Influence of morphine on levels of type Ⅱ inhibitory guanine nucleotide binding protein in primary hippocampal neurons

BACKGROUND： The pharmacological action of opioid drugs is related to signal transduction of inhibitory guanine nucleotide binding protein. OBJECTIVE： To quantitatively and qualitatively analyze the influence of morphine on levels of type Ⅱ inhibitory guanine nucleotide binding protein （Gi2 protein） in primary cultured hippocampal neurons at different time points. DESIGN, TIME AND SETTING： A randomized controlled study, which was performed at the Department of Neurobiology, Changzheng Hospital, Second Military Medical University of Chinese PLA between September 2002 and March 2004. MATERIALS： Cerebral hippocampal neurons were obtained from newborn SD rats at 1 2 days of age. Biotin-antibody Ⅱ-avidin fluorescein isothiocyanate （Avidin-FITC） was purchased from Sigma Company （USA） and the Gi2 protein polyclonal antibody from Santa Cruz Biochemistry Company （USA）. METHODS： Seven days after culture, mature hippocampal neurons were randomly divided into six groups： 4-, 8-, 16-, 24-, and 48-hour morphine groups, and a blank control group. Neurons in the morphine groups received morphine （10 μ mol/L）, which could cause alterations of G-protein mRNA and cAMP expression in the prefrontal cortex. Neurons in the blank control group were given the same volume of saline. MAIN OUTCOME MEASURES： Gi2 protein levels were detected by an immunofluorescence technique, and were analyzed by the image analytic system with the use of green fluorescence intensity. RESULTS： Gi2 protein levels in hippocampal neurons gradually decreased in the 4-, 8-, 16-, 24-, and 48-hour morphine groups. In particular, Gi2 protein levels in the 16-, 24-, and 48-hour morphine groups were significantly lower than that in the blank control group （P 〈 0.05 0.01）. CONCLUSION： Morphine may decrease Gi2 protein level in primary hippocampal neurons, and the decreasing trend is positively related to morphine-induced time.

Inhibitory Kinetics of p-Substituted Benzaldehydes on Polyphenol Oxidase from the Fifth Instar of Pieris Rapae L.

Polyphenol oxidase （PPO） is the enzyme responsible for enzymatic browning during the growth of insects. It is also involved in defense reactions and is related with immunities in insects. PPO a metalloenzyme oxidase, catalyzes the oxidation of o-diphenol to o-quinone. The present paper describes the effects of benzaldehyde and its p-substituted derivatives on the activity of PPO from the fifth instar of Pieris rapae L. PPO from the fifth instar of Pieris rapae L. was purified using ammonium sulfate fractionation and chromatography on Sephadex G-100. The enzyme kinetics was characterized using L-3,4-dihydroxyphenylalanine （L-DOPA） as substrate. The results show that benzaldehyde, phydroxybenzaldehyde, p-chlorobenzaldehyde, and p-cyanobenzaldehyde can inhibit the PPO activity for the oxidation of L-DOPA. The inhibitor concentration leading to 50% activity lost, IC50, was estimated to be 5.90, 5.62, 2.83, and 2.91 mmol/L for the four tested inhibitors, respectively. Kinetic analyses show that the inhibitory effects of these compounds are reversible. Benzaldehyde, phydroxybenzaldehyde, and p-chlorobenzaldehyde are noncompetitive inhibitors while p-cyanobenzaldehyde is a mixed-type inhibitor. The inhibition constants were determined for all four inhibitors. p-chlorobenzaldehyde and p-cyanobenzaldehyde were more potent inhibitors than the other compounds. These results provide a basis for developing PPO inhibition-based pesticides.