Publications

The proliferative and transforming properties of m2 and m5 muscarinic acetylcholine receptors and a series of wild-type, chimeric, and mutant G proteins were measured alone or in combination in NIH 3T3 cells to determine which G proteins mediate these signals and to what extent these signals can be influenced by changing the stoichiometry of receptors and G proteins. Responses were measured using the focus-forming assay and a novel assay called R-SAT (Receptor Selection and Amplification Technology) in which proliferative responses are monitored using a reporter gene. Individually, GTPase-deficient mutants (*) of G alpha q and G alpha 12, wild-type G alpha q, and m5 were active in R-SAT. G alpha 12* and m5 also induced focus formation. m2 was inactive in both assays. The ability of m5 to induce foci was significantly reduced by coexpression of G alpha q*. Synergistic effects of receptor/ G protein combinations were not observed in focus-forming assays but were readily detected by R-SAT. Coexpression of G alpha q with m5 induced constitutive activity in R-SAT and increased the potency of agonists at m5 by 90-fold. G alpha q also evoked agonist-dependent responses from m2 but not constitutive activity. Agonist potency was increased 10-fold at m2 and decreased 15-fold at m5 when these receptors were coexpressed with G alpha qi5, a chimeric G protein containing the five C-terminal residues of G alpha i2, compared with coexpression with G alpha q. Both G alpha q and G alpha qi5 had biphasic effects on the proliferative responses to m5 and m2, respectively, inhibiting responses at high agonist concentrations. Coexpression of G alpha 12 or G alpha 12i5 had no effect on the concentration-response relationships of m5, but both elicited weak responses from m2. We conclude that although G alpha 12 is a more potent oncogene, G alpha q transduces m5-driven cellular responses. The demonstrations that proliferative responses can be elicited from a nonmitogenic receptor by altering the type and concentration of available G proteins and that constitutive responses can be induced by G proteins imply that both the magnitude and type of receptor-initiated signal can be regulated at the level of G proteins in vivo.

OBJECTIVES

An investigation was conducted to determine whether ongoing transmission of Mycobacterium tuberculosis was occurring in a California state prison.

METHOD

Prison pharmacy records were used to identify cases of active tuberculosis (TB).

RESULTS

Ten of the 18 cases of active TB treated at the facility during 1991 were diagnosed at the prison that same year (an incidence of 184 per 100,000). Three inmates were infectious for a total of 7 months while imprisoned. The prevalence of TB skin test-positivity among inmates was 30%, and the incidence of new infection attributable to incarceration was 5.9 per 100 inmates per year.

CONCLUSIONS

Transmission of M. tuberculosis may be occurring in the California prison system.

Psychological factors can play a role in asthma symptoms and may play a role in how individuals manage asthma. Because poor self-management of asthma has been linked to poor outcomes, it is important to understand perceived control of asthma--the individual's perceived ability to deal with asthma and its exacerbations effectively. This study used data from an ongoing panel study of adults with asthma (n = 601). The 11-item Perceived Control of Asthma Questionnaire (PCAQ) demonstrated internal consistency (Cronbach's alpha = 0.74) and excellent construct validity, correlating strongly with asthma severity, quality of life, and Medical Outcomes Study Short Form (SF-36) measures of health status (p < 0.05). After controlling for demographics and asthma severity, each 6-point decrement in PCAQ score was significantly associated with increased risk of hospitalization (OR = 1.4 [95% CI: 1.1 to 1.8]), frequent activity restriction (OR = 1.5 [1.2 to 1.8]), and, among those with labor force participation (n = 551), asthma-related cessation of employment (OR = 1.7 [1.1 to 2.4]). The PCAQ is a short, easy to administer, reliable, and valid measure of perceived control of asthma. It is strongly associated with adverse asthma outcomes even taking into account demographic characteristics and asthma severity, suggesting that patient-centered interventions focusing on perceived control might improve asthma outcomes.

STUDY HYPOTHESIS

Physicians may consult references such as Physicians' Desk Reference (PDR) for overdose management advice. Although PDR recommendations are approved by the US Food and Drug Administration (FDA), we hypothesized that they are often outdated and potentially hazardous.

METHODS

We surveyed physicians who consulted our poison center during a 1-month period with regard to their use of the PDR for overdose information and also compared PDR overdose treatment recommendations with those of five current major toxicology references. For the PDR overdose information review we examined data from the American Association of Poison Control Centers to identify pharmaceutical categories with the largest number of deaths. We reviewed the four leading drugs with at least 1,000 reported exposures in each category and identified 20 PDR-listed brand-name products for analysis. We obtained the consensus from five current toxicology references on contraindicated treatments, ineffective treatments, and specific recommended treatments or antidotes. Finally, we compared the overdose management advice provided in the 1994 PDR with the toxicology reference consensus.

RESULTS

Forty of 80 of physicians surveyed (50%) reported use of the PDR for overdose information in the preceding 12 months. Of the 20 PDR entries, 16 (80%) had at least one deficiency, and 5 (25%) had two or more deficiencies. Thirteen (65%) omitted an indicated specific treatment, three (15%) recommended contraindicated treatments, and four (20%) advised ineffective treatments with potential for harm. Only four entries (20%) had no deficiencies by our survey criteria.

CONCLUSION

We found serious discrepancies in overdose treatment advice in the PDR compared with a consensus of current toxicology references. Altogether, four of five PDR entries were deficient, and almost half advised ineffective or frankly contraindicated therapies. Despite FDA approval, the use of PDR overdose advice in a serious poisoning case could result in unnecessary morbidity or mortality.

The male-specific P450 enzyme CYP 2C11, whose expression is developmentally and hormonally regulated, is the major steroid 16alpha-hydroxylase of the untreated rat liver. The enzyme metabolizes a host of substrates, including mechanism-based inactivators, such as 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) and spironolactone (SPL). Structural and functional characterization of the specific mode of such inactivation, however, requires sufficient quantities of the fully purified enzyme. Although several laboratories including our own have isolated and purified the enzyme from male rats, the yields are typically low and of the order of 1%. For these reasons, we chose to heterologously express the enzyme in Escherichia coli. The full-length cDNA was excised from the yeast vector pD2M1 and cloned into the plasmid vector pCW after appropriate modifications for optimal expression in E. coli. The enzyme was isolated and purified from E. coli membranes in relatively high yields (approximately 60%) and relatively high specific content (19 nmol/mg protein). The purified recombinant enzyme had spectral and functional characteristics comparable to those reported for the native rat liver enzyme, including mechanism-based inactivation by DDEP and SPL. Studies with 14C-heme-labeled enzyme indicated that the major mode of DDEP inactivation was via heme-N-ethylation. On the other hand, studies with radiolabeled SPL-SH (the proximal inactivating deacetylated metabolite of SPL) revealed that although both [22-14C]SPL-SH and SPL-35SH inactivated the enzyme, only SPL-35SH was found to irreversibly radiolabel the 2C11 protein. The latter findings thus suggest that during mechanism-based inactivation of 2C11, the thiol moiety of SPL-SH is oxidatively activated to a species that attacks the 2C11 protein during or after cleavage from the thiosteroid. Thus, these modes of mechanism-based 2C11 inactivation by DDEP and SPL-SH considerably differ from the corresponding modes of P450 3A inactivation by these agents, wherein heme modification of the protein predominates.

In infants, vaccines consisting of a carrier protein conjugated to the bacterial capsular polysaccharide (PRP) are far more protective against Hemophilus influenzae type b (Hib) disease than unconjugated PRP. To determine the tolerability and immunogenicity of Hib conjugate vaccines in the elderly, we vaccinated 30 volunteers, aged 69-84 years, with either PRP conjugated to an outer membrane protein complex (PRP-OMP), or PRP oligomers conjugated to CRM197, a nontoxic, mutant diphtheria toxin (HbOC). Prior to vaccination, 40% of subjects had serum anti-PRP antibody levels < 1.0 microgram ml-1. Four weeks following vaccination, all subjects had concentrations > 1.0 microgram ml-1, a level generally considered to be protective. The post-vaccination geometric mean concentrations were 35.5 and 50.1 micrograms ml-1 for the PRP-OMP and HbOC groups, respectively (0.05 < P < 0.10). Subjects in the HbOC group, but not the PRP-OMP group, showed, on average, ten fold increases in IgG antibody to diphtheria toxoid after conjugate vaccination. Side-effects of vaccination were mild except in one subject given HbOC, who developed extensive erythema and swelling of the injected arm.

Interleukin 1 alpha (IL-1 alpha) is a pleiotropic cytokine involved in the immune response, inflammatory processes, and hematopoiesis, and acts as a mitogen for several malignant cell types, including acute leukemia and Kaposi sarcoma cells. These diverse activities have been exclusively attributed to the plasma membrane receptor-binding, 17-kDa C-terminal component (mature IL-1 alpha) that results from proteolytic processing of the 31- to 33-kDa precursor protein. No biologic function has been ascribed to the unusually large, 16-kDa N-terminal propiece formed as a result of proteolytic processing of IL-1 alpha. We report that the IL-1 alpha N-terminal propiece is concentrated by means of a nuclear localization sequence within the nuclei of both transfected and leukemic cell lines. Overexpression of this component in glomerular mesangial cells, a model perivascular myofibroblast cell type capable of IL-1 alpha synthesis and processing, results in malignant transformation to a spindle cell-type tumor. The functionally bipartite nature of the IL-1 alpha precursor represents a unique combination of the C-terminal, classical cytokine and an N-terminal nuclear oncoprotein. These findings suggest that nuclear transport of the IL-1 alpha N-terminal component may represent a critical component in the transformation of IL-1 alpha-producing cells in the bone marrow or the perivascular area to a malignant phenotype.