Novel chromophores and buried charges control color in mFruits

mFruits are second-generation monomeric red fluorescent proteins (mRFPs) that have improved brightness and photostability compared to the first-generation mRFP1. The emission and excitation maxima are distributed over the remarkably large ranges of about 550-650 and 540-590 nm, respectively; however, the variations in the spectra can be traced to a few key amino acids. Spectroscopic and atomic resolution crystallographic analyses of three representatives, mOrange, mStrawberry, and mCherry, reveal that different mechanisms operate to establish the excitation and emission maxima. Evidently, they all undergo the second oxidation step to produce an acylimine linkage in the polypeptide backbone. In comparison to the progenitor DsRed, direct covalent modification to this linkage (mOrange) and indirect modification of the chromophore environment (mStrawberry and mCherry) produce strong blue- and red-shifted variants. The blue shift of mOrange is induced by an unprecedented covalent modification of the protein backbone. The electron-density map indicates the formation of a third heterocycle, 2-hydroxy-dihydrooxazole, upon the reaction of Thr 66 Ogamma with the polypeptide backbone, which in turn reduces the conjugation of the carbonyl at position 65 with the rest of the chromophore. In mStrawberry and mCherry, the movement of charged Lys 70 and protonation of Glu 215 are proposed to modify the chromophore electron-density distribution, inducing the red shift. pH-dependent spectral shifts of mCherry and mStrawberry appear to result from the titration of Glu 215, although, for mStrawberry, partial cyclization of Thr 66 may contribute at high pH.     

Nitrite inhibition of aerobic bacteria

Nitrite was shown to inhibit active transport, oxygen uptake, and oxidative phosphorylation byPseudomonas aeruginosa. The evidence strongly suggested that nitrite exerted its inhibitory effect by oxidizing ferrous iron of an electron carrier(s), such as cytochrome oxidase, to ferric iron. The inhibitory effect of nitrite was readily reversible by washing the cells. Glucose transport byStreptococcus faecalis andS. lactis was not inhibited by nitrite, presumably because these species lack cytochromes and because glucose is transported by the phosphoenolpyruvate: phosphotransferase system rather than by active transport.     

The role of calcium in monoamine-induced depression of cerebral cortical neurones

The calcium antagonists, lanthanum, verapamil and manganese, have been shown to antagonize the depressant actions of ionto-phoretically applied monoamines (noradrenaline, 5-hydroxytryptamine, dopamine and histamine), but not of γ-aminobutyric acid, on cerebral cortical neurones. Cocaine and ethanol, which also affect membrane calcium fluxes, have a similar antagonistic action. These findings suggest that calcium ions are essential for, and perhaps mediate, the characteristic depressant actions of the monoamines on cerebral cortical neurones.     

N-(1-pyrene)maleimide: a fluorescent cross-linking reagent.

N-(1-Pyrene)maleimide is nonfluorescent in aqueous solution but forms strongly fluorescent adducts with sulfhydryl groups of organic compounds or proteins. The conjugation reactions of N-(1-pyrene)maleimide are relatively fast and can be monitored by the increase in fluorescence intensity of the pyrene chromophore. In cases where primary amino groups are also present in the system, we have observed a red shift of the emission spectra of the fluorescent adducts subsequent to the initial conjugation, as characterized by the disappearance of three emission peaks at 376, 396, and 416 nm, and the appearance of two new peaks at 386 and 405 nm. Model studies with N-(1-pyrene)maleimide adducts of L-cysteine and cysteamine indicate that the spectral shift is the result of an intramolecular aminolysis of the succinimido ring in the adducts. Evidence from both chemical analysis and nuclear magnetic resonance studies of the addition products supports this reaction scheme. N-(1-Pyrene)maleimide adducts of N-acetyl-L-cysteine and beta-mercaptoethanol, which have no free amino group, do not exhibit a spectral shift. Among several protein conjugates only the N-(1-pyrene)maleimide adduct of bovine serum albumin (PM-BSA) shows the spectral shift resembling that of PM-cysteine. N-(1-Pyrene)maleimide reacts with the sulfhydryl group of the single cysteine residue at position 34 in BSA. The finding that the alpha-amino group of the N-terminus in PM-BSA is blocked after the spectral shift is completed strongly suggests that N-(1-pyrene)maleimide cross-links the N-terminus and the cysteine residue in BSA. The relative proximity of the sulfhydryl and amino groups is very critical in the cross-linking as demonstrated by the observation that the spectral shift observed with PM-BSA can be prevented by addition of denaturing reagents such as 1% sodium dodecyl sulfate immediately after labeling, and by the failure of PM-glutathione to undergo the intramolecular aminolysis. Since the intramolecular rearrangement of PM adducts is associated with characteristic fluorescence changes, N-(1-pyrene)maleimide can serve as a fluorescent cross-linking reagent which provides information about the spatial proximity of sulfhydryl and amino groups in proteins.     

Tolerance of low oxygen stress in insecticide-resistant and susceptible populations of mosquitofish (Gambusia affinis)

The tolerance of low oxygen stress in insecticide-resistant and susceptible populations of mosquitofish ($\text{Gambusia}$$\text{affinis}$) was investigated. Dissolved oxygen was reduced chemically with formaldehyde and sodium sulfite treatment. Available oxygen was reduced physically through barriers, both by preventing oxygen exchange at the air-water interface and by preventing the fish from contacting the surface of the water. In all cases, the tolerance to low oxygen stress was reduced in the insecticide-resistant population compared to the susceptible population. The results indicate that although tolerance of pesticides in the resistant population has been dramatically increased by environmental selective pressures, tolerance to other stresses has not been concomitantly increased.