Cytidinediphosphate diacylglycerol synthase-Mediated phosphatidic acid metabolism is crucial for early embryonic development of Arabidopsis.

Embryonic development is a key developmental event in plant sexual reproduction; however, regulatory networks of plant early embryonic development, particularly the effects and functional mechanisms of phospholipid molecules are still unknown due to the limitation of sample collection and analysis. We innovatively applied the microspore-derived in vitro embryogenesis of Brassica napus and revealed the dynamics of phospholipid molecules, especially phosphatidic acid (PA, an important second messenger that plays an important role in plant growth, development, and stress responses), at different embryonic developmental stages by using a lipidomics approach. Further analysis of Arabidopsis mutants deficiency of CDS1 and CDS2 (cytidinediphosphate diacylglycerol synthase, key protein in PA metabolism) revealed the delayed embryonic development from the proembryo stage, indicating the crucial effect of CDS and PA metabolism in early embryonic development. Decreased auxin level and disturbed polar localization of auxin efflux carrier PIN1 implicate that CDS-mediated PA metabolism may regulate early embryogenesis through modulating auxin transport and distribution. These results demonstrate the dynamics and importance of phospholipid molecules during embryo development, and provide informative clues to elucidate the regulatory network of embryogenesis.

Construction of a high-quality genomic BAC library for Chinese peanut cultivar Zhonghua 8 with high oil content.

BACKGROUND: Arachis hypogaea L. (2n = 4× = 40, AABB) is one of the most important oil and economic crop plants in the word. This species has the largest genome size of about 2,813 Mb among the oil crop species. Zhonghua 8 is a peanut cultivar planted widely in central China and has several superior traits including high oil content, high yield and disease resistance. A high-quality BAC library of Zhonghua 8 was constructed for future researches on the genomics of Chinese peanut cultivars. RESULTS: A Hin d III-digested genomic BAC (bacterial artificial chromosome) library was constructed with the genomic DNA from leaves of Zhonghua 8. This BAC library consists of 160,512 clones and the average insert is estimated about 102 kb ranging from 30 to 150 kb. The library represents about 5.55× haploid genome equivalents, and provides a 99.71% probability of finding specific genes. The empty-vector rate is under 5 percent detected from 200 randomly selected clones. Probing of 384 clones with the psbA gene of barley chloroplast and the atp6 gene of rice mitochondrion indicated that the contamination with organellar DNA is insignificant. Successive subculture of three clones showed that the inserts are stable in one hundred generations. CONCLUSIONS: This study presented the construction of a high-quality BAC library for the genome of Chinese cultivated peanut. Many essential experiences were summarized in the present study. This BAC library can serve as a substantial platform for development of molecular marker, isolation of genes and further genome research.

The effect of N-acetylcysteine in the nucleus accumbens on neurotransmission and relapse to cocaine.

BACKGROUND: Relapse to cocaine seeking has been linked with low glutamate in the nucleus accumbens core (NAcore) causing potentiation of synaptic glutamate transmission from prefrontal cortex (PFC) afferents. Systemic N-acetylcysteine (NAC) has been shown to restore glutamate homeostasis, reduce relapse to cocaine seeking, and depotentiate PFC-NAcore synapses. Here, we examine the effects of NAC applied directly to the NAcore on relapse and neurotransmission in PFC-NAcore synapses, as well as the involvement of the metabotropic glutamate receptors 2/3 (mGluR2/3) and 5 (mGluR5). METHODS: Rats were trained to self-administer cocaine for 2 weeks and following extinction received either intra-accumbens NAC or systemic NAC 30 or 120 minutes, respectively, before inducing reinstatement with a conditioned cue or a combined cue and cocaine injection. We also recorded postsynaptic currents using in vitro whole cell recordings in acute slices and measured cystine and glutamate uptake in primary glial cultures. RESULTS: NAC microinjection into the NAcore inhibited the reinstatement of cocaine seeking. In slices, a low concentration of NAC reduced the amplitude of evoked glutamatergic synaptic currents in the NAcore in an mGluR2/3-dependent manner, while high doses of NAC increased amplitude in an mGluR5-dependent manner. Both effects depended on NAC uptake through cysteine transporters and activity of the cysteine/glutamate exchanger. Finally, we showed that by blocking mGluR5 the inhibition of cocaine seeking by NAC was potentiated. CONCLUSIONS: The effect of NAC on relapse to cocaine seeking depends on the balance between stimulating mGluR2/3 and mGluR5 in the NAcore, and the efficacy of NAC can be improved by simultaneously inhibiting mGluR5.

The role of arabinogalactan proteins binding to Yariv reagents in the initiation, cell developmental fate, and maintenance of microspore embryogenesis in Brassica napus L. cv. Topas.

Arabinogalactan proteins (AGPs) are extracellular proteoglycans involved in plant growth and development. The addition of beta-D-glucosyl Yariv reagent (betaGlcY), a synthetic phenylglycoside that specifically reacts with AGPs, to the culture medium notably disturbed microspore embryogenesis in a concentration-dependent manner. The initiation of microspore embryogenesis was clearly inhibited by 30 microM betaGlcY and completely inhibited by 50 microM betaGlcY. The transfer of microspore-derived embryos at different developmental stages into NLN6 medium containing 50 microM betaGlcY prohibited their normal development, as approximately 21.24, 43.99, and 59.73%, respectively, of the treated globular-, heart-, and torpedo-stage embryos exhibited numerous root hair-like structures. Both heart-stage and torpedo-stage embryos showed a rapid growth of roots with a large number of clustered root hairs. Some root hair-like structures were also observed on the apical portions of embryos. Microscopy of the treated embryos revealed that the basic patterns of cells at both the radial and apical-basal axes were greatly altered, such that the cells lost their ability to carry out programmed embryogenesis. These results show that the betaGlcY-AGP interaction modulates the developmental fate of embryonic cells, especially epidermal cells, and thereby strongly affects root generation and development. Immunofluorescence microscopy revealed that both JIM8 and JIM13 binding to AGP co-localize with betaGlcY-binding sites. Thus, AGPs binding to betaGlcY, co-localized with Jim8- and Jim13-binding protein, appear to play a crucial role in the initiation of Brassica microspore embryogenesis and the maintenance of cell differentiation during embryonic development. In addition, these proteins may also be involved in the regulation of root generation.

Protective effects of iptakalim, a novel ATP-sensitive potassium channel opener, on global cerebral ischemia-evoked insult in gerbils.

AIM: To investigate the protective role of iptakalim, a novel ATP sensitive potassium channel opener, on global cerebral ischemia-evoked insult in gerbils and glutamate-induced PC12 cell injury. METHODS: Global cerebral ischemia was induced by occluding the bilateral common carotid arteries in gerbils for 5 min. The open field maze and T-maze were employed to investigate the experimental therapeutic value of iptakalim on ischemic brain insult (n=8). The pyramidal cells in the hippocampal CA1 regions were counted to assess the protective effects of iptakalim. Glutamate released from the gerbil hippocampus and PC12 cells were determined by HPLC. Intracellular calcium was measured by Fluo-3 AM with A Bio-Rad Radiance 2100TM confocal system in conjunction with a Nikon TE300 microscope. Astrocyte glutamate uptake measurements were determined by liquid scintillation counting. RESULTS: Iptakalim (0.5-4.0 mg/kg per day, ip) could reduce the high locomotor activity evoked by ischemia and improve global cerebral ischemia-induced working memory impairments. Histological studies revealed that iptakalim could increase the survival neuron in the hippocampus CA1 zone in a dose-dependent manner. Moreover, iptakalim could reverse ischemia-evoked increases of glutamate in the hippocampus of gerbils. In an in vitro study, iptakalim protected PC12 cells against glutamate-induced excitotoxicity, reduced the [Ca(2+)](i) increases, and enhanced the glutamate uptake activity of primary cultured astrocytes. CONCLUSIONS: Iptakalim plays a key role in preventing global cerebral ischemia-evoked insults in gerbils and glutamate-induced PC12 cell injury by anti-excitotoxicity. Iptakalim might be a promising novel candidate for the prevention and/or treatment of stroke.

Cocaine-induced reinstatement requires endogenous stimulation of mu-opioid receptors in the ventral pallidum.

The projection from the nucleus accumbens to the ventral pallidum regulates the reinstatement of cocaine seeking in rats extinguished from cocaine self-administration. This projection coexpresses GABA and enkephalin, posing a role for mu-opioid receptors in the ventral pallidum in mediating the reinstatement of cocaine seeking. Rats were extinguished from cocaine self-administration, and the reinstatement of active lever pressing by cocaine was blocked by intra-ventral pallidum administration of the mu receptor antagonist Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) (0.03-3.0 microg). Conversely, stimulating mu receptors with morphine (1-30 microg) in the ventral pallidum reinstated cocaine seeking. The ability of intra-ventral pallidum morphine to reinstate lever pressing was blocked by co-microinjection of the mu antagonist CTAP and was augmented by systemic cocaine administration. The reinstatement of cocaine seeking was associated with reduced extracellular GABA in the ventral pallidum, and the reduction in GABA was also prevented by blocking mu receptors with CTAP (10 microm). Although immunoblotting revealed that neither the total tissue concentration nor the membrane insertion of mu receptors in the ventral pallidum was altered by withdrawal from cocaine, the capacity of morphine (0.01-10 microm) to reduce ventral pallidum levels of extracellular GABA was augmented in rats extinguished from cocaine self-administration. These data are consistent with the reinstatement of cocaine seeking being modulated in part by coreleased enkephalin and GABA from the accumbens-ventral pallidal projection, a modulation that may involve the inhibition of GABA release by presynaptic mu receptors.

Simultaneous determination of histamine and polyamines by capillary zone electrophoresis with 4-fluor-7-nitro-2,1,3-benzoxadiazole derivatization and fluorescence detection.

Capillary zone electrophoresis (CZE) with fluorescence detection was applied to the simultaneous determination of histamine and polyamines including spermine, spermidine, diaminopropane, putrescine, cadaverine, diaminohexane with 4-fluor-7-nitro-2,1,3-benzoxadiazole (NBD-F) as the fluorescent derivatization reagent. The seven NBD-F labeled amines was separated within 200 s using 85 mM phosphate running buffer at pH 3.0. The concentration limits of these amines ranged from 5.1 x 10(-8) M for spermine to 2.1 x 10(-8) M for histamine. The relative standard deviations for migration time and peak height were less than 1.5% and 6.0%, respectively. The method was successfully applied to the analysis of biogenic amines in the lysate of tobacco mesophyll protoplasts, and spermidine and putrescine were detected in the lysate with satisfying recovery.

Targeting ischemic stroke with a novel opener of ATP-sensitive potassium channels in the brain.

During cerebral ischemia, the opening of neuronal ATP-sensitive potassium channels (K(ATP) channels) affords intrinsic protection by regulating membrane potential. To augment this endogenous mechanism, we have synthesized iptakalim, a K(ATP) opener. Through K(ATP) channel activation, iptakalim affected multiple pathways of the glutamatergic system, limiting glutamate release and receptor actions, which are involved in excitotoxicity during ischemic damage. The molecule readily penetrated the brain and showed low toxicity in animal experiments. In different animal models of stroke as well as in cell cultures, iptakalim provided significant neuroprotection, not only in promoting behavioral recovery but also in protecting neurons against necrosis and apoptosis. This compound thus has promise as a neuroprotective drug for the treatment of stroke and other forms of neuronal damage.

Neuroadaptations in cystine-glutamate exchange underlie cocaine relapse.

Repeated cocaine treatment and withdrawal produces changes in brain function thought to be involved in relapse to drug use. Withdrawal from repeated cocaine reduced in vivo extracellular glutamate in the nucleus accumbens of rats by decreasing the exchange of extracellular cystine for intracellular glutamate. In vivo restoration of cystine/glutamate exchange by intracranial perfusion of cystine or systemically administered N-acetylcysteine normalized the levels of glutamate in cocaine-treated subjects. To determine if the reduction in nonvesicular glutamate release is a mediator of relapse, we examined cocaine-primed reinstatement of drug seeking after cocaine self-administration was stopped. Reinstatement was prevented by stimulating cystine/glutamate exchange with N-acetylcysteine and restoring extracellular glutamate. Thus, withdrawal from repeated cocaine increases susceptibility to relapse in part by reducing cystine/glutamate exchange, and restoring exchanger activity prevents cocaine-primed drug seeking.

[Studies on callus growth and phillyrin accumulation of Forsythia suspensa].

OBJECTIVE: To investigate the effects of physical and chemical factors on callus growth and phillyrin contents of F. suspensa. METHOD: The cell growth index and phyllirin yield in different culture condition such as different plant hormones mixed, mediums, light and dark were compared. HPLC was used to examine phillyrin contents. RESULT AND CONCLUSION: Growth cycle of cells is twenty-eight days. During the course of callus growth, the processes of phillyrin biosynthesis were parallel with the cell growth. The optimum medium is MS. The optimum hormones concentrations are 1 mg.L-1 2,4-D, 0.5 mg.L-1 6-BA and 0.5 mg.L-1KT. The cell culture in light is more suitable than that in dark.