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.     

Mirex-induced liver enlargement in rats is dependent upon an intact pituitary-adrenalcortical axis

The effect of prior hypophysectomy upon mirex-induced liver hypertrophy in male Sprague-Dawley rats was examined. Mirex had no effect upon adrenal weight, liver weight, plasma glucose or plasma corticosterone in hypophysectomized rats. However, daily corticosterone supplements (20 mg/kg body weight, sc) given to mirex-treated hypophysectomized animals yielded a 52% increase in liver weight to body weight ratios over those observed in mirex-treated hypophysectomized animals not receiving supplement. In intact rats, both liver weight to body weight ratios and plasma ACTH were significantly increased over controls 2 days after mirex treatment. These results indicate that mirex-induced liver enlargement not only requires corticosterone, but that the response is dependent upon an intact pituitary-adrenalcortical axis.     

Physiological effects of crude oil exposure in the striped mullet, Mugil cephalus

Juvenile mullet ($\text{Mugil cephalus}$) were exposed to a surface slick of Empire Mix crude oil for a three week period in a simulated estuarine ecosystem. Liver weight to body weight ratios were increased in the mullet from the oil-treated ponds when compared to those from the control ponds. Activities of alkaline phosphatase, which were elevated in gill and muscle of oil-treated mullet, and β-glucuronidase, which was elevated in the muscle of oil-treated mullet, may be related to the degree of stress the animals were experiencing. Malic dehydrogenase, which was depressed in the livers and elevated in the muscle of oil-treated organisms, indicate changes in aerobic metabolism in response to the stress of crude oil exposure. Muscle acetylcholinesterase was not affected by oil exposure.     

Kinetics of interaction of 2-amino-6-mercapto-9-beta-ribofuranosylpurine 5'-triphosphate with bovine brain tubulin

The binding of the guanine nucleotide analogue 2-amino-6-mercapto-9-beta-ribofuranosylpurine 5'-triphosphate (S6-GTP) to tubulin from which the associated proteins and exchangeably bound nucleotide have been removed produces about a 15% decrease in intrinsic tubulin fluorescence. Using a fluorescence stopped-flow technique, we have examined the kinetics and mechanism of this process. Analysis of the data reveals that the binding is complex, involving at least one conformational change subsequent to nucleotide binding. The bimolecular association rate constant for binding of S6-GTP to tubulin is approximately 6 X 10(5) M-1 s-1, suggesting that the orientation requirements are stringent. The kinetic parameters for dissociation of GDP, S6-GTP, and S6-GDP from the exchangeable nucleotide binding site have also been determined. S6-GDP and GDP were found to have comparable rates of dissociation; S6-GTP dissociated approximately twice as slowly as either GDP or S6-GDP. Glycerol produces a significant decrease in the rates of nucleotide dissociation. The mechanism whereby glycerol produces such an effect is not known; however, it may involve slight changes in the conformation of the tubulin protomer.     

Amphiphilic conjugates of human brain natriuretic peptide designed for oral delivery: in vitro activity screening

Congestive heart failure (CHF) is a complex syndrome involving altered neurohormonal levels and impaired cardiac and renal function. In recent years, intravenous administration of exogenous human brain-type natriuretic peptide (hBNP) has become an important therapy in treating patients with acutely decompensated CHF. However, reports during the past year suggest that hBNP could play a prominent role in the chronic treatment of CHF patients as well. We are currently developing conjugates of hBNP suitable for oral delivery to provide a patient-friendly treatment option for chronic heart failure patients. In this report, we present in vitro activity results obtained from hBNP conjugates featuring a variety of rationally designed amphiphilic oligomers. Mapping studies revealed that the hydrophobic/hydrophilic balance of the oligomer impacted the regioselectivity of conjugation. Additionally, the regiochemistry and extent of conjugation had a significant impact on activity. Many monoconjugates retained activity comparable to native peptide and are currently under evaluation in subsequent in vivo screens.     

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.     

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.     

Expression of the alpha and beta tubulin genes of the African trypanosome in Escherichia coli

The African trypanosome, Trypanosoma brucei, contains multiple genes for both alpha- and beta-tubulins, which code for similar if not identical proteins. Studies of the structure and function of trypanosome microtubules have been limited due to the difficulties in obtaining sufficient amounts of purified tubulin. To produce large amounts of purified tubulin for studies of structure and function and to begin developing a system for producing systematic alterations of tubulin structure we have cloned and expressed the alpha- and beta-tubulin genes of T. brucei in Escherichia coli to produce the unfused proteins. Controlled high-level expression of both alpha- and beta-tubulin was achieved using a plasmid vector, pOTS, in which expression is controlled by phage lambda promoter/operator and a temperature-sensitive lambda repressor. The tubulins produced are insoluble, as has been found for many other proteins expressed to high levels in E. coli; they are readily purified to near homogeneity by chromatography on DEAE-cellulose in 7 M urea. N-terminal analysis of the purified proteins indicates that they are initiated correctly and that the N-formyl group is removed from the initiating methionine. This factor will probably prove important in the reconstitution of biologically active tubulin.