A Quick Primer in Fluorescence-Based Equilibrium and Pre-steady State Methods for Determining Protein-Nucleotide Affinities.

Biomolecular interactions facilitate the biochemical processes that sustain life. Proteins, RNAs, and ribonucleoprotein complexes perform cellular functions that range from catalyzing the formation or cleavage of bonds to being structural building blocks, both of which are only possible through the interaction with their respective biomolecular partner(s). Having access to the parameters that describe these interactions is important for our understanding of the principles that underlie enzymatic and nonenzymatic processes. Here we describe two fluorescence-based approaches to determine two key parameters, the affinity and the rate of association/dissociation of a protein and a ligand. Considerations are provided to expand the described approach to other experimental systems.

Cellular roles of the human Obg-like ATPase 1 (hOLA1) and its YchF homologs.

P-loop NTPases comprise one of the major superfamilies of nucleotide binding proteins, which mediate a variety of cellular processes, such as mRNA translation, signal transduction, cell motility, and growth regulation. In this review, we discuss the structure and function of two members of the ancient Obg-related family of P-loop GTPases: human Obg-like ATPase 1 (hOLA1), and its bacterial/plant homolog, YchF. After a brief discussion of nucleotide binding proteins in general and the classification of the Obg-related family in particular, we discuss the sequence and structural features of YchF and hOLA1. We then explore the various functional roles of hOLA1 in mammalian cells during stress response and cancer progression, and of YchF in bacterial cells. Finally, we directly compare and contrast the structure and function of hOLA1 with YchF before summarizing the future perspectives of hOLA1 research. This review is timely, given the variety of recent studies aimed at understanding the roles of hOLA1 and YchF in such critical processes as cellular-stress response, oncogenesis, and protein synthesis.

Health behaviours associated with indoor tanning based on the 2012/13 Manitoba Youth Health Survey. / Comportements en matière de santé associés au bronzage artificiel d'après l'Enquête sur la santé des jeunes du Manitoba de 2012-2013.

INTRODUCTION: Although indoor tanning causes cancer, it remains relatively common among adolescents. Little is known about indoor tanning prevalence and habits in Canada, and even less about associated behaviours. This study explores the prevalence of adolescent indoor tanning in Manitoba and its association with other demographic characteristics and health behaviours. METHODS: We conducted secondary analyses of the 2012/13 Manitoba Youth Health Survey data collected from Grade 7 to 12 students (n = 64 174) and examined associations between indoor tanning (whether participants had ever used artificial tanning equipment) and 25 variables. Variables with statistically significant associations to indoor tanning were tested for collinearity and grouped based on strong associations. For each group of highly associated variables, the variable with the greatest effect upon indoor tanning was placed into the final logistic regression model. Separate analyses were conducted for males and females to better understand sex-based differences, and analyses were adjusted for age. RESULTS: Overall, 4% of male and 9% of female students reported indoor tanning, and prevalence increased with age. Relationships between indoor tanning and other variables were similar for male and female students. Binary logistic regression models indicated that several variables significantly predicted indoor tanning, including having part-time work, being physically active, engaging in various risk behaviours such as driving after drinking for males and unplanned sex after alcohol/drugs for females, experiencing someone say something bad about one's body shape/size/appearance, identifying as trans or with another gender, consuming creatine/other supplements and, for females only, never/rarely using sun protection. CONCLUSION: Indoor tanning among adolescents was associated with age, part-time work, physical activity and many consumption behaviours and lifestyle risk factors. Though legislation prohibiting adolescent indoor tanning is critical, health promotion to discourage indoor tanning may be most beneficial if it also addresses these associated factors.

INTRODUCTION: Bien que le bronzage artificiel cause le cancer, il demeure relativement courant chez les adolescents. Nous en savons peu sur les habitudes en matière de bronzage artificiel ainsi que sur la prévalence de ce dernier au Canada, et nous en savons encore moins sur les comportements associés. Cette étude se penche sur la prévalence du bronzage artificiel chez les adolescents du Manitoba ainsi que sur son association avec d'autres caractéristiques individuelles et comportements en matière de santé. MÉTHODOLOGIE: Nous avons effectué des analyses secondaires des données recueillies dans le cadre de l'Enquête sur la santé des jeunes du Manitoba (2012-2013) auprès d'élèves de la 7e à la 12e année (n = 64 174) et nous avons examiné les associations entre le bronzage artificiel (selon si les répondants avaient déjà utilisé des appareils de bronzage artificiel) et 25 variables. Les variables présentant un lien statistiquement significatif avec le bronzage artificiel ont fait l'objet de tests pour déterminer toute colinéarité potentielle et elles ont été regroupées en fonction de la force de leurs associations. Pour chaque groupe de variables dont l'association avait une signification statistique, la variable ayant l'effet de plus grande ampleur sur le bronzage artificiel a été intégrée au modèle final de régression logistique. Des analyses séparées ont été réalisées pour les garçons et les filles afin de mieux comprendre les différences fondées sur le sexe, et les analyses ont été ajustées en fonction de l'âge. RÉSULTATS: Globalement, 4 % des garçons et 9 % des filles ont indiqué avoir eu recours au bronzage artificiel, et la prévalence augmentait avec l'âge. Les associations entre le bronzage artificiel et les autres variables étaient similaires chez les garçons et les filles. Les modèles de régression logistique binaire ont révélé que plusieurs variables prédisaient de façon significative le recours au bronzage artificiel, notamment l'occupation d'un emploi à temps partiel, l'activité physique, l'adoption de divers comportements à risque comme conduire un véhicule sous l'emprise de l'alcool (chez les garçons) et le fait d'avoir des relations sexuelles non planifiées après avoir consommé de l'alcool ou de la drogue (chez les filles), être victime de commentaires négatifs sur sa silhouette, sa taille ou son apparence, se déclarer comme transgenre ou d'un autre genre, consommer de la créatine ou autres suppléments et, pour les filles, utiliser rarement ou ne jamais utiliser de protection solaire. CONCLUSION: Le bronzage artificiel chez les adolescents a été associé à l'âge, au travail à temps partiel, à l'activité physique et à de nombreux comportements de consommation et de facteurs de risque liés au mode de vie. Même si la législation interdisant l'accès des adolescents aux salons de bronzage est essentielle, les activités de promotion de la santé visant à décourager le bronzage artificiel pourraient porter davantage fruit si elles s'attaquaient aussi à ces facteurs connexes.

The conserved GTPase HflX is a ribosome splitting factor that binds to the E-site of the bacterial ribosome.

Using a combination of biochemical, structural probing and rapid kinetics techniques we reveal for the first time that the universally conserved translational GTPase (trGTPase) HflX binds to the E-site of the 70S ribosome and that its GTPase activity is modulated by peptidyl transferase centre (PTC) and peptide exit tunnel (PET) binding antibiotics, suggesting a previously undescribed mode of action for these antibiotics. Our rapid kinetics studies reveal that HflX functions as a ribosome splitting factor that disassembles the 70S ribosomes into its subunits in a nucleotide dependent manner. Furthermore, our probing and hydrolysis studies show that the ribosome is able to activate trGTPases bound to its E-site. This is, to our knowledge, the first case in which the hydrolytic activity of a translational GTPase is not activated by the GTPase activating centre (GAC) in the ribosomal A-site. Furthermore, we provide evidence that the bound state of the PTC is able to regulate the GTPase activity of E-site bound HflX.

Introducing a class of standardized and interchangeable parts utilizing programmed ribosomal frameshifts for synthetic biology applications.

Synthetic biology and the rational design of biological devices depend on the availability of standardized and interchangeable biological parts with diverse range of functions. Reliable access to different reading frames during translation has largely been overlooked as functionality for bioengineering applications. Here we report the construction and initial characterization of the first member of such a class of biological parts that conforms to the BioBrick Standard (RFC25), allowing its interchangeable use in biological devices. Using our standardized frameshifting signal consisting of a UUUAAAG slippery sequence, a 6 nt spacer and an engineered pseudoknot based on the infectious bronchitis virus pseudoknot PK401 embedded in a dual reporter construct, we confirm that the frameshifting activity is comparable to the previously published frequency despite the introduced sequence changes. The frameshifting activity is demonstrated using SDS-PAGE and fluorescence spectroscopy. Standardized programmable ribosomal frameshift parts with specific frameshifting frequencies will be of utility for applications such as double coding DNA sequences by expanding the codable space into the -1 frame. Programmed shifting into the -1 frame to bypass a stop codon allows labeling of a protein pool with a fixed stoichiometry of fusion protein, as well as the construction of multi-enzyme expression constructs with specific expression ratios. A detailed understanding of the structural basis of programmed frameshifting will provide the opportunities to rationally design frameshifting elements with a wide range of applications in synthetic biology, including signals that are regulated by small ligands.

The ribosome modulates the structural dynamics of the conserved GTPase HflX and triggers tight nucleotide binding.

The universally conserved GTPase HflX is a putative translation factor whose GTPase activity is stimulated by the 70S ribosome as well as the 50S but not the 30S ribosomal subunit. However, the details and mechanisms governing this interaction are only poorly understood. In an effort to further elucidate the functional mechanism of HflX, we examined its interaction with the 70S ribosome, the two ribosomal subunits (50S and 30S), as well as its ability to interact with guanine nucleotides in the respective ribosomal complexes using a highly purified in vitro system. Binding studies reported here demonstrate that HflX not only interacts with 50S and 70S particles, but also with the 30S subunit, independent of the nucleotide-bound state. A detailed pre-steady-state kinetic analysis of HflX interacting with a non-hydrolyzable analog of mant-GTP, coupled with an enzymatic probing assay utilizing limited trypsinolysis, reveal that HflX·GTP exists in a structurally distinct 50S- and 70S-bound form that stabilizes GTP binding up to 70 000-fold and that may represent the "GTPase-activated" state. This activation is likely required for efficient GTP-hydrolysis, and may be similar to that observed in elongation factor G. Results reported here address the surprising low affinity of free HflX for GTP and suggest that cellular HflX will mainly exist in the HflX·GTP·ribosome-bound form. A minimal model for the functional cycle of HflX is proposed.

Primary amenorrhoea caused by congenital absence of the uterus.

The authors discuss the case of a 21-year-old female of Afro-Caribbean origin, who presented with primary amenorrhoea, and was found to have congenital absence of the uterus and fallopian tubes in the presence of a normal vaginal cavity.

Rectal bioavailability of delta-9-tetrahydrocannabinol from various esters.

The bioavailability of delta-9-tetrahydrocannabinol (delta 9-THC) from suppository formulations containing several polar esters was studied. The esters tested were the hemisuccinate, N-formyl alaninate, N-methyl carbamate, and methoxy acetate. These esters were administered to monkeys in both lipophilic and hydrophilic suppository bases, namely, Witepsol H15 and polyethylene glycol, respectively. Each suppository contained a dose equivalent to 10 mg delta 9-THC. Blood samples were analyzed for both delta 9-THC and its carboxylic acid metabolite (ll-nor-delta 9-THC-9-COOH) using gas chromatography/mass spectrometry. The data showed that, with the exception of the hemisuccinate, no delta 9-THC or its metabolite was detected in the blood samples using the Witepsol H15. Using polyethylene glycol, low levels of delta 9-THC and its metabolite were detected in blood for all esters tested. The levels, however, were lower than those observed with delta 9-THC hemisuccinate using Witepsol H15. Subsequent studies in the conscious dog using the hemisuccinate in Witepsol H15 showed 67% bioavailability of delta 9-THC with a linear response in the dose range equivalent to 5-20 mg of delta 9-THC. No significant bioavailability differences were found when delta 9-THC hemisuccinate ester was administered in various lipophilic bases (Hydrokote 25, Kaomel, Suppocire AIML, and Witepsol H15).

Rectal bioavailability of delta-9-tetrahydrocannabinol from the hemisuccinate ester in monkeys.

Oral administration of delta-9-tetrahydrocannabinal (delta 9-THC) was shown to result in low and erratic bioavailability, while the drug showed no bioavailability from various suppository formulations. delta 9-THC-Hemisuccinate was formulated as a prodrug for delta 9-THC in suppositories using Witepsol H15 base. The bioavailability of delta 9-THC from this formulation was evaluated in monkeys. The plasma levels of delta 9-THC and its metabolite 11-nor-delta 9-THC-9-COOH were determined using GC/MS analysis. The calculated bioavailability of delta 9-THC from this formulation was found to be 13.5%. Non-compartmental analysis of the plasma concentration data using statistical moments showed the mean residence time (MRT) for delta 9-THC in the body to be 3 h following iv administration of delta 9-THC or its hemisuccinate ester (3.4 and 2.7 h, respectively), as compared with 5.8 h following rectal administration of the delta 9-THC hemisuccinate. The observed rectal bioavailability of delta 9-THC from suppositories containing the hemisuccinate ester as a prodrug is of significant importance in developing an alternative approach to oral administration of the drug.

Comparison of serum progesterone levels in dogs after administration of progesterone by vaginal tablet and vaginal suppositories.

Serum progesterone levels from a vaginal tablet formulation and six different vaginal suppository formulations, each containing 25 mg of progesterone, were evaluated in mongrel dogs. Bioavailabilities relative to an intravenous dose of progesterone were calculated. The vaginal tablet was found to have a significantly higher bioavailability compared with the vaginal suppositories.

Development and evaluation of enteric-coated penicillamine tablets.

Commercially available 250-mg penicillamine tablets were converted into enteric-coated tablets. Based on in vitro dissolution and disintegration tests, tablets coated with five layers of a cellulose acetate phthalate formulation by a modified pan coating technique were judged to be superior to other coated tablets. These tablets resisted disintegration in simulated gastric fluid over a 4-h period and disintegrated in an average of 21 min in simulated intestinal fluid. Enteric-coated penicillamine tablets were tested in vivo in nine weanling pigs divided into three groups: a negative control group, a test group dosed with enteric-coated penicillamine tablets, and a positive control group dosed with uncoated tablets. The incidence of GI tract bleeding, as determined by daily occult blood tests of the stools, was significantly less in the animals receiving the enteric-coated tablets when compared with the positive control group. The enteric-coated dosage form appeared to decrease GI tract irritation caused by penicillamine. Plasma concentration-time curves for penicillamine in the pigs were similar in shape to those reported in humans. Atypical double peaks occur in both species. Relative bioavailability of the enteric-coated tablet was found to be 67% when compared with the uncoated tablet. This apparent reduction is probably due to a large intrasubject variation in areas under the plasma concentration-time curves and not to a dosage form effect.

Cannabinoids in glaucoma II: the effect of different cannabinoids on intraocular pressure of the rabbit.

Thirty-two different cannabinoids were tested for their ability to reduce intraocular pressure (IOP) in the rabbit. These included many of delta 9- and delta 8-THC derivatives and metabolites along with other natural and synthetic cannabinoids. In addition, some non-cannabinoid constituents of Cannabis were screened using the same model. All compounds were administered intravenously, while only a few were tested topically in mineral oil. Water soluble derivatives of delta 9- and delta 8-THC were prepared and tested topically in aqueous solution. The data revealed that certain derivatives of delta 9-and delta 8-THC were more active in lowering IOP than the parent cannabinoids. In addition, compounds other than delta 9- and delta 8-THC and their derivatives were shown to have activity.

Toxicological evaluation of poison oak urushiol and its esterified derivative.

Poison oak urushiol was compared with its esterified derivative for toxicity after oral administration to rats and guinea pigs. No hematological or pathological changes were noted and there were no differences seen in clinical chemistry measurements when compared to clinical biochemical and hematological reference values of normal experimental animals. Comparative LD50 studies in mice and tissue reactivity studies in rabbits indicated that acetylated urushiols were substantially less toxic than free urushiols. However, neither poison oak urushiol or poison oak urushiol acetate appeared to produce any tissue toxicity not related to a direct irritant effect. The free urushiol produced a much greater degree of skin irritation than did the urushiol acetate. Whether or not an animal had been sensitized to urushiol apparently had no effect on organ toxicity.

Biological effects of nonalkaloid-containing fractions of Erythroxylon coca.

Water soluble nonalkaloid fractions of Erythroxylon coca were screened in mice for their effects on oxygen utilization and central nervous system (CNS) activity. The fractions were screened in dogs for cardiovascular, blood glucose, and respiratory changes. No CNS effects were demonstrated in mice; however, there was a reduction in the oxygen utilization rate. Intravenous administration of the extract to dogs produced hyperglycemia, a reduction in heart rate, and a decrease in blood pressure. No substantial change in the respiratory rate and tidal or minute volumes were observed.

Pharmacokinetic interactions of tolazamide and oxyphenbutazone in dogs.

The described pharmacokinetic analysis involved two separate studies on nine dogs randomly assigned to three groups of three dogs each. In the first study, the effect of varying the dosage of tolazamide was examined. The second study concerned the effect of varying the dosage of oxyphenbutazone on tolazamide. A 3 x 3 Latin square was used to study both effects. Each group received each treatment, with a minimum of 1 week separating each session. THe pharmacokinetics of tolazamide followed a two-compartment open model. The hybrid rate constants, alpha and beta, were not significantly different at the three dosages when measured by a three-way analysis of variance. The only significant difference at the three dosage levels of tolazamide was in the apparent volume of distribution. In the pharmacokinetic interaction associated with intravenous administration of one dose of tolazamide and three doses of oxyphenbutazone, the apparent volume of distribution and the hybrid rate constant alpha did not change significantly while the hybrid rate constant for tolazamide, beta, seemed to decrease with increasing oxyphenbutazone.

Cannabinoids in glaucoma: a primary screening procedure.

A procedure was developed for screening of cannabinoids for their ability to reduce intraocular pressure (IOP) using normal rabbits. Eight animals per group were used for statistical significance of data. A negative control group was used for every screen as well as a positive control with 1.5 mg/kg delta 9-THC given intravenously (I.V.). All compounds were tested by I.V. injection and IOP measurements were taken periodically for 5 hours. Data were analyzed by a computer program which takes into account the change in IOP of the control group. Following this procedure we found that delta 8-THC, delta 9-THC, cannabinol, and nabilone were active while cannabidiol was inactive.

Immunological studies of poisonous anacardiaceae: effect of vehicle on absorption of 3-n-pentadecylcatechol and its diacetate ester derivative after oral feeding in rats.

Tritium-labeled 3-n-pentadecylcatechol and its diacetate ester were fed to Sprague-Dawley rats. Both compounds were dissolved in ethanol and in corn oil vehicles and administered by gavage. The rats were placed in metabolic cages, and the urine and feces was determined by liquid scintillation counting, and the percentage of the administered dose was utilized as a measure of absorption. While there was no difference between the absorption of either compound, absorption was affected by the vehicle. Approximately 30% of the administered radioactivity appeared in the urine when ethanol was the vehicle, but about half that amount (14%) was excreted in the urine when the compounds were dissolved in corn oil. A subsequent bile cannulation study showed that the balance of the radioactivity found in the feces was not a result of biliary excretion. The majority of the activity recovered from urine and feces was eliminated within 48 hr after dosing. These data indicate that oil is a poor vehicle for GI absorption of urushiol components.