Effect of smoke-free policies on the behaviour of social smokers.

OBJECTIVE: To test the hypothesis that proposed amendments to the Occupational Safety and Health Act making all enclosed workplaces in Western Australia smoke free would result in a decrease in cigarette consumption by patrons at nightclubs, pubs, and restaurants without adversely affecting attendance. DESIGN: Cross sectional structured interview survey. PARTICIPANTS AND SETTING: Patrons of several inner city pubs and nightclubs in Perth were interviewed while queuing for admission to these venues. OUTCOME MEASURES: Current social habits, smoking habits; and how these might be affected by the proposed regulations. Persons who did not smoke daily were classified as "social smokers." RESULTS: Half (50%) of the 374 patrons interviewed were male, 51% currently did not smoke at all, 34.3% smoked every day, and the remaining 15.7% smoked, but not every day. A clear majority (62.5%) of all 374 respondents anticipated no change to the frequency of their patronage of hospitality venues if smoke-free policies became mandatory. One in five (19.3%) indicated that they would go out more often, and 18.2% said they would go out less often. Half (52%) of daily smokers anticipated no change to their cigarette consumption, while 44.5% of daily smokers anticipated a reduction in consumption. A majority of social smokers (54%) predicted a reduction in their cigarette consumption, with 42% of these anticipating quitting. CONCLUSIONS: One in nine (11.5%) of smokers say that adoption of smoke-free policies would prompt them to quit smoking entirely without a significant decrease in attendance at pubs and nightclubs. There can be few other initiatives as simple, cheap, and popular that would achieve so much for public health.

Lck phosphorylates the activation loop tyrosine of the Itk kinase domain and activates Itk kinase activity.

The Tec family tyrosine kinase Itk has been implicated in T cell receptor (TCR) signaling, yet its precise role and mechanism of activation remain undefined. To investigate these issues, we examined the biochemical response of Itk to TCR stimulation. We found that Itk is tyrosine-phosphorylated after TCR cross-linking and that this phosphorylation depends on the presence of functional Lck. To determine if this Lck dependence results from direct phosphorylation of Itk by Lck, we generated recombinant Itk and Lck using a baculovirus expression system and used these proteins in subsequent biochemical analyses. We found that Lck phosphorylates Itk upon co-expression in insect cells and, further, that this phosphorylation of Itk results in increased Itk in vitro kinase activity. The major site of Lck phosphorylation on Itk was mapped to the conserved tyrosine (Tyr511) in the activation loop of the Itk kinase domain. Substitution of this tyrosine with phenylalanine abolishes Itk kinase activity in insect cells, indicating that phosphorylation at this site plays a critical role in regulating Itk function.

Proteasome inhibition enhances the stability of mouse Cu/Zn superoxide dismutase with mutations linked to familial amyotrophic lateral sclerosis.

Point mutations occurring within the Cu/Zn superoxide dismutase (SOD1) gene have been implicated in the etiology of some cases of familial amyotrophic lateral sclerosis (FALS). In order to better understand the functional consequences of these mutations, we have introduced FALS mutations into the mouse SOD1 gene and studied the expression of the mutant templates in stably transformed cell lines. Pulse-chase analyses of lysates derived from cell lines stably expressing the Cu/Zn SOD isoforms indicate that the FALS mutant Cu/Zn SOD proteins are turned over more rapidly than wild-type SOD. Protease inhibitors specific for the major intracellular proteolytic activities were used to characterize the degradative pathways involved in the turnover of mutant Cu/Zn SOD. Inhibition of the chymotrypsin-like activity of the proteasome (also known as multicatalytic proteinase or ubiquitin, ATP-dependent proteinase) by a synthetic dipeptide aldehyde led to a significant increase in levels of the mutant Cu/Zn SOD implicating this proteolytic pathway in the turnover of the FALS mutant SOD proteins.

Motor neurons in Cu/Zn superoxide dismutase-deficient mice develop normally but exhibit enhanced cell death after axonal injury.

The discovery that some cases of familial amyotrophic lateral sclerosis (FALS) are associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) has focused much attention on the function of SOD1 as related to motor neuron survival. Here we describe the creation and characterization of mice completely deficient for this enzyme. These animals develop normally and show no overt motor deficits by 6 months in age. Histological examination of the spinal cord reveals no signs of pathology in animals 4 months in age. However Cu/Zn SOD-deficient mice exhibit marked vulnerability to motor neuron loss after axonal injury. These results indicate that Cu/Zn SOD is not necessary for normal motor neuron development and function but is required under physiologically stressful conditions following injury.

Systemic dexamethasone administration increases septal Trk autophosphorylation in adult rats via an induction of nerve growth factor.

Nerve growth factor (NGF) maintains cholinergic neurons in various animals models of neurodegeneration and is thus a potential treatment for certain neurodegenerative disorders such as Alzheimer's disease. Because NGF does not cross the blood-brain barrier, we have proposed elevating endogenous levels of NGF in the central nervous system with small molecules that induce NGF expression, as an alternative strategy. The present studies were conducted to determine whether pharmacologically elevated levels of NGF are sufficient to cause subsequent stimulation of its high affinity receptor, as measured by increased levels of Trk phosphorylation. Dexamethasone (0.5-20 mg/kg, intraperitoneally) caused a time- and dose-dependent increase in NGF mRNA and NGF protein in the hippocampus and septum of adult male Sprague-Dawley rats. Exogenously administered NGF (1 microgram, intracerebroventricularly) led to a rapid (30 min) and transient increase in Trk phosphorylation in the septum, which has high levels of NGF-specific TrkA. Similarly, dexamethasone led to an increase in Trk phosphorylation only within the septum. Dexamethasone-mediated Trk phosphorylation was dose and time dependent, with peak increases being observed 12 hr after injection, concurrently with peak increases in NGF protein. These data demonstrate an increase in activation of the high affinity NGF receptor with a compound that elevates levels of NGF in the central nervous system, and they support the strategy of discovering a pharmacological agent that induces NGF as a method for treating neurodegenerative disorders.

Chronic 1,25-dihydroxyvitamin D3-mediated induction of nerve growth factor mRNA and protein in L929 fibroblasts and in adult rat brain.

We have proposed that elevating levels of nerve growth factor (NGF) in the CNS is a rational strategy for treating certain neurodegenerative disorders. The present studies were conducted to determine: (1) if pharmacologically induced levels of NGF could be sustained for an extended time, and (2) if correlations exist between increases in NGF mRNA and NGF protein in L929 cells and in vivo. Short-term treatment of L929 cells with 1,25-dihydroxyvitamin D3 resulted in a two-fold increase in both NGF mRNA and NGF protein. These increases were sustained for up to 48 h with continuous exposure to 1,25-dihydroxyvitamin D3. In rats, 1,25-dihydroxyvitamin D3 (2.5 nmol; i.c.v.) induced NGF mRNA transiently, with peak two-fold increases observed 4 h post-injection. In contrast to L929 cells, 1,25-dihydroxyvitamin D3 did not elicit an increase in NGF protein after a single administration in vivo. However, consistent with long-term exposure in L929 cells, chronic 6 day infusion of 1,25-dihydroxyvitamin D3 resulted in induction of both NGF mRNA and NGF protein in the brain. These results indicate that 1,25-dihydroxyvitamin D3-mediated NGF induction in cultured L929 cells may predict of NGF induction in vivo, suggesting that L929 cells may have utility in studying underlying mechanisms of NGF induction by 1,25-dihydroxyvitamin D3. On the basis of NGF's ability to increase cholinergic function in animal models of cholinergic degeneration, these results are supportive of a role for NGF inducers as potential drugs for neurodegenerative disorders.

Pharmacological induction of nerve growth factor mRNA in adult rat brain.

Three structurally unrelated compounds, all of which induce nerve growth factor (NGF) in cell culture systems, were assessed for their ability to induce NGF mRNA in adult rat brain using a highly sensitive RNAse protection assay. Interleukin-1 beta (0.5-1 pmol) and 1,25-dihydroxyvitamin D3 (25-25,000 pmol) were extremely potent inducers of NGF mRNA, being respectively at least 50,000 and 4000 times more potent than 4-methylcatechol. These compounds elicited an approximate twofold increase in NGF mRNA in both the hippocampus and cortex, without altering beta-actin mRNA levels after a single intracerebroventricular injection. The duration of NGF induction was dependent on the compound administered. For example, the elevation of NGF mRNA elicited by interleukin-1 beta peaked at 8 h and lasted for at least 24 h. In contrast, the induction of NGF after 1,25-dihydroxyvitamin D3 and 4-methylcatechol administration peaked between 4 and 8 h and was not apparent 24 h after injection. These results demonstrate induction of NGF mRNA in vivo by administration of physiological or pharmacological agents and differentiate these agents by potency and duration of action. Further, these findings indicate that pharmacological induction of NGF may be a viable strategy for the treatment of neurodegenerative disorders such as Alzheimer's disease.

Induction of NGF by isoproterenol, 4-methylcatechol and serum occurs by three distinct mechanisms.

Evidence is provided that isoproterenol, 4-methylcatechol and serum induce NGF by three separate mechanisms. Isoproterenol and 4-methylcatechol induced NGF and NGF mRNA in mouse fibroblast L929 cells in either the presence or absence of serum. Propranolol prevented NGF induction by isoproterenol, but not by 4-methylcatechol or serum. All possible combinations of these inducers resulted in additive increases in the levels of NGF and NGF mRNA.