbB above 30 micrograms/dl. Males had significantly higher PbB than females (geometric mean 6.0 vs. 3.2 micrograms/dl; p < 0.0001). The PbB at age 21 was 53% lower than in the same individuals at age 11 (geometric mean 4.8 vs. 10.2 micrograms/dl; p < 0.001; n = 480) and the correlation between corresponding values was weak (r = 0.19; p < 0.001). PbB at age 21 showed significant associations with high risk occupational activities, recreational exposure, domicile close to a main road, smoking, and male sex. Blood lead concentrations continue to fall in New Zealand, but occupational and recreational activities remain a significant source of lead exposure.

Chlamydia pneumoniae serological status is not associated with asthma in children or young adults.

BACKGROUND: The factors that cause the allergic sensitization and inflammation in asthma still remain to be clarified. A role for Chlamydia pneumoniae has been suggested although serological studies have produced conflicting findings. This study aims to clarify the relationship between asthmatic variables and C. pneumoniae serological status. METHODS: A case-control study was undertaken on an asthma-enriched subset from a longitudinal birth cohort. In all, 198 subjects (96 with self-reported asthma) had C. pneumoniae serology (microimmunofluorescence [MIF] IgG, IgA) undertaken at age 11 and age 21 and assessment made in relation to a number of asthma variables. RESULTS: The only statistically significant finding was in subjects self-reporting asthma at age 21 who had evidence of lower IgG titres (P = 0.046), a finding in the opposite direction to that expected from the hypothesis. Subjects with high IgG titres (> or =128) were less likely to have reported ever having asthma; odds ratio (OR) = 0.29, (95% CI: 0.10-0.87). No association existed between symptoms suggestive of asthma in the previous 12 months and either IgG (P = 0.127) or IgA (P = 0.189) antibody titres at age 21. Likewise, no association was found between symptoms suggestive of asthma in the previous two years and C. pneumoniae IgG antibody titre (P = 0.81) at age 11. There was no evidence of an association with any of the other variables examined at either age 11 or age 21. These included use of inhaled steroids, serum IgE levels, airway responsiveness, skin test evidence of atopy, or smoking status. CONCLUSION: The results of this study suggest that C. pneumoniae infection when diagnosed by MIF serology is not a major risk factor for the development of asthma in children and young adults. The study has not, however, addressed the role this organism may play in specific asthmatic subsets or asthma exacerbations.

Reduction of salbutamol-induced drinking by putative and proven antidepressants.

Salbutamol-induced drinking was determined in rats after 4 days of twice daily treatment with the conventional and putative antidepressants desipramine, clenbuterol or salbutamol. Drinking was also determined 21 h after a single dose of the abovementioned drugs. Both chronic and acute administration of the drugs resulted in a significant reduction in the salbutamol-induced fluid intake. The results suggest that these drugs decrease beta 2-adrenoceptor activity, but that repeated administration is not a prerequisite for the effect.