Cyclic AMP-Dependent Protein Phosphorylation in Isolated Neuronal Growth Cones from Developing Rat Forebrain

We have shown recently that neuronal growth cones isolated from developing rat forebrain possess an appreciable activity of adenylate cyclase, which produces cyclic AMP and can be stimulated by various neurotransmitter receptor agonists and by forskolin. To investigate cyclic AMP-mediated biochemical mechanisms in isolated growth cones, we have centered the present study on cyclic AMP-dependent protein phosphorylation. One-dimensional gel electrophoretic analysis showed that cyclic AMP analogs increased incorporation of 32P into several phosphoproteins in molecular mass ranges of 50-58 and 76-82 kilodaltons, including those of 82, 76, and 51 kilodaltons. Two-dimensional electrophoresis, using isoelectric focusing in the first dimension, resolved phosphorylated alpha- and beta-tubulin species, actin, a very acidic protein (isoelectric point 4.0) with a molecular mass of 93 kilodaltons, and two proteins (x and x') closely neighboring beta-tubulin. Two other phosphoproteins seen in the gels had molecular masses of 56 and 51 kilodaltons (respective isoelectric points, 4.5 and 4.4) and, along with the 93-kilodalton phosphoprotein, were highly enriched in the isolated growth cones. Only the tubulin and actin species were major proteins in the isolated growth cones. Cyclic AMP analogs enhanced incorporation of 32P into phosphoproteins x and x', and, as assessed by immunoprecipitation, into beta-tubulin. Peptide digest experiments suggested that phosphoproteins x and x' are unrelated to beta-tubulin. Nonequilibrium two-dimensional electrophoresis resolved many phosphoproteins, of which a 79- and 75-kilodalton doublet, a 74-kilodalton species, and a 58-kilodalton doublet showed enhanced incorporation of 32P in the presence of cyclic AMP.     

神经生长抑制因子研究进展

神经生长抑制因子(neuronal growth inhibitory factor, GIF) 又名金属硫蛋白-Ⅲ (metallothionein-Ⅲ,MT-Ⅲ),特异分布于中枢神经系统(CNS),是神经系统中第一个被鉴定的具有神经元生长抑制功能的蛋白. GIF一级序列、高级结构、金属结合特性类似于其他MTs,基因结构也与其他MTs高度同源,但表达调控途径相异. GIF可能以其β结构域的CPCP区,与脑组织提取物中的相关因子结合,进而表现其生物学功能. 有研究认为GIF与阿尔茨海默等脑相关疾病均有密切关系.     

Coupling of Nerve Growth Factor to Its Receptor: Inhibition by Anti-GM3 Ganglioside Antibody

1. Normal differentiation of PC 12 cells and dorsal root ganglionic neurons in culture need nerve growth factor (NGF) for their neurite outgrowth.2. An antibody against GM₃ ganglioside was found to inhibit the nerve growth factor mediated neurite formation of both the cells in vitro significantly.3. Further analysis revealed that the binding of ¹²⁵I-NGF to live PC 12 cells could be markedly inhibited by anti-GM₃ antibody in a dose dependent manner.4. Scatchard analysis revealed that in the presence of anti-GM₃ antibody only some low affinity binding sites were available for NGF—high affinity binding sites were totally blocked.5. These results further strengthen the hypothesis that anti-GM₃ antibody affects neuronal cell growth by interfering with the coupling of growth factors to their cell surface receptors.     

Inhibitory Effect of Bacterial Attachment on Candidal Growth Due to Adherence with Mannose-Sensitive Pili

A bacterial strain with affinity to Candida albicans was successfully obtained from a natural environment. An uncovered Petri dish containing a suspension of heat-killed C. albicans cells was allowed to stand in a laboratory for several days. Some bacteria which had adhered to the candidal cells were tested for their ability to agglutinate the cells. A bacterial strain, designated later as CAB-1, was found to agglutinate candidal cells through bridging by mannose-sensitive pili. CAB-1 showed similar bacteriological characteristics to those of Citrobacter freundii by ID test. The adherence of CAB-1 to candidal cell was precisely presented by scanning electron microscopy. The inhibitory effect of CAB-1 attachment to candidal cells on the growth of Candida was also preliminarily confirmed.     

Neurite Outgrowth Stimulated by L1 Requires Calcium Influx into Neurons but is Not Associated with Changes in Steady State Levels of Calcium in Growth Cones

L1, NCAM and N-cadherin are cell adhesion molecules (CAMs), present on neuronal growth cones, which promote cell-contact dependent axonal growth by activating a second messenger pathway in neurons that requires calcium influx through L- and N- type calcium channels. In the present study we show that two of these CAMs, (L1 and N-cadherin) can stimulate neurite regeneration from axotomised adult dorsal root ganglion (DRG) neurons cultured in vitro and that this response can be fully inhibited by agents that block or negate the effect of calcium influx into the neurons. However although the response required calcium influx into neurons, it was not associated with an increase in the steady state levels of calcium in neuronal growth cones. These results suggest that small localised changes, or increases in the rate of calcium cycling, in growth cones and/or filopodia, are more important for regulating axonal growth than changes in the steady-state level of calcium.     

Neurite Outgrowth Stimulated by L1 Requires Calcium Influx into Neurons but is Not Associated with Changes in Steady State Levels of Calcium in Growth Cones

L1, NCAM and N-cadherin are cell adhesion molecules (CAMs), present on neuronal growth cones, which promote cell-contact dependent axonal growth by activating a second messenger pathway in neurons that requires calcium influx through L- and N- type calcium channels. In the present study we show that two of these CAMs, (L1 and N-cadherin) can stimulate neurite regeneration from axotomised adult dorsal root ganglion (DRG) neurons cultured in vitro and that this response can be fully inhibited by agents that block or negate the effect of calcium influx into the neurons. However although the response required calcium influx into neurons, it was not associated with an increase in the steady state levels of calcium in neuronal growth cones. These results suggest that small localised changes, or increases in the rate of calcium cycling, in growth cones and/or filopodia, are more important for regulating axonal growth than changes in the steady-state level of calcium.     

Topography and Nanomechanics of Live Neuronal Growth Cones Analyzed by Atomic Force Microscopy

Neuronal growth cones are motile structures located at the end of axons that translate extracellular guidance information into directional movements. Despite the important role of growth cones in neuronal development and regeneration, relatively little is known about the topography and mechanical properties of distinct subcellular growth cone regions under live conditions. In this study, we used the AFM to study the P domain, T zone, and C domain of live Aplysia growth cones. The average height of these regions was calculated from contact mode AFM images to be 183 ± 33, 690 ± 274, and 1322 ± 164 nm, respectively. These findings are consistent with data derived from dynamic mode images of live and contact mode images of fixed growth cones. Nano-indentation measurements indicate that the elastic moduli of the C domain and T zone ruffling region ranged between 3-7 and 7-23 kPa, respectively. The range of the measured elastic modulus of the P domain was 10-40 kPa. High resolution images of the P domain suggest its relatively high elastic modulus results from a dense meshwork of actin filaments in lamellipodia and from actin bundles in the filopodia. The increased mechanical stiffness of the P and T domains is likely important to support and transduce tension that develops during growth cone steering.     

Growth and Reproductive Responses of Daphnia to Cyanobacterial Blooms on the Potomac River

We examined reproductive responses of the cladoceran Daphnia parvula to algal assemblages from the Potomac River, during and after Cyanobacterial blooms. Algal assemblages were manipulated by filtration, dilution, or additions of nutritious algae, in order to determine causes for growth inhibition. There was no evidence for lethal toxic effects or food limitation of population growth rates during the algal bloom, although growth was food limited later in the year. However, growth rates were lower in the experiment conducted during the Cyanobacteria bloom than in later experiments, and the results suggest an inhibitory factor associated with the particulate material. Results from filtered treatments indicate that picoplankton present in hypereutrophic waters can support Daphnia growth and reproduction.     

Neuronal differentiation in PC12 cells is accompanied by diminished inducibility of Hsp 70 and Hsp60 in response to heat and ethanol

The stress response of PC12 cells was characterized by evaluating the production of heat shock proteins of the 70 kDa (Hsp70), 60 kDa (Hsp60) and 90 kDa (Hsp90) families by western blot analysis. Induction of Hsp synthesis was elicited by brief exposure to elevated temperatures or by addition of ethanol to the cultures. Normal PC12 cells responded to stress with rapid up-regulation of Hsp70 and Hsp60 production. However, fully differentiated PC12 cells (induced by nerve growth factor, NGF) failed to produce Hsp70 or Hsp60 in response to heat or ethanol treatment. The disappearance of the heat shock response of the cells was directly related to the extent of neuronal differentiation. The cellular levels of the constitutive proteins, Hsc70 and Hsp90, were not altered by differentiation of the cells. Production of Hsps was restored in the differentiated cells by removal of NGF which coincided with the loss of neurite expression and retraction of processes.     

Selective Changes in Cell Bodies and Growth Cones of Nerve Growth Factor-Differentiated PC12 Cells Induced by Chemical Hypoxia

Abstract: Cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was measured in differentiated PC12 cells to test whether chemical hypoxia selectively alters intracellular Ca²⁺ in growth cones and cell bodies. Hypoxia increased [Ca²⁺]_i and exaggerated its response to K⁺ depolarization in both parts of the cells. [Ca²⁺]_i in the cell bodies was greater than that in the growth cones under resting conditions and in response to K⁺ or hypoxia. Ca²⁺-channel blockers selectively altered these responses. The L-channel blocker nifedipine reduced [Ca²⁺]_i following K⁺ depolarization by 67% in the cell bodies but only 25% in the growth cones. In contrast, the N-channel blocker ω-conotoxin GVIA (ω-CgTX) diminished K⁺-induced changes in [Ca²⁺]_i only in the growth cones. During hypoxia, nifedipine was more effective in the cell bodies than in the growth cones. During hypoxia, ω-CgTX diminished K⁺-induced changes by 50–75% in both parts of the cell, but only immediately after depolarization. The combination of nifedipine and ω-CgTX diminished the [Ca²⁺]_i response to K⁺ with or without hypoxia by >90% in the cell body and 70% in the growth cones. Thus, the increased Ca²⁺ entry with K⁺ during hypoxia is primarily through L channels in the cell bodies, whereas in growth cones influx through L and N channels is about equal. The results show that chemical hypoxia selectively alters Ca²⁺ regulation in the growth cone and cell body of the same cell.     

Growth and morphology of neuronal cell lines cultured in perfusion

Summary To optimize culture conditions and gain a more reliable culturing system for studies of metabolic properties of neuronal cells, a simplified perfusion chamber was developed. It consists of two parts: a perfusion block and a standard plastic culture dish. To confirm the suitability of this chamber for continuous culturing of anchorage-dependent cells, the growth and morphology of the four neuronal cell lines glioma C6 and glioma 138MG, neuroblastoma C1300, clones N1E115 and N18 were followed for 4 d using both traditional and perfusion techniques. A marked increase in growth and a decrease in the degree of morphological differentiation were obtained with the latter technique compared to the former. This work was supported by grants from the National Swedish Board for Technical Development (Grant 81-5009), the Swedish Work Environmental Foundation (Grant 76-53), and Ollie and Elof Ericssons Foundation for Scientific Research.     

Neuronal differentiation of PC12 cells as a result of prevention of cell death by bcl-2

Rat pheochromocytoma PC12 cells die when cultured in serum-free medium. Neurotrophic factors can rescue PC12 cells from cell death, and induce neuronal differentiation. To further investigation the relationship among cell death, survival, and differentiation, the bcl-2 cDNA, which is known to prevent apoptosis in various types of cells, was transfected into PC12 cells. Six monoclonal bcl-2-transfected cell lines were isolated and confirmed to express mRNA and protein product of bcl-2. The wild-type and bcl-2-transfected PC12 cells were kept to adhere to collagen-coated dishes at the inintiation of serum-free experiments to avoid cellular damage due to detachment of the cells by triturtion. Even under the conditions, the control PC12 cells mostly died within 24 h, when cultured in serum-free medium whereas those expressing Bcl-2 survived even for 7 days in serum-free medium. Moreover, outgrowth of long processes in thebcl-2-transfected cells was only observed under the condition to keep the cells attached to the dishes in serum-free medium without any additive neurotrophic or growth factors. Neurofilament medium protein, which is a neuron-specific cytoskeletal component, was also expressed in the differentited cells, suggesting that the long processes in bcl-2-transfected PC12 cells are neurites. However, neuronal differentiation of PC12 cells expressing Bcl-2 was not observed when cultured in serum-containing medium. Accordingly, survival of PC12 cells expressing Bcl-2 under the condition which cells usually die may be accompanied with neuronal differentiation. 1994 John Wiley & Sons, Inc.     

Growth inhibition of activated sludge by humus

Humus at 0.5 to 1 mg l–1 inhibited growth of activated sludge by about 55% in batch and long-term repeated batch cultures without decreasing sugar utilization. The growth inhibition was considerable when concentrations of substrates in the medium supplied per unit weight of activated sludge were low.     

Induction of Growth Inhibition and Differentiation of Human Leukemia HL-60 Cells by a Tunisian Gerboui Olive Leaf Extract

Cancer protection associated with the consumption of olive products is well established, but not for leukemia. The protective effects of olive (Olea europaea L.) leaves were investigated by incubating human promyelocytic leukemia HL-60 cells with olive leaf extracts (OLEs) from seven principal Tunisian olive varieties, namely, Chemchali, Chemlali, Chétoui, Gerboui, Sayali, Zalmati and Zarrazi. The results showed significant growth inhibition of HL-60 cells incubated for 48 h with a 100-fold dilution of each OLE which had been obtained by incubating 10 g of dried leaves in 100 ml of 70% ethanol for one week with subsequent ultrafiltration. DNA fragmentation was observed in the cells incubated for 19 h with a 100-fold dilution of the Chemchali, Chemlali and Zalmati extracts. The results of a nitroblue tetrazolium (NBT) assay revealed NBT reduction, a differentiation marker, by the OLE-treated cells after an overnight incubation. The Gerboui extract showed the highest NBT reduction ability at more than 90%. An HPLC analysis revealed the presence of apigenin 7-glucoside in the extract, which was found in subsequent experiments to be responsible for the Gerboui extract-mediated cell differentiation.     

Increased Intracellular Cyclic AMP Differentially Modulates Nerve Growth Factor Induction of Three Neuronal Recognition Molecules Involved in Neurite Outgrowth

The relative expression of the immunoglobulin superfamily members Thy-1 and L1 and the neural cell adhesion molecule (N-CAM) in PC12 cells grown in the presence of nerve growth factor (NGF), cholera toxin, or both has been quantified. Whereas NGF treatment induced increases in the cell surface expression of all three glycoproteins, treatment with cholera toxin resulted in the specific induction of L1. During the first few days of culture, cholera toxin acted synergistically with NGF to promote increases in neuritic outgrowth and the synthesis and cell surface accumulation of the 140- and 180-kilodalton subunits of N-CAM. In contrast, over the same period of culture, cholera toxin inhibited the NGF induction of Thy-1 and L1. Over longer periods of culture (3-5 days), cholera toxin inhibited the NGF induction of N-CAM and neurite outgrowth. A similar pattern of synergistic and inhibitory responses was observed when differentiation was induced by fibroblast growth factor (FGF) rather than NGF or when cholera toxin was replaced with forskolin. These data suggest that intracellular cyclic AMP can differentially modulate cell surface glycoprotein expression induced by either NGF or FGF. Of the three cell surface glycoproteins we have studied, temporal changes in N-CAM expression correlate best with the morphological differentiation status of PC12 cells.     

G-蛋白 Rab3a 对神经生长抑制因子 (GIF) 抑制神经元细胞生长作用的影响

为了深入研究 Rab3a 和神经生长抑制因子 (GIF) 的相互作用,在大鼠海马神经元细胞原代培养体系中,用 MTT 还原法定量研究了 Rab3a 对 GIF 神经生长活性的影响,发现 Rab3a 可以替代脑提取物而使 GIF 发挥神经生长抑制活性 . 接着又利用多克隆抗体方法研究了 GIF 与 Rab3a 的相互作用,结果说明 Rab3a 是 GIF 发挥神经生长抑制活性所必需的蛋白质,并且它们的相互作用受空间位置的因素影响较大 . 在随后对它们相互作用的机理和可能的生物学意义进行了讨论 .     

Isoform-Specific Effects of Transforming Growth Factors-β on Degeneration of Primary Neuronal Cultures Induced by Cytotoxic Hypoxia or Glutamate

Abstract: The transforming growth factors-β (TGFs-β) are multifunctional peptide-growth factors that have been localized in neuronal and glial cells of the C'NS of mice. rats, and chick embryos. We tested the TGF-β isoforms 1. 2. and 3 for their protective ctlects against neuronal degeneration caused by cytotoxic hypoxia or by the excitatory amino acid i -glutamate. A cytotoxic hypoxia was induced in cultured chick embryo telencephalic neurons by adding I m.l/ sodium cyanide to the culture medium tor a period of 30min. I reatment with TGF-81 (1-30 ng/ml) led to a statistically significant increase in cell viability, neuronal ATP levels. and protein content of the cultures assessed 72 h after the toxic insult. TGF-33 was able to reduce the cyanide-induced neuronal damage at concentrations of 0.3 and 1 ng/ ml. whereas TGF-33, only showed neuroprotective activity at concentrations of 30 and 50 ng/ml. Both pre- and posttreatment with TGF-31, also prevented the degeneration of cultured chick embryo telencephalic neurons that had been exposed to I mM L-glutamate in a buffered salt solution for a period of 60 min. Furthermore, TGF-β1 (0.3-3 ng/ml). and to a lesser extent TGF-β3 (0.1-1 ng/ml). significantly reduced excitotoxic injury of cultured neurons from rat cerebral cortex that had been exposed to serum-free culture medium supplemented with 1 m.M L-glutamate. These results demonstrate that the TGFs-β are able to prevent the degeneration of primary neuronal cultures, which was caused by energy depletion and activation of glutamate receptors, in an isoform-specific manner.     

瑞香狼毒根中活性物质的分离鉴定及作用机理

以瑞香狼毒根为研究材料,模式植物拟南芥为受体,采用实验室活体生物实验方法研究了瑞香狼毒根提取物及不同极性溶剂萃取物对7 d龄拟南芥幼苗的生长抑制作用;采用活性跟踪的化合物分离方法,分析了其中的活性化合物成分,并通过拟南芥DR5-GUS转基因株系,研究了单体化合物对拟南芥生长发育及根系生长素分布的影响.结果显示,瑞香狼毒根乙醇提取物对拟南芥有很强的生长抑制作用,其乙酸乙酯和氯仿萃取物的抑制活性最为显著,从氯仿萃取物中分离得到两种香豆素类化合物伞形花内酯(1)和西瑞香素(2),其中化合物1能够显著抑制拟南芥幼苗生长及根系发育,且明显降低了根部内源生长素的分布水平;化合物2也有较为明显的抑制活性.研究表明,氯仿和乙酸乙酯萃取物是瑞香狼毒植物毒活性的主要有效部位;伞形花内酯(1)和西瑞香素(2)是瑞香狼毒植物毒活性的有效成分,化合物1对拟南芥生长发育的抑制作用与生长素途径密切相关.     

Intrauterine Growth Retardation (Malnutrition by Vascular Ligation) Induces Modifications in Fatty Acid Composition of Neurons and Oligodendrocytes

Intrauterine growth retardation (IUGR) induced by ligation of one uterine artery on day 17 of pregnancy in the rat lead to major abnormalities in the fatty acid content of neurons and oligodendrocytes but not in astrocytes. In neurons from IUGR rats, monounsaturated fatty acids were decreased; in the polyunsaturated series, ω-3 fatty acids were increased and Ω-6 fatty acids were decreased. In oligodendrocytes, monounsaturated fatty acids were also decreased, but the modifications in polyunsaturated fatty acids were the opposite of those in neurons: Ω-3 being decreased and w-6 increased. Although the animals received a normal diet after birth, the alterations were still present in adulthood. In addition, fatty acid composition of brain cells is a very indicative criterion of brain maturation.