Biomarkers of Recent Cannabis Use in Blood, Oral Fluid and Breath.

Proving driving under the influence of cannabis (DUIC) is difficult. Establishing a biomarker of recent use to supplement behavioral observations may be a useful alternative strategy. We determined whether cannabinoid concentrations in blood, oral fluid (OF) or breath could identify use within the past 3 h-likely the period of the greatest impairment. In a randomized trial, 191 frequent (≥4/week) and occasional (<4/week) cannabis users smoked one cannabis (placebo [0.02%], or 5.9% or 13.4% Δ9-tetrahydrocannabinol [THC]) cigarette ad libitum. Blood, OF and breath samples were collected prior to and up to 6 h after smoking. Samples were analyzed for 10 cannabinoids in OF, 8 in blood and THC in breath. Frequent users had more residual THC in blood and were more likely to be categorized as 'recently used' prior to smoking; this did not occur in OF. Per se limits ranging from undetectable to 5 ng/mL THC in blood offered limited usefulness as biomarkers of recent use. Cannabinol (CBN, cutoff = 1 ng/mL) in blood offered 100% specificity but only 31.4% sensitivity, resulting in 100% positive predictive value (PPV) and 94.0% negative predictive value (NPV) at 4.3% prevalence; however, CBN may vary by cannabis chemovar. A 10 ng/mL THC cutoff in OF exhibited the overall highest performance to detect its use within 3 h (99.7% specificity, 82.4% sensitivity, 92.5% PPV and 99.2% NPV) but was still detectable in 23.2% of participants ∼4.4 h post-smoking, limiting specificity at later time points. OF THC may be a helpful indicator of recent cannabis intake, but this does not equate to impairment. Behavioral assessment of impairment is still required to determine DUIC. This study only involved cannabis inhalation, and additional research evaluating alternative routes of ingestion (i.e., oral) is needed.

The determination of protonation states in proteins.

The protonation states of aspartic acids and glutamic acids as well as histidine are investigated in four X-ray cases: Ni,Ca concanavalin A at 0.94 A, a thrombin-hirugen binary complex at 1.26 A resolution and two thrombin-hirugen-inhibitor ternary complexes at 1.32 and 1.39 A resolution. The truncation of the Ni,Ca concanavalin A data at various test resolutions between 0.94 and 1.50 A provided a test comparator for the ;unknown' thrombin-hirugen carboxylate bond lengths. The protonation states of aspartic acids and glutamic acids can be determined (on the basis of convincing evidence) even to the modest resolution of 1.20 A as exemplified by our X-ray crystal structure refinements of Ni and Mn concanavalin A and also as indicated in the 1.26 A structure of thrombin, both of which are reported here. The protonation-state indication of an Asp or a Glu is valid provided that the following criteria are met (in order of importance). (i) The acidic residue must have a single occupancy. (ii) Anisotropic refinement at a minimum diffraction resolution of 1.20 A (X-ray data-to-parameter ratio of approximately 3.5:1) is required. (iii) Both of the bond lengths must agree with the expectation (i.e. dictionary values), thus allowing some relaxation of the bond-distance standard uncertainties required to approximately 0.025 A for a '3sigma' determination or approximately 0.04 A for a '2sigma' determination, although some variation of the expected bond-distance values must be allowed according to the microenvironment of the hydrogen of interest. (iv) Although the F(o) - F(c) map peaks are most likely to be unreliable at the resolution range around 1.20 A, if admitted as evidence the peak at the hydrogen position must be greater than or equal to 2.5 sigma and in the correct geometry. (v) The atomic B factors need to be less than 10 A(2) for bond-length differentiation; furthermore, the C=O bond can also be expected to be observed with continuous 2F(o) - F(c) electron density and the C-OH bond with discontinuous electron density provided that the atomic B factors are less than approximately 20 A(2) and the contour level is increased. The final decisive option is to carry out more than one experiment, e.g. multiple X-ray crystallography experiments and ideally neutron crystallography. The complementary technique of neutron protein crystallography has provided evidence of the protonation states of histidine and acidic residues in concanavalin A and also the correct orientations of asparagine and glutamine side chains. Again, the truncation of the neutron data at various test resolutions between 2.5 and 3.0 A, even 3.25 and 3.75 A resolution, examines the limits of the neutron probe. These various studies indicate a widening of the scope of both X-ray and neutron probes in certain circumstances to elucidate the protonation states in proteins.

A method for identification of inhibitors of the phosphorylation reactions of bacterial response regulator proteins using (31)P nuclear magnetic resonance spectroscopy.

Bacterial response regulators are attractive targets for antibacterial drug development, yet random screening against these targets has failed as yet to identify chemicals that constitute viable leads. Alternative methods to provide leads for drug development based on identification and optimization of low affinity ligands from NMR screens have been described. However, leads from these processes still require verification in a bioassay, which is often problematic if compounds have unfavorable optical and solubility properties. A simple method, based on using NMR to observe the activity of the target, is described. It has the advantages of being able to characterize both low affinity leads and a wider selection of compounds in a structure activity relationships series, without the problems affecting a fluorescence assay. In this example we use (31)P to monitor the turnover of a bacterial response regulator, but the generic approach could be applied to other nuclei and thus a range of biological systems.

Emotion expression processes in children's peer interaction: the role of peer rejection, aggression, and gender.

The goal of the current study was to investigate sociometric status, aggression, and gender differences in children's expression of anger, happiness, and sadness. Participants were 111 second-grade African American boys and girls, half rejected and half average sociometric status, and half aggressive and half nonaggressive as assessed by their peers. Children interacted with a confederate in two standardized competitive game paradigms. Participants' expressions of anger, happiness, and sadness were observationally coded across facial, verbal intonation, and nonverbal modalities. Rejected children expressed more facial and verbal anger than average-status children. Rejected children also expressed more nonverbal happiness than average children, but only during turns of the game that were favorable to the participant. Finally, boys expressed more facial, verbal, and nonverbal anger than girls.

The dyadic nature of social information processing in boys' reactive and proactive aggression.

The correlation between boys' social cognitions and their aggressive behavior toward peers was examined as being actor driven, partner driven, or dyadic relationship driven. Eleven groups of 6 familiar boys each (N = 165 dyads) met for 5 consecutive days to participate in play sessions and social-cognitive interviews. With a variance partitioning procedure, boys' social-cognitive processes were found to vary reliably across their dyadic relationships. Furthermore, mixed models regression analyses indicated that hostile attributional biases toward a particular peer were related to directly observed reactive aggression toward that peer even after controlling for actor and partner effects, suggesting that these phenomena are dyadic or relationship oriented. On the other hand, the relation between outcome expectancies for aggression and the display of proactive aggression appeared to be more actor driven and partner driven that dyadic.

Proactive and reactive aggression in delinquent adolescents: relations to aggression outcome expectancies.

Investigated whether the relation between aggression and the tendency to expect positive outcomes for aggressive behavior is specific to the proactive subtype of aggression (as opposed to the reactive subtype). In a sample of 86 incarcerated adolescent boys ages 13 to 18, we measured outcome expectancies for aggression using audiotaped hypothetical vignettes. For each participant, staff members completed proactive and reactive aggression rating scales. Regression analyses revealed that the relation between aggression and outcome expectancies was indeed specific to proactive aggression. Furthermore, this finding was supported regardless of whether outcome expectancies were assessed using vignettes describing proactive-aggressive behavior or those describing reactive-aggressive behavior. We discuss these findings and argue that interventions to reduce proactive or reactive aggression should differ from each other by addressing the specific social cognitive processes involved in each type of aggression.

It takes two to fight: a test of relational factors and a method for assessing aggressive dyads.

Observations of aggressive interactions in boys' laboratory play groups were used to evaluate the relative importance of relational and individual factors in accounting for aggressive acts. A classroom peer-rating method for identifying mutually aggressive dyads was validated in 11 5-session play groups, composed of 2 mutually aggressive boys and 4 randomly selected male classmates from 11 predominately African American 3rd-grade classrooms. When the social relations model was used, relationship effects accounted for equally as much of the variance in total aggression and proactive aggression as either actor or target effects. Mutually aggressive dyads displayed twice as much total aggression as randomly selected dyads. Members of mutually aggressive dyads attributed greater hostile intentions toward each other than did randomly selected dyads, which may serve to explain their greater aggression toward each other. The importance of studying relational factors, including social histories and social-cognitive processes, is discussed.

Social-cognitive and behavioral correlates of aggression and victimization in boys' play groups.

A contrived play group procedure was utilized to examine the behavioral and social-cognitive correlates of reactive aggression, proactive aggression, and victimization via peers. Eleven play groups, each of which consisted of six familiar African-American 8-year-old boys, met for 45-min sessions on five consecutive days. Social-cognitive interviews were conducted following the second and fourth sessions. Play group interactions were videotaped and examined by trained observers. High rates of proactive aggression were associated with positive outcome expectancies for aggression/assertion, frequent displays of assertive social behavior, and low rates of submissive behavior. Reactive aggression was associated with hostile attributional tendencies and frequent victimization by peers. Victimization was associated with submissive behavior, hostile attributional bias, reactive aggression, and negative outcome expectations for aggression/assertion. These results demonstrate that there is a theoretically coherent and empirically distinct set of correlates associated with each of the examined aggression subtypes, and with victimization by peers.

Identification of the epitopes of calcitonin gene-related peptide (CGRP) for two anti-CGRP monoclonal antibodies by 2D NMR.

The interactions between calcitonin gene-related peptide and FAB fragments prepared from two different high-affinity anti-CGRP monoclonal antibodies (CB3 and CD1) have been studied at physiological pH using the ability of 1H NMR to detect selectively regions of dynamic flexibility. The 37-residue peptide retains considerable flexibility in regions of its sequence when bound to both antibodies; in each case, more than half of the residues can be seen to have linewidths little perturbed from those of the free peptide. However the regions where substantial broadening of resonances occur, attributed to substantially reduced motional freedom of the peptide resulting from interactions within the antibody combining site, differ greatly in the two cases. In the complex with CB3 the results indicate that the restricted residues lie exclusively within the C-terminal half of the peptide, and include residues 25 to 32 and the terminal two residues (36 and 37). By contrast, in the complex with CD1, the conformationally restricted residues appear to lie predominantly within the N-terminal half of the CGRP molecule, particularly residues 4-16, although several residues in the middle section of the sequence (22-31) have reduced conformational freedom. These findings, consistent with the results from immunological assays, add considerably to our knowledge of the epitopes.

Conformation of the cytoplasmic domain of phospholamban by NMR and CD.

Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy have been used to characterize the conformation of the putative cytoplasmic domain of phospholamban (PLB), an oligomeric membrane-bound protein which regulates the activity of the cardiac sarcoplasmic reticulum Ca(2+)-dependent ATPase. In aqueous solution the 25-residue peptide adopts a number of rapidly interconverting conformers with no secondary structural type obviously predominating. However, in trifluoroethanol (TFE) the conformation, while still highly dynamic, is characterized by a high proportion of helical structures. Evidence for this is provided by alpha CH chemical shifts and low NH chemical shift temperature coefficients, small NH-alpha CH intraresidue scalar coupling constants, a substantial number of distinctive interresidue nuclear Overhauser effects (NOEs) [dNN(i, i + 1), d alpha N(i, i + 3), d alpha beta(i, i + 3) and d alpha N(i, i + 4)] and characteristic CD bands at 190 (positive), 206 (negative) and 222 nm (negative). The helicity is interrupted around Pro-21. The activity of PLB is regulated by phosphorylation at either Ser-16 or Thr-17. CD shows that phosphorylation at Ser-16 by the cAMP-activated protein kinase causes about an 11% decrease in alpha-helical content in TFE.

On the role of the C-terminus of alpha-calcitonin-gene-related peptide (alpha CGRP). The structure of des-phenylalaninamide37-alpha CGRP and its interaction with the CGRP receptor.

alpha-Calcitonin-gene-related peptide (alpha CGRP) lacking its C-terminal phenylalaninamide residue was found not to bind to its receptor as did full-length (amidated) alpha CGRP. Investigation of the structure of these peptides by c.d. and n.m.r. revealed no significant difference, so it seemed that the effect of deleting the C-terminal phenylalaninamide on the biological activity of alpha CGRP was not by disruption of the peptide's structure. Thus the C-terminal phenylalaninamide is an important factor in this ligand-receptor interaction, and the group itself may interact directly with the receptor.

Cefotaxime and metabolite disposition in two pediatric continuous ambulatory peritoneal dialysis patients.

OBJECTIVE: To characterize the pharmacokinetics of cefotaxime and desacetylcefotaxime in pediatric patients undergoing continuous ambulatory peritoneal dialysis (CAPD) after intraperitoneal administration of cefotaxime. DESIGN: Case series. SETTING: Ambulatory children from Children's Hospital nephrology clinic, Columbus, Ohio. PATIENT POPULATION: Two adolescents without peritonitis. METHODS: A single intraperitoneal dose of cefotaxime 500 mg per 1 L dianeal was given during CAPD. Cefotaxime and desacetyl-cefotaxime were measured in plasma, urine, and dialysate by HPLC. RESULTS: Maximum plasma concentration (Cmax) of cefotaxime was 11.94 and 13.08 mg/L and that of desacetylcefotaxime 5.73 and 5.33 mg/L. Time to reach maximum concentration (Tmax) of cefotaxime was 2.22 and 4.08 h, and that of desacetylcefotaxime was 5.33 and 5.73 h after instillation of the intraperitoneal cefotaxime dose. Systemic absorption of cefotaxime was 56.6 and 64.8 percent. Total clearance of cefotaxime was 62 and 79 mL/min/1.73 m2. Nonrenal clearance accounted for nearly 95 percent; renal and CAPD clearance contributed approximately 5 percent of the total clearance. Renal and CAPD clearance measurements of desacetylcefotaxime were similar to those for cefotaxime. Cefotaxime half-life was 1.83 and 2.49 h and desacetylcefotaxime half-life was 8.14 and 11.0 h. CONCLUSIONS: Cefotaxime was well absorbed and therapeutic serum concentrations were achieved after intraperitoneal administration. Renal and CAPD clearances for cefotaxime and desacetylcefotaxime were low. Cefotaxime nonrenal clearance was unaffected. Further studies are needed to establish appropriate intraperitoneal dosing guidelines of cefotaxime in pediatric CAPD patients.

Functional size measurements of the ubiquinol oxidase activity of the cytochrome o terminal oxidase complex of Escherichia coli.

The functional molecular mass for ubiquinol-1 oxidation by the Escherichia coli terminal oxidase, cytochrome o, was determined by radiation-inactivation analysis of membranes from a cytochrome d-deficient mutant. The functional molecular mass for ubiquinol-l oxidase activity was found to be 38.3 +/- 2.65 kDa.

Solution structures of calcitonin-gene-related-peptide analogues of calcitonin-gene-related peptide and amylin.

Near-u.v. and far-u.v. c.d. spectra of human alpha-calcitonin-gene-related peptide (h alpha CGRP), analogues and fragments of CGRP and amylin were recorded in aqueous solution and in trifluoroethanol (TFE)/water mixtures. All peptides contained significant amounts of alpha-helix in aqueous solution, and this amount increased on adding TFE. The helical content was unaffected by pH and salt. However, amylin contained much less helix than CGRP and the c.d. spectrum was more temperature-sensitive. A band in the near-u.v. c.d. spectrum of CGRP (but not present in the spectrum of amylin) was attributed to the disulphide bond in CGRP. The intensity of this band was pH-dependent and titrated with a pKa of 6.5, suggesting the involvement of histidine ionization.

Initial dyadic peer interaction of attention deficit-hyperactivity disorder and normal boys.

The present study assessed the frequency and patterns of play duration and verbal behavior of medicated attention deficit-hyperactivity disorder (ADHD) boys in an initial social encounter with a normal, same-age peer. Eight pairs each of previously unacquainted ADHD/normal boys and normal/normal boys were videotaped as they interacted in a free-play setting (N = 32). The ADHD/normal dyads engaged in more solitary play and less associative play than the normal/normal dyads. Sequential analyses of the ADHD/normal dyads' play patterns revealed that they had problems in their progression along the play hierarchy, in sustaining associative play, and in avoiding withdrawal after rough and tumble play. In comparison to the normal/normal dyads, the ADHD/normal dyads also differed in the quality of verbal interaction as seen in their lower levels of verbal reciprocity and affective expression. Process explanations for the problems ADHD boys display in an initial social encounter and the implications of these difficulties for diminished socialization opportunities were discussed.

Effects of iron-limitation of Escherichia coli on growth, the respiratory chains and gallium uptake.

The effects of iron limitation on growth, the composition and function of the respiratory chains, and gallium uptake in Escherichia coli have been studied. Decreasing the iron concentration in a defined medium using Chelex resin gave lower growth yields in both continuous culture and prolonged batch culture. In the former, iron-limited (entering [Fe] less than or equal to 2.0 microM) cells exhibited diminished respiration rates, respiration-driven proton translocation quotients, and levels of non-haem iron and cytochromes. The cellular concentration of haemoprotein b-590 (a cytochrome alpha 1-like hydroperoxidase) decreased 20-fold on iron limitation, whilst a CO-binding pigment with an absorption maximum in the dithionite-treated form near 500 nm appeared. Gallium(III) (9 microM) added to iron-limited, but not iron-sufficient, cultures diminished growth yields further; cells grown with low entering concentrations of iron took up less gallium than iron-sufficient cells. These results are attributed to the interference by gallium(III) with siderophore-mediated metal uptake. Gallium also stimulated iron uptake and was itself accumulated by iron-sufficient cells, suggesting that gallium(III) also affects the iron transport system(s) of low affinity.

Purification and partial characterization of two cytochrome oxidases (caa3 and o) from the thermophilic bacterium PS3.

Two cytochrome oxidases, cytochrome aa3 (EC 1.9.3.1) and cytochrome o, have been purified from the membranes of a thermophilic bacterium, PS3. The enzymes were solubilized with Triton X-100 and purified to apparent homogeneity on anion-exchange columns. The properties of the three-subunit cytochrome oxidase complex caa3 obtained here are compared with the same enzyme isolated by Sone, N. and Yanagita, Y. (1982) (Biochim. Biophys. Acta 682, 216-226). On storage, the purified caa3 enzyme undergoes denaturation; a shoulder at 432 nm seen in (CO-reduced)-minus-reduced difference spectra may be due in part to denaturation products of the enzyme. The purified cytochrome o is more stable. At room temperature, the reduced-minus-oxidized difference spectrum shows absorbance maxima at 427 and 559 nm; at 77 K, its alpha-band is split into 554 and 557 nm components. At room temperature, the CO-reduced-minus-reduced spectrum shows troughs at 430 nm and 560 nm. Dissociating polyacrylamide gel electrophoresis suggests that the purified cytochrome o is composed of one type of subunit with an apparent molecular mass of 47 000-48 000. Metal analysis of the purified enzyme demonstrated the lack of copper. Both oxidases, purified in the presence of Triton X-100, exist in highly polydisperse forms.

Nitrate, but not silver, ions induce spectral changes in Escherichia coli cytochrome d.

The absorbance maximum (630 nm) of reduced cytochrome d in Escherichia coli membrane particles was diminished by 160 microM AgNO3 or NaNO3 and accompanied by the formation of a species with an absorption maximum at 640-645 nm. Nitrite, trioxodinitrate and nitric oxide elicited qualitatively similar, but faster, changes in the spectrum of cytochrome d, suggesting that formation of a nitrosyl complex may be involved in all cases. In direct contrast to an earlier report, silver ions (160 microM) were without effect on the alpha-bands of reduced cytochromes d, b or a 1.