Sensitive determination of alpha-methyltryptamine (AMT) and 5-methoxy-N,N-diisopropyltryptamine (5MeO-DIPT) in whole blood and urine using gas chromatography-mass spectrometry

We devised a sensitive and simple method to determine alpha-methyltryptamine (AMT) and 5-methoxy-N,N-diisopropyltryptamine (5MeO-DIPT) in whole blood and urine, using gas chromatography-mass spectrometry (GC-MS). AMT and 5MeO-DIPT were extracted using an Extrelut column with an internal standard, bupivacaine, followed by derivatization with acetic anhydride. The derivatized extract was used for GC-MS analysis of EI-SIM mode. The calibration curves of AMT and 5MeO-DIPT were linear in the concentration range from 10 to 750 ng/ml in both blood and urine samples. The method detection limit (MDL) of AMT and 5MeO-DIPT were 1 ng/ml each in whole blood and 5 ng/ml each in urine. This method should be most useful to accurately determine the presence of these drugs in blood and urine in clinical and forensic cases.     

Detection and determination of reticuline and N-methylcoculaurine in the Annonaceae family using liquid chromatography-tandem mass spectrometry

In Guadeloupe, the French West Indies, there is a high incidence of atypical parkinsonism or progressive supranuclear palsy, and all of the investigated patients had taken herbal tea or tropical fruits of the Annonaceae family. Local inhabitants consume the fruits, and also drink tea made from the leaves. In the present study, we used liquid chromatography-tandem mass spectrometry (LC/MS/MS) with multiple reaction monitoring (MRM) to detect low-molecular-weight neurotoxic benzylisoquinoline derivatives in the Annonaceae family. We detected reticuline and N-methylcoculaurine in every Annona muricata sample examined, except for pulp and seed. They were not detected in sweetsop fruits. Norreticuline was not detected in any sample. These three compounds were toxic to SH-SY5Y neuroblastoma cells and inhibited mitochondrial respiratory complex I. It is possible that uptake of the benzylisoquinoline derivatives reticuline and N-methylcoculaurine and their accumulation in the brain may be related to the pathogenesis of the local endemic disease.     

Relationship between temperament and fatness in 11-year-old children and 17-year-old adolescents from Wrocław,Poland

Childhood obesity is increasing globally, and Poland is no exception. Studies indicate that relationship between obesity and psychological well-being is a complex issue and this needs further research. The objective of the present cross sectional study was to analyze the relationship between some temperament components and fatness among children in two developmental periods, approximately before and after adolescence. Participants included 122 children aged 11 years (57 boys and 65 girls), and 153 adolescents aged 17 years (64 boys and 89 girls) from 6 primary and 4 secondary schools in Wroc?aw, Poland. Height, weight, triceps-, subscapular and abdominal skinfold thicknesses were measured. Temperament was assessed by a questionnaire of Buss and Plomin (1984) in two versions: EAS-C for children completed by parents and EAS-D for youth and adults. Physical activity (PAL) was also assessed by a questionnaire. Mean, median and standard deviation (SD) were calculated and Student's t tests were performed to test for significance of differences between groups. Chi squared (χ²) statistic was used to test the significance of differences in frequencies. Analyses of covariance (ANCOVA) were performed to show the effect of the social - psychological factors on fatness. Controlling for socioeconomic status and physical activity level, activity, as a component of temperament had a significant effect on body fatness. The only component of temperament, which significantly influenced level of fatness in girls, was emotionality. These relationships differed according to sex and the two age groups concerned.     

Growth and development in school-age children from Rostov region,Russia: Comparison between urban and rural settings

The purposes of the current study were: (1) to describe growth and physical development and establish norms for schoolchildren from Rostov region in Russia; (2) to compare major characteristics of development between urban and rural children by sex and age.Nearly 200,000 children (198,712) aged between 7 and 17 years from 232 urban and rural schools of Rostov region (Southern Federal District of Russia) participated in the study. School age is a period of intensive growth and physiological and psychological development. Irregularities of personal development are caused by a multitude of factors, such as sex differences, heredity, socio-economic status of a family, standard of living, particular environmental conditions, and lifestyle.It has been established that children from the Southern Federal District of Russia had body mass index values higher than age-appropriate norms for all Russians (Total Russian, Rudnev et al., 2014) and World Health Organization charts. Children from urban settings were taller and heavier than children from rural settings.Sex is one of the most influential factors which play key role in determining specific characteristics of growth and personal development. According to our results, boys and girls both had similar age-related changes in weight and height, but their respective dynamics differed. Girls’ height and weight values accelerated at the age 10 to 12 years and plateaued after the age fourteen, whereas in boys height and weight steadily increased with age, showing slight acceleration at the age 12 to 13 years, and reached a plateau by the age of seventeen.     

Evaluation of the contribution of D9S1120 to anthropological studies in Native American populations

The D9S1120 locus exhibits a population-specific allele of 9 repeats (9RA) in all Native American and two Siberian populations currently studied, but it is absent in other worldwide populations. Although this feature has been used in anthropological genetic studies, its impact on the evaluation of the structure and genetic relations among Native American populations has been scarcely assessed. Consequently, the aim of this study was to evaluate the anthropological impact of D9S1120 when it was added to STR population datasets in Mexican Native American groups. We analyzed D9S1120 by PCR and capillary electrophoresis (CE) in 1117 unrelated individuals from 13 native groups from the north and west of Mexico. Additional worldwide populations previously studied with D9S1120 and/or 15 autosomal STRs (Identifier kit) were included for interpopulation analyses. We report statistical results of forensic importance for D9S1120. On average, the modal alleles were the Native American-specific allele 9RA (0.3254) and 16 (0.3362). Genetic distances between Native American and worldwide populations were estimated. When D9S1120 was included in the 15 STR population dataset, we observed improvements for admixture estimation in Mestizo populations and for representing congruent genetic relationships in dendrograms. Analysis of molecular variance (AMOVA) based on D9S1120 confirms that most of the genetic variability in the Mexican population is attributable to their Native American backgrounds, and allows the detection of significant intercontinental differentiation attributed to the exclusive presence of 9RA in America. Our findings demonstrate the contribution of D9S1120 to a better understanding of the genetic relationships and structure among Mexican Native groups.     

Determination of NG,NG-dimethyl-L-arginine, an endogenous NO synthase inhibitor, by gas chromatography-mass spectrometry

A fully validated gas chromatographic-mass spectrometric (GC-MS) method for the accurate and precise quantification of NG,NG-dimethyl-L-arginine (asymmetric dimethylarginine, ADMA), an endogenous inhibitor of the NO synthase, in cell culture supernatants and in small volumes of plasma is described. ADMA was concentrated by solid phase extraction and converted to its methyl ester pentafluoropropionic amide derivative. The derivatives were analyzed without any further purification. Using gas chromatography-chemical ionization mass spectrometry, fragment ions at m/z 634 and m/z 640 were obtained for ADMA and for NG,NG-[2H6]-dimethyl-L-arginine ([2H6]-ADMA) as internal standard, respectively. [2H6]-ADMA was synthesized by reaction of L-ornithine fastened at bromcyan-agarose with dimethylamine. The limit of detection of the method was 2 fmol, while the limit of quantitation for cell culture supernatants was 0.05 microM. The method was validated in a concentration range of 0-1.2 microM in cell culture medium and 0-2 microM in 50 microl aliquots of human plasma. The precision was > or =97% and the accuracy was determined to be > or =94%. This method is fast, rugged and an alternative to high performance liquid chromatography (HPLC) analysis of ADMA in cell culture supernatants and small volumes of human plasma.     

Sex-specific reliability and multidimensional stability of responses to tests assessing neuromuscular function

The objective of this study was to estimate sex-specific effects in the test–retest cross-reliability of peripheral and central changes in nonlinear and linear measures of a surface electromyography signal during a brief (5 second) and sustained (2 minute) isometric maximal voluntary contraction, combined with superimposed electrical stimulation involving the ankle plantar flexors over five identical trials. In this study, we repeated the testing protocol used in our previous study of 10 women (age 20.9, SD = 0.3 years) (Bernecke et al., 2015) in a group of 10 men (age 21.2, SD = 0.4 years). Despite the central (sex effect; p < 0.05, η_p² > 0.71, SP > 70%) and peripheral fatigability (sex effect; p < 0.01, η_p² > 0.8, SP > 90%) during sustained isometric maximal voluntary contraction, and lower reliability for central activation ratio during brief (intraclass correlation coefficient [ICC] = 0.95 for men and ICC = 0.82 for women) and sustained maximal voluntary contraction (ICC > 0.82 for men and ICC > 0.66 for women) over ankle plantar flexors expressed in women more than in men, all the ICCs of all indices measured by tests assessing neuromuscular function across the five identical test–retest trials were found as meaningful (correlation significance of p < 0.05 was reached) and no significant differences were found between trials for any of the measured variables. In conclusion, the present study demonstrated greater central and peripheral fatigue for female participants following sustained (2 minute) isometric maximal voluntary contraction of the plantar flexor muscles for all repeated trials and indicated an acceptable agreement between measurements of the characteristic variables made using the three different devices (dynamometry, electrical stimulation, and surface electromyography) over time for both sexes.     

Characterization and kinetic analysis of enzyme–substrate recognition by three recombinant lactococcal tripeptidases

Tripeptidases from Lactococcus lactis subsp. lactis (L9PepTR), L. lactis subsp. cremoris (L6PepTR), and L. lactis subsp. hordniae (hTPepTR) were cloned, overexpressed, purified, and characterized. Although these enzymes contained three to seven naturally occurring amino acid differences, both metal-binding and catalytic sites were highly conserved. The k_cat values of hTPepTR were approximately 1.5- to 2-fold higher than those of L9PepTR, while, for L6PepTR, they were approximately 0.8- to 1.4-times the L9PepTR values. The K_m of tripeptidase from subsp. lactis (L9PepTR) was considerably larger when glycine was the amino acid located at both the N- and C-terminus of the peptide substrate. In addition, the K_m values of L9PepTR increased in the following order for YGG, LGG, FGG, SGG, and α-aminoisobutyrylglycylglycine, while the k_cat/K_m decreased in the same order. These results suggest that the dipole moment and steric hindrance of the N-terminal amino acid side chain may be the most important factors controlling substrate specificity.     

Effects of atrazine on osmoregulation in the Chinese mitten crab,Eriocheir sinensis

This study integrates results from acute contamination with atrazine of isolated perfused gills, and from in vivo chronic contamination of euryhaline Chinese mitten crabs, Eriocheir sinensis, acclimated to freshwater. Atrazine 1 mg/l in contact with the basolateral membrane (IN) increases the transepithelial potential difference (TEP) from -20.8 +/- 4.9 to -29.7 +/- 3.8 mV in isolated perfused posterior gills (P < 0.01). This effect is only partially explained by a modification of Na(+) and Cl(-) active influxes. No TEP modification is detected when atrazine is added (OUT) indicating that molecular mechanisms located on the basolateral membrane are likely to be the only ones affected. Another explanation would be that cuticular barrier prevents atrazine penetration into the gill. Haemolymph osmolarity, Na(+) and Cl(-) concentrations of crabs living in freshwater contaminated with atrazine 1 mg/l during 14 days are not significantly modified. We conclude that although atrazine can disturb osmoregulatory mechanisms of isolated gills, this pollutant would be of minor importance in affecting osmoregulatory capacities of the Chinese mitten crab in natural conditions.     

Corrigendum to “Dihydropyrimidine dehydrogenase: a flavoprotein with four iron–sulfur clusters” [Biochim. Biophys. Acta 1701 (2004) 61–74]

Lixelle is a direct hemoperfusion-type adsorption column that was developed to selectively eliminate β2-microglobulin (β2-m) from the circulating blood of patients with dialysis-related amyloidosis (DRA). The adsorbent in Lixelle comprises porous cellulose beads to which hydrophobic hexadecyl alkyl chain is covalently bound. One milliliter of wet Lixelle beads eliminates more than 1 mg of β2-m in vitro. In hemodialysis patients who were treated with Lixelle, Lixelle improved joint pain, nocturnal awakening, pinch strength, motor terminal latency, and their activity of daily living. The adsorbent adsorbs β2-m selectively but not specifically, as well as inflammatory cytokines such as interleukin-1β and IL-6 which are considered to be involved in the development of DRA. Lixelle treatments reduce the circulating levels of β2-m and inflammatory cytokines, thereby improving the symptoms of patients with DRA.     

Stir bar sorptive extraction and thermal desorption-gas chromatography-mass spectrometry for the measurement of 4-nonylphenol and 4-tert-octylphenol in human biological samples

Alkylphenols, 4-nonylphenol (NP) and 4-tert-octylphenol (OP), in human urine and plasma samples were analyzed using stir bar sorptive extraction (SBSE) in combination with thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). The method involved correction by stable isotopically labeled surrogate standards, 4-(1-methyl)octylphenol-d5 (m-OP-d5) and deuterium 4-tert-octylphenol (OP-d). A biological sample was extracted for 60 min at room temperature (25 degrees C) using a stir bar coated with a 500 microm thick polydimethylsiloxane (PDMS) layer. Then, the stir bar was analyzed by TD-GC-MS in the selected ion monitoring (SIM) mode without any derivatization step. The average recoveries in human urine and plasma samples spiked with NP and OP at levels of 0.5 and 10 ng ml-1 were between 95.8 and 99.8% with correction using the added surrogate standards. The limits of quantitation were 0.2 ng ml-1 for NP and 0.02 ng ml-1 for OP. We measured the background levels of NP and OP in five human urine and three human plasma samples from healthy volunteers. NP and OP were not detected in all human urine samples (N.D. < 0.2 ng ml-1 for NP, and N.D. < 0.02 ng ml-1 for OP). However, 0.2-0.3 ng ml-1 for NP and 0.1-0.2 ng ml-1 for OP in human plasma samples were observed by this method.     

Production in vitro of toxic macromolecules by strains of Beauveria bassiana, and purification of a chitosanase-like protein secreted by a melanizing isolate

The production of macromolecular insecticidal toxins in Adamek's medium by two selected strains of Beauveria bassiana was investigated. Filtrates and dialysates of the melanizing strain 618 were toxic when injected into the lepidopteran insect Galleria mellonella. Separation by DEAE chromatography revealed that peaks eluted respectively with 100 and 200 mM NaCl (P 100 and P 200) had an insecticidal activity and induced cuticular blackening. A hydrophilic protein, Bclp, which causes the formation of brownish spots of the integument, was purified from P 200 by means of chromatographic and electrophoretic methods. Bclp exhibited clear sequence homologies with fungal chitosanases of Fusarium solani. It has a molecular mass of 28 kDa, a pHI of 4 and is thermolabile. Injection of Bclp causes the same cytoxic effects and alterations of the cuticule as those observed during mycosis, and may contribute to the virulence of strain 618. Comparatively, the most obvious characteristic of the weakly melanizing strain 101 is the lack of significant toxic activity of its P 200, which does not contain Bclp. However, this strain secretes other insecticidal molecules active on lepidopterans, presently unidentified.