Nitrophorins 1-4 (NP1-4) are ferriheme proteins from the blood-sucking insect Rhodnius prolixus that transport nitric oxide (NO) to the victim, sequester histamine, and inhibit blood coagulation. Here, we report kinetic and thermodynamic analyses for ligand binding by all four proteins and their reduction potentials. All four undergo biphasic association and dissociation reactions with NO. The initial association is fast (1.5-33 microM(-)(1) s(-)(1)) and similar to that of elephant metmyoglobin. However, unlike in metmyoglobin, a slower second phase follows ( approximately 50 s(-)(1)), and the stabilized final complexes are resistant to autoreduction (E degrees = +3 to +154 mV vs normal hydrogen electrode). NO dissociation begins with a slow, pH-dependent step (0.02-1.4 s(-)(1)), followed by a faster phase that is again similar to that of metmyoglobin (3-52 s(-)(1)). The equilibrium dissociation constants are quite small (1-850 nM). NP1 and NP4 display larger release rate constants and smaller association rate constants than NP2 and NP3, leading to values for K(d) that are about 10-fold greater. The results are discussed in light of the recent crystal structures of NP1, NP2, and NP4, which display open, polar distal pockets, and of NP4-NO, which displays an NO-induced conformational change that leads to expulsion of solvent and complete burial of the NO ligand in a now nonpolar distal pocket. Taken together, the results suggest that tighter NO binding in the nitrophorins is due to the trapping of the molecule in a nonpolar distal pocket rather than through formation of particularly strong Fe-NO or hydrogen bonds.

Estimation of genomic breeding values is the key step in genomic selection (GS). Many methods have been proposed for continuous traits, but methods for threshold traits are still scarce. Here we introduced threshold model to the framework of GS, and specifically, we extended the three Bayesian methods BayesA, BayesB and BayesCp on the basis of threshold model for estimating genomic breeding values of threshold traits, and the extended methods are correspondingly termed BayesTA, BayesTB and BayesTCp. Computing procedures of the three BayesT methods using Markov Chain Monte Carlo algorithm were derived. A simulation study was performed to investigate the benefit of the presented methods in accuracy with the genomic estimated breeding values (GEBVs) for threshold traits. Factors affecting the performance of the three BayesT methods were addressed. As expected, the three BayesT methods generally performed better than the corresponding normal Bayesian methods, in particular when the number of phenotypic categories was small. In the standard scenario (number of categories ¼ 2, incidence ¼ 30%, number of quantitative trait loci ¼ 50, h 2 ¼ 0.3), the accuracies were improved by 30.4%, 2.4%, and 5.7% points, respectively. In most scenarios, BayesTB and BayesTCp generated similar accuracies and both performed better than BayesTA. In conclusion, our work proved that threshold model fits well for predicting GEBVs of threshold traits, and BayesTCp is supposed to be the method of choice for GS of threshold traits.

Liquid chromatography-electrochemical detection (LCEC) methods and instrumentation have been successfully applied for the trace determination of at least three separate platinum (Pt) anti-cancer (neoplastic) agents. All of these compounds have proven clinically effective in the treatment of human neoplasms. The three Pt derivatives studied were: cis-dichloro diammine platinum (cis-Pt); cis-diammine-1, 1-cyclobutane dicarboxylate platinum (CBDCA); and cis-dichloro-trans-dihydroxy diisopropylamine platinum (CHIP). The parent compound, cis-Pt (CDDP), can be determined via both oxidative and reductive LCEC, with differences in the minimum limits of detection. Calibration plots and minimum detection limits have been determined for all three derivatives. Both CBDCA and CHIP can be determined via direct LCEC, but the minimum detection limits for CBDCA are not practical for stability or clinical studies. A new method of derivatization for CBDCA and related Pt compounds has been developed, wherein this can be quantitatively converted to cis-Pt, and the final derivative is then determined as for the parent cis-Pt. CDDP can be determined via reductive LCEC at the 100-ppb level in plasma. The final methods of LCEC analysis have now been applied to a variety of stability studies with all three Pt drugs, in water, plasma, and saline infusion solutions. It is suggested that these LCEC methods are directly applicable and amenable to "real world" clinical settings and cancer-patient samples.