胚胎干细胞体外分化为多巴胺能神经元

近年来,胚胎干细胞在体外分化为多巴胺能神经元方面取得了重大突破,这对神经发生的基础性研究和神经细胞移植具有重要意义。现对胚胎干细胞体外定向诱导分化为多巴胺能神经元的方法、相关细胞因子及检测鉴定等方面进行了分析和比较,并探讨了当前存在的问题和今后发展的方向。     

Aurintricarboxylic Acid Protects Hippocampal Neurons from Glutamate Excitotoxicity In Vitro

Abstract: Aurintricarboxylic acid (ATA), an endonuclease inhibitor, has been shown to protect several cell types from an apoptotic form of cell death. We tested ATA for protective effects against glutamate excitotoxicity in 2-week-old cultured hippocampal neurons. Cell viability was determined 24 h after glutamate exposure either by trypan blue exclusion or by measurement of lactate dehydrogenase release. When ATA was added during exposure to glutamate, there was a dramatic increase in the number of viable neurons compared with cultures that did not receive ATA. If ATA was added after glutamate exposure, the rate of survival approached 100%. Several cellular processes may be the targets for ATA action. If the mechanisms of ATA protection are similar for excitotoxicity and apoptosis, then these distinct forms of cell death may share a common intracellular pathway.     

In Vitro Laser Ablation of Natural Marine Biofilms

We studied the efficiency of pulsed low-power laser irradiation of 532 nm from an Nd:YAG (neodymium-doped yttrium-aluminum-garnet) laser to remove marine biofilm developed on titanium and glass coupons. Natural biofilms with thicknesses of 79.4 ± 27.8 μm (titanium) and 107.4 ± 28.5 μm (glass) were completely disrupted by 30 s of laser irradiation (fluence, 0.1 J/cm²). Laser irradiation significantly reduced the number of diatoms and bacteria in the biofilm (paired t test; P < 0.05). The removal was better on titanium than on glass coupons.     

l-Deprenyl Protects Mesencephalic Dopamine Neurons from Glutamate Receptor-Mediated Toxicity In Vitro

Abstract: l -Deprenyl is a relatively selective inhibitor of monoamine oxidase (MAO)-B that delays the emergence of disability and the progression of signs and symptoms of Parkinson's disease. Experimentally, deprenyl has also been shown to prevent neuronal cell death in various models through a mechanism that is independent of MAO-B inhibition. We examined the effect of deprenyl on cultured mesencephalic dopamine neurons subjected to daily changes of feeding medium, an experimental paradigm that causes neuronal death associated with activation of the NMDA subtype of glutamate receptors. Both deprenyl (0.5–50 µ M ) and the NMDA receptor blocker MK-801 (10 µ M ) protected dopamine neurons from damage caused by medium changes. The nonselective MAO inhibitor pargyline (0.5–50 µ M ) was not protective, indicating that protection by deprenyl was not due to MAO inhibition. Deprenyl (50 µ M ) also protected dopamine neurons from delayed neurotoxicity caused by exposure to NMDA. Because deprenyl had no inhibitory effect on NMDA receptor binding, it is likely that deprenyl protects from events occurring downstream from activation of glutamate receptors. As excitotoxic injury has been implicated in neurodegeneration, it is possible that deprenyl exerts its beneficial effects in Parkinson's disease by suppressing excitotoxic damage.     

Susceptibility of Hippocampal and Cortical Neurons to Argon-Mediated In Vitro Ischemia

Abstract: Neurons from cerebral cortex and hippocampal CA1 sector exhibit a striking difference in vulnerability to transient ischemia. To establish whether this difference is due to the inherent (pathoclitic) properties of these neurons, the ischemic susceptibility was studied in primary cortical and hippocampal cultures by using a new model of argon-induced in vitro ischemia. Neuronal cultures were exposed at 37°C for 10–30 min to argon-equilibrated glucose-free medium. During argon equilibration, P o ₂ declined to <2.5 torr within 1 min and stabilized shortly later at ∼1.3 torr. After 30 min of in vitro ischemia, total adenylate was <45% and ATP content <15% of control in both types of culture. Cytosolic calcium activity increased from 15 to 50 n M . Reoxygenation of cultures after in vitro ischemia led to delayed neuronal death, the severity of which depended on the duration of in vitro ischemia but not on the type of neuronal cultures. Energy charge of adenylate transiently returned to ∼90% of control after 3 h, but ATP content recovered only to 40% and protein synthesis to <35%. Cytosolic calcium activity continued to rise after ischemia and reached values of ∼500 n M after 3 h. The new argon-induced in vitro ischemia model offers major advantages over previous methods, but despite this improvement it was not possible to replicate the differences in cortical and hippocampal vulnerability observed in vivo. Our study does not support the hypothesis that selective vulnerability is due to an inherent pathoclitic hypersensitivity.     

Corticosterone Induces Dysregulation of Iron Metabolism in Hippocampal Neurons In Vitro

Iron is required for neuronal function but in excess generates neurodegeneration. Although the iron homeostasis machinery in neurons has been described extensively, little is known about the influence of corticosterone on the iron homeostasis in neurons. In this study, we characterized the response of hippocampal neurons to a model of progressive corticosterone condition. We found that increasing extracellular corticosterone-induced iron accumulation killed a large proportion of neurons. Iron concentrations were significantly increased in the corticosterone-treated cells. In the hippocampal neurons, corticosterone decreased expression of ferritin and increased expression of transferrin receptor1 (TfR1), iron regulatory protein1 (IRP1), and divalent metal transporter 1. Corticosterone-induced elevation of IRP1 expression can cause increase of TfR1 and decrease of ferritin expression, which further leads to iron accumulation in hippocampal neurons and subsequently increases the oxidative damage of the neurons; it is indicated that corticosterone might be an important reason for iron deposition-caused neurodegenerative diseases.     

Robust Synchrony and Rhythmogenesis in Endocrine Neurons via Autocrine Regulations In Vitro and In Vivo

Episodic pulses of gonadotropin-releasing hormone (GnRH) are essential for maintaining reproductive functions in mammals. An explanation for the origin of this rhythm remains an ultimate goal for researchers in this field. Some plausible mechanisms have been proposed among which the autocrine-regulation mechanism has been implicated by numerous experiments. GnRH binding to its receptors in cultured GnRH neurons activates three types of G-proteins that selectively promote or inhibit GnRH secretion (Krsmanovic et al. in Proc. Natl. Acad. Sci. 100:2969–2974, 2003). This mechanism appears to be consistent with most data collected so far from both in vitro and in vivo experiments. Based on this mechanism, a mathematical model has been developed (Khadra and Li in Biophys. J. 91:74–83, 2006) in which GnRH in the extracellular space plays the roles of a feedback regulator and a synchronizing agent. In the present study, we show that synchrony between different neurons through sharing a common pool of GnRH is extremely robust. In a diversely heterogeneous population of neurons, the pulsatile rhythm is often maintained when only a small fraction of the neurons are active oscillators (AOs). These AOs are capable of recruiting nonoscillatory neurons into a group of recruited oscillators while forcing the nonrecruitable neurons to oscillate along. By pointing out the existence of the key elements of this model in vivo, we predict that the same mechanism revealed by experiments in vitro may also operate in vivo. This model provides one plausible explanation for the apparently controversial conclusions based on experiments on the effects of the ultra-short feedback loop of GnRH on its own release in vivo.     

Excitotoxic Death of a Subset of Embryonic Rat Motor Neurons In Vitro

Abstract : We have used cultures of purified embryonic rat spinal cord motor neurons to study the neurotoxic effects of prolonged ionotropic glutamate receptor activation. NMDA and non-NMDA glutamate receptor agonists kill a maximum of 40% of the motor neurons in a concentration- and time-dependent manner, which can be blocked by receptor subtype-specific antagonists. subunit-specific antibodies stain all of the motor neurons with approximately the same intensity and for the same repertoire of subunits, suggesting that the survival of the nonvulnerable population is unlikely to be due to the lack of glutamate receptor expression. Extracellular Ca²⁺ is required for excitotoxicity, and the route of entry initiated by activation of non-NMDA, but not NMDA, receptors is L-type Ca²⁺ channels. Ca²⁺ imaging of motor neurons after application of specific glutamate receptor agonists reveals a sustained rise in intracellular Ca²⁺ that is present to a similar degree in most motor neurons, and can be blocked by appropriate receptor/channel antagonists. Although the lethal effects of glutamate receptor agonists are seen in only a subset of cultured motor neurons, the basis of this selectivity is unlikely to be simply the glutamate receptor phenotype or the level/pattern of rise in agonist-evoked intracellular Ca²⁺.     

Active Transport of Nicotine by the Isolated Choroid Plexus In Vitro

Abstract: In vitro , the transport of [¹⁴C]nicotine into the isolated choroid plexus, the anatomical locus of the blood–CSF barrier, was studied. The isolated rabbit choroid plexus accumulated [¹⁴C]nicotine by two processes: an active saturable transport process and a nonsaturable process. The [¹⁴C]nicotine accumulation process by choroid plexus was not due to binding or intracellular metabolism of the [¹⁴C]nicotine. The [¹⁴C]nicotine accumulation process in isolated choroid plexus was inhibited by weak bases, including tolazoline and lidocaine, but not by the weak acid probenecid. The accumulation process was decreased 60% by iodoacetate and dinitrophenol and by low temperatures. These results are consistent with previous autoradiographic evidence showing the choroid plexus concentrated [¹⁴C]nicotine in vivo , and suggest that the choroid plexus may transfer nicotine between blood and CSF in vivo .     

In Vitro Action of Carbenicillin Against Bacteria Isolated from Clinical Material

More than 500 bacteria isolated from patient material were tested against carbenicillin (disodium alpha-carboxybenzylpenicillin) by diffusion and dilution modalities. The same bacteria, which included Pseudomonas aeruginosa, Escherichia coli, Klebsiella-Aerobacter-Enterobacter group, various species of Proteus, Staphylococcus aureus and epiddermidis, enterococci, pneumococci, Streptococcus pyogenes, etc., were examined for susceptibility to other antibiotics commonly used with special emphasis on ampicillin and cephalothin. The responses of pyocine-typed P. aeruginosa were the most remarkable. The majority of these bacteria displayed susceptibility to carbenicillin by both the dilution and the diffusion techniques. The concentrations of this antibiotic used in the laboratory were of the same order of magnitude as that of the other drugs. The laboratory behavior of the other bacteria, toward this new semisynthetic penicillin derivative approximated their response to ampicillin and cephalothin.     

An Improved Medium For Plasmodium chabaudi In Vitro Erythrocyte Invasion Assays

ABSTRACT. RPMI-1640 is routinely used as the basal medium for the in vitro maintenance of malaria parasites. In this study we tested several commercially available nutritional media in a Plasmodium chabaudi chabaudi erythrocyte invasion assay and showed that three media, BME Basal Medium—modified, Dulbecco's Modified Eagle Medium, and William's Medium E, improved the level of merozoite invasion when compared with RPMI-1640. These media improve the rate of maturation of newly invaded rings to young trophozoites. Radioisotope incorporation by trophozoites maintained in these three media was also improved when compared to trophozoites maintained in RPMI-1640. BME Basal Medium—modified, or a combination of three parts BME Basal Medium—modified with one part William's Medium E, supported higher levels of erythrocyte invasion by merozoites. We suggest that either of these media replace the currently used RPMI-1640 for in vitro studies on P. c. chabaudi.     

FGF-2 Low Molecular Weight Selectively Promotes Neuritogenesis of Motor Neurons In Vitro

In this study, we screened in vitro the different capabilities of trophic factors with promising effect for enhancing selective regeneration and thus promoting specific reinnervation of target organs after peripheral nerve regeneration. We found that FGF-2 (18 kDa) was the trophic factor that exerted the most selective effect in promoting neurite outgrowth of spinal motoneurons both in terms of elongation and arborization. The mechanism underlying this effect on neuritogenesis seems related to FGF-2 enhancing the interaction between FGFR-1 and PSA-NCAM. The interaction of these two receptors is important during the early stages of neuritogenesis and pathfinding, while integrin alpha7B subunit seems to play a role during neurite stabilization.     

In Vivo Quantification of Peroxisome Tethering to Chloroplasts in Tobacco Epidermal Cells Using Optical Tweezers

Peroxisomes are highly motile organelles that display a range of motions within a short time frame. In static snapshots, they can be juxtaposed to chloroplasts, which has led to the hypothesis that they are physically interacting. Here, using optical tweezers, we tested the dynamic physical interaction in vivo. Using near-infrared optical tweezers combined with TIRF microscopy, we were able to trap peroxisomes and approximate the forces involved in chloroplast association in vivo in tobacco (Nicotiana tabacum) and observed weaker tethering to additional unknown structures within the cell. We show that chloroplasts and peroxisomes are physically tethered through peroxules, a poorly described structure in plant cells. We suggest that peroxules have a novel role in maintaining peroxisome-organelle interactions in the dynamic environment. This could be important for fatty acid mobilization and photorespiration through the interaction with oil bodies and chloroplasts, highlighting a fundamentally important role for organelle interactions for essential biochemistry and physiological processes.A combination of genetically encoded fluorescent probes, advances in light microscopy, and interdisciplinary approaches has revolutionized our understanding of organelle transport. Organelle movement in highly vacuolated leaf epidermal cells appears erratic, with individual organelles undergoing a range of movements within a relatively short time frame: they stop-go, change direction (trajectory), and move at varying speeds. The use of pharmacological inhibitors indicated a role for actin, and therefore myosins, in this process; however, myosin-organelle specificity is poorly characterized (Madison and Nebenführ, 2013; Tamura et al., 2013; Buchnik et al., 2015). Therefore, we are still at a relatively rudimentary stage in the understanding of the molecular and physical control, and interaction, of organelles in plant cells compared with that known in other model systems (Hammer and Sellers, 2012; Prinz, 2014). However, it is clear that organelle movement plays important roles in physological processes in plants; reduced movement effects growth and development, and movement is correlated with responses to extracellular stresses such as pathogens and heavy metals (for refs., see Sparkes, 2011; Madison and Nebenführ, 2013; Buchnik et al., 2015). Organelle interactions in other systems have important roles in calcium and lipid exchange, setting a precedent for physiologically important roles in plants (Prinz, 2014). However, characterization of the molecular factors required to physically tether organelles, as opposed to those that function in the exchange of molecules at the interaction site, is challenging. Monitoring organelle interactions in highly vacuolated plant epidermal cells is further complicated by the constraints imposed by the large central vacuole. Static snapshots provided through electron microscopy of highly vacuolated cells, where the vacuole can effectively push organelles together, giving the impression of direct interaction between organelles, is not a suitable method to determine dynamic interactions. Other techniques, such as the laser-induced shockwave by explosion method used by Oikawa et al. (2015), works globally without directly manipulating the individual organelle. Here, using optical tweezers with submicron precision, we provide a means to assess and quantify the dynamic interaction between peroxisomes and chloroplasts in vivo in leaf epidermal cells.Peroxisomes are responsible for several biochemical reactions, including the glyoxylate cycle and β-oxidation, which provides an energy source for germination in oilseeds. They also produce and scavenge free radicals, synthesize jasmonic acid and indole-3-acetic acid, and are required for photorespiration (for refs., see Hu et al., 2012). The photorespiratory pathway spans peroxisomes, chloroplasts, and mitochondria, where phosphoglycolate produced in the chloroplast is converted back to 3-phosphoglycerate. It has been suggested that functional connectivity between these organelles accounts for the close association observed in ultrastructural micrographs (Frederick and Newcomb, 1969). Several Arabidopsis (Arabidopsis thaliana) pex10 (peroxisomal membrane protein) mutants show altered chloroplast-peroxisome juxtaposition with a defect in photorespiration, while others do not (Schumann et al., 2007; Prestele et al., 2010). Both CLUMPED CHLOROPLASTS1 (CLMP1) and CHLOROPLAST UNUSUAL POSITIONING1 (CHUP1) encode for proteins that localize to the chloroplast, with CHUP1 playing a role in chloroplast-actin formation (Oikawa et al., 2003, 2008; Schmidt von Braun and Schleiff, 2008; Yang et al., 2011). While CHUP1 and CLMP1 affect chloroplast positioning, they have differential effects on peroxisome and mitochondrial location; clmp1 causes chloroplast clustering without affecting mitochondria or peroxisome location (Yang et al., 2011), whereas chup1 was reported to affect peroxisome location (Oikawa et al., 2003). In vitro analysis through density centrifugation highlighted chloroplast sedimentation with peroxisomes under certain conditions (Schnarrenberger and Burkhard, 1977), although this does not necessarily reflect the organelle interaction in live cells. Peroxisome proteomics studies have been hampered by difficulties in isolating pure peroxisomal fractions (Bussell et al., 2013). This could be indicative of interaction, where associated membranes are isolated together, or sticky nonspecific contaminating chloroplast membranes. The work by Oikawa et al. (2015) provides insight into the physiological processes controlling peroxisome-chloroplast interaction (photosynthesis dependent), but they did not determine the effective baseline force required to move peroxisomes that were not next to chloroplasts under control or altered environmental conditions. Comparisons between the relative forces required to move peroxisomes next to chloroplasts versus those that are not next to chloroplasts are critical in understanding and probing the physical interaction between the two organelles, the hypothesis being that tethering would increase the force required to move peroxisomes compared with organelles that are not tethered. Since peroxisomes have diverse biochemical roles that affect a wide range of physiological processes throughout the plant life cycle (Hu et al., 2012), an understanding of if and how peroxisomes may interact with other subcellular structures is likely to be an important consideration for efficient peroxisome function.Peroxisomes are highly pleomorphic, dynamic organelles bounded by a single membrane (Hu et al., 2012), whose movement is driven by acto-myosin-dependent processes (Jedd and Chua, 2002; Mano et al., 2002; Mathur et al., 2002; Avisar et al., 2008; Sparkes et al., 2008). Tubular emanations termed peroxules (Scott et al., 2007) can extend from the main peroxisome body, yet it is unclear what function they may play. Formation is quite frequent in hypocotyl cells (Cutler et al., 2000; Mano et al., 2002; Sinclair et al., 2009), can occur around chloroplasts in cotyledonary leaf pavement cells (Sinclair et al., 2009), and is not always from the trailing edge of the peroxisome (Sinclair et al., 2009). Exogenous addition of hydroxyl reactive oxygen species (ROS), or exposure to UV light, induces peroxule formation (Sinclair et al., 2009). It has been suggested that they represent an increased surface area for increased biochemical function or might represent a morphological precursor for peroxisome division (Jedd and Chua, 2002). Based on subcellular coalignment, a retro-flow model for the potential exchange of luminal content between the endoplasmic reticulum (ER) and peroxisome through the peroxule has been suggested (Sinclair et al., 2009; Barton et al., 2013). However, these studies, as with many others, interpret the close association between organelles to indicate physical connectivity between organelles, whereas, in fact, in highly vacuolated leaf epidermal cells, organelles can be closely packed within the cytoplasm due to mere spatial constrictions generated through the large central vacuole. This is further complicated by the highly motile, and seemingly stochastic, nature of acto-myosin-driven organelle movement, resulting in frequent apparent organelle collisions that may not reflect a functional requirement for organelle interaction.Optical trapping provides a highly specific and sensitive means to measure physical connectivity between organelles. By focusing an infrared beam, it allows the user to trap objects that have a significantly different refractive index from the surrounding medium. Upon trapping, the user can then move the trapped object relative to its original position to gain an understanding of whether the movement affects the position and motion of other structures (such as other organelles) that may be physically attached to the trapped organelle. For example, unlike the ER, Golgi bodies are amenable to trapping. By trapping and micromanipulating (i.e. precisely moving) the Golgi, a physical association between the ER and the Golgi was determined in a qualitative manner (Sparkes et al., 2009b). Here, we have developed a system to generate quantitative measures for organelle interaction by standardizing and automating how far we move the trapped organelle (which we call the translation step) at a defined speed and assessing how trapping efficiency alters in response to the power of the laser trap itself. By using these parameters, we can then model the forces imparted on the organelle, providing further insight into the tethering processes.Our results indicate that peroxisomes are amenable to being trapped, that they physically interact with chloroplasts in leaf epidermal cells, and, surprisingly, that peroxisomes are also tethered to other unknown structures within the cell. This approach highlights that organelle interactions within plant cells are not random but regulated through tethering. In addition, we provide a novel role for peroxules and a simple biophysical model to describe peroxisome motion during the trapping process.     

Calcitriol Imparts Neuroprotection In Vitro to Midbrain Dopaminergic Neurons by Upregulating GDNF Expression

During development a tightly controlled signaling cascade dictates the differentiation, maturation and survival of developing neurons. Understanding this signaling mechanism is important for developing therapies for neurodegenerative illnesses. In previous work we have sought to understand the complex signaling pathways responsible for the development of midbrain dopamine neurons using a proteomic approach. One protein we have identified as being expressed in developing midbrain tissue is the vitamin D receptor. Therefore we investigated the effect of the biologically active vitamin D₃ metabolite, calcitriol, on primary fetal ventral mesencephalic cultures of dopamine neurons. We observed a dose responsive increase in numbers of rat primary dopamine neurons when calcitriol was added to culture media. Western blot data showed that calcitriol upregulated the expression of glial derived neurotrophic factor (GDNF). Blocking GDNF signaling could prevent calcitriol’s ability to increase numbers of dopamine neurons. An apoptosis assay and cell birth dating experiment revealed that calcitriol increases the number of dopamine neurons through neuroprotection and not increased differentiation. This could have implications for future neuroprotective PD therapies.     

Muscarinic Agonists Cause Calcium Influx and Calcium Mobilization in Forebrain Neurons In Vitro

We have examined the effects of the muscarinic agonist carbachol on the intracellular free Ca2+ concentration ([Ca2+]i) in primary cultures of neurons from rat forebrain using the Ca2+-sensitive fluorescent dye fura-2. Addition of carbachol increased the [Ca2+]i in approximately 60% of cells studied. Oxotremorine-M, but not pilocarpine, mimicked the effects of carbachol. The response was reduced by 60% on removal of extracellular Ca2+, a finding suggesting that muscarinic receptor activation causes Ca2+ influx in addition to intracellular Ca2+ mobilization. Tetrodotoxin and nitrendipine also significantly reduced the response to carbachol. These studies suggest that the changes in [Ca2+]i produced by activation of muscarinic receptors result in part from mobilization of intracellular Ca2+ and that influx through voltage-sensitive Ca2+ channels also provides a significant contribution to the net [Ca2+]i change observed.     

In Vitro Nitrogen Fixation by Two Actinomycete Strains Isolated from Casuarina Nodules

Acetylene reduction activity was demonstrated in pure cultures of two actinomycete strains isolated from nodules of Casuarina equisetifolia. This activity was comparable to that of free-living Rhizobium strains, but appeared to be less sensitive to pO₂ and more sensitive to the presence of combined nitrogen.     

The Formation of Stromules In Vitro from Chloroplasts Isolated from Nicotiana benthamiana

Stromules are stroma-containing tubules that have been observed to emanate from the main plastidic body in vivo. These structures have been shown to require cytoskeletal components for movement. Though numerous studies have shown a close association with the endoplasmic reticulum, nucleus, mitochondria, and other plastids, the mechanism of formation and their overall function remain unknown. A limiting factor in studying these structures has been the lack of a reconstituted system for in vitro stromule formation. In this study, stromule formation was induced in vitro by adding a plant extract fraction that is greater than 100 kDa to a population of isolated chloroplasts. Kinetic measurements show that stromule formation occurs within ~10 seconds after the addition of the plant extract fraction. Heat inactivation and apyrase treatment reveal that the stromule stimulating compound found in the extract fraction is a protein or protein complex 100 kDa or greater. The formation of the stromules in vitro with isolated chloroplasts and a concentrated fraction of cell extract opens an avenue for the biochemical dissection of this process that has heretofore been studied only in vivo.