Flooding-induced membrane damage, lipid oxidation and activated oxygen generation in corn leaves

Flooding effects on membrane permeability, lipid peroxidation and activated oxygen metabolism in corn (Zea mays L.) leaves were investigated to determine if activated oxygens are involved in corn flooding-injury. Potted corn plants were flooded at the 4-leaf stage in a controlled environment. A 7-day flooding treatment resulted in a significant increase in chlorophyll breakdown, lipid peroxidation (malondialdehye content), membrane permeability, and the production of superoxide (O ₂ ^- ) and hydrogen peroxide (H₂O₂) in corn leaves. The effects were much greater in older leaves than in younger ones. Spraying leaves with 8-hydroxyquinoline (an O ₂ ^- scavenger) and sodium benzoate (an .OH scavenger) reduced the oxidative damage and enhanced superoxide dismutase (SOD) activity. A short duration flooding treatment elevated the activities of SOD, catalase, ascorbate peroxidase (AP), and glutathione reductase (GR), while further flooding significantly reduced the enzyme activities but enhanced the concentrations of ascorbic acid and reduced form glutathione (GSH). It was noted that the decline in SOD activity was greater than that in H₂O₂ scavengers (AP and GR). The results suggested that O ₂ ^- induced lipid peroxidation and membrane damage, and that excessive accumulation of O ₂ ^- is due to the reduced activity of SOD under flooding stress.     

Calcium-dependent Modulation of the Agonist Affinity of the Mammalian Olfactory Cyclic Nucleotide-gated Channel by Calmodulin and a Novel Endogenous Factor

The calcium-dependent modulation of the affinity of the cyclic nucleotide-gated (CNG) channels for adenosine 3′,5′-cyclic monophosphate (cAMP) was studied in enzymatically dissociated rat olfactory receptor neurons, by recording macroscopic cAMP-activated currents from inside-out patches excised from their dendritic knobs. Upon intracellular addition of 0.2 mm Ca²⁺ (0.2 Ca) the concentration of cAMP required for the activation of half-maximal current (EC₅₀) was reversibly increased from 3 μm to about 30 μm. This Ca²⁺-induced affinity shift was insensitive to the calmodulin antagonist, mastoparan, was abolished irreversibly by a 2-min exposure to 3 mm Mg²⁺+ 2 mm EGTA (Mg + EGTA), and was not restored by the application of calmodulin (CAM). Addition of CAM plus 0.2 mm Ca²⁺ (0.2 Ca + CAM), further reversibly shifted the cAMP affinity from 30 μm to about 200 μm. This affinity shift was not affected by Mg + EGTA exposure, but was reversed by mastoparan. Thus, the former Ca²⁺-only effect must be mediated by an unknown endogenous factor, distinct from CAM. Removal of this factor also increased the affinity of the channel for CAM. The affinity shift induced by Ca²⁺-only was maintained in the presence of the nonhydrolyzable cAMP analogue, 8-bromo-cAMP and the phosphatase inhibitor, microcystin-LR, ruling out modulation by phosphodiesterases or phosphatases. Our results indicate that the olfactory CNG channels are modulated by an as yet unidentified factor distinct from CAM. Received: 26 December 1995/Revised: 14 March 1996     

The fluidity of the nuclear envelope lipid does not affect the rate of nucleocytoplasmic RNA transport in mammalian liver

The effects of in vitro and in vivo modifications of nuclear envelope lipid on DNA leakage and on ATP-stimulated RNA release from isolated rat liver nuclei were investigated. The modifications included corn-oil feeding of the animals to alter the fatty acid composition of the lipids, phospholipase treatment of the isolated nuclei, and extraction of the total lipid with Triton X-100. Significant changes in lipid composition and approximate order parameter values of the spin-label 5-doxylstearate resulted, but there was no significant effect on RNA transport rate. It was concluded that the nuclear envelope lipid does not play any important part in nucleocytoplasmic RNA transport in mammalian liver.     

鲫鱼视网膜谷氨酸受体和GABA受体在爪蟾卵母细胞中的表达

我们以两栖类卵母细胞为功能表达系统,通过注射鲫鱼（Ｃａｒａｓｓｉｕｓｃａｒａｓｓｉｕｓ）视网膜ｍＲＮＡ,利用电压箝及药物灌流手段,系统地研究了鲫鱼视网膜内氨基酸受体的类型和特征,结果如下：（１）Ｇｌｕ受体：ＫＡ可以诱发明显的去极化电流,而且Ｄｉａｚｏｘｉｄｅ能增强ＫＡ诱导的反应,这提示鲫鱼视网膜内某些Ｃｌｕ受体是ＡＭＰＡ选择性亚型（ＡＭＰＡ－ｐｒｅｆｅｒｒｉｎｇｓｕｂｔｙｐｅ）。（２）ＣＡＢＡ受体：ＧＡＢＡ能诱发一个快速、光滑的内向电流,绝大部分对ＧＡＢＡ的反应可被ｂｉｃｕｃｕｌｌｉｎｅ所压抑,而ＧＡＢＡ＿Ｂ受体的激动剂ｂａｃｌｏｆｅｎ则无任何作用,这提示,鲫鱼视网膜内大部分是ＧＡＢＡ＿Ａ受体。     

The γ subunits of the native GABAA/benzodiazepine receptors

Subunit-specific antibodies to all the γ subunit isoforms described in mammalian brain (γ₁, γ_2S, γ_L, and γ₃) have been made. The proportion of GABA_A receptors containing each γ subunit isoform in various brain regions has been determined by quantitative immunoprecipitation. In all tested regions of the rat brain, the γ₁, and γ₃ subunits are present in considerable smaller proportion of GABA_A receptor than the γ₂ subunit. Immunocytochemistry shows that γ₁ immunoreactivity concentrates in the stratum oriens and stratum radiatum of the CA1 region of the hippocampus. In the dentate gyrus, γ₁ immunoreactivity concentrates on the outer 2/3 of the molecular layer coinciding with the localization of the axospinous synapses of the perforant pathway. In contrast, γ₃ immunoreactivity concentrates on the basket cells and other GABAergic local circuit neurons of the hilus. These cells are also rich in γ_2S. In the cerebellu, γ₁ immunolabeling was localized on the Bergmann glia. The γ_2S and γ_2L subunits are differentially expressed in various brain regions. Thus the γ_2S is highly expressed in the olfactory bulb and hippocampus whereas the γ_2L is very abundant in inferior colliculus and cerebellum, particularly in Purkinje cells, as immunocytochemistry, in situ hybridization and immunoprecipitation techniques have revealed. The γ_2S and γ_2L coexist in some brain areas and cell types. Moreover, the γ_2S and γ_2L subunits can coexist in the same GABA_A receptor pentamer. We have shown that this is the case in some GABA_A receptors expressed in cerebellar granule cells. These GABA_A receptors also have α and β subunits forming the pentamer. Immunoblots have shown that the rat γ₁, γ_2S, γ_2L and γ₃ subunits are peptides of 47, 45, 47 and 44 kDa respectively. Results also indicate that there are aging-related changes in the expression of the γ_2S and γ_2L subunits in various brain regions which suggest the existence of aging-related changes in the subunit composition of the GABA_A receptors which in turn might lead to changes in receptor pharmacology. The results obtained with the various γ subunit isoforms are discussed in terms of the high molecular and binding heterogeneity of the native GABA_A receptors in brain. Special issue dedicated to Dr. Kinya Kuriyama     

Increased phosphorylation of the NR1 subunit of the NMDA receptor following cerebral ischemia

The effects of transient cerebral ischemia on phosphorylation of the NR1 subunit of the NMDA receptor by protein kinase C (PKC) and protein kinase A (PKA) were investigated. Adult rats received 15 min of cerebral ischemia followed by various times of recovery. Phosphorylation was examined by immunoblotting hippocampal homogenates with antibodies that recognized NR1 phosphorylated on the PKC phosphorylation sites Ser890 and Ser896, the PKA phosphorylation site Ser897, or dually phosphorylated on Ser896 and Ser897. The phosphorylation of all sites examined increased following ischemia. The increase in phosphorylation by PKC was greater than by PKA. The ischemia-induced increase in phosphorylation was predominantly associated with the population of NR1 that was insoluble in 1% deoxycholate. Enhanced phosphorylation of NR1 by PKC and PKA may contribute to alterations in NMDA receptor function in the postischemic brain.     

Immobilization of catalase via adsorption onto -histidine grafted functional pHEMA based membrane

Poly(2-hydroxyethylmethacrylate) (pHEMA) based flat sheet membrane was prepared by UV-initiated photopolymerization technique. The membrane was then grafted with -histidine. Catalase immobilization onto the membrane from aqueous solutions containing different amounts of catalase at different pH was investigated in a batch system. The maximum catalase immobilization capacity of the pHEMA–histidine membrane was 86 μg cm⁻². The activity yield was decreased with the increase of the enzyme loading. It was observed that there was a significant change between V_max value of the free catalase and V_max value of the adsorbed catalase on the pHEMA–histidine membrane. The K_m value of the immobilized enzyme was higher 1.5 times than that of the free enzyme. Optimum operational temperature was 5°C higher than that of the free enzyme and was significantly broader. It was observed that enzyme could be repeatedly adsorbed and desorbed without loss of adsorption capacity or enzyme activity.     

Ceramide inhibits the outward potassium current in rat pinealocytes

CD1 mice lacking the CB1 receptors (knockout, KO) were compared with wild-type littermates for their ability to degrade N-arachidonoylethanolamine (anandamide, AEA) through a membrane transporter (AMT) and a fatty acid amide hydrolase (FAAH). The regional distribution and age-dependence of AMT and FAAH activity were investigated. Anandamide membrane transporter and FAAH increased with age in knockout mice, whereas they showed minor changes in wild-type animals. Remarkably, they were higher in all brain areas of 6-month-old knockout versus wild-type mice, and even higher in 12-month-old animals. The molecular mass (approximately 67 kDa) and isoelectric point (approximately 7.6) of mouse brain FAAH were determined and the FAAH protein content was shown to parallel the enzyme activity. The kinetic constants of AMT and FAAH in the cortex of wild-type and knockout mice at different ages suggested that different amounts of the same proteins were expressed. The cortex and hippocampus of wild-type and knockout mice contained the following N-acylethanolamines: AEA (8% of total), 2-arachidonoylglycerol (5%), N-oleoylethanolamine (20%), N-palmitoylethanolamine (53%) and N-stearoylethanolamine (14%). These compounds were twice as abundant in the hippocampus as in the cortex. Minor differences were observed in AEA or 2-arachidonoylglycerol content in knockout versus wild-type mice, whereas the other compounds were lower in the hippocampus of knockout versus wild-type animals.     

Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing

In the present study, we provide evidence for the production of reactive oxygen species (ROS) during cryopreservation of bovine spermatozoa. Cooling and thawing of spermatozoa cause an increase in the generation of superoxide radicals. Although nitric oxide production remains unaltered during sperm cooling from 22-4 degrees C, a sudden burst of nitric oxide radicals is observed during thawing. Increase in lipid peroxidation levels have been observed in frozen/thawed spermatozoa and appears to be associated with a reduction in sperm membrane fluidity as detected by spin labeling studies. The data presented provide strong evidence that oxygen free radicals are produced during freezing and thawing of bovine spermatozoa and suggest that these reactive oxygen species may be a cause for the decrease in sperm function following cryopreservation. Mol. Reprod. Dev. 59: 451-458, 2001.