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Background: Gilles de la Tourette Syndrome is a neurodevelopmental disorder that is caused by the interaction of environment with a complex genetic background. The genetic etiology of the disorder remains, so far, elusive, although multiple promising leads have been recently reported. The recent implication of the histamine decarboxylase (HDC) gene, the key enzyme in histamine production, raises the intriguing hypothesis of a possible role of histaminergic dysfunction leading to TS onset., Methods: Following up on the finding of a nonsense mutation in a single family with TS, we investigated variation across the HDC gene for association with TS. As a result of a collaborative international effort, we studied a large sample of 520 nuclear families originating from seven European populations (Greek, Hungarian, Italian, Polish, German, Albanian, Spanish) as well as a sample collected in Canada., Results and Conclusions: Interrogating 12 tagging SNPs (tSNP) across the HDC region, we find strong over-transmission of alleles at two SNPs (rs854150 and rs1894236) in the complete sample, as well as a statistically significant associated haplotypes. Analysis of individual populations also reveals signals of association in the Canadian, German and Italian samples. Our results provide strong support for the histaminergic hypothesis in TS etiology and point to a possible role of histamine pathways in neuronal development.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is prone to inactivation from non-productive binding of sugar-phosphates. Reactivation of Rubisco requires conformational remodeling by a specific chaperone, Rubisco activase. Rubisco activase from tobacco and other plants in the family Solanaceae is an inefficient activator of Rubisco from non-Solanaceae plants and from the green alga Chlamydomonas reinhardtii. To determine if the Rubisco small subunit plays a role in the interaction with Rubisco activase, a hybrid Rubisco (SSNT) composed of tobacco small subunits and Chlamydomonas large subunits was constructed. The SSNT hybrid, like other hybrid Rubiscos containing plant small subunits, supported photoautotrophic growth in Chlamydomonas, but growth in air was much slower than for cells containing wild-type Rubisco. The kinetic properties of the SSNT hybrid Rubisco were similar to the wild-type enzyme, indicating that the poor growth in air was probably caused by disruption of pyrenoid formation and the consequent impairment of the CO2concentrating mechanism. Recombinant Rubisco activase from Arabidopsis activated the SSNT hybrid Rubisco and hybrid Rubiscos containing spinach and Arabidopsis small subunits at rates similar to the rates with wild-type Rubisco. However, none of the hybrid Rubiscos was activated by tobacco Rubisco activase. That replacement of Chlamydomonas small subunits with plant small subunits does not affect the species-specific interaction between Rubisco and Rubisco activase suggests that the association is not dominated by the small subunits that surround the Rubisco central solvent channel. Therefore, the geometry of a side-on binding mode is more consistent with the data than a top-on or ring-stacking binding mode.

Background: To detect attention deficit hyperactivity disorder (ADHD) in treatment seeking substance use disorders (SUD) patients, a valid screening instrument is needed., Objectives: To test the performance of the Adult ADHD Self-Report Scale V 1.1(ASRS) for adult ADHD in an international sample of treatment seeking SUD patients for DSM-IV-TR; for the proposed DSM-5 criteria; in different subpopulations, at intake and 1-2 weeks after intake; using different scoring algorithms; and different externalizing disorders as external criterion (including adult ADHD, bipolar disorder, antisocial and borderline personality disorder)., Methods: In 1138 treatment seeking SUD subjects, ASRS performance was determined using diagnoses based on Conner's Adult ADHD Diagnostic Interview for DSM-IV (CAADID) as gold standard., Results: The prevalence of adult ADHD was 13.0% (95% CI: 11.0-15.0%). The overall positive predictive value (PPV) of the ASRS was 0.26 (95% CI: 0.22-0.30), the negative predictive value (NPV) was 0.97 (95% CI: 0.96-0.98). The sensitivity (0.84, 95% CI: 0.76-0.88) and specificity (0.66, 95% CI: 0.63-0.69) measured at admission were similar to the sensitivity (0.88, 95% CI: 0.83-0.93) and specificity (0.67, 95% CI: 0.64-0.70) measured 2 weeks after admission. Sensitivity was similar, but specificity was significantly better in patients with alcohol compared to (illicit) drugs as the primary substance of abuse (0.76 vs. 0.56). ASRS was not a good screener for externalizing disorders other than ADHD., Conclusions: The ASRS is a sensitive screener for identifying possible ADHD cases with very few missed cases among those screening negative in this population., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Background: Declining activity in children over the past decades is thought to be one of the main risk factors for an early development of exercise intolerance and obesity. Taking this background into account, this prospective study investigated the seasonal change of children's physical activity and its association with objective measures of exercise capacity., Methods: A total of 96 children from two schools in Munich (42 girls, age 12.4 ± 0.8 y) underwent a cardiopulmonary exercise test (CPET) and an assessment of their daily activities (school sport, club sport, leisure sport) twice. Baseline testing was conducted in summer 2011. Follow-up examination was performed during winter 2012., Results: From summer to winter, self-reported sporting activity decreased from 10.6 ± 4.1 to 8.5 ± 4.3 h/wk (P < 0.001) as school sport (P < 0.001) and leisure sport activities (P = 0.002) decreased, but the activity associated with club sport did not (P = 0.700). In parallel, peak oxygen uptake (VO2) declined from 102.0 ± 17.5 to 96.9 ± 17.9 % of predicted (P < 0.001). This decline in VO2 was associated with a reduction in overall sporting activity (r = 0.234; P < 0.032)., Conclusion: Enhancing sporting activity in children during winter might be important to maintaining their exercise capacity.

Attention deficit/hyperactivity disorder (ADHD) is an increasingly recognized comorbid condition in subjects with substance use disorders (SUDs). This paper describes the methods and study population of the International ADHD in Substance Use Disorders Prevalence (IASP) study. Objectives of the IASP are to determine the prevalence of ADHD in adult treatment seeking patients with SUD in different countries and SUD populations, determine the reliability and validity of the Adult ADHD Self-report Scale V 1.1 (ASRS) as ADHD screening instrument in SUD populations, investigate the comorbidity profile of SUD patients with and without ADHD, compare risk factors and protective factors in SUD patients with and without a comorbid diagnosis of ADHD, and increase our knowledge about the relationship between ADHD and the onset and course of SUD. In this cross-sectional, multi-centre two stage study, subjects were screened for ADHD with the ASRS, diagnosed with the Conner's Adult ADHD Diagnostic Interview for DSM-IV (CAADID), and evaluated for SUD, major depression, bipolar disorder, anti social personality disorder and borderline personality disorder. Three thousand five hundred and fifty-eight subjects from 10 countries were included. Of these 40.9% screened positive for ADHD. This is the largest international study on this population evaluating ADHD and comorbid disorders., (Copyright © 2013 John Wiley & Sons, Ltd.)

Background: Heroin dependence is a debilitating psychiatric disorder with complex inheritance. Since the dopaminergic system has a key role in rewarding mechanism of the brain, which is directly or indirectly targeted by most drugs of abuse, we focus on the effects and interactions among dopaminergic gene variants., Objective: To study the potential association between allelic variants of dopamine D2 receptor (DRD2), ANKK1 (ankyrin repeat and kinase domain containing 1), dopamine D4 receptor (DRD4), catechol-O-methyl transferase (COMT) and dopamine transporter (SLC6A3) genes and heroin dependence in Hungarian patients., Methods: 303 heroin dependent subjects and 555 healthy controls were genotyped for 7 single nucleotide polymorphisms (SNPs) rs4680 of the COMT gene; rs1079597 and rs1800498 of the DRD2 gene; rs1800497 of the ANKK1 gene; rs1800955, rs936462 and rs747302 of the DRD4 gene. Four variable number of tandem repeats (VNTRs) were also genotyped: 120 bp duplication and 48 bp VNTR in exon 3 of DRD4 and 40 bp VNTR and intron 8 VNTR of SLC6A3. We also perform a multivariate analysis of associations using Bayesian networks in Bayesian multilevel analysis (BN-BMLA)., Findings and Conclusions: In single marker analysis the TaqIA (rs1800497) and TaqIB (rs1079597) variants were associated with heroin dependence. Moreover, -521 C/T SNP (rs1800955) of the DRD4 gene showed nominal association with a possible protective effect of the C allele. After applying the Bonferroni correction TaqIB was still significant suggesting that the minor (A) allele of the TaqIB SNP is a risk component in the genetic background of heroin dependence. The findings of the additional multiple marker analysis are consistent with the results of the single marker analysis, but this method was able to reveal an indirect effect of a promoter polymorphism (rs936462) of the DRD4 gene and this effect is mediated through the -521 C/T (rs1800955) polymorphism in the promoter.

Carotid to femoral pulse wave velocity (PWV) is associated with an increase in cardiovascular morbidity and all-cause mortality. Noninvasive approach has made this method applicable for the examination of larger populations. This study aimed to obtain reference values of PWV measured with the Vicorder device. PWV was obtained using the oscillometric Vicorder in 318 healthy, normotensive patients (165 women, 28.7±17.6 years, range 6-83 years). A plethysmographic sensor was placed over the right carotid region to pick up the carotid pulse wave and a blood pressure cuff was placed around the upper thigh to trace the femoral pulse wave. Path length was defined as the distance from the suprasternal notch to the top of the thigh cuff. Mean PWV was 6.1±1.4 m/s and significantly increased with age (r=.842; P<.0001). PWV was associated with mean arterial pressure (r=.546; P<.0001) and body mass index (r=.396; P<.0001). In a multiple linear regression model, age, mean arterial pressure, and body height emerged as independent markers for PWV. This study established reference values for carotid to femoral PWV derived by oscillometric measures that can now be used for risk stratification., (© 2012 Wiley Periodicals, Inc.)

Mutations in the gene OCA2 are responsible for oculocutaneous albinism type 2, but polymorphisms in and around OCA2 have also been associated with normal pigment variation. In Europeans, three haplotypes in the region have been shown to be associated with eye pigmentation and a missense SNP (rs1800407) has been associated with green/hazel eyes (Branicki et al. in Ann Hum Genet 73:160-170, 2009). In addition, a missense mutation (rs1800414) is a candidate for light skin pigmentation in East Asia (Yuasa et al. in Biochem Genet 45:535-542, 2007; Anno et al. in Int J Biol Sci 4, 2008). We have genotyped 3,432 individuals from 72 populations for 21 SNPs in the OCA2-HERC2 region including those previously associated with eye or skin pigmentation. We report that the blue-eye associated alleles at all three haplotypes were found at high frequencies in Europe; however, one is restricted to Europe and surrounding regions, while the other two are found at moderate to high frequencies throughout the world. We also observed that the derived allele of rs1800414 is essentially limited to East Asia where it is found at high frequencies. Long-range haplotype tests provide evidence of selection for the blue-eye allele at the three haplotyped systems but not for the green/hazel eye SNP allele. We also saw evidence of selection at the derived allele of rs1800414 in East Asia. Our data suggest that the haplotype restricted to Europe is the strongest marker for blue eyes globally and add further inferential evidence that the derived allele of rs1800414 is an East Asian skin pigmentation allele.

Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) activase functions as a mechano-chemical motor protein using the energy from ATP hydrolysis to contort the structure of its target protein, Rubisco. This action modulates the activation state of Rubisco by removing tightly-bound inhibitory sugar-phosphates from Rubisco's catalytic sites, thereby restoring the sites to catalytic competence. This chapter reports methods developed for assaying the two activities of Rubisco activase: ATP hydrolysis and Rubisco activation.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a multifunctional enzyme that catalyzes the fixation of CO2 and O2 in photosynthesis and photorespiration, respectively. As the rate-limiting step in photosynthesis, improving the catalytic properties of Rubisco has long been viewed as a viable strategy for increasing plant productivity. Advances in biotechnology have made this goal more attainable by making it possible to modify Rubisco in planta. To properly evaluate the properties of Rubisco, it is necessary to isolate the enzyme in pure form. This chapter describes procedures for rapid and efficient purification of Rubisco from leaves of several species.

Rubisco activase is a molecular chaperone that modulates the activation state of Rubisco by catalyzing the ATP-dependent removal of tightly-bound inhibitory sugar-phosphates from Rubisco's catalytic sites. This chapter reports methods developed for the purification of native and recombinant Rubisco activase from leaves and bacterial cells, respectively.

The stems and roots of the semiarid shrub guayule, Parthenium argentatum, contain a significant amount of natural rubber. Rubber accumulates in guayule when plants are vegetatively and reproductively dormant, complicating the relationship between growth/reproduction and product synthesis. To evaluate the factors regulating the partitioning of carbon to rubber, carbon assimilation and partitioning were measured in guayule plants that were grown under simulated summer- and winter-like conditions and under winter-like conditions with CO(2) enrichment. These conditions were used to induce vegetatively active and dormant states and to increase the source strength of vegetatively dormant plants, respectively. Rates of CO(2) assimilation, measured under growth temperatures and CO(2) , were similar for plants grown under summer- and winter-like conditions, but were higher with elevated CO(2) . After 5 months, plants grown under summer-like conditions had the greatest aboveground biomass, but the lowest levels of non-structural carbohydrates and rubber. In contrast, the amount of resin in the stems was similar under all growth conditions. Emission of biogenic volatile compounds was more than three-fold higher in plants grown under summer- compared with winter-like conditions. Taken together, the results show that guayule plants maintain a high rate of photosynthesis and accumulate non-structural carbohydrates and rubber in the vegetatively dormant state, but emit volatile compounds at a lower rate when compared with more vegetatively active plants. Enrichment with CO(2) in the vegetatively dormant state increased carbohydrate content but not the amount of rubber, suggesting that partitioning of assimilate to rubber is limited by sink strength in guayule., (Copyright © Physiologia Plantarum 2010.)

Direct amplitude and phase shaping of mid-infrared femtosecond pulses is realized with a calomel-based acousto-optic programmable dispersive filter transparent between 0.4 and 20 μm. The shaped pulse electric field is fully characterized with high accuracy, using chirped-pulse upconversion and time-encoded arrangement spectral phase interferometry for direct electric field reconstruction techniques. Complex mid-infrared pulse shapes at a center wavelength of 4.9 μm are generated with a spectral resolution of 14 cm(-1), which exceeds by a factor of 5 the reported experimental resolutions of calomel-based filters.

In plants, isoprene plays a dual role: (a) as thermo-protective agent proposed to prevent degradation of enzymes/membrane structures involved in photosynthesis, and (b) as reactive molecule reducing abiotic oxidative stress. The present work addresses the question whether suppression of isoprene emission interferes with genome wide transcription rates and metabolite fluxes in grey poplar (Populus x canescens) throughout the growing season. Gene expression and metabolite profiles of isoprene emitting wild type plants and RNAi-mediated non-isoprene emitting poplars were compared by using poplar Affymetrix microarrays and non-targeted FT-ICR-MS (Fourier transform ion cyclotron resonance mass spectrometry). We observed a transcriptional down-regulation of genes encoding enzymes of phenylpropanoid regulatory and biosynthetic pathways, as well as distinct metabolic down-regulation of condensed tannins and anthocyanins, in non-isoprene emitting genotypes during July, when high temperature and light intensities possibly caused transient drought stress, as indicated by stomatal closure. Under these conditions leaves of non-isoprene emitting plants accumulated hydrogen peroxide (H(2)O(2)), a signaling molecule in stress response and negative regulator of anthocyanin biosynthesis. The absence of isoprene emission under high temperature and light stress resulted transiently in a new chemo(pheno)type with suppressed production of phenolic compounds. This may compromise inducible defenses and may render non-isoprene emitting poplars more susceptible to environmental stress.

Background: Candidate genes of the dopaminergic system have been reported as key elements in shaping human temperament. Catechol-O-methyltransferase (COMT) plays a vital role in dopamine inactivation, and the Val(158)Met single nucleotide polymorphism (rs4680) in its gene has been recently associated with the Novelty Seeking (NS) temperament scale of the Temperament and Character Inventory in studies of healthy adults, as well as methamphetamine abusers., Method: Our goal was to examine the association between temperament dimensions of the Temperament and Character Inventory and the COMT Val(158)Met variation in a Hungarian sample of 117 heroin-dependent patients and 124 nondependent controls., Results: Case-control analysis did not show any significant difference in allele or genotype distributions. However, dimensional approach revealed an association between the COMT Val(158)Met and NS (P = .01): both controls and opiate users with Met/Met genotypes showed higher NS scores compared to those with the Val allele. The NS scores are also significantly higher among opiate users; however, no interaction was found between group status and COMT genotype., Conclusion: Association of the COMT polymorphism and NS temperament scale has been shown for heroin-dependent patients and controls regardless of group status., (Copyright 2010 Elsevier Inc. All rights reserved.)

Inhibition of photosynthesis by heat has been linked to the instability of the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) chaperone, Rubisco activase. Examination of the recombinant enzyme showed that ADP and ATP protected against inactivation, whereas Mg(2+) promoted inactivation. Heating caused aggregation of Rubisco activase characterized by disruption of secondary structure content and formation of insoluble protein. In contrast, incubation at room temperature without nucleotide caused the active approximately 660 kDa protein to form a soluble, but inactive aggregate of > 2 x 10(6) Da. Circular dichroism (CD) spectroscopy and fluorescence established that structural perturbations in the aggregate did not reduce alpha-helical content significantly. Differences in the thermal stability between wild type and mutant Rubisco activase were observed for the recombinant proteins and when the proteins were expressed in transgenic Arabidopsis. That the sensitivity of these plants to heat differs indicates that the thermal instability of Rubisco activase is a main determinant of the temperature-sensitivity of photosynthesis., (Published by Elsevier Inc.)

The polymorphic inversion on 17q21, sometimes called the microtubular associated protein tau (MAPT) inversion, is an approximately 900 kb inversion found primarily in Europeans and Southwest Asians. We have identified 21 SNPs that act as markers of the inverted, i.e., H2, haplotype. The inversion is found at the highest frequencies in Southwest Asia and Southern Europe (frequencies of approximately 30%); elsewhere in Europe, frequencies vary from < 5%, in Finns, to 28%, in Orcadians. The H2 inversion haplotype also occurs at low frequencies in Africa, Central Asia, East Asia, and the Americas, though the East Asian and Amerindian alleles may be due to recent gene flow from Europe. Molecular evolution analyses indicate that the H2 haplotype originally arose in Africa or Southwest Asia. Though the H2 inversion has many fixed differences across the approximately 900 kb, short tandem repeat polymorphism data indicate a very recent date for the most recent common ancestor, with dates ranging from 13,600 to 108,400 years, depending on assumptions and estimation methods. This estimate range is much more recent than the 3 million year age estimated by Stefansson et al. in 2005., (Copyright (c) 2010 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

To investigate the interactive effects of increasing [CO(2)] and heat wave occurrence on isoprene (IE) and methanol (ME) emissions, Platanus orientalis was grown for one month in ambient (380 micromol mol(-1)) or elevated (800 micromol mol(-1)) [CO(2)] and exposed to high temperature (HT) (38 degrees C/4 h). In pre-existing leaves, IE emissions were always higher but ME emissions lower as compared to newly-emerged leaves. They were both stimulated by HT. Elevated [CO(2)] significantly reduced IE in both leaf types, whereas it increased ME in newly-emerged leaves only. In newly-emerged leaves, elevated [CO(2)] decreased photosynthesis and altered the chloroplast ultrastructure and membrane integrity. These harmful effects were amplified by HT. HT did not cause any unfavorable effects in pre-existing leaves, which were characterized by inherently higher IE rates. We conclude that: (1) these results further prove the isoprene's putative thermo-protective role of membranes; (2) HT may likely outweigh the inhibitory effects of elevated [CO(2)] on IE in the future.

Heroin dependence is a disorder with complex inheritance. It is influenced by multiple genetic and environmental factors. This paper gives an overview on the specific risk factors in the background of heroin addiction, especially within the dopaminergic system, which is one of the most important components of the brain's reward system. In connection with the development of heroin addiction the role of the dopamine D2 and D4 receptors, the dopamine transporter and the catechol-O-methyltransferase genes is discussed. Certain polymorphisms of the most extensively studied dopamine D2 receptor gene show strong association with heroin dependence. Dopamine D4 receptor and catechol-O-methyltransferase genes are also associated with the disease, but some results are still controversial. Only few studies have been done in association with the dopamine transporter gene and substance misuse and no convincing results have been found. To unravel the contradictions and better understand the pathogenesis of the disease more research needs to be conducted.

Summary: Black poplar (Populus nigra L.) plants grown at 25 and 35 degrees C were subjected to drought stress to assess the combined impact of two consequences of global climate change--rising temperature and drought--on isoprene biosynthesis and emission. At both temperatures, photosynthesis was inhibited by moderate drought, but isoprene emission only decreased when drought was prolonged. The mRNA transcript level, protein concentration and activity of isoprene synthase (ISPS) changed in concert with isoprene emission during drought stress. However, ISPS activity decreased before isoprene emission during drought development, indicating a tighter control of the emission at a transcriptional or post-transcriptional level under moderate drought stress, and at both temperatures. A residual isoprene emission was measured when photosynthesis was totally inhibited after 35 days of drought. This photosynthesis-independent emission of isoprene was probably dependent on a cytosolic carbon supply as all the properties of ISPS were drastically inhibited. Isoprene emission was associated with dark respiration during the entire drought stress period, and at both temperatures, indicating that the two processes are sustained by, but do not compete for, the same carbon source. Isoprene emission was directly related to phosphoenolpyruvate carboxylase activity in plants grown at 25 degrees C and inversely related in plants grown at 35 degrees C, suggesting a strong temperature control on the regulation of the pyruvate flowing from the cytosol to the plastidic isoprenoid biosynthetic pathway under drought stress and recovery. In re-watered plants, the temperature control on isoprene emission was suppressed, despite complete recovery of photosynthesis and ISPS activity similar to levels in plants subjected to mild drought stress. Our results reveal the overriding effects of drought on isoprene emission, possibly affecting protein level or substrate supply. These effects may largely offset the predicted impact of rising temperatures on the emission of isoprene in terrestrial ecosystems.

Gilles de la Tourette syndrome (GTS) is a complex disorder with a clear genetic component but no clearly identified genes with variation of etiologic relevance. Various candidate regions and genes show some evidence of affecting risk, though clearly not all patients/families can be explained by any one of them. Resequencing one candidate gene, SLITRK1, has identified four new variants. Including them, we have typed over 2,300 normal individuals from 44 populations for 11 SNPs spanning the gene. The unusual global pattern seen is that one non-ancestral haplotype is the single most common haplotype worldwide. Other haplotypes appear to result from accumulation of mutations with no evidence of historical recombination. Although there is no evidence of selection, the haplotype frequency variation seen around the world will need to be considered in any future association studies of this locus with GTS or any other neuropsychiatric disorder., ((c) 2007 Wiley-Liss, Inc.)

The ADH1B Arg47His polymorphism has been convincingly associated with alcoholism in numerous studies of several populations in Asia and Europe. In a review of literature from the past 30 years, we have identified studies that report allele frequencies of this polymorphism for 131 population samples from many different parts of the world. The derived ADH1B*47His allele reaches high frequencies only in western and eastern Asia. To pursue this pattern, we report here new frequency data for 37 populations. Most of our data are from South and Southeast Asia and confirm that there is a low frequency of this allele in the region between eastern and western Asia. The distribution suggests that the derived allele increased in frequency independently in western and eastern Asia after humans had spread across Eurasia.

Several studies suggested a possible link between substance use disorder and attention deficit hyperactivity syndrome (ADHD). The ADHD Rating Scale (ADHD-RS) completed by parents is a tool for diagnosing ADHD in childhood. We adapted this questionnaire for a self-report retrospective scale to estimate the presence of childhood ADHD symptoms in adults. This retrospective questionnaire was completed by 121 heroin dependent patients and 85 age- and sex-matched healthy controls. The ADHD Rating Scale Retrospective Questionnaire is a novel tool for assessing ADHD symptoms that demonstrated high validity. Our results showed strong gender difference in the prevalence of ADHD symptoms, since male subjects obtained higher mean scores of both attention-deficit and hyperactivity scales than females in both the control and the heroin dependent population. Besides, mean score of both scales were higher in the clinical population as a higher portion of substance abusers reported symptoms of childhood ADHD than controls. These results support the hypothesis that untreated childhood ADHD could be a risk factor for developing substance use disorder.

In past studies, it was hypothesized that reductions in chloroplast isoprene emissions at high atmospheric CO(2) concentrations were caused by competition between cytosolic and mitochondrial processes for the same substrate, possibly phosphoenolpyruvate (PEP). We conducted field and laboratory experiments using leaves of white poplar (Populus alba L.) to identify whether an inverse relationship occurs between the dark respiration rate (a mitochondrial process) and the isoprene emission rate. Field experiments that were carried out in a free-air CO(2)-enriched (FACE) facility showed no clear effect of elevated CO(2) on either isoprene emission rate or respiration rate by leaves. In young, not yet fully expanded leaves, low isoprene emission and high dark respiration rates were measured in both ambient and elevated CO(2). In these leaves, isoprene emission was inversely correlated with dark respiration. It is possible to interpret from these results that, in young leaves, high rates of growth respiration compete with isoprene biosynthesis for the same substrate. However, it is also possible that the negative correlation reflects the contrasting reductions in growth respiration and increases in expression of the enzyme isoprene synthase at this final stage of leaf maturation. In contrast to our observations on young leaves, respiration rate and isoprene emission rate were positively correlated in older, fully expanded leaves (8 and 11 from apex). A positive correlation was also found between respiration rate and isoprene emission rate when these parameters were modulated using different ozone exposure, growth light intensity, growth temperature and exposure to different leaf temperatures in laboratory experiments. These data show that competition for substrate between isoprene biosynthesis and leaf respiration does not determine the rate of isoprene emission in most circumstances that affect both processes. A negative correlation was observed across all experiments between isoprene emission rate and the activity of phosphoenolpyruvate carboxylase (PEPc), a cytosolic enzyme that competes with isoprene biosynthesis for substrate. The cytosolic metabolite, PEP, occurs at a metabolic branch point from which substrate flows into three processes: (1) the production of pyruvate for mitochondrial respiration, (2) the production of oxaloacetate (OAA) by PEPc for anabolic support of mitochondrial respiration and (3) transport into the chloroplast to support chloroplastic demands for pyruvate, including isoprenoid biosynthesis. The results of our observations suggest that only the second process competes for substrate with isoprenoid synthesis, while the partitioning of PEP between mitochondrial respiration and chloroplast isoprenoid biosynthesis is controlled in a way that retains balance in substrate demand.

The mechanism uncoupling isoprene emission and photosynthesis under drought was investigated in Populus alba saplings. Isoprene emission, incorporation of 13C into the isoprene molecule, isoprene synthase (ISPS) activity, concentration and gene expression, and photosynthesis were measured as a function of the fraction of transpirable soil water (FTSW) and in plants recovering from drought. Photosynthesis sharply declined below FTSW30 (a FTSW of 30%) and its inhibition was not caused by metabolic factors. A decline in isoprene emission was only evident towards the FTSW endpoint. 13C incorporation into isoprene was lower when photosynthesis was constrained by drought. ISPS activity was inhibited by mild drought, while ISPS gene expression and concentration declined in concert with isoprene emission at the FTSW endpoint. Following rewatering, isoprene emission was higher than, and photosynthesis was similar to, prestress values. ISPS activity and concentration, and 13C incorporation into isoprene, also rapidly recovered to prestress values, while ISPS gene expression remained low in rewatered plants. Our experiment revealed a larger contribution of alternative carbon sources to isoprene emission only when photosynthesis was dramatically constrained by drought. Isoprene emission was likely controlled at the posttranscriptional level under severe drought.

Among the volatile organic compounds (VOCs) emitted by plants, some are characteristic of stress conditions, but their biosynthesis and the metabolic and environmental control over the emission are still unclear. We performed experiments to clarify whether (1) the emission following wounding can occur at distance from the wounding site, from VOC pools subjected to metabolic signals; and (2) the emission of biogenic VOCs generated by membrane damage (e.g. consequent to wounding or ozone exposure) can also be induced by exposure to high light and high temperature, recurrent in nature. In Phragmites australis, leaf cutting caused large and rapid bursts of acetaldehyde both at the cutting site and on parts of the cut leaf distant from the cutting site. This emission was preceded by a transient stomatal opening and did not occur in conditions preventing stomatal opening. This suggests the presence of a large pool of leaf acetaldehyde whose release is under stomatal control. VOCs other than isoprene, particularly acetaldehyde and (E)-2-hexenal, one of the C-6 compounds formed by the denaturation of membrane lipids, were released by leaves exposed to high temperature and high light. The high-temperature treatment (45 degrees C) also caused a rapid stimulation and then a decay of isoprene emission in Phragmites leaves. Isoprene recovered to the original emission level after suspending the high-temperature treatment, suggesting a temporary deficit of photosynthetically formed substrate under high temperature. Emission of C-6 compounds was slowly induced by high temperature, and remained high, indicating that membrane denaturation occurs also after suspending the high-temperature treatment. Conversely, the emission of C-6 compounds was limited to the high-light episode in Phragmites. This suggests that a membrane denaturation may also occur in conditions that do not damage other important plant processes such as the photochemistry of photosynthesis of photoinhibition-insensitive plants. In the photoinhibition-sensitive Arabidopsis thaliana mutant NPQ1, a large but transient emission of (E)-2-hexenal was also observed a few minutes after the high-light treatment, indicating extensive damage to the membranes. However, (E)-2-hexenal emission was not observed in Arabidopsis plants fumigated with isoprene during the high-light treatment. This confirms that isoprene can effectively protect cellular membranes from denaturation. Our study indicates that large, though often transient, VOC emissions by plants occur in nature. In particular, we demonstrate that VOCs can be released by much larger tissues than those wounded and that even fluctuations of light and temperature regularly observed in nature can induce their emissions. This knowledge adds information that is useful for the parameterization of the emissions and for the estimate of biogenic VOC load in the atmosphere.

It was investigated whether the methyl-erythritol phosphate (MEP) pathway that generates volatile isoprenoids and carotenoids also produces foliar abscisic acid (ABA) and controls stomatal opening. When the MEP pathway was blocked by fosmidomycin and volatile isoprenoid emission was largely suppressed, leaf ABA content decreased to about 50% and leaf stomatal conductance increased significantly. No effect of fosmidomycin was seen in leaves with constitutively high rates of stomatal conductance and in plant species with low foliar ABA concentration. In all other cases, isoprene emission was directly associated with foliar ABA, but ABA reduction upon MEP pathway inhibition was also observed in plant species that do not emit isoprenoids. Stomatal closure causing a midday depression of photosynthesis was also associated with a concurrent increase of isoprene emission and ABA content. It is suggested that the MEP pathway generates a labile pool of ABA that responds rapidly to environmental changes. This pool also regulates stomatal conductance, possibly when coping with frequent changes of water availability. MEP pathway inhibition by leaf darkening, and its down-regulation by exposure to elevated CO2, was also associated with a reduction of foliar ABA content. However, stomatal conductance was reduced, indicating that stomatal aperture is not regulated by the MEP-dependent foliar ABA pool, under these specific cases.

Background: The human dopamine D4 receptor (DRD4) gene has been studied extensively as a candidate gene for certain psychological traits and several behavioural and psychiatric disorders. Both the 5' regulatory region and the coding sequence contain a number of polymorphisms. The promoter variants have received particular attention in the past few years due to their possible role in the regulation of gene transcription. Previously, the -521C/T SNP was shown to influence promoter activity. The aim of this study is to perform an in-depth analysis of this effect in the context of various neural cell lines., Results: Endogenous mRNA expression of the DRD4 gene was demonstrated in two neuroblastoma (SK-N-F1, IMR32) and one retinoblastoma cell line (Y79) by RT-PCR. In addition, very low DRD4 mRNA levels were also detected in HeLa cells. The transcriptional activity of a series of 5' promoter deletion mutants was determined by transient transfection of luciferase reporter constructs. The activity profile of these promoter fragments was similar in each of the cell lines tested. The highest luciferase reporter activity was obtained with a construct containing promoter sequences between nucleotides -668 to -389, while a putative silencer region was localised spanning from nucleotide -1571 to -800. Surprisingly, the -521 C/T polymorphism had no significant effect on transcriptional activity of the reporter construct with the highest activity (-668 to -389) in any of the three cell lines tested., Conclusion: Our results do not confirm previous data assigning different transcriptional activities to the -521 C/T alleles of the human DRD4 promoter. Furthermore, these findings highlight the need for further characterization of the 5' regulatory region of the DRD4 gene and identification of additional functional promoter polymorphic sites, especially in the context of haplotype.

Dopamine D4 receptor (DRD4) and serotonin transporter (SERT) gene polymorphisms were studied, as possible genetic risk factors for substance dependence. The case-control study involved a large cohort (n = 362) of healthy Caucasian population, and an initial sample of 73 substance dependent patients (including a subgroup of 53 heroin dependents). Improved methods were applied for genotype detection of the DRD4 polymorphisms (exon 3 48 bp VNTR; -521 C/T SNP and 120 bp duplication in the 5' flanking region) and the SERT gene polymorphisms (5-hydroxytriptamin transporter linked polymorphic region [5-HTTLPR] in the 5' flanking region and the intron 2 VNTR [STin2]). Association between the -521 C/T SNP of the DRD4 promoter region and substance dependence was significant in the subgroup of heroin dependents (p = 0.044). The other analyzed polymorphisms did not show any significant association, but an interaction between -521 C/T SNP of DRD4 and the 5-HTTLPR polymorphisms was observed. Association between the -521 CC vs. CT or TT genotypes and heroin dependence was enhanced in the presence of short (s or 14-repeat) 5-HTTLPR allele (p 0.01). The odds ratio of 2.14 observed for the -521 CC genotype increased to 4.82 in double homozygotes of -521 CC and 5-HTTLPR ss, emphasizing the importance of combined analysis of polymorphisms in the dopaminergic and serotonergic systems in heroin dependence. However, due to the limited size of our sample these results should be interpreted with caution.

Possible mechanisms behind the light-induced wilting of dark-germinated pea (Pisum sativum L.) epicotyls were studied. Illumination with photosynthetically active radiation caused a fast turgor loss and wilting in the middle segments of the epicotyls accompanied by accumulation of water in the intercellular cavities. During this process, room temperature fluorescence emission spectra showed gradual bleaching of porphyrin-type pigments, which was lessened by incubating the epicotyls with excess ascorbate before illumination. Detection of singlet oxygen and lipid peroxidation products in the illuminated epicotyls suggested the occurrence of porphyrin-photosenzitized membrane damage as a cause of disordered water status and sequential wilting.

The capacity of ultraviolet (UV) radiation to generate reactive oxygen species (ROS) in spinach leaves was studied with fluorescent sensors. Leaf segments were exposed to short-term (15-30 min), narrow-band UV irradiation of various wavelengths between 290-390 nm corresponding to equal numbers of photons and, depending on the wavelength, 18-36 μmol m -2 s -1 quantum flux. This caused 5-30% loss of photosynthesis measured as loss of variable chlorophyll fluorescence. In accordance with previous results, photosynthesis was less affected by longer than by shorter wavelength UV. UV-irradiated leaves were probed for two major representatives of ROS, namely the superoxide radical anion, a product of electron transfer to oxygen, and singlet oxygen, which is usually formed in photosensitised reactions between oxygen and triplet dyes. Both ROS were detected, but the efficacy of UV in promoting their production was different. Formation of superoxide radicals was positively correlated with loss of photosynthesis; more radicals were observed in leaves exposed to shorter UV wavelengths than in leaves irradiated at 370-390 nm, suggesting a functionally damaged electron transport as a possible site of oxygen reduction. Singlet oxygen was, however, mainly inducible in leaves exposed to longer UV wavelengths and was barely induced by 290-300-nm irradiation. The mechanism of singlet oxygen generation by UV radiation in the leaf, reported for the first time, is not clear, but the process does not appear to be linked to the UV-induced damage of photosynthesis.

In order to understand the physiological functions of reactive oxygen species (ROS) generated in leaves, their direct measurement in vivo is of special importance. Here we report experiments with two dansyl-based ROS sensors, the singlet oxygen specific DanePy and HO-1889NH, which is reactive to both singlet oxygen and superoxide radicals. Here we report in vivo detection of (1)O(2) and O(2)(-*) by fluorescence quenching of two dansyl-based ROS sensors, the (1)O(2) specific DanePy and HO-1889NH, which was reactive with both (1)O(2) and O(2)(-*). The ROS sensors were administered to spinach leaves through a pinhole, and then the leaves were exposed to either excess photosynthetically active radiation or UV (280-360 nm) radiation. Microlocalization of the sensors' fluorescence and its ROS-induced quenching was followed with confocal laser scanning microscopy and with fluorescence imaging. These sensors were specifically localized in chloroplasts. Quenching analysis indicated that the leaves exposed to strong light produced (1)O(2), but hardly any O(2)(-*). On the other hand, the dominant ROS in UV-irradiated leaves was O(2)(-*), while (1)O(2) was minor.

Ultrafast, membrane-mediated restriction digestion of DNA molecules followed by rapid gel microchip electrophoresis of the resulting fragments is described. Combination of restriction endonuclease digestion on small pore-size microfibrous membranes with sample loading and electrophoresis analysis in a multilane (up to 96) format resulted in very fast restriction digest based microscale DNA analysis. Complete digestion of several nanogram target DNA was accomplished on the microporous membrane at room temperature just in a few minutes with a single or a combination of various restriction enzymes, using only submicroliter quantities of samples and reagents. The reaction mixture containing membrane also served as sample loading device for the subsequent gel microchip electrophoresis based analysis. This work establishes methods for high-speed, high-throughput DNA analysis, featuring extremely low sample and reagent consumption, and fast restriction digestion in combination with sample loading and rapid gel microchip analysis of the resulting fragments. The entire restriction digestion, sample loading and electrophoresis analysis process required less than 20 min.

Decreased activity of plasma cholinesterase is responsible for prolonged apnea during anesthesia using neuromuscular blockers such as suxamethonium and mivacurium. More than 20 mutations have been identified so far in the BCHE gene resulting in impaired plasma cholinesterase activity. Biochemical tests are not always able to differentiate between pathological and normal sera; hence in some cases unanticipated complications can still occur during anesthesia even after measurements of enzyme activity and dibucaine numbers within the normal range. Therefore, molecular genetic testing is required for the accurate diagnosis of this deficiency. Here we present a study of plasma cholinesterase activity and BCHE genotyping of patients with a history of prolonged neuromuscular block and most of their pedigrees. All four exons of the BCHE gene were directly sequenced from samples and a number of mutations responsible for the reduction of plasma cholinesterase activity were identified. In most cases the atypical mutation in exon 2 (nt 209A --> G, Asp70 --> Gly) was found together with the K-variant mutation in exon 4 (nt 1615G --> A, Ala539 --> Thr), which is in good agreement with previous data suggesting that these mutations along with two others (at nt -116 and nt 1914) are in linkage disequilibrium., (Copyright 2001 Elsevier Science.)

Photoactivation of the water splitting enzyme was performed with 13 different synthetic manganese complexes and characterized by oxygen evolution yield, thermoluminescence and chlorophyll fluorescence induction kinetics. The efficiency of different compounds in photoactivation correlated with the rate of linear electron transport in the presence of these compounds. The organic ligands, associated with the manganese ions, do not prevent the photoactivation of the water splitting complex (WOC). Photoactivation with different manganese complexes depended on the number of the Mn-ions in the complex, their valence state and the nature of their donor atoms. The most efficient restorations were achieved by using tetrameric complexes having a dimer+dimer structure, complexes containing Mn(II) ions, and having 4-6 oxygen and 0-2 nitrogen atoms as donor atoms. Further, the effectiveness of photoactivation depended largely on the structure of the complexes. Our data support the notion that WOC in intact thylakoids requires the cooperation and well determined arrangement of all four manganese ions, and argue against the hypothesis that two manganese ions are sufficient for water splitting. Photoactivation by some complexes led to anomalous flash-oxygen patterns, which are explained by a modified/perturbed water splitting complex.

The -521C/Tsingle nucleotide polymorphism (SNP) in the promoter region of the dopamine D4 receptor gene (DRD4) has recently been detected in oriental (Japanese) individuals and related to novelty seeking and schizophrenia. Here, we report the analysis of the -521C/T polymorphism in a Caucasian (Hungarian) population using two independent genotyping methods. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) procedure utilized the Fspl restriction site around the -521 position. An additional, nonpolymorphic cleavage site was also included into the amplified region to serve as an internal standard for verifying the completion of the digestion. As another independent method, a tetraprimer system for single-tube allele-specific PCR (SAS-PCR) was developed to generate -521C and -521T specific PCR products with different fragment sizes. Consequently, genotyping with SAS-PCR is based on the gel-electrophoretic separation of the allele-specific double-stranded DNA (dsDNA) fragments. 119 healthy Hungarian individuals were genotyped for -521C/T polymorphism of the dopamine D4 promoter region, using both methods. Similar allele frequencies were found (-521C allele: 0.43; -521T allele: 0.57) as reported earlier for the Japanese population.

Applicability of modern microfabrication technology to electrophoresis microchips initiated a rapidly moving interdisciplinary field in analytical chemistry. Electric field mediated separations in microfabricated devices (electrophoresis microchips) are significantly faster than conventional gel electrophoresis, usually completed in seconds to minutes. Electrophoretic separation of DNA molecules on microfabricated devices proved to have the potential to improve the throughput of analysis by orders of magnitude. The flexibility of electrophoresis microchips allows the use of a plethora of separation matrices and conditions. In this paper, we report on electric field mediated separation of fluorescent intercalator-labeled dsDNA fragments in polyvinylpyrrolidone matrix-filled microchannel structures. The separations were detected in real time by a confocal, single-point laser-induced fluorescence/photomultiplier setup. Effects of the sieving matrix concentration (Ferguson plot), migration characteristics (reptation plot), separation temperature (Arrhenius plot), as well as applied electric field strength and intercalator concentration on the separation of DNA fragments are thoroughly discussed.

Rapid molecular diagnosis of 21-hydroxylase deficiency by detecting the most common mutation in the 21-hydroxylase gene is presented using primer extension and capillary electrophoresis with a polyvinyl pyrrolidone matrix. DNA samples were subjected to polymerase chain reaction (PCR) in order to amplify a 422 bp fragment of the CYP21 gene containing the single nucleotide polymorphism (SNP) site. This product served as a template in the primer extension reaction using a fluorescently labeled primer in close proximity to the SNP. ddGTP was used to block the extension if the mutation was present and the other three dNTPs to enable elongation of the primer. Fast analysis of the resulting fragments was performed by capillary electrophoresis using 10% polyvinylpyrrolidone as sieving and wall coating matrix. The Cy5-labeled primer and the two possible primer extension products (mutant and wild type) were completely separated in 90 s.

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders, causing impaired secretion of cortisol and aldosterone from the adrenal cortex, with subsequent overproduction of adrenal androgens. The most common enzyme defect causing CAH is steroid 21-hydroxylase deficiency. To determine the mutational spectrum in the Hungarian CAH population, the CYP21 active gene was analyzed using PCR. A total of 297 Hungarian patients with 21-hydroxylase deficiency are registered in the 2nd Department of Pediatrics, Budapest, Hungary, and their clinical status was evaluated. Blood samples for CYP21 genotype determination could be obtained from 167 patients (representing 306 unrelated chromosomes and 56.2% of the total group of patients). Eight of the most common mutations were screened [In2 (intron 2 splice mutation), I172N, Del (Del: apparents large gene conversion), Q318X, R356W, 1761Tins, ClusterE6, V281L] using allele-specific amplification. The most frequent mutation in the Hungarian CAH population was found to be In2. Our results have shown a good genotype/phenotype correlation in case of most mutations; the In2 mutation is associated mostly with the severe form of the disease, whereas I172N was expressed in a wide spectrum of phenotypes. 1999)

Growing evidence shows the correlation between the allelic type of the dopamine D4 receptor and the human novelty-seeking personality trait. A sensitive, ultrathin agarose gel electrophoresis-based, high-throughput screening method was developed for genotyping the dopamine D4 receptor (D4DR) exon III 48 base pair repeat polymorphism. The efficiency of the method was increased by reamplification nested polymerase chain reaction (PCR) - of the 48 base pair repeat containing the PCR product with internal primers. The nested PCR fragments were analyzed by ultrathin layer agarose gel electrophoresis with an automated real-time laser-induced fluorescent detection system. Noncovalent affinity complexation was accomplished during the separation process by the addition of a very low concentration of intercalation dye, To-Pro-3 (2 nM) to the gel buffer system. This resulted in instant fluorescent labeling of the migrating PCR fragments. This method can readily facilitate genetic association studies between dopamine receptor genotypes and some human behavioral and neuropsychiatric disorders.

Interleukin-2 (IL-2) production induced by heat--inactivated Staphylococcus aureus (SAU) was enhanced by simultaneous addition of phorbol myristate acetate (PMA). The effect was optimal at a concentration of 10 ng/ml SAU; in the presence of 10 ng/ml PMA, the amount of SAU required for maximal IL-2 production was lower. The kinetics of SAU and of SAU plus PMA-induced IL-2 production were similar. Stimulated mononuclear cells produced interferon (IFN) in addition to IL-2. The titre of accompanying IFN was decreased in cultures stimulated with the SAU plus PMA combination. Plastic nonadherent sheep erythrocyte-positive cells were the most active in the SAU-induced IL-2 production. In contrast, the bulk of the IFN activity was produced by the nonadherent E rosette-nonforming cells. Neutralization of IFN with specific antibodies and pH 2 treatment indicated that SAU-induced IFN consisted mainly of alpha-IFN.