Prediction of exposure-driven myelotoxicity of continuous infusion 5-fluorouracil by a semi-physiological pharmacokinetic-pharmacodynamic model in gastrointestinal cancer patients.

PURPOSE: To describe 5-fluorouracil (5FU) pharmacokinetics, myelotoxicity and respective covariates using a simultaneous nonlinear mixed effect modelling approach. METHODS: Thirty patients with gastrointestinal cancer received 5FU 650 or 1000 mg/m2/day as 5-day continuous venous infusion (14 of whom also received cisplatin 20 mg/m2/day). 5FU and 5-fluoro-5,6-dihydrouracil (5FUH2) plasma concentrations were described by a pharmacokinetic model using NONMEM. Absolute leukocyte counts were described by a semi-mechanistic myelosuppression model. Covariate relationships were evaluated to explain the possible sources of variability in 5FU pharmacokinetics and pharmacodynamics. RESULTS: Total clearance of 5FU correlated with body surface area (BSA). Population estimate for total clearance was 249 L/h. Clearances of 5FU and 5FUH2 fractionally changed by 77%/m2 difference from the median BSA. 5FU central and peripheral volumes of distribution were 5.56 L and 28.5 L, respectively. Estimated 5FUH2 clearance and volume of distribution were 121 L/h and 96.7 L, respectively. Baseline leukocyte count of 6.86 × 109/L, as well as mean leukocyte transit time of 281 h accounting for time delay between proliferating and circulating cells, was estimated. The relationship between 5FU plasma concentrations and absolute leukocyte count was found to be linear. A higher degree of myelosuppression was attributed to combination therapy (slope = 2.82 L/mg) with cisplatin as compared to 5FU monotherapy (slope = 1.17 L/mg). CONCLUSIONS: BSA should be taken into account for predicting 5FU exposure. Myelosuppression was influenced by 5FU exposure and concomitant administration of cisplatin.

Ergothioneine stands out from hercynine in the reaction with singlet oxygen: Resistance to glutathione and TRIS in the generation of specific products indicates high reactivity.

The candidate vitamin ergothioneine (ET), an imidazole-2-thione derivative of histidine betaine, is generally considered an antioxidant. However, the precise physiological role of ET is still unresolved. Here, we investigated in vitro the hypothesis that ET serves specifically to eradicate noxious singlet oxygen (1O2). Pure 1O2 was generated by thermolysis at 37°C of N,N'-di(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide 1,4-endoperoxide (DHPNO2). Assays of DHPNO2 with ET or hercynine (= ET minus sulfur) at pH 7.4 were analyzed by LC-MS in full scan mode to detect products. Based on accurate mass and product ion scan data, several products were identified and then quantitated as a function of time by selected reaction monitoring. All products of hercynine contained, after a [4+2] cycloaddition of 1O2, a carbonyl at position 2 of the imidazole ring. By contrast, because of the doubly bonded sulfur, we infer from the products of ET as the initial intermediates a 4,5-dioxetane (after [2+2] cycloaddition) and hydroperoxides at position 4 and 5 (after Schenck ene reactions). The generation of single products from ET, but not from hercynine, was fully resistant to a large excess of tris(hydroxymethyl)aminomethane (TRIS) or glutathione (GSH). This suggests that 1O2 markedly favors ET over GSH (at least 50-fold) and TRIS (at least 250-fold) for the initial reaction. Loss of ET was almost abolished in 5mM GSH, but not in 25mM TRIS. Regeneration of ET seems feasible, since some ET products - by contrast to hercynine products - decomposed easily in the MS collision cell to become aromatic again.

Control measures following a case of imported Lassa fever from Togo, North Rhine Westphalia, Germany, 2016.

In a patient transferred from Togo to Cologne, Germany, Lassa fever was diagnosed 12 days post mortem. Sixty-two contacts in Cologne were categorised according to the level of exposure, and gradual infection control measures were applied. No clinical signs of Lassa virus infection or Lassa specific antibodies were observed in the 62 contacts. Thirty-three individuals had direct contact to blood, other body fluids or tissue of the patients. Notably, with standard precautions, no transmission occurred between the index patient and healthcare workers. However, one secondary infection occurred in an undertaker exposed to the corpse in Rhineland-Palatinate, who was treated on the isolation unit at the University Hospital of Frankfurt. After German authorities raised an alert regarding the imported Lassa fever case, an American healthcare worker who had cared for the index patient in Togo, and who presented with diarrhoea, vomiting and fever, was placed in isolation and medevacked to the United States. The event and the transmission of Lassa virus infection outside of Africa underlines the need for early diagnosis and use of adequate personal protection equipment (PPE), when highly contagious infections cannot be excluded. It also demonstrates that larger outbreaks can be prevented by infection control measures, including standard PPE.

A novel mode of operation of SLC22A11: Membrane insertion of estrone sulfate versus translocation of uric acid and glutamate.

Estrone sulfate alias estrone-3-sulfate (E3S) is considerably larger and much more hydrophobic than typical substrates of SLC22 transporters. It is puzzling that many otherwise unrelated transporters have been reported to transport E3S. Here we scrutinized the mechanism of transport of E3S by SLC22A11 (alias OAT4), by direct comparison with uric acid (UA), an important physiological substrate. Heterologous expression of SLC22A11 in human 293 cells gave rise to a huge unidirectional efflux of glutamate (Glu) and aspartate, as determined by LC-MS/MS. The uptake of E3S was 20-fold faster than the uptake of UA. Yet, the outward transport of Glu was inhibited by extracellular E3S, but not by UA. The release of E3S after preloading was trans-stimulated by extracellular dehydroepiandrosterone sulfate (DHEAS), but neither by UA nor 6-carboxyfluorescein (6CF). The equilibrium accumulation of E3S was enhanced 3-fold by replacement of chloride with gluconate, but the opposite effect was observed for UA. These results establish that SLC22A11 provides entirely different transport mechanisms for E3S and UA. Therefore, E3S must not be used as a substitute for UA to assay the function of SLC22A11. In equilibrium accumulation experiments, the transporter-mediated uptake was a linear function of the concentration of UA and 6CF. By contrast, in the same concentration range the graph for E3S was hyperbolic. This suggests that SLC22A11 inserts E3S into a small volume with limited capacity, the plasma membrane. Our data support the notion that the reverse process, extraction from the membrane, is also catalyzed by the carrier.

Knockout of the ergothioneine transporter ETT in zebrafish results in increased 8-oxoguanine levels.

Ergothioneine (ET) is a natural compound that humans and other vertebrates must absorb from dietary sources. In general, ET is considered an intracellular antioxidant. However, the precise physiological purpose of ET and the consequences of ET deficiency are still unclear. The ergothioneine transporter ETT (human gene symbol SLC22A4) is a highly specific transporter for the uptake of ET. Here, we sought to identify and knock out ETT from zebrafish (Danio rerio) to determine the function of ET. We cloned and assayed three related proteins from zebrafish, only one of which catalyzed the uptake of ET. RT-PCR analysis revealed that the protein is strongly expressed in the skin, brain, kidney, intestine, and eye. In ETT-knockout animals generated by retroviral insertion into exon 1, ET content was reduced by more than 1000-fold compared to the wild type. Thus, ETT is the sole transporter responsible for uptake of ET into zebrafish. ETT-knockout fish did not exhibit obvious differences in morphology or behavior. In whole-fish homogenates, an increase in 4-hydroxy-2,3-trans-nonenal and malondialdehyde was observed, but only after stress caused by incubation with Pb(2+) or Cu(2+). Comparison of unstressed fish at the level of small molecules by LC-MS difference shading revealed a 3.8-fold increase in 8-oxoguanine (8-oxo-7,8-dihydroguanine) in the skin of ETT-knockout animals. Our knockout represents a new model for examining the consequences of complete absence of ET. Based on the phenotype observed here, we hypothesize that the specific purpose of ET could be to eliminate singlet oxygen.

SLC22A13 catalyses unidirectional efflux of aspartate and glutamate at the basolateral membrane of type A intercalated cells in the renal collecting duct.

In vertebrates, SLC22A13 is an evolutionarily conserved transport protein of the plasma membrane. In humans and rat, it is principally expressed in the kidney. The precise localization and physiological function are unknown. In the present study, immunohistochemistry revealed that expression of SLC22A13 is confined to the basolateral membrane of type A intercalated cells in rat kidney. Double-staining confirmed that SLC22A13 co-localizes with anion exchanger 1. LC-MS difference shading showed that heterologous expression of human and rat SLC22A13 in HEK (human embryonic kidney)-293 cells stimulates efflux of guanidinosuccinate, aspartate, glutamate and taurine. Time courses of uptake of [3H]aspartate and [3H]glutamate revealed that SLC22A13 counteracted endogenous uptake. By contrast, OAT2 (organic anion transporter 2), a bidirectional glutamate transporter, increased accumulation of [3H]glutamate. Thus SLC22A13 catalyses unidirectional efflux. Velocity of efflux of standard amino acids was measured by LC-MS/MS. Expression of SLC22A13 strongly stimulated efflux of aspartate, taurine and glutamate. When the intracellular concentrations of aspartate and taurine were increased by pre-incubation, velocities of efflux increased linearly. We propose that in type A intercalated cells, SLC22A13 compensates luminal exit of protons by mediating the basolateral expulsion of the anions aspartate and glutamate. In this context, unidirectional efflux is essential to avoid anion re-entering. Loss of SLC22A13 function could cause distal tubular acidosis.

Benzoic acid and specific 2-oxo acids activate hepatic efflux of glutamate at OAT2.

The liver is the principal source of glutamate in blood plasma. Recently we have discovered that efflux of glutamate from hepatocytes is catalyzed by the transporter OAT2 (human gene symbol SLC22A7). Organic anion transporter 2 (OAT2) is an integral membrane protein of the sinusoidal membrane domain; it is primarily expressed in liver and much less in kidney, both in rats and humans. Many years ago, Häussinger and coworkers have demonstrated in isolated perfused rat liver that benzoic acid or specific 2-oxo acid analogs of amino acids like e.g. 2-oxo-4-methyl-pentanoate ('2-oxo-leucine') strongly stimulate release of glutamate (up to 7-fold); '2-oxo-valine' and the corresponding amino acids were without effect. The molecular mechanism of efflux stimulation has remained unclear. In the present study, OAT2 from human and rat were heterologously expressed in 293 cells. Addition of 1 mmol/l benzoic acid to the external medium increased OAT2-specific efflux of glutamate up to 20-fold; '2-oxo-leucine' was also effective, but not '2-oxo-valine'. Similar effects were seen for efflux of radiolabeled orotic acid. Expression of OAT2 did not increase uptake of benzoic acid; thus, benzoic acid is no substrate, and trans-stimulation can be excluded. Instead, further experiments suggest that increased efflux of glutamate is caused by direct interaction of benzoic acid and specific 2-oxo acids with OAT2. We propose that stimulators bind to a distinct extracellular site and thereby accelerate relocation of the empty substrate binding site to the intracellular face. Increased glutamate efflux at OAT2 could be the main benefit of benzoate treatment in patients with urea cycle defects.

OAT2 catalyses efflux of glutamate and uptake of orotic acid.

OAT (organic anion transporter) 2 [human gene symbol SLC22A7 (SLC is solute carrier)] is a member of the SLC22 family of transport proteins. In the rat, the principal site of expression of OAT2 is the sinusoidal membrane domain of hepatocytes. The particular physiological function of OAT2 in liver has been unresolved so far. In the present paper, we have used the strategy of LC (liquid chromatography)-MS difference shading to search for specific and cross-species substrates of OAT2. Heterologous expression of human and rat OAT2 in HEK (human embryonic kidney)-293 cells stimulated accumulation of the zwitterion trigonelline; subsequently, orotic acid was identified as an excellent and specific substrate of OAT2 from the rat (clearance=106 µl·min⁻¹·mg of protein⁻¹) and human (46 µl·min⁻¹·mg of protein⁻¹). The force driving uptake of orotic acid was identified as glutamate antiport. Efficient transport of glutamate by OAT2 was directly demonstrated by uptake of [³H]glutamate. However, because of high intracellular glutamate, OAT2 operates as glutamate efflux transporter. Thus expression of OAT2 markedly increased the release of glutamate (measured by LC-MS) from cells, even without extracellular exchange substrate. Orotic acid strongly trans-stimulated efflux of glutamate. We thus propose that OAT2 physiologically functions as glutamate efflux transporter. OAT2 mRNA was detected, after laser capture microdissection of rat liver slices, equally in periportal and pericentral regions; previous reports of hepatic release of glutamate into blood can now be explained by OAT2 activity. A specific OAT2 inhibitor could, by lowering plasma glutamate and thus promoting brain-to-blood efflux of glutamate, alleviate glutamate exotoxicity in acute brain conditions.

Paraoxonase-1 is a major determinant of clopidogrel efficacy.

Clinical efficacy of the antiplatelet drug clopidogrel is hampered by its variable biotransformation into the active metabolite. The variability in the clinical response to clopidogrel treatment has been attributed to genetic factors, but the specific genes and mechanisms underlying clopidogrel bioactivation remain unclear. Using in vitro metabolomic profiling techniques, we identified paraoxonase-1 (PON1) as the crucial enzyme for clopidogrel bioactivation, with its common Q192R polymorphism determining the rate of active metabolite formation. We tested the clinical relevance of the PON1 Q192R genotype in a population of individuals with coronary artery disease who underwent stent implantation and received clopidogrel therapy. PON1 QQ192 homozygous individuals showed a considerably higher risk than RR192 homozygous individuals of stent thrombosis, lower PON1 plasma activity, lower plasma concentrations of active metabolite and lower platelet inhibition. Thus, we identified PON1 as a key factor for the bioactivation and clinical activity of clopidogrel. These findings have therapeutic implications and may be exploited to prospectively assess the clinical efficacy of clopidogrel.

Do activities of cytochrome P450 (CYP)3A, CYP2D6 and P-glycoprotein differ between healthy volunteers and HIV-infected patients?

BACKGROUND: In inflammation and infection, cytochrome P450 (CYP) enzyme activities are down-regulated. Information on possible discrepancies in activities of CYP enzymes and drug transporters between HIV-infected patients and healthy people is limited. METHODS: We used midazolam, dextromethorphan and digoxin as in vivo phenotyping probes for CYP3A (CYP3A4/5), CYP2D6 and P-glycoprotein activities, respectively, and compared these activities between 12 healthy Caucasian volunteers and 30 treatment-naive HIV-infected patients. RESULTS: Among the HIV-infected patients, the overall CYP3A activity (apparent oral midazolam clearance) was approximately 50% of the activity observed in healthy volunteers (point estimate 0.490, 90% confidence interval [CI] 0.377-0.638). The CYP2D6 activity (plasma ratio area under the curve [AUC]; AUC(dextromethorphan)/AUC(dextrorphan)) was essentially unchanged (point estimate 1.289, 90% CI 0.778-2.136). P-glycoprotein activity was slightly lower in patients (digoxin maximum concentration point estimate 1.304, 90% CI 1.034-1.644). CONCLUSIONS: The overall CYP3A activity was approximately 50% lower in HIV-infected patients than in healthy volunteers. The CYP2D6 activity was highly variable, but, on average was not different between groups, whereas a marginally lower P-glycoprotein activity was observed in treatment-naive HIV-infected patients.

Substrate discrimination by ergothioneine transporter SLC22A4 and carnitine transporter SLC22A5: gain-of-function by interchange of selected amino acids.

ETT (originally designated as OCTN1; human gene symbol SLC22A4) and CTT (OCTN2; SLC22A5) are highly specific transporters of ergothioneine and carnitine, respectively. Despite a high degree of sequence homology, both carriers discriminate precisely between substrates: ETT does not transport carnitine, and CTT does not transport ergothioneine. Our aim was to turn ETT into a transporter for carnitine and CTT into a transporter for ergothioneine by a limited number of point mutations. From a multiple alignment of several mammalian amino acid sequences, those positions were selected for conversion that were momentously different between ETT and CTT from human but conserved among all orthologues. Mutants were expressed in 293 cells and assayed for transport of ergothioneine and carnitine. Several ETT mutants clearly catalyzed transport of carnitine, up to 35% relative to wild-type CTT. Amazingly, complementary substitutions in CTT did not provoke transport activity for ergothioneine. In similar contrast, carnitine transport by CTT mutants was abolished by very few substitutions, whereas ergothioneine transport by ETT mutants was maintained even with the construct most active in carnitine transport. To explain these results, we propose that ETT and CTT use dissimilar pathways for conformational change, in addition to incongruent substrate binding sites. In other words, carnitine is excluded from ETT by binding, and ergothioneine is excluded from CTT by turnover movement. Our data indicate amino acids critical for substrate discrimination not only in transmembrane segments 5, 7, 8, and 10, but also in segments 9 and 12 which were hitherto considered as unimportant.

Decreased anxiety in mice lacking the organic cation transporter 3.

The organic cation transporter 3 (OCT3; synonymous: extraneuronal monoamine transporter, EMT, Slc22a3) encodes an isoform of the organic cation transporters and is expressed widely across the whole brain. OCTs are a family of high-capacity, bidirectional, multispecific transporters of organic cations. These also include serotonin, dopamine and norepinephrine making OCTs attractive candidates for a variety of neuropsychiatric disorders including anxiety disorders. OCT3 has been implicated in termination of monoaminergic signalling in the central nervous system. Interestingly, OCT3 mRNA is however also significantly up-regulated in the hippocampus of serotonin transporter knockout mice where it might serve as an alternative reuptake mechanism for serotonin. The examination of the behavioural phenotype of OCT3 knockout mice thus is paramount to assess the role of OCT3. We have therefore subjected mice lacking the OCT3 gene to a comprehensive behavioural test battery. While cognitive functioning in the Morris water maze test and aggression levels measured with the resident-intruder paradigm were in the same range as the respective control animals, OCT3 knockout animals showed a tendency of increased activity and were significantly less anxious in the elevated plus-maze test and the open field test as compared to their respective wild-type controls arguing for a role of OCT3 in the regulation of fear and anxiety, probably by modulating the serotonergic tone in limbic circuitries.

Uptake of mIBG and catecholamines in noradrenaline- and organic cation transporter-expressing cells: potential use of corticosterone for a preferred uptake in neuroblastoma- and pheochromocytoma cells.

For imaging of neuroblastoma and phaeochromocytoma, [(123)I]meta-iodobenzylguanidine ([(123)I]mIBG) is routinely used, whereas [(18)F]6-fluorodopamine ([(18)F]6-FDA) is sporadically applied for positron emission tomography in pheochromocytoma. Both substances are taken up by catecholamine transporters (CATs). In competition, some other cell types are able to take up catecholamines and related compounds probably by organic cation (OCT) [extraneuronal monoamine (EMT)] transporters (OCT1, OCT2, OCT3=EMT). In this study, we investigated the uptake of radioiodine-labeled meta-iodobenzylguanidine (mIBG) as well as [(3)H]dopamine (mimicring 6-fluorodopamine) and [(3)H]noradrenaline. SK-N-SH (neuroblastoma) and PC-12 (phaeochromocytoma) cells were used and compared with HEK-293 cells transfected with OCT1, OCT2 and OCT3, respectively. In order to gain a more selective uptake in CAT expressing tumor cells, different specific inhibitors were measured. Uptake of mIBG into OCT-expressing cells was similar or even better as into both CAT-expressing cell lines, whereas dopamine and noradrenaline uptake was much lower in OCT-expressing cells. In presence of corticosterone (f.c. 10(-4) M], catecholamine and mIBG uptake into SK-N-SH and PC-12 cells was only slightly reduced. In contrast, this process was significantly inhibited in OCT2 and OCT3 transfected HEK-293 as well as in Caki-1 cells, which naturally express OCT3. We conclude that the well-known corticosteroid corticosterone might be used in combination with [(18)F]6-FDA or [(123)I]mIBG to improve specific imaging of neuroblastoma and pheochromocytoma and to reduce irradiation dose to nontarget organs in [(131)I]mIBG treatment.

In vivo role of cytochrome P450 2E1 and glutathione-S-transferase activity for acrylamide toxicokinetics in humans.

Acrylamide, a potential food carcinogen in humans, is biotransformed to the epoxide glycidamide in vivo. Both acrylamide and glycidamide are conjugated with glutathione, possibly via glutathione-S-transferases (GST), and bind covalently to proteins and nucleic acids. We investigated acrylamide toxicokinetics in 16 healthy volunteers in a four-period change-over trial and evaluated the respective role of cytochrome P450 2E1 (CYP2E1) and GSTs. Participants ingested self-prepared potato chips containing acrylamide (1 mg) without comedication, after CYP2E1 inhibition (500 mg disulfiram, single dose) or induction (48 g/d ethanol for 1 week), and were phenotyped for CYP2E1 with chlorzoxazone (250 mg, single dose). Unchanged acrylamide and the mercapturic acids N-acetyl-S-(2-carbamoylethyl)-cysteine (AAMA) and N-acetyl-S-(2-hydroxy-2-carbamoylethyl)-cysteine (GAMA) accounted for urinary excretion [geometric mean (percent coefficient of variation)] of 2.9% (42), 65% (23), and 1.7% (65) of the acrylamide dose in the reference period. Hemoglobin adducts increased clearly following the acrylamide test-meal. The cumulative amounts of acrylamide, AAMA, and GAMA excreted and increases in AA adducts changed significantly during CYP2E1 blockade [point estimate (90% confidence interval)] to the 1.34-fold (1.14-1.58), 1.18-fold (1.02-1.36), 0.44-fold (0.31-0.61), and 1.08-fold (1.02-1.15) of the reference period, respectively, but were not changed significantly during moderate CYP2E1 induction. Individual baseline CYP2E1 activity, CYP2E1*6, GSTP1 313A>G and 341T>C single nucleotide polymorphisms, and GSTM1-and GSTT1-null genotypes had no major effect on acrylamide disposition. The changes in acrylamide toxicokinetics upon CYP2E1 blockade provide evidence that CYP2E1 is a major but not the only enzyme mediating acrylamide epoxidation in vivo to glycidamide in humans. No obvious genetic risks or protective factors in xenobiotic-metabolizing enzymes could be determined for exposed subjects.

The carnitine transporter SLC22A5 is not a general drug transporter, but it efficiently translocates mildronate.

In addition to its function as carnitine transporter, novel organic cation transporter type 2 (OCTN2; human gene symbol SLC22A5) is widely recognized as a transporter of drugs. This notion is based on several reports of direct measurement of drug accumulation. However, a rigorous, comparative, and comprehensive analysis of transport efficiency of OCTN2 has not been available so far. In the present study, OCTN2 orthologs from human, rat, and chicken were expressed in 293 cells using an inducible expression system. Uptake of trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide (ASP(+)), cephaloridine, ergothioneine, gabapentin, mildronate, pyrilamine, quinidine, spironolactone, tetraethylammonium, verapamil, and vigabatrin was determined by liquid chromatography/mass spectrometry. For reference, uptake of carnitine was measured in parallel. Our results indicate that OCTN2-mediated uptake of drugs was not significantly different from zero or, with tetraethylammonium and ergothioneine, was minute relative to carnitine. The carnitine congener mildronate, by contrast, was transported very efficiently. Thus, OCTN2 is not a general drug transporter but a highly specific carrier for carnitine and closely related molecules. Transport parameters (cellular accumulation, transporter affinity, sodium dependence) were similar for mildronate and carnitine. Efficiency of transport of mildronate was even higher than that of carnitine. Hence, our results establish that OCTN2 is a key target of the cardioprotective agent mildronate because it controls, as integral protein of the plasma membrane, cellular entry of mildronate and enables efficient access to intracellular targets. The highest levels of human OCTN2 mRNA were detected by real-time reverse transcription-polymerase chain reaction in kidney, ileum, breast, small intestine, skeletal muscle, and ovary but also in some heart and central nervous system tissues.

Acute and chronic cannabinoid treatment differentially affects recognition memory and social behavior in pubertal and adult rats.

Although cannabis belongs to the most widely used drugs among adolescents, little is known about its acute and lasting neurobehavioral effects during critical developmental periods. In the present study we investigated acute and long-term behavioral effects of the cannabinoid agonist WIN 55,212-2 (WIN) in pubertal and adult rats. Chronic WIN (1.2 mg/kg)/vehicle treatment was extended over 25 days throughout puberty, from postnatal day (pd) 40 to pd 65, or for a similar time period in adult rats (> pd 80). All animals were tested at three time points for object/social recognition memory, social interaction and spontaneous social behavior. First, acute cannabinoid effects were investigated directly after the first injection. Additionally, behavioral performance was retested 24 hours and 15 days after cessation of WIN treatment. Chronic pubertal WIN treatment induced persistent object/social recognition deficits, indicating a general impairment in short-term information processing. Lasting disturbances in social behavior, social play and self-grooming were also found. Furthermore, behavioral deficits seen after acute WIN administration were more pronounced in pubertal than in adult rats. These results confirm our recent findings that chronic pubertal cannabinoid treatment leads to lasting behavioral alterations in adulthood, and they show that acute cannabinoid administration induces more severe behavioral deficits in pubertal rats than in mature animals. We therefore conclude that an immature brain is more susceptible to the acute and chronic effects of exogenous cannabinoids than an adult organism, which might be explained by an overactive endocannabinoid system and concomittant maturational disturbances in further neurotransmitter systems during pubertal development.

Simultaneous fitting of real-time PCR data with efficiency of amplification modeled as Gaussian function of target fluorescence.

BACKGROUND: In real-time PCR, it is necessary to consider the efficiency of amplification (EA) of amplicons in order to determine initial target levels properly. EAs can be deduced from standard curves, but these involve extra effort and cost and may yield invalid EAs. Alternatively, EA can be extracted from individual fluorescence curves. Unfortunately, this is not reliable enough. RESULTS: Here we introduce simultaneous non-linear fitting to determine - without standard curves - an optimal common EA for all samples of a group. In order to adjust EA as a function of target fluorescence, and still to describe fluorescence as a function of cycle number, we use an iterative algorithm that increases fluorescence cycle by cycle and thus simulates the PCR process. A Gauss peak function is used to model the decrease of EA with increasing amplicon accumulation. Our approach was validated experimentally with hydrolysis probe or SYBR green detection with dilution series of 5 different targets. It performed distinctly better in terms of accuracy than standard curve, DART-PCR, and LinRegPCR approaches. Based on reliable EAs, it was possible to detect that for some amplicons, extraordinary fluorescence (EA > 2.00) was generated with locked nucleic acid hydrolysis probes, but not with SYBR green. CONCLUSION: In comparison to previously reported approaches that are based on the separate analysis of each curve and on modelling EA as a function of cycle number, our approach yields more accurate and precise estimates of relative initial target levels.

Novel mutations of the extraneuronal monoamine transporter gene in children and adolescents with obsessive-compulsive disorder.

Obsessive-compulsive disorder (OCD) is a disease of complex aetiology with a marked genetic component. Impact of the serotonergic system has been reported but the contribution of additional transmitter systems to the pathogenesis seems likely. The extraneuronal monoamine transporter, EMT (SLC22A3), is implicated in non-neuronal termination of noradrenergic signalling in the central nervous system and a candidate gene for a variety of neuropsychiatric disorders. We conducted a case-control study of 84 Caucasian children and adolescents with OCD according to DSM-IV criteria, and healthy adults by comprehensive sequencing of the EMT gene. Additionally, targeted genotype analysis was done with patient-parent trios. Known polymorphisms and frequent haplotypes were not associated with OCD in the present sample. Transmission disequilibrium test was negative for the presumptive cryptic splice site 1233G>A polymorphism. However, we identified two novel independent mutations exclusively in affected patients. A thus far unknown -106/107delAG mutation was detected in three male patients of unaffected parents but was not prevalent in 204 healthy subjects (p=0.024). In a luciferase reporter assay the mutant allele conferred increased promoter activity by 36%. Furthermore, we describe the first non-synonymous substitution in the EMT gene, Met370Ile, in a family of affected female members that co-segregated with the disease. The residue exhibits a high degree of inter-species conservation. Heterologous expression of mutant cDNA revealed a 40% decline of transport capacity for norepinephrine. Rare mutations in the EMT gene suggest a causative or modulating role in genetic subtypes of OCD.