Nicotine-stimulated release of [3H]norepinephrine from fetal rat locus coeruleus cells in culture

Acute nicotine administration stimulated [3H]norepinephrine ([3H]NE) release from cultured fetal locus coeruleus (LC) cells. The effect was concentration dependent, with an EC50 of 0.9 microM, and was abolished by removal of calcium from, or addition of tetrodotoxin (500 nM) to, the assay buffer. Other nicotinic receptor agonists stimulated [3H]NE release, with the rank order of potency being (+)-epibatidine > (-)-nicotine > 1,1-dimethyl-4-phenylpiperazinium (DMPP). Whereas (-)-nicotine and (+/-)-epibatidine exhibited equal maximal responses, DMPP was a partial agonist and (-)-cytisine had no agonist activity. Nicotine-stimulated release of [3H]NE was blocked by nicotinic receptor antagonists, with an order of potency of mecamylamine > lobeline > cytisine > methyllycaconitine > dihydro-beta-erythroidine. The pharmacological profile of this nicotinic receptor is largely consistent with that described previously for an alpha4beta2 subunit combination, although discrepancies in the efficacies of agonists were observed. No additivity in NMDA- and nicotine-stimulated [3H]NE release was observed, suggesting a common signal transduction mechanism. However, the pharmacological characteristics of MK-801 blockade of nicotine-induced responses were not consistent with those of an NMDA receptor. We therefore conclude that nicotine directly releases [3H]NE from LC cells and does not act indirectly via activation of glutamate release.     

Regulation of SNARE complex assembly by an N-terminal domain of the t-SNARE Sso1p

The fusion of intracellular transport vesicles with their target membranes requires the assembly of SNARE proteins anchored in the apposed membranes. Here we use recombinant cytoplasmic domains of the yeast SNAREs involved in Golgi to plasma membrane trafficking to examine this assembly process in vitro. Binary complexes form between the target membrane SNAREs Sso1p and Sec9p; these binary complexes can subsequently bind to the vesicle SNARE Snc2p to form ternary complexes. Binary and ternary complex assembly are accompanied by large increases in alpha-helical structure, indicating that folding and complex formation are linked. Surprisingly, we find that binary complex formation is extremely slow, with a second-order rate constant of approximately 3 M(-1) s(-1). An N-terminal regulatory domain of Sso1p accounts for slow assembly, since in its absence complexes assemble 2,000-fold more rapidly. Once binary complexes form, ternary complex formation is rapid and is not affected by the presence of the regulatory domain. Our results imply that proteins that accelerate SNARE assembly in vivo act by relieving inhibition by this regulatory domain.     

The genetic effects of environmental lead

Distribution and metabolism of the thyroid hormone 3,5, 3'-l-triiodothyronine (T3) were studied in several ways to gain insights into these processes in the warm water fish tilapia Oreochromis mossambicus. Trace doses of 125I-labeled T3 (T*3)1 were injected intraarterially, extraarterially, or intraperitoneally in freshwater-reared male tilapia to explore plasma clearance kinetic responses to these different input modalities. Multicompartmental analysis of the plasma clearance data indicated a kinetic distribution of T*3 much like that reported for the rat and human, with about 2% of total body T*3 in plasma, 5% in rapidly exchanging tissues such as kidney and liver, and 93% in slowly exchanging tissues such as muscle. However, plasma clearance rates (PCR, 5.37 mL/h . 100 g body wt) and plasma appearance rates (PAR3 = PCR x [T3] plasma = 36.3 ng/h . 100 g body wt) were quite different than these indices in rat and human and 5 to 50 times larger than values reported for rainbow trout. On a whole-body basis, normalized for body weight, the tilapia we studied produced and accumulated much more T3 than rat, human, or rainbow trout. Enzymatic and chromatographic analyses of the plasma clearance data samples indicated substantial production of labeled glucuronide, but not sulfate, conjugates of iodothyronines (TiG) of unknown origin appearing in plasma. The TiG appeared beginning a few hours postinjection, peaked at 6 hours, and yielded a predicted steady-state TiG level of 8.3% of the T3 level in plasma. In contrast, in published studies, no conjugates were detected in rainbow trout plasma from 2 to 24 h after iv injection of T*3, T*4, or reverse-T*3, although conjugates of all were present in bile. To our knowledge, although T3 and T4 sulfate conjugates are present in the sera of several mammals, this is the first quantification of iodothyronine glucuronides reported in blood of any species under normal conditions. This might have physiological significance for the tilapia, with T3G providing a reversible storage form of T3 in blood, as has been suggested for sulfate conjugates of T3 and T4 in blood of several mammals.     

Rare association of human herpesvirus 6 DNA with human papillomavirus DNA in cervical smears of women with normal and abnormal cytologies

We investigated by nested PCR the possible association of human herpesvirus 6 (HHV-6) and human papillomavirus (HPV) genomes in the cervixes of 109 women with normal and abnormal cytological smears. HPV DNA was detected in 8.33% of 24 women with normal cytologies and in 41.1% of 85 women with abnormal cytologies; the proportion of HPV DNA was directly related to the severity of the lesions. HHV-6 DNA was found in only one patient, who had a cytological pattern of koilocytosis. The HHV-6 genome was classified by restriction enzyme analysis as variant B. The study indicates that detection of the HHV-6 genome in the cervixes of women with a wide spectrum of gynecological complaints is a rare event and rules out the possible association between HHV-6 and HPV genomes in cervical cancer lesions.     

Ubiquitin and apoptosis in the corpus luteum of the marmoset monkey (Callithrix jacchus)

The thiazolidinedione analogue troglitazone is an antidiabetic agent that improves insulin resistance in rodents and humans. Although coronary artery disease is common in patients with the insulin resistance syndrome, the effects of troglitazone on smooth muscle cells (SMC) have not been fully elucidated. We therefore examined the effects of troglitazone on cell growth and glucose uptake in human aortic SMC. Mitogen-activated protein (MAP) kinase activity and glucose transporter (Glut) 1 mRNA levels were also studied. In the absence of troglitazone, insulin (10(-7) M) caused a 2-fold increase of DNA synthesis in SMC and troglitazone suppressed the increase of DNA synthesis in a dose-dependent manner. This growth suppression was accompanied by inhibition of MAP kinase activity. On the other hand, troglitazone significantly increased Glut 1 mRNA and enhanced glucose uptake in SMC. These results suggest that troglitazone affects the insulin signaling pathways in SMC and suppresses growth while promoting glucose uptake. Our findings support the application of troglitazone as an inhibitor of SMC proliferation in patients with insulin resistance.     

Tumor cell cytotoxicity of a novel metal chelator

We have synthesized a novel six-coordinate metal chelator from the triamine cis-1,3,5-triaminocyclohexane by the addition of a 2-pyridylmethyl pendant arm on each nitrogen, which we term tachpyr. The experiments described here were designed to explore whether this compound exhibits potential antitumor activity. When added to MBT2 or T24 cultured bladder cancer cells, tachpyr was profoundly cytotoxic, with an IC50 of approximately 4.6 micromol/L compared with 70 micromol/L for desferioxamine. To explore the mode of action of tachpyr, several metal complexes were prepared, including Fe(II), Ca(II), Mn(II), Mg(II), Cu(II), and Zn(II) tachpyr complexes. Of these, the Zn(II), Cu(II), and Fe(II) complexes were without toxic effect, whereas the Ca(II), Mn(II), and Mg(II) complexes remained cytotoxic. To further probe the role of Zn(II) and Cu(II) chelation in the cytotoxicity of tachpyr, sterically hindered tachpyr derivatives were prepared through N-alkylation of tachpyr. These derivatives were unable to strongly bind Fe(III) or Fe(II) but were able to bind Zn(II) and Cu(II). When added to cells, these sterically hindered tachpyr derivatives were nontoxic, consistent with a role of iron depletion in the cytotoxic mechanism of tachpyr. Further, the addition of tachpyr to proliferating cultures resulted in an early and selective inhibition of ferritin synthesis, an iron storage protein whose translation is critically dependent on intracellular iron pools. Taken together, these experiments suggest that tachpyr is a cytotoxic metal chelator that targets intracellular iron, and that the use of tachpyr in cancer therapy deserves further exploration.     

Protective factors in adolescent health behavior

The role of psychosocial protective factors in adolescent health-enhancing behaviors--healthy diet, regular exercise, adequate sleep, good dental hygiene, and seatbelt use--was investigated among 1,493 Hispanic, White, and Black high school students in a large, urban school district. Both proximal (health-related) and distal (conventionality-related) protective factors have significant positive relations with health-enhancing behavior and with the development of health-enhancing behavior. In addition, in cross-sectional analyses, protection was shown to moderate risk. Key proximal protective factors are value on health, perceived effects of health-compromising behavior, and parents who model health behavior. Key distal protective factors are positive orientation to school, friends who model conventional behavior, involvement in prosocial activities, and church attendance. The findings suggest the importance of individual differences on a dimension of conventionality-unconventionality. Strengthening both proximal and distal protective factors may help to promote healthful behaviors in adolescence.