A method for amending loose smokeless tobacco with menthol for administration in clinical studies.

INTRODUCTION: Menthol has long been incorporated as a flavor additive in tobacco products and can impact use behaviors. Despite its inclusion in some of the most popular flavored smokeless tobacco (ST) products (e.g., "mint" flavored products), few studies have systematically investigated the impact of menthol on ST use behaviors in prospective empirical studies. Rigorous investigation of ST menthol content on behavioral and physiological outcomes requires ST products with stable and precise levels of menthol; however, commercial product composition variability prevents product comparisons when evaluating the effects of systematic changes in menthol content on clinical outcomes. METHODS: We developed amended loose moist snuff ST products by treating commercially available, unflavored loose ST with an ethanol-based menthol spiking solution or a nonmentholated ethanol control solution to develop test products with different levels of menthol: 0, 1, 3, and 5 mg menthol/g tobacco. We evaluated the stability of menthol content in these products over 24 months and evaluated menthol exposure associated with the products through pharmacokinetic analysis of plasma menthol-glucuronide in human participants (n=22). RESULTS: Menthol content of the amended products was on target, homogenous, and stable for up to 24 months. Menthol exposure (menthol-glucuronide Cmax and AUC) significantly differed between each test product. CONCLUSIONS: These data suggest that stable products with nonoverlapping menthol content can be developed using a menthol spiking solution and can be subsequently administered for clinical assessments of mentholated loose ST. IMPLICATIONS: The results from this study suggest that a menthol spiking solution can be used to mentholate unflavored, loose ST to a target menthol content. With this method, the ST menthol content was stable for at least 24 months, and the products exposed users to menthol in a dose-dependent manner. This method yielded loose ST products with precise, stable levels of menthol to allow systematic evaluation of ST menthol content on clinical outcomes. The method may have applications for systematically evaluating changes in other tobacco product ingredients.

Insight into the Initial Stages of the Folding Process in Onconase Revealed by UNRES.

The unfolded state of proteins presents many challenges to elucidate the structural basis for biological function. This state is characterized by a large degree of structural heterogeneity which makes it difficult to generate structural models. However, recent experiments into the initial folding events of the 104-residue ribonuclease homologue onconase (ONC) were able to identify the regions in the protein that participate in the initial folding of this protein. Therefore, to gain additional structural insight into the unfolded state of proteins, this study utilized molecular dynamics simulations using the UNited-RESidue (UNRES) force field to evaluate whether there is a good agreement between the experimentally determined initial structures and the structures identified by computer simulations along a folding pathway. Indeed, these UNRES simulations accurately identified the two regions experimentally observed to form the initial native structure along the folding pathway of ONC. In addition, these regions are determined to be chain folding initiation sites (CFIS) according to methods developed previously. Subsequent self-organization maps (SOM) analysis has revealed key structural states involved in these early folding events.

Expanding the Biological Importance of Protein Structures: Insight into Dynamic Biological Function from Protein Folding Theory Analyses.

While recent developments in the determination of the three-dimensional structure of proteins have rapidly progressed, there remains a difficult challenge of studying proteins that exhibit dynamic behavior as part of their biological functions in environments considerably different than how their three-dimensional structure was determined. This study investigates the dynamic behavior of Bax, a member of the Bcl-2 family of proteins, during the regulation of apoptosis in the context of its published three-dimensional structure. The location of Bax in live cells is an equilibrium between the cytosol and outer-mitochondrial membrane. However, the regions of Bax that have been determined to be responsible for this equilibrium are shown to be inaccessible to engage in these interactions, namely, the C-terminal helix, according to the solved three-dimensional structure. Therefore, the analyses that have been applied to identify chain folding initiation sites (CFIS) and propose unfolding pathways have also been applied to the three-dimensional structure of Bax to provide a rationale for how Bax can engage in the dynamic behavior that is part of its biological function. The analyses identified regions in Bax that contribute to its stability and regions that could be susceptible to conformational changes, including the C-terminal helix, and, consequently, dynamic behavior. Experimental observations confirmed the classification of these regions. Consequently, the utilization of methods to identify CFIS on three-dimensional structures can be an effective tool to help expand our knowledge about the biological function of proteins that exhibit dynamic behavior.

Analysis of Nicotine and Non-nicotine Tobacco Constituents in Aqueous Smoke/Aerosol Extracts by UHPLC and Ultraperformance Convergence Chromatography-Tandem Mass Spectrometry.

The non-nicotine constituents of tobacco may alter the reinforcing effects of nicotine, but the quantitative and qualitative profiles of these chemicals in tobacco products such as electronic cigarettes (e-cigarettes), cigars, and waterpipe tobacco are not well characterized. The objective of this work was to develop and validate analytical methods to utilize saline both as an extraction solvent for smoke condensates from cigarettes, little cigars, and waterpipe tobacco and aerosols from e-cigarettes and as a delivery vehicle of nicotine and non-nicotine constitents for nonclinical pharmacological studies. Ultrahigh-performance liquid chromatography was used to analyze nicotine and acetaldehyde, and a novel ultraperformance convergence chromatography-tandem mass spectrometry method was developed to analyze anabasine, anatabine, cotinine, myosmine, nornicotine, harmane, and norharmane. Linearity was confirmed for each standard curve with correlation coefficients (r) ≥ 0.99, and relative errors (RE) for the standards were ≤±10% over the calibration ranges. Method validation was performed by preparing triplicate samples in saline to mimic the composition and concentration of each analyte in the smoke or aerosol condensate and were used to determine method accuracy and precision. Relative standard deviation values were ≤15% and mean RE ≤15% for each analyte at each concentration level. Selectivity of the methods was demonstrated by the absence of peaks in blank vehicle or diluent samples. Storage stability was assessed over ∼45 days. Precision (%RSD ≤ 13) and recovery (percent of day 0 ≥ 80%) indicated that the saline formulations of all four products could be considered stable for up to ∼45 days at 4-8 °C. Therefore, the use of saline both as an extraction solvent and as a delivery vehicle adds versatility and improved performance in the study of the pharmacological effects of constituents from mainstream smoke and aerosols generated from cigarettes, little cigars, waterpipes, and e-cigarettes.

Comparison of cigarette, little cigar, and waterpipe tobacco smoke condensate and e-cigarette aerosol condensate in a self-administration model.

The pharmacological effects of tobacco products are primarily mediated by nicotine; however, research suggests that several non-nicotine tobacco constituents may alter the reinforcing effects of nicotine. This study evaluated the reinforcing effects of aqueous solutions of smoke/aerosol condensate from cigarettes, little cigars, electronic cigarettes (e-cigarettes), and waterpipe tobacco in a self-administration procedure to determine if abuse liability of these tobacco products differed. Adult male Sprague-Dawley rats (n = 64 total) were trained to self-administer intravenous nicotine (30 µg/kg/infusion) on a fixed ratio 5 schedule of reinforcement. Following nicotine dose-effect assessment (1, 7.5, 15, and 30 µg/kg/infusion), rats were given access to smoke/aerosol condensate derived from their assigned tobacco product. Rats responded for smoke/aerosol condensate containing 1, 7.5, 15, and 30 µg/kg/infusion nicotine, with the ratio of nicotine:non-nicotine constituents held constant across doses for each tobacco product. Responding for nicotine or smoke/aerosol condensate was also assessed on a progressive ratio schedule of reinforcement. Cigarette, little cigar, and e-cigarette smoke/aerosol condensates shifted the nicotine dose-effect curve leftward, whereas waterpipe tobacco smoke condensate shifted the dose-effect curve rightward. Smoke/aerosol condensate from all tobacco products produced similar levels of responding compared to nicotine alone during the progressive ratio phase. Results suggest that non-nicotine constituents in cigarettes, little cigars, and e-cigarettes differentially enhance nicotine's reinforcing potency. In contrast, waterpipe tobacco blunted nicotine's reinforcing potency, suggesting that it may contain unique constituents that dampen nicotine's reinforcing effects.

Microinjection of Live Mammalian Cells: A Delivery Method that Provides Added Versatility to the Study of Cellular Function.

Microinjection is a technique that allows for the delivery of a diverse array of compounds and biomolecules into live mammalian cells. As a result, the behavior of injected biomolecules and their resulting effects can be observed in the native environments of mammalian cells in real time. This capability allows for more accurate observations and conclusions about biological systems of interest. This chapter discusses the protocol and guidelines to successfully microinject live mammalian cells to maintain their viability. This chapter outlines considerations into the preparation of samples and the microscopy setup as well as a description of a stepwise presentation of the methods for proper execution of a microinjector. The presentation of the technique serves as a starting point to understanding how to apply microinjection to a cellular system compatible with a wide array of live-cell imaging techniques.

Bcl-2 proteins bid and bax form a network to permeabilize the mitochondria at the onset of apoptosis.

The most critical step in the initiation of apoptosis is the activation of the Bcl-2 family of proteins to oligomerize and permeabilize the outer-mitochondrial membrane (OMM). As this step results in the irreversible release of factors that enhance cellular degradation, it is the point of no return in programmed cell death and would be an ideal therapeutic target. However, the arrangement of the Bcl-2 proteins in the OMM during permeabilization still remains unknown. It is also unclear whether the Bcl-2 protein, Bid, directly participates in the formation of the oligomers in live cells, even though it is cleaved and translocates to the OMM at the initiation of apoptosis. Therefore, we utilized confocal microscopy to measure Förster resonance energy transfer (FRET) efficiencies in live cells to determine the conformation(s) and intermolecular contacts of Bid within these Bcl-2 oligomers. We found that Bid adopts an extended conformation, which appears to be critical for its association with the mitochondrial membrane. This conformation is also important for intermolecular contacts within the Bid oligomer. More importantly for the first time, direct intermolecular contacts between Bid and Bax were observed, thereby, confirming Bid as a key component of these oligomers. Furthermore, the observed FRET efficiencies allowed us to propose an oligomeric arrangement of Bid, Bax, and possibly other members of the Bcl-2 family of proteins that form a self-propagating network that permeabilizes the OMM.

Acquiring snapshots of the orientation of trans-membrane protein domains using a hybrid FRET pair.

One challenge in studying the function of membrane-embedded proteins is determining the orientation of key domains in the context of the changing and dynamic membrane environment. We describe a confocal microscopy setup that utilizes external electric field pulses to direct dipicrylamine (DPA) to a membrane leaflet. The detection of FRET between DPA and a fluorescent probe attributes it to the inner or outer leaflet of a membrane. By utilizing short acquisition times and confocal imaging, this attribution could be made even in changing membrane environments. Our setup adds versatility to the study of the biological activity of membrane-embedded proteins.

Conformational rearrangements in the pro-apoptotic protein, Bax, as it inserts into mitochondria: a cellular death switch.

The B-cell lymphoma 2 (Bcl-2) family of proteins regulates the activation of apoptosis through the mitochondria pathway. Pro- and anti-apoptotic members of this family keep each other in check until the correct time to commit to apoptosis. The point of no return for this commitment is the permeabilization of the outer mitochondrial membrane. Translocation of the pro-apoptotic member, Bax, from the cytosol to the mitochondria is the molecular signature of this event. We employed a novel method to reliably detect Förster resonance energy transfer (FRET) between pairs of fluorophores to identify intra-molecular conformational changes and inter-molecular contacts in Bax as this translocation occurs in live cells. In the cytosol, our FRET measurement indicated that the C-terminal helix is exposed instead of tucked away in the core of the protein. In addition fluorescence correlation spectroscopy (FCS) showed that cytosolic Bax diffuses much slower than expected, suggesting possible complex formation or transient membrane interaction. Cross-linking the C-terminal helix (α9) to helix α4 reduced the potential of those interactions to occur. After translocation, our FRET measurements showed that Bax molecules form homo-oligomers in the mitochondria through two distinct interfaces involving the BH3 domain (helix α2) and the C-terminal helix. These findings have implications for possible contacts with other Bcl-2 proteins necessary for the regulation of apoptosis.

Single color FRET based measurements of conformational changes of proteins resulting from translocation inside cells.

Translocation of proteins to different parts of the cell is necessary for many cellular mechanisms as a means for regulation and a variety of other functions. Identifying how these proteins undergo conformational changes or interact with various partners during these events is critical to understanding how these mechanisms are executed. A protocol is presented that identifies conformational changes in a protein that occur during translocation while overcoming challenges in extracting distance information in very different environments of a living cell. Only two samples are required to be prepared and are observed with one optical setup. Live-cell FRET imaging has been applied to identify conformational changes between two native cysteines in Bax, a member of the Bcl-2 family of proteins that regulates apoptosis. Bax exists in the cytosol and translocates to the mitochondria outer membrane upon apoptosis induction. The distance, r, between the two native cysteines in the cytosolic structure of Bax necessitates the use of a FRET donor-accepter pair with R0~r as the most sensitive probe for identifying structural changes at these positions. Alexa Fluor 546 and Dabcyl, a dark acceptor, were used as FRET pairs - resulting in single color intensity variations of Alexa-546 as a measure of FRET efficiency. An internal reference, conjugated to Bax, was employed to normalize changes in fluorescence intensity of Alexa Fluor 546 due to inherent inhomogeneities in the living cell. This correction allowed the true FRET effects to be measured with increased precision during translocation. Normalization of intensities to the internal reference identified a FRET efficiency of 0.45±0.14 in the cytosol and 0.11±0.20 in the mitochondria. The procedure for the conjugation of the internal reference and FRET probes as well as the data analysis is presented.

Identification of formation of initial native structure in onconase from an unfolded state.

In the oxidative folding of onconase, the stabilization of intermediates early in the folding process gives rise to efficient formation of its biologically active form. To identify the residues responsible for the initial formation of structured intermediates, the transition from an ensemble of unstructured three-disulfide species, 3S(U), to a single structured three-disulfide intermediate species, des-[30-75] or 3S(F), at pH 8.0 and 25 °C was examined. This transition was first monitored by far-UV circular dichroism spectroscopy at pH 8.0 and 25 °C, showing that it occurs with the formation of secondary structure, presumably because of native interactions. The time dependence of formation of nativelike structure was then followed by nuclear magnetic resonance spectroscopy after we had arrested the transition at different times by lowering the pH to 3 and then acquiring (1)H-(15)N heteronuclear single-quantum coherence spectra at pH 3 and 16 °C to identify amide hydrogens that become part of nativelike structure. H/D exchange was utilized to reduce the intensity of resonances from backbone amide hydrogens not involved in structure, without allowing exchange of backbone amide hydrogens involved in initial structure. Six hydrogen-bonding residues, namely, Tyr38, Lys49, Ser82, Cys90, Glu91, and Ala94, were identified as being involved in the earliest detectable nativelike structure before complete formation of des-[30-75] and are further stabilized later in the formation of this intermediate through S-S/SH interchange. By observing the stabilization of the structures of these residues by their neighboring residues, we have identified the initial, nativelike structural elements formed in this transition, providing details of the initial events in the oxidative folding of onconase.

Effects of tyrosine mutations on the conformational and oxidative folding of ribonuclease a: a comparative study.

Ribonuclease A (RNase A) undergoes more rapid conformational folding with its disulfide bonds intact than during oxidative folding from its reduced form. In this study, the effects of the mutants Y92G, Y92A, and Y92L on both the conformational and oxidative folding pathways were examined to determine the role of native interactions in different types of conformational searches for the biologically active structure of a protein. These mutations did not affect the overall conformational folding pathway of RNase A. However, in the mutants Y92G and Y92A, a key structured disulfide-bonded species, des-[65-72], involved in the oxidative folding pathway of RNase A, was destabilized. These results demonstrate the importance of native interactions in the folding process, namely, protection of a native (40-95) disulfide bond by a nearby tyrosyl-prolyl stacking interaction, when disulfide bonds are allowed to undergo SH/S-S reshuffling.

Oxidative folding pathway of onconase, a ribonuclease homologue: insight into oxidative folding mechanisms from a study of two homologues.

The oxidative folding pathways of two four-disulfide proteins of the ribonuclease family, ONC and RNase A, which have similar three-dimensional folds but only 30% sequence homology, are compared. In this study, a mechanism for the oxidative folding pathway of ONC is proposed. In particular, the kinetic roles and thermodynamic characteristics of key intermediates along the oxidative folding pathway, specifically, the structured intermediates, I(1), I(2), and I(3), previously identified as des-[19-68,30-75], des-[30-75], and des-[19-68], respectively, are discussed. In addition, the effects of temperature on the oxidative folding pathway have been examined. Differences in the folding mechanism between ONC and RNase A are attributed to the differences in their amino acid sequences and related inter-residue interactions, including differences in hydrophobic interactions. Compared to RNase A, ONC utilizes more efficient interactions along the oxidative folding pathway to adopt its native fold more rapidly.

Dissimilarity in the oxidative folding of onconase and ribonuclease A, two structural homologues.

The oxidative folding of frog onconase (ONC), a member of the ribonuclease A family, was examined and shows markedly different behavior compared to its structural homologue bovine pancreatic ribonuclease A (RNase A) under similar conditions. Application of a reduction pulse (using a small amount of reduced dithiothreitol) during the oxidative regeneration of ONC indicated the survival of the native protein along with three other (structured) species, I(1), I(2) and I(3), with the rest of the unstructured species being converted to fully reduced protein. Mass spectrometry indicates that I(1) has two disulfide bonds, whereas I(2) and I(3) have three disulfide bonds each. A disulfide mapping method, based on cyanylation, was used to identify I(2) and I(3) as des-[30-75] and des-[19-68], respectively. On enzymatic digestion using trypsin, I(1) was identified as des-[19-68, 30-75]. Differences in the intermediates that are generated during the oxidative folding of the two structural homologues, RNase A and ONC, demonstrate that regenerative pathways are not necessarily influenced by tertiary structure. This indicates that the lack of a disulfide bond in ONC, analogous to the (65-72) disulfide bond in RNase A which plays an important role in its oxidative regeneration, does not adversely affect the oxidative folding of ONC.

Trimethylamine-N-oxide modulates the reductive unfolding of onconase.

The physiological osmolyte trimethylamine-N-oxide (TMAO) stabilizes proteins by decreasing the entropy of the unfolded state through a solvophobic effect. Our studies on the effect of TMAO on the reductive unfolding of onconase (ONC) to form its reductive intermediate, des [30-75], indicate that TMAO diminishes the reductive unfolding rate of the protein although it does not significantly affect the stability of the native protein relative to its denatured state. Since the reductive unfolding of ONC is a local event, our studies provide direct evidence for a TMAO-induced local structural change that reduces the rate of redox-dependent protein unfolding. The implications of our findings for protein folding/unfolding are discussed.