Investigating the transfer rate of waterpipe additives to smoke as an integral part of toxicological risk assessments.

Waterpipe, also known as hookah, narghile or narghila, shisha or hubbly bubbly, is a tobacco-smoking device. Waterpipe tobacco is heated and consumed by a process of inhaling tobacco smoke, that bubbles through water before being inhaled. To date, limited studies have examined the transfer of waterpipe additives from tobacco to smoke. This study was designed to investigate the filtration ability of water in the waterpipe's bowl to define exposure to additives in waterpipe smoke, which is an essential requirement to perform toxicological risk assessments of waterpipe additives. Within this study, a standard smoking protocol (ISO 22486) was used to evaluate the transfer of > 40 additives from experimental and commercially available samples. These results are the first to provide such an extensive dataset of information showing transfer rates varying between 6% and 61% depending on the additive. Various physicochemical parameters of the additives including water solubility, partition coefficient, molecular weight, boiling point, and vapor pressure were also evaluated to seek to identify any correlation to transfer rate that may be later used to predict transfer. The amount of additive transfer from waterpipe tobacco to the smoke was found to be moderately correlated to vapor pressure (Pearson correlation coefficient = 0.33) with subsequent multivariate analysis using step-wise selection indicating 39% of the transfer rate variance can be explained collectively by the additive boiling point, molecular weight, vapor pressure and water solubility. These findings underscore the complexity of additive transfer and highlight the necessity of exposure assessment for meaningful waterpipe additive risk assessments.

A review of efferent cholinergic synaptic transmission in the vestibular periphery and its functional implications.

It has been over 60 years since peripheral efferent vestibular terminals were first identified in mammals, and yet the function of the efferent vestibular system remains obscure. One reason for the lack of progress may be due to our deficient understanding of the peripheral efferent synapse. Although vestibular efferent terminals were identified as cholinergic less than a decade after their anatomical characterization, the cellular mechanisms that underlie the properties of these synapses have had to be inferred. In this review we examine how recent mammalian studies have begun to reveal both nicotinic and muscarinic effects at these terminals and therefore provide a context for fast and slow responses observed in classic electrophysiological studies of the mammalian efferent vestibular system, nearly 40 years ago. Although incomplete, these new results together with those of recent behavioral studies are helping to unravel the mysterious and perplexing action of the efferent vestibular system. Armed with this information, we may finally appreciate the behavioral framework in which the efferent vestibular system operates.

The alpha9/alpha10-containing nicotinic ACh receptor is directly modulated by opioid peptides, endomorphin-1, and dynorphin B, proposed efferent cotransmitters in the inner ear.

UNLABELLED: Opioid peptides have been detected in the auditory and vestibular efferent neurons where they colocalize with the major neurotransmitter, acetylcholine. We investigated the function of opioids to modulate neurotransmission mediated by hair cell's alpha9/alpha10-containing nicotinic acetylcholine receptors (alpha9/alpha10nAChRs). The endogenous opioid peptides, endomorphin-1 (mu agonist) and dynorphin B (kappa agonist), but not a delta agonist [D-Pen2,D-Pen-5]enkephalin, inhibited the acetylcholine-evoked currents in frog saccular hair cells and rat inner hair cells. This inhibition was noncompetitive, voltage-independent, and was accompanied by an acceleration of the rate of current decay. Selective mu- and kappa-opioid receptor antagonists did not block the inhibition, although partial reduction by naloxone was observed. All opioid antagonists tested also reduced the acetylcholine response. Endomorphin-1 and dynorphin B inhibited the acetylcholine-evoked currents in alpha9/alpha10-expressing Xenopus oocytes. Because oocytes lack opioid receptors, it provides strong evidence for the direct interaction of opioid peptides with alpha9/alpha10nAChR. CONCLUSION: alpha9/alpha10nAChR is a target for modulation by endomorphin-1 and dynorphin B, efferent cotransmitters in the inner ear.

The effect of proteolytic enzymes on the alpha9-nicotinic receptor-mediated response in isolated frog vestibular hair cells.

In frog vestibular organs, efferent neurons exclusively innervate type II hair cells. Acetylcholine, the predominant efferent transmitter, acting on acetylcholine receptors of these hair cells ultimately inhibits and/or facilitates vestibular afferent firing. A coupling between alpha9-nicotinic acetylcholine receptors (alpha9nAChR) and apamin-sensitive, small-conductance, calcium-dependent potassium channels (SK) is thought to drive the inhibition by hyperpolarizing hair cells thereby decreasing their release of transmitter onto afferents. The presence of alpha9nAChR in these cells was demonstrated using pharmacological, immunocytochemical, and molecular biological techniques. However, fewer than 10% of saccular hair cells dissociated using protease VIII, protease XXIV, or papain responded to acetylcholine during perforated-patch clamp recordings. When present, these responses were invariably transient, small in amplitude, and difficult to characterize. In contrast, the majority of saccular hair cells ( approximately 90%) dissociated using trypsin consistently responded to acetylcholine with an increase in outward current and concomitant hyperpolarization. In agreement with alpha9nAChR pharmacology obtained in other hair cells, the acetylcholine response in saccular hair cells was reversibly antagonized by strychnine, curare, tetraethylammonium, and apamin. Brief perfusions with either protease or papain permanently abolished the alpha9-nicotinic response in isolated saccular hair cells. These enzymes when inactivated became completely ineffective at abolishing the alpha9-nicotinic response, suggesting an enzymatic interaction with the alpha9nAChR and/or downstream effector. The mechanism by which these enzymes render saccular hair cells unresponsive to acetylcholine remains unknown, but it most likely involves proteolysis of alpha9nAChR, SK, or both.

Morphine inhibits an alpha9-acetylcholine nicotinic receptor-mediated response by a mechanism which does not involve opioid receptors.

Nicotinic acetylcholine (nACh) receptors are known to be targets for modulation by a number of substances, including the opiates. It is known that acetylcholine (ACh) coexists with opioid peptides in cochlear efferent neurons, and such a colocalization has been proposed for the vestibular system. In the present study we test the hypothesis that morphine, an opioid receptor agonist with a broad spectrum of selectivity, modulates alpha9nACh receptor-mediated responses in frog vestibular hair cells. Morphine dose-dependently and reversibly inhibited ACh-induced currents as recorded by the perforated patch-clamp method. In the presence of morphine the ACh dose-response curve was shifted to the right in a parallel fashion, suggesting a competitive interaction. However, naloxone did not antagonize the inhibition produced by morphine. To test the hypothesis that morphine could interact with the alpha9nACh receptor without the involvement of opioid receptors, experiments were performed using Xenopus laevis oocytes injected with the alpha9nACh receptor cRNA. The currents activated by ACh in Xenopus oocytes, a system that lacks opioid receptors, were also dose-dependently inhibited by morphine. We conclude that morphine inhibits the alpha9nACh receptor-mediated response in hair cells and Xenopus oocytes through a mechanism which does not involve opioid receptors but may be a direct block of the alpha9nACh receptor.

A role for chloride in the hyperpolarizing effect of acetylcholine in isolated frog vestibular hair cells.

Acetylcholine (ACh) is the dominant transmitter released from inner ear efferent neurons. In frog vestibular organs, these efferent neurons synapse exclusively with type II hair cells. Hair cells isolated from the frog saccule hyperpolarize following the application of 50 microM ACh, thereby demonstrating the presence of an ACh receptor. A role for Cl(-) in the response of hair cell-bearing organs to efferent nerve activation or ACh application was suggested some years ago. Perfusion with solutions in which most of the Cl(-) was replaced by large impermeant anions decreased the cholinergic inhibition of afferent firing in the cat and turtle cochleas, and frog semicircular canal. Our previous work in the intact organ demonstrated that substitution of large impermeant anions for Cl(-) or use of Cl(-) channel blockers reduced the effect of ACh on saccular afferent firing. Using the perforated-patch clamping technique, replacement of Cl(-) by methanesulfonate, iodide, nitrate, or thiocyanate attenuated the hyperpolarizing response to ACh in hair cells isolated from the frog saccule. The chloride channel blockers picrotoxin and 4,4'-dinitrostilbene-2,2'-disulfonic acid were also tested and found to inhibit the ACh response. Thus, the present work demonstrates that the effects of Cl(-) substitutions or Cl(-) channel blockers on the ACh response in the intact saccule can be explained completely by effects on the hair cell. Evidence is also presented for the presence of the messenger RNA for a calcium-dependent chloride channel in all hair cells but especially saccular hair cells. This channel may be involved in the response to ACh. The precise role for chloride in this response, whether as a distinct ion current, as a transported ion, or as a permissive ion for other components, is discussed.

An examination of antecedent traumas and psychiatric comorbidity among male inmates with PTSD.

Despite substantially higher rates of posttraumatic stress disorder (PTSD) among male inmates than among men in the general population, there is a dearth of research on PTSD among incarcerated men. The current study addresses traumatic events that precede PTSD and psychiatric disorders that are comorbid with PTSD in an inmate sample. Seeing someone seriously injured or killed, being sexually abused, and being physically assaulted were the three most commonly reported antecedent traumas to PTSD. Lifetime and current rates of mood disorders, anxiety disorders, and antisocial personality disorder were elevated among inmates with a diagnosis of PTSD. Two hundred and thirteen inmates participated in the study. Sixty-nine participants (33%) met lifetime DSM-III-R criteria for PTSD, and 45 (21%) met current criteria. The findings are compared with general population samples, and implications of the findings are discussed.

Psychological impact of childhood sexual abuse on male inmates: the importance of perception.

OBJECTIVE: The current study examined the association between childhood sexual victimization and adult psychiatric disorders among male inmates. It further assessed the association between the perception of an event (as sexual abuse or not) and psychiatric diagnoses. METHOD: A sample of 211 randomly-selected male inmates were interviewed. The Diagnostic Interview Schedule (Version III-R) was used to assess psychiatric diagnoses. An additional questionnaire assessing childhood sexual abuse and perception of childhood sexual abuse was also administered. RESULTS: Forty percent of the inmates met standard criteria for childhood sexual abuse, which far exceeded rates found in the general population. Significant differences were found between inmates who had a history of childhood sexual abuse and those who did not for a variety of psychiatric diagnoses. Forty-one percent of those who met criteria for childhood sexual abuse did not consider themselves to have been abused. Those who did not consider themselves to have been abused had higher rates of alcohol abuse/dependence, while those who considered themselves to have been abused had higher rates of posttraumatic stress and obsessive-compulsive disorder. CONCLUSIONS: This study emphasizes the importance of perception or "cognitive appraisal" of the sexual experience (as abusive or not) and the need for further study regarding the potential mediating role of cognitive appraisal. Other implications of these findings include the need for primary prevention programs designed to reduce childhood sexual abuse, and inmate rehabilitation programs with an emphasis on the connection between victimization and criminality.

The metabotropic glutamate receptors of the vestibular organs.

This research sought to test the presence and function of metabotropic excitatory amino acid receptors (mGluR) in the frog semicircular canal (SCC). The mGluR agonist +/- 1-aminocyclopentane-trans-1,3-dicarboxylate (ACPD) produced an increase in afferent firing rates of the ampullar nerve of the intact posterior canal. This increase was not due to a stimulation of cholinergic efferent terminals or the acetylcholine (ACh) receptor, since atropine, in concentrations which blocked the response to exogenous acetylcholine, did not affect the response to ACPD. Likewise, ACPD effects were not due to stimulation of postsynaptic NMDA receptors, since the NMDA antagonist D(-)-2-amino-5-phosphonopentanoate (AP-5) did not affect the response to ACPD, reinforcing the reported selectivity of ACPD for mGluRs. When the SCC was superfused with artificial perilymph known to inhibit hair cell transmitter release (i.e. low Ca-high Mg), ACPD failed to increase afferent firing. This suggests that the receptor activated by ACPD is located on the hair cell. Pharmacological evidence suggested that the mGluRs involved in afferent facilitation belong to Group I (i.e. subtypes 1 and 5). In fact, the Group III agonist AP-4 had no effect, and the ACPD facilitatory effect was blocked by the Group I mGluR antagonists (S)-4-carboxyphenylglycine (CPG) and (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA). Additional pharmacological evidence supported the presence of Group I mGluRs. Interestingly, the mGluR antagonists, AIDA and 4CPG, by themselves did not affect the resting firing rates of ampullar afferents. This may suggest that the mGluRs are not involved in resting activity but perhaps only in evoked activity (as suggested in Guth et al. (1991) Hear. Res. 56, 69-78). In addition, the mRNA for the mGluR1 has been detected in hair cells of both SCC, utricle, and saccule. In summary, the evidence points to an mGluR localized to the hair cell (i.e. an autoreceptor) which may be activated to produce a positive feedback augmentation of evoked but not resting transmitter release and thus affect afferent activity.

Mechanisms and effects of transepithelial polarization in the isolated semicircular canal.

Previous studies have shown that galvanic stimulation of semicircular canal organs can modulate their afferent discharge. However, it has not been resolved whether this modulation derived from direct stimulation of hair cells, afferent nerve fibers, some combination of the two, or some as yet unknown path. This problem is addressed in the present study. Experiments were designed first to determine the gross current path necessary for the DC current to modulate afferent firing. These led to the conclusion that the current path had to flow between endolymph and perilymph across the neuroepithelium. Next, the various components in this established path were considered: the afferents, the hair cells, between the hair cells, or some combination of the three. These experiments led to the conclusion that the current pathway was across the hair cells causing transmitter release and thus affecting afferent activity.

Cloning of the cDNA and nucleotide sequence of a skeletal muscle protease from myopathic hamsters.

A neutral protease with an estimated Mr of about 26 kD and responsible for cleavage ofmyosin LC2 was isolated from hamster skeletal muscle. Complementary DNAs were generated by RT-PCR using total hamster muscle RNA and degenerate oligonucleotide primers based on the sequences of two internal peptides. The nucleotide sequences of the resultant cDNAs were subsequently determined and the complete amino acid sequence of the protease deduced. Although the hamster protein shared 63-85% identity in nucleotide and amino acid sequences with rat and mouse mast cell proteases, it had a higher degree of specificity for myosin LC2 than mast cell proteases which also digested myosin LC1 and myosin heavy chains. As a result, the hamster protease was designated mekratin because of its unique enzymatic specificities to distinguish it from other mast cell proteases. A polyclonal antibody was raised specific to the hamster muscle and human cardiac muscle mekratins without apparent cross-reaction with rat mast cell proteases. We have earlier demonstrated the presence in excess of a neutral protease that specifically cleaves LC2 in human hearts obtained at end stage idiopathic dilated cardiomyopathy (IDC). Western analyses revealed that heart tissue from patients with IDC contained 5-10 fold more mekratin than control samples. Furthermore, the level of the protease in human IDC tissues was similar to that seen in myopathic hamster skeletal muscle. No bands were recognized by the antibody when IDC myofibrils were probed due to the removal of soluble proteins during sample preparation. Thus, these results strongly suggest that the anti-mekratin antibody will provide positive identification of IDC in many cases and diagnosis by exclusion may be replaced.

Alboaggregins A and B. Structure and interaction with human platelets.

Viper venoms contain a variety of platelet binding proteins including those which bind to platelet GPIb/GPIX. Most of these proteins inhibit von Willebrand factor mediated platelet agglutination. Here we report the primary structures of unique members of this family, alboaggregins A and B, isolated from Trimeresurus albolabris, which have the ability to stimulate platelet agglutination and aggregation. Four chains of alboaggregin A and two chains of alboaggregin B share a high degree of homology and all cysteines in both alboaggregins are conserved. Both alboaggregins caused similar agglutination of fixed platelets. Alboaggregin A induced platelet aggregation and release reaction with EC50 = 10 and 30 nM, respectively, which is 20-fold lower than those for alboaggregin B. These observations suggest that the dimeric structure of alboaggregin B is sufficient to mediate its binding to GPIb and induce agglutination of platelets whereas aggregation and release reaction are significantly enhanced by tetrameric structure of alboaggregin A.

A role for chloride in the suppressive effect of acetylcholine on afferent vestibular activity.

Afferents of the frog semicircular canal (SCC) respond to acetylcholine (ACh) application (0.3-1.0 mM) with a facilitation of their activity while frog saccular afferents respond with suppression (Guth et al., 1994). All recordings are of resting (i.e., non-stimulated) multiunit activity as previously reported (Guth et al., 1994). Substitution of 80% of external chloride (Cl-) by large, poorly permeant anions of different structures (isethionate, methanesulfonate, methylsulfate, and gluconate) reduced the suppressive effect of ACh in the frog saccular afferents. This substitution did not affect the facilitatory response of SCC afferents to ACh. Chloride channel blockers were also used to test further whether Cl- is involved in the ACh suppressive effect. These included: niflumic and flufenamic acids, picrotoxin, 5-nitro-2-(-3-phenylpropylamino)benzoic acid (NPPB), and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). As with the Cl- substitutions, all of these agents reduced the suppressive response to ACh in the saccule, but not the facilitatory response seen in the SCC. The suppressive effect of ACh on saccular afferents is considered to be due to activation of a nicotinic-like receptor (Guth et al., 1994; Guth and Norris, 1996). Taking into account the effects of both Cl- substitutions and Cl- channel blockers, we conclude that changes in Cl- availability influence the suppressive effect of ACh and that therefore Cl- may be involved in this effect.

The prevalence of mental illness among inmates in a rural state.

A limited number of recent empirical studies suggest that inmates suffer from high rates of serious mental illness. Different explanations are offered depending on the type of institution: jail or prison. The literature is based largely on urban samples and does not offer comparisons of rates across types of institution within a single study. The present study examined a random sample of 213 jail and prison inmates in a rural state using the Diagnostic Interview Schedule (III-R). Among jail inmates there was little evidence of high rates of serious mental illness, suggesting the criminalization of mental illness may not be as evident in rural settings as urban areas. Among prison inmates, however, high rates of mental disorders were found, supporting previous findings in urban and rural jurisdictions. Implications of the findings are discussed in the context of a consolidated correctional system.

Ketorolac causes the release of methionine-enkephalin in rats.

Ketorolac is a nonsteroidal anti-inflammatory drug (NSAID) that is indicated for the short-term management of moderately severe, acute pain, that causes analgesia equivalent to that caused by morphine. It has been shown experimentally that the analgesia produced by ketorolac in mice can be diminished by pretreatment with naloxone. This observation suggests that ketorolac produces some of its analgesia by interacting with opioid receptors. However, ketorolac does not directly interact with opioid receptors (Lopez et al., 1987). The present experiments demonstrate that the analgesia produced by ketorolac may be caused by the release of the endogenous opioid, methionine-enkephalin.

Human cardiac myosin light chains: sequence comparisons between myosin LC1 and LC2 from normal and idiopathic dilated cardiomyopathic hearts.

The primary structures of light chains isolated from the human myocardium with idiopathic dilated cardiomyopathy (IDC) were determined and compared with the sequence structures of myosin light chains obtained from control human heart myosin. Sequences were determined by chemical analysis and the identity of N-terminal residues established by mass spectrometry. The N-terminal residues in essential (ELC) and regulatory (RLC) light chains were blocked and were identified to be trimethyl alanine. The amino acid sequences of ELC and RLC from control human myosin revealed a high degree of homology with those purified from rat and chicken cardiac myosin. Comparison with a published partial chemical sequence of the human heart myosin light chains revealed significant variations. However, there was very good agreement with published sequences obtained by molecular biological techniques. Sequences of the light chains from cardiomyopathic myosin revealed no difference in the primary structures when compared with control human heart myosin light chains indicating IDC had no influence on, nor was caused by, altered myosin light chain gene expression.

Purification and partial characterization of draculin, the anticoagulant factor present in the saliva of vampire bats (Desmodus rotundus).

From the saliva of the vampire bat Desmodus rotundus, we isolated an unknown anticoagulant protein which we have named draculin. Its molecular mass as determined by non-reduced SDS-PAGE is about 83 kDa. The reduced polypeptide shows a slower migration. HPLC in a molecular sieve matrix yields a single, symmetrical peak corresponding to 88.5 kDa. Isoelectric focusing shows an acidic protein with pI = 4.1-4.2. Aminoacid analysis is compatible with a single chain polypeptide of about 80 kDa. Cyanogen bromide cleavage yields a single 16-aminoacid peptide, corresponding to the amino-terminus of the native molecule. Draculin inhibits the activated form of coagulation factors IX and X. It does not act on thrombin, trypsin, chymotrypsin and does not express fibrinolytic activity. The inhibition is immediate and not readily reversible, with a stoichiometry of about two molecules of draculin per molecule of factor IXa or Xa. Surprisingly, the inhibitory activity against either factor is not affected by the presence of the other. Draculin binds quantitatively to either immobilised factor Xa or factor IXa. Our preliminary interpretation is that there are two forms of draculin that hardly differ in structure. Both bind to factor Xa and to factor IXa but one form inhibits factor Xa and the other inhibits factor IXa. When added to plasma, draculin increases the lag phase as well as the height of the peak of thrombin generation.

Primary structure control of recombinant proteins using high-performance liquid chromatography, mass spectrometry and microsequencing.

The conformity of two recombinant proteins (a von Willbrand factor fragment and human serum albumin, consisting of respectively 289 and 585 amino acids) has been examined by HPLC combined with mass spectrometry and microsequencing, on both intact material and fragment peptides obtained by proteolytic cleavage. These studies confirmed that the primary structure of the recombinant proteins corresponds to that predicted from their gene, particularly the integrity of their N and C termini, and, in the case of albumin, the agreement between the observed disulfide bond pattern and the published model. Furthermore, the structure of an albumin-related compound could be elucidated. Application of LC-MS for batch-to-batch quality control is also under discussion.

Isolation, characterization and amino acid sequence of echicetin beta subunit, a specific inhibitor of von Willebrand factor and thrombin interaction with glycoprotein Ib.

Echicetin is a dimeric protein isolated from the venom of Echis carinatus that is a potent inhibitor of von Willebrand Factor and thrombin binding to glycoprotein Ib. Here, we report isolation and amino acid sequence of the beta subunit of echicetin that contains 123 amino acids, including 7 cysteines, and shows similarity with amino acid sequences of botrocetin and Factor IXa/Xa binding protein. We provide evidence that biological activity of echicetin which resides in this beta subunit is relatively resistant to reduction of the molecule.

Structural requirements of platelet chemokines for neutrophil activation.

Using recombinantly expressed proteins and synthetic peptides, we examined the structural/functional features of the platelet chemokines, neutrophil-activating peptide-2 (NAP-2) and platelet factor 4 (PF4); that were important in their activation of neutrophils. Previous studies with the chemokine interleukin-8 (IL-8) had shown that the N-terminal region preceding the first cysteine residue was critical in defining neutrophil-activating properties. We examined whether NAP-2 and PF4 had similar structural requirements. In the Ale-glu-leu-arg (AELR) N-terminus of NAP-2, substitution of E or R abolished Ca2+ mobilization and elastase secretion. Unlike the parent molecule PF4, AELR/PF4, the hybrid formed by replacing the N-terminal sequence of PF4 before the first cysteine residue with the homologous sequence of NAP-2, stimulated Ca2+ mobilization and elastase secretion. Furthermore, the effect of amino acid substitutions in the ELR motif differed from those seen with NAP-2 in that conserved substitutions of E or R in NAP-2 abolished activity, but only reduced neutrophil activation in the hybrid. These studies show that just as with IL-8, the N-termini of NAP-2 and PF4 are critical for high-level neutrophil-activating function. Desensitization studies provided information on receptor binding. NAP-2, which binds almost exclusively to the type 2 IL-8 receptor (IL-8R), did not desensitize neutrophils to activation by IL-8 because IL-8 could bind to and activate via both type 1 and 2 IL-8R. AELR/PF4 appears to bind to both types of receptors because it desensitized neutrophils to NAP-2 activation; but was not desensitized by NAP-2, and because it desensitized to and was desensitized by IL-8. Thus, although NAP-2 and AELR/PF4 share approximately 60% amino acid homology, they have different receptor affinities. Studies were performed to define the role of the C-termini of these platelet chemokines in receptor binding. Heparin and a monoclonal antibody specific for the heparin-binding domain of PF4 both inhibited Ca2+ mobilization and elastase release, further suggesting that the C-terminus of these chemokines is important in receptor binding. Synthetic NAP-2(51-70) failed to mobilize Ca2+, whereas PF4(47-70) and PF4(58-70) induced Ca2+ mobilization and secretion of elastase at high concentrations. Pertussis toxin inhibited neutrophil activation by 40% to 50%, establishing a role for G-protein-coupled receptors such as the IL-8Rs in activation by the PF4 C-terminal peptides.(ABSTRACT TRUNCATED AT 400 WORDS)