Observations on the use of medetomidine/ketamine and its reversal with atipamezole for chemical restraint in the mouse.

Ketamine and medetomidine produced chemical restraint for minor procedures in mice. Male mice required 50 mg/kg ketamine, 10 mg/kg medetomidine intraperitoneally (i.p.), and females a higher dose of ketamine (75 mg/kg i.p.). The onset of restraint effects, judged by loss of righting reflex, was more rapid in males than females. The effects were reversed using atipamezole (1-2.5 mg/kg). Recovery following administration of atipamezole was more rapid in males than females. We conclude that ketamine/medetomidine, followed by reversal with atipamezole, is an effective technique for chemical restraint in the mouse.

[Anesthesia in experimental animals. Basic principles]. / Anestesia en animales de experimentación. Bases elementales.

The use of experimental animals often requires anesthesia to provide immobility, analgesia and sufficiently lowered levels of consciousness. In addition to ethical reasons, animals require anesthesia not only for ethical reasons but also because pain or stress can alter the quality of research results. European Union rules contemplating the use of anesthesia, analgesia and other procedures to eliminate the suffering of experimental animals were adopted for use in Spain on March 14, 1980. Among the preoperatory considerations to be kept in mind is the possibility of diseases carried by animals, some of which can be transmitted to humans. Management and restraint of animals must also be planned before administering drugs. Recognition of pain, and its adequate analgesic treatment is important throughout surgical procedures. Different species of experimental animals require different types of administration. Some drugs, types and routes of administration and monitoring requirements are the same as those used in humans. Great differences exist, however, specifically in the large number of drugs used in animals but not in humans, and in techniques such as tracheal intubation that involve special difficulties.

Thermostability of native and pegylated Myceliophthora thermophila laccase in aqueous and mixed solvents.

A commercial preparation of laccase (EC 1.10.3.2), cloned from Myceliophthora thermophila and expressed in Aspergillus oryzae (MtL), was purified and modified by conjugation with poly(ethylene glycol) (M(r) = 5000) and is labeled PEG-MtL. Native enzyme was found to have a molecular mass of 80 kDa, as determined by gel filtration, and 110 kDa, by SDS-PAGE. The oxidative dimerization of 2,6-dimethoxyphenol (DMP) to produce the corresponding dibenzoquinone was catalyzed by MtL in a manner comparable to that for a diffusion-controlled reaction (k(cat)/K(M) approximately = 10(8) M(-)(1) s(-)(1) and E(a) approximately = 18 kJ M(-)(1)). PEG-MtL was found, by TNBS titration, to have blocked 54% of lysine groups; its hydrodynamic and charge properties were different from those of MtL. Catalytic efficiency (k(cat)/K(M)) of PEG-MtL was similar to that of MtL with DMP as substrate; however, k(cat)/K(M) was 2-fold reduced for the reaction in which 2',2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is oxidized to form a radical cation. E(a) values were similar in both enzyme preparations when assayed in buffered solutions. Far-UV CD spectra were similar for MtL and PEG-MtL and consistent with a protein rich in beta-sheet structure with negligible content of alpha-helices. A blue shift of near-UV CD spectrum for PEG-MtL as compared to MtL was consistent with the decreased polarity of the tyrosyl side chains upon PEG conjugation. Also the blue band of the copper active site was shifted from lambda approximately 610 nm (MtL) to lambda approximately 575 nm (PEG-MtL). Scanning microcalorimetry showed small denaturation enthalpies (6.3 and 7.5 J g(-)(1) for MtL and PEG-MtL, respectively), indicating the high stability of the beta-sheet folding pattern of laccases. However, PEG-MtL proved to be more stable, its half-denaturation temperature being 2 degrees C higher than that of MtL. In 30% alcohol, pegylated laccase showed slower enzyme-activity decay rates than the unmodified enzyme; this behavior was caused by a decrease in the activation entropy of the denaturation reaction. Results can be explained by entropic stabilization by PEG conjugation because of the restricted motion of some surface amino acid side chains, which results in a more stable active site.