Oxidized low density lipoprotein (oxLDL) induces apoptosis in macrophages, smooth muscle cells, and endothelial cells. To elucidate the molecular mechanism of oxLDL-induced cytotoxicity and determine its tissue specificity, we have used Chinese hamster ovary (CHO)-K1 cells expressing human CD36 (CHO/CD36). Expression of CD36 rendered these cells susceptible to killing by oxLDL. This cytotoxicity was due to the induction of apoptosis. Therefore, CD36 expression is the only requirement for oxLDL-induced apoptosis. Oxysterols apparently mediate the cytotoxicity of oxLDL in macrophage foam cells and endothelial cells. 25-Hydroxycholesterol, at concentrations higher than 1 μg/ml, killed CHO-K1 cells, by apoptosis, in medium supplemented with serum as a source of cholesterol. These effects were not seen in a 25-hydroxycholesterol-resistant CHO/CD36 mutant (OX(R)), which was otherwise capable of undergoing apoptosis in response to staurosporine. This mutant was also resistant to killing by oxLDL, suggesting that oxysterols are at least partially responsible for the toxic effects of oxLDL. Oxysterol-induced apoptosis did not involve regulation of sterol regulatory element-binding protein proteolysis or the cholesterol biosynthetic pathway. 25-Hydroxycholesterol stimulated calcium uptake by CHO-K1 cells within 2 min after addition. Treatment of CHO or THP-1 (macrophage) cells with the calcium channel blocker nifedipine prevented 25-hydroxycholesterol induction of apoptosis. OX(R) showed no enhanced calcium uptake in response to 25- hydroxycholesterol.

C-reactive protein (CRP) performs two recognition functions that are relevant to cardiovascular disease. First, in its native pentameric conformation, CRP recognizes molecules and cells with exposed phosphocholine (PCh) groups, such as microbial pathogens and damaged cells. PCh-containing ligand-bound CRP activates the complement system to destroy the ligand. Thus, the PCh-binding function of CRP is defensive if it occurs on foreign pathogens because it results in the killing of the pathogen via complement activation. On the other hand, the PCh-binding function of CRP is detrimental if it occurs on injured host cells because it causes more damage to the tissue via complement activation; this is how CRP worsens acute myocardial infarction and ischemia/reperfusion injury. Second, in its nonnative pentameric conformation, CRP also recognizes atherogenic low-density lipoprotein (LDL). Recent data suggest that the LDL-binding function of CRP is beneficial because it prevents formation of macrophage foam cells, attenuates inflammatory effects of LDL, inhibits LDL oxidation, and reduces proatherogenic effects of macrophages, raising the possibility that nonnative CRP may show atheroprotective effects in experimental animals. In conclusion, temporarily inhibiting the PCh-binding function of CRP along with facilitating localized presence of nonnative pentameric CRP could be a promising approach to treat atherosclerosis and myocardial infarction. There is no need to stop the biosynthesis of CRP.

The environmental arylamine mutagens are implicated in the etiology of various sporadic human cancers. Arylamine-modified dG lesions were studied in two fully paired 11-mer duplexes with a-G*CN-sequence context, in which G* is a C8-substituted dG adduct derived from fluorinated analogs of 4-aminobiphenyl (FABP), 2-aminofluorene (FAF) or 2-acetylaminofluorene (FAAF), and N is either dA or dT. The FABP and FAF lesions exist in a simple mixture of 'stacked' (S) and 'B-type' (B) conformers, whereas the N-acetylated FAAF also samples a 'wedge' (W) conformer. FAAF is repaired three to four times more efficiently than FABP and FAF. A simple A-to-T polarity swap in the G*CA/G*CT transition produced a dramatic increase in syn-conformation and resulted in 2-to 3-fold lower nucleotide excision repair (NER) efficiencies in Escherichia coli. These results indicate that lesion-induced DNA bending/thermodynamic destabilization is an important DNA damage recognition factor, more so than the local S/B-conformational heterogeneity that was observed previously for FAF and FAAF in certain sequence contexts. This work represents a novel 3′-next flanking sequence effect as a unique NER factor for bulky arylamine lesions in E. coli.

The environmental arylamine mutagens are implicated in the etiology of various sporadic human cancers. Arylamine-modified dG lesions were studied in two fully paired 11-mer duplexes with a-G*CN-sequence context, in which G* is a C8-substituted dG adduct derived from fluorinated analogs of 4-aminobiphenyl (FABP), 2-aminofluorene (FAF) or 2-acetylaminofluorene (FAAF), and N is either dA or dT. The FABP and FAF lesions exist in a simple mixture of 'stacked' (S) and 'B-type' (B) conformers, whereas the N-acetylated FAAF also samples a 'wedge' (W) conformer. FAAF is repaired three to four times more efficiently than FABP and FAF. A simple A-to-T polarity swap in the G*CA/G*CT transition produced a dramatic increase in syn-conformation and resulted in 2-to 3-fold lower nucleotide excision repair (NER) efficiencies in Escherichia coli. These results indicate that lesion-induced DNA bending/thermodynamic destabilization is an important DNA damage recognition factor, more so than the local S/B-conformational heterogeneity that was observed previously for FAF and FAAF in certain sequence contexts. This work represents a novel 3′-next flanking sequence effect as a unique NER factor for bulky arylamine lesions in E. coli.

The mechanism of action of C-reactive protein (CRP) in protecting mice against lethal Streptococcus pneumoniae infection is unknown. The involvement of the phosphocholine (PCh)-binding property of CRP in its antipneumococcal function previously has been explored twice, with conflicting results. In this study, using three different intravenous sepsis mouse models, we investigated the role of the PCh-binding property of CRP by employing a CRP mutant incapable of binding to PCh. The ability of wild-type CRP to protect mice against infection was found to differ in the three models; the protective ability of wild-type CRP decreased when the severity of infection was increased, as determined by measuring mortality and bacteremia. In the first animal model, in which we used 25 μg of CRP and 107 CFU of pneumococci, both wild-type and mutant CRP protected mice against infection, suggesting that the protection was independent of the PCh-binding activity of CRP. In the second model, in which we used 25 μg of CRP and 5×107 CFU of pneumococci, mutant CRP was not protective while wild-type CRP was, suggesting that the protection was dependent on the PCh-binding activity of CRP. In the third model, in which we used 150 μg of CRP and 107 CFU of pneumococci, mutant CRP was as protective as wild-type CRP, again indicating that the protection was independent of the PCh-binding activity of CRP. We conclude that both PCh-dependent and PCh-independent mechanisms are involved in the CRP-mediated decrease in bacteremia and the resulting protection of mice against pneumococcal infection.

No description available.

Flumazenil (FMZ, Flumazepil, Anexate, RO 15-1788) is a benzodiazepine antagonist useful in reversal of benzodiazepine sedation or intoxication. It reverses respiratory and CNS depression from benzodiazepine overdosage as well as the effects of short acting, more potent benzodiazepines such as midazolam used in surgery. This investigation evaluates standard chromatographic (TLC, GLC) and immunological (EMIT, FPIA) methods for the detection of FMZ in urine. FMZ was extracted and detected to a minimum concentration of 250 ng/ml using the TOXI-LAB TLC system. Acid hydrolysis (FMZ ≤ 100 μg/ml) did not produce aminobenzophenones which could be detected by TLC/UV visualization. Specimens containing ≤ 300 ng/ml FMZ exhibited no cross reactivity and concentrations of 5 to 100 μg/ml exhibited cross reactivity of less than 0.2% with the Abbott TDx Benzodiazepine Assay. Specimens containing up to 100 μg/ml FMZ did not produce a positive response with the Syva EMIT st Urine Benzodiazepine Assay. Confirmation of FMZ was made by GLC/FID. FMZ and flunitrazepam (internal standard) were extracted from basified urine (pH 9.2) with n-butyl chloride and separated using a glass, 6'x 2mm ID, 3% SP2250DA column at 250°C with He (flow rate 40 ml/min). FMZ eluted at 11.31 min with a retention index of 0.908 relative to flunitrazepam. Data indicate that FMZ can be detected at nanogram concentrations by TLC and GC. The lack of cross reactivity in EMIT and FPIA assays and the lack of production of aminobenzophenones with acid hydrolysis decrease the possibility of false positives and the need for confirmation of true negative specimens. Broad use of FMZ in chemical dependency treatment programs will generate an increased need for the detection of FMZ in biological fluids.

5-Hydroxytryptamine (5-HT) 5-HT(2c) receptors mediate the enhanced oral activity response to the dopamine (DA) D1 agonist, (±)-1-phenyl-2,3,4,5- tetrahydro-1H-3-benzazepine-7,8-diol (SKF 38393) in neonatal 6- hydroxydopamine (6-OHDA)-lesioned rats. To study the possible involvement of 5-HT fibers on this process, the effect of the 5-HT neurotoxin, 5,7- dihydroxytryptamine (5,7-DHT) was determined. Rats were treated at 3 days after birth with desipramine (20 mg/kg i.p., base) 1 hr before intracerebroventricular vehicle, 6-OHDA (134 μg, base) and/or 5,7-DHT (50 μg, base). Oral activity was observed at 7 weeks and later. In neonatal 6- OHDA-lesioned rats oral activity dose-effect responses were increased by SKF 38393, 1-(3-chlorophenyl)-piperazine (m-CPP) and pilocarpine-respective DA D1, 5-HT(2c) and muscarinic receptor agonists. Rats treated neonatally with 5,7-DHT had agonist-induced responses resembling that of the saline control group. However, in rats treated neonatally with both 5,7-DHT and 6-OHDA, oral activity responses were reduced at nearly all doses of SKF 38393 and pilocarpine vs. that of the 6-OHDA group. The response of the 5,7-DHT + 6- OHDA group to m-CPP was reduced at only the 1.0 mg/kg dose. Of a variety of stereotyped behaviors that were observed in separate test sessions, m-CPP- induced grooming activity was less in the 5,7-DHT + 6-OHDA group vs. the 6- OHDA group. SKF 38393-induced arousal, rearing activity and immobility were also reduced in the 5,7-DHT + 6-OHDA vs. 6-OHDA group. Striatal DA content was reduced >98%, whereas 5-HT content was elevated >2-fold in the 6-OHDA group. Striatal 5-HT content was reduced by >85% after 5,7-DHT. These findings indicate that neonatal 5-HT fiber destruction does not produce enhanced m-CPP-induced oral activity in rats. In contrast, this lesion appears to attenuate DA D1 and muscarinic receptor supersensitivity. These findings indicate that 5-HT fibers have an important influence on the process of DA D1 and muscarinic receptor supersensitivity.

The pharmacological effects of substance P (SP), neurokinin A (NKA), an amino terminal fragment of SP and related tachykinin receptor agonists on renal resistance vessels were assessed in isolated rat kidney perfused at constant rate (3 ml min-1 g-1 of tissue) with a modified Krebs-Ringer bicarbonate buffer. At a basal perfusion pressure (PP) of 75 ± 6 mm Hg (n = 5), bolus injections of SP (1-33.3 nmol) had no significant vasoactive effect. After a sustained increase in base-line PP (134 ± 10 mm Hg) produced by 1 μM phenylephrine, SP evoked a dose-dependent increase in PP. The largest dose of SP increased PP by 60 ± 5 mm Hg. Physalaemin and kassinin had similar effects as SP but caused a smaller increase in PP at the largest dose. NKA was less potent than the other tachykinins. The vasoconstrictor response to SP was not blocked by 1 μM phentolamine when angiotensin II (0.22-0.26 μM) was used to increase basal tone (n = 5). Thus, the response to SP is not mediated by norepinephrine. The N-terminal SP fragment, SP(1- 7), had no effect on PP, which suggested that the pressor response to SP is C-terminal dependent and tachykinin receptor mediated. The selective NK-1 receptor agonist, [Sar9,Met(O2)11]SP, had no significant effect on PP. By contrast, the selective NK-2 and NK-3 receptor agonist, GR64349 and [MePhe7]NKB, produced concentration-dependent pressor responses and were more potent than SP (116 ± 8 and 134 ± 15 mm Hg increases in PP at 33.3 nmol, respectively). These results suggest that the vasoconstrictor effect of tachykinins in the isolated rat kidney is mediated by NK-2 and NK-3 receptors.

No description available.