An alternative mechanism of bioluminescence color determination in firefly luciferase

Beetle luciferases (including those of the firefly) use the same luciferin substrate to naturally display light ranging in color from green (lambda(max) approximately 530 nm) to red (lambda(max) approximately 635 nm). In a recent communication, we reported (Branchini, B. R., Murtiashaw, M. H., Magyar, R. A., Portier, N. C., Ruggiero, M. C., and Stroh, J. G. (2002) J. Am. Chem. Soc. 124, 2112-2113) that the synthetic adenylate of firefly luciferin analogue D-5,5-dimethylluciferin was transformed into the emitter 5,5-dimethyloxyluciferin in bioluminescence reactions catalyzed by luciferases from Photinus pyralis and the click beetle Pyrophorus plagiophthalamus. 5,5-Dimethyloxyluciferin is constrained to exist in the keto form and fluoresces mainly in the red. However, bioluminescence spectra revealed that green light emission was produced by the firefly enzyme, and red light was observed with the click beetle protein. These results, augmented with steady-state kinetic studies, were taken as experimental support for mechanisms of firefly bioluminescence color that require only a single keto form of oxyluciferin. We report here the results of mutagenesis studies designed to determine the basis of the observed differences in bioluminescence color with the analogue adenylate. Mutants of P. pyralis luciferase putative active site residues Gly246 and Phe250, as well as corresponding click beetle residues Ala243 and Ser247 were constructed and characterized using bioluminescence emission spectroscopy and steady state kinetics with adenylate substrates. Based on an analysis of these and recently reported (Branchini, B. R., Southworth, T. L., Murtiashaw, M. H., Boije, H., and Fleet, S. E. (2003) Biochemistry 42, 10429-10436) data, we have developed an alternative mechanism of bioluminescence color. The basis of the mechanism is that luciferase modulates emission color by controlling the resonance-based charge delocalization of the anionic keto form of the oxyluciferin excited state.     

Effectiveness of Gemzar-Cisplatin therapy in stage IIIA-N2, IIIB and IV non-small cell lung cancer

120 chemotherapy naive patients were treated with gemcitabine 1250 mg/m2 iv. days 1 and 8 and cisplatin 70 mg/m2 iv. on day 1 between May 1999 and June 2001. The treatments were administered in 21 cycles. The median age of the patients was 53.1 years, the male/female ratio 65%-35%. Performance status was: WHO 0: 26%, WHO 1: 74%. The staging of patients were: IIIA-N2 23%, IIIB 37%, IV 40%. By histology the tumors were: 53.3% adenocarcinoma, 40% squamous cell carcinoma, 2.5% adenosquamous carcinoma, 0.8% macrocellular carcinoma and 3% non-small cell carcinoma (not categorised). We evaluated 413 cycles of chemotherapy. The median number of cycles was 3.44. The primary endpoint of the study was the median survival and time to progression, and the response rate. The results are the following: RR 40% (PR 37.5%, CR 2.5%), MR 13.3%, SD 25%, PD 22%. The time to progression (TTP) in the SD+MR group: 29.8 weeks, in the RR group: 34.1 weeks, mean of all patients: 28.1 weeks. The survival time was estimated by Kaplan-Meier curves. The median survival (MS) of all treated patients was: 54.9 weeks, in the PD group: 34.4 weeks, in the SD+MR group: 59.1 weeks, in the PR+CR group: 62.1 weeks. Conclusion: gemcitabine and cisplatin combination is a very well tolerated therapeutic regimen in the 1st line treatment of NSCLC. Furthermore, this treatment improves the RR and the survival of the patients as well.     

Status report on the chemotherapy of ovarian cancer at special cancer centers in Hungary (2002-2003)

Data on the first-line treatment of ovarian cancer in special centers of Hungary 2002 and 2003 are presented, involving 283 and 416 patients, respectively. Patients' age, clinical stage and histological type of the tumor were highly similar to literature data, while grades were different. Surgical effectiveness in case of IIIc staged tumors with >1 cm residual mass was 37%. The ratio of interval laparotomy was about 15%. Overall response rates of the first-line treatment of ovarian cancer was 82%, while the rate of complete remissions was 60%. The authors provide detailed analysis of factors that can improve the chemotherapy of ovarian cancer in Hungary.     

Vitamin D Deficiency Reduces Vascular Reactivity of Coronary Arterioles in Male Rats

Background: Vitamin D deficiency (VDD) may be considered an independent cardiovascular (CV) risk factor, and it is well known that CV risk is higher in males. Our goal was to investigate the pharmacological reactivity and receptor expression of intramural coronary artery segments of male rats in cases of different vitamin D supply. Methods: Four-week-old male Wistar rats were divided into a control group (n = 11) with optimal vitamin D supply (300 IU/kgbw/day) and a VDD group (n = 11, <0.5 IU/kgbw/day). After 8 weeks of treatment, intramural coronary artery segments were microprepared, their pharmacological reactivity was examined by in vitro microangiometry, and their receptor expression was investigated by immunohistochemistry. Results: Thromboxane A₂ (TXA₂)-agonist induced reduced vasoconstriction, testosterone (T) and 17-β-estradiol (E2) relaxations were significantly decreased, a significant decrease in thromboxane receptor (TP) expression was shown, and the reduction in estrogen receptor-α (ERα) expression was on the border of significance in the VDD group. Conclusions: VD-deficient male coronary arteries showed deteriorated pharmacological reactivity to TXA₂ and sexual steroids (E2, T). Insufficient vasoconstrictor capacity was accompanied by decreased TP receptor expression, and vasodilator impairments were mainly functional. The decrease in vasoconstrictor and vasodilator responses results in narrowed adaptational range of coronaries, causing inadequate coronary perfusion that might contribute to the increased CV risk in VDD.     

Synthesis of novel steroid-tetrahydroquinoline hybrid molecules and D-homosteroids by intramolecular cyclization reactions

Steroidal aryliminium salts were prepared from D-seco-pregnene aldehyde 2b, and their BF3.OEt2-catalyzed reactions were studied. The nature of the substituent R1 in the anilines 3-6 essentially influenced the chemoselectivity. Using unsubstituted 3, 4-methoxy- (4) or 4-bromoaniline (5), different tetrahydroquinoline derivatives 7a-13a via intramolecular hetero Diels-Alder reaction were formed. In the case of 4-nitroaniline (6) the N-arylamino-D-homopregnane (14a) were also obtained. We assume, that an intramolecular Prins reaction led to this type of fluoro-D-homosteroid. The main products represent a new class of tetrahydroquinolino-androstenes.     

Altered nitric oxide production in mouse brain after administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridin or methamphetamine

Several studies have demonstrated the involvement of reactive nitrogen and oxygen species (RNOS) in the neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridin (MPTP) and methamphetamine (METH), so the contribution of altered nitric oxide synthase (NOS) enzyme function can be suspected. In this study, about 50% increase in nitric oxide (NO) production in the mouse striatum was found between 4 and 12 h after a single MPTP injection, allowing an increased peroxynitrite (ONOO⁻) formation in the target brain region. However, METH injection induced a rapid decrease of NO formation both in mouse striatum and hippocampus, reaching its minimum level at 2 h, and restored to the control value after 6 h in the striatum and 12 h in the hippocampus. The uncoupled function of NOS with increased superoxide (O₂⁻) production after METH injection is suggested.     

Cardiovascular effects of BRX-005 comparison to bimoclomol

Concentration-dependent effects of BRX-005, the novel heat shock protein coinducer, cardioprotective and vasoprotective agent, on intracellular calcium transients and contractility were studied in Langendorff-perfused guinea pig hearts loaded with the fluorescent calcium indicator dye Fura-2. BRX-005 increased peak left ventricular pressure, the rate of force development and relaxation significantly in a concentration-dependent manner. The amplitude of [Ca2+]i transients was left unaltered by the drug. In contrast to BRX-005, bimoclomol increased both contractility and the amplitude of [Ca2+]i transients. In canine ventricular cardiomyocytes high concentrations of BRX-005 had no effect on depolarization, whereas bimoclomol suppressed action potential upstroke markedly. In guinea pig pulmonary artery preparations precontracted with phenylephrine, BRX-005 induced concentration-dependent relaxation. This effect of BRX-005 was independent of the integrity of endothelium indicating that vasorelaxant effect of the drug develops directly on vascular smooth muscle.