Skin flaps inhibit both the current of injury at the amputation surface and regeneration of that limb in newts

For over two decades, we have been investigating a strong (ca. 20-100 microA/cm2), outwardly directed electric current driven through the limb stump for the first few days following amputation in regenerating salamanders. This current is driven through the stump in a proximal/distal direction by the amiloride-sensitive transcutaneous voltage of the intact skin of the stump. Limb regeneration can be manipulated by several technique that manipulate this physiology, demonstrating that the ionic current is necessary, but not sufficient, for normal regeneration of the amphibian limb. Here, we demonstrate that a full thickness graft of skin covering the forelimb stump of newts strikingly inhibits the regeneration of the limb, and that this procedure is also highly correlated to a suppression of peak outwardly directed stump currents in those animals that fail to regenerate.     

Effects of amiloride analogues on adult Notophthalmus viridescens limb stump currents

We have previously investigated the relevance to limb regeneration of epidermally driven, Na+-dependent limb stump currents by blocking epidermal Na+ channels with amiloride, 3,5-diamino-6-chloro-N-(diaminomethylene)pyrazinecarboxamide. In order to reduce Notophthalmus viridescens stump currents more effectively than with amiloride, we have examined six amiloride analogues. Of these, only benzamil, 3,5-diamino-6-chloro-N-[(benzylamino)aminomethylene]pyrazinecarboxamide, was more effective than amiloride. The concentration of benzamil that reduced stump currents to half their initial value was 0.034 microM, while this concentration for amiloride was 0.42 microM. We also found a contribution of calcium ions to these currents. When immersed in water whose Ca2+ concentration decreased stepwise from 1 to 0 mM, stump currents decreased significantly, although to a variable extent, depending on the batch of newts. With 30 microM benzamil and 0.5 mM calcium (in water that also contained 1.5 mM NaCl and 0.06 mM KCl) stump currents could be reduced to very low levels and, in many cases, changed to incurrents.     

Salamander limb regeneration involves the activation of a multipotent skeletal muscle satellite cell population

In contrast to mammals, salamanders can regenerate complex structures after injury, including entire limbs. A central question is whether the generation of progenitor cells during limb regeneration and mammalian tissue repair occur via separate or overlapping mechanisms. Limb regeneration depends on the formation of a blastema, from which the new appendage develops. Dedifferentiation of stump tissues, such as skeletal muscle, precedes blastema formation, but it was not known whether dedifferentiation involves stem cell activation. We describe a multipotent Pax7⁺ satellite cell population located within the skeletal muscle of the salamander limb. We demonstrate that skeletal muscle dedifferentiation involves satellite cell activation and that these cells can contribute to new limb tissues. Activation of salamander satellite cells occurs in an analogous manner to how the mammalian myofiber mobilizes stem cells during skeletal muscle tissue repair. Thus, limb regeneration and mammalian tissue repair share common cellular and molecular programs. Our findings also identify satellite cells as potential targets in promoting mammalian blastema formation.     

Electric currents in Xenopus tadpole tail regeneration

Xenopus laevis tadpoles can regenerate tail, including spinal cord, after partial amputation, but lose this ability during a specific period around stage 45. They regain this ability after stage 45. What happens during this “refractory period” might hold the key to spinal cord regeneration. We hypothesize that electric currents at amputated stumps play significant roles in tail regeneration. We measured electric current at tail stumps following amputation at different developmental stages. Amputation induced large outward currents leaving the stump. In regenerating stumps of stage 40 tadpoles, a remarkable reversal of the current direction occurred around 12-24 h post-amputation, while non-regenerating stumps of stage 45 tadpole maintained outward currents. This reversal of electric current at tail stumps correlates with whether tails regenerate or not (regenerating stage 40—inward current; non-regenerating stage 45—outward current). Reduction of tail stump current using sodium-free solution decreased the rate of regeneration and percentage regeneration. Fin punch wounds healed normally at stages 45 and 48, and in sodium-free solution, suggesting that the absence of tail re-growth at stage 45 is regeneration-specific rather than a general inhibition of wound healing. These data suggest that electric signals might be one of the key players regulating regeneration.     

Effects of semiconductor substrate and glia-free culture on the development of voltage-dependent currents in rat striatal neurones

An essential requirement for successful long-term coupling between neuronal assemblies and semiconductor devices is that the neurones must be able to fully develop their electrogenic repertoire when growing on semiconductor (silicon) substrates. While it has for some time been known that neurones may be cultured on silicon wafers insulated with SiO2 and Si3N4, an electrophysiological characterisation of their development under such conditions is lacking. The development of voltage-dependent membrane currents, especially of the rapid sodium inward current underlying the action potential, is of particular importance because the conductance change during the action potential determines the quality of cell-semiconductor coupling. We have cultured rat striatal neurones on either glass coverslips or silicon wafers insulated with SiO2 and Si3N4 using both serum-containing and serum-free media. We here report evidence that not only serum-free culture media but also growth on semiconductor surfaces may negatively affect the development of voltage-dependent currents in neurones. Furthermore, using surface-charge measurements with the atomic force microscope, we demonstrate a reduced negativity of the semiconductor surface compared to glass. The reduced surface charge may affect cellular development through an effect on the binding and/or orientation of extracellular matrix proteins, such as laminin. Our findings therefore suggest that semiconductor substrates are not entirely equivalent to glass in terms of their effects on neuronal cell growth and differentiation.     

Relationship between innervation and forelimb regenerative capacity in the postmetamorphic pond frog Rana brevipoda porosa.

The limb regenerative capacity and the quantity of innervation (the percentage of a cross-sectional area of amputation forelimb stump occupied by nerves) in the pond frog, Rana brevipoda porosa, was investigated in postmetamorphic froglets and adults of various sizes by means of amputating forelimbs through the zeugopodium. Nearly all the amputated limbs of newly metamorphosed froglets, 18-19 mm in snout-vent length, showed heteromorphic regeneration. However, the larger the body size, the lower the presence of limb regeneration. Limb regenerative capacity was completely lost in froglets and adults with snout-vents larger than 35 mm. The quantity of innervation of limbs was highest in newly metamorphosed froglets, gradually decreasing with growth. The nerve quantity in adults with a snout-vent length between 60-67 mm was approximately half that of the froglets. When the nerve supply was augmented by deviating ipsilateral sciatic nerve bundles to the forelimb stump, almost all limbs, which were usually non-regenerative with normal innervation, regenerated heteromorphically. These results show that the decline in limb regenerative capacity during postmetamorphic growth is in part attributable to the reduction in innervation levels to below the threshold level required for regeneration.     

Generation of mononucleate cells from post-mitotic myotubes proceeds in the absence of cell cycle progression

The remarkable regenerative ability of adult urodele amphibians depends in part of the plasticity of differentiated cells at the site of injury. Limb regeneration proceeds by formation of a mesenchymal growth zone or blastema under the wound epidermis at the end of the stump. Previous work has shown that when cultured post-mitotic newt myotubes are introduced into the blastema, they re-enter the cell cycle and undergo conversion to mononucleate cells which divide and contribute to the regenerate [11, 13]. In order to investigate the interdependence of these two aspects of plasticity, we have blocked cell cycle progression of the myotubes either by X-irradiation or by transfection of the CDK4/6 inhibitor p16. In each case, the efficacy of the block was evaluated in culture after activation of S phase re-entry by serum stimulation. The experimental myotubes were implanted into limb blastemas along with a differentially labelled control population of myotubes containing an equivalent number of nuclei. X-irradiated myotubes gave rise to mononucleate cells in the limb blastema, and the progeny were blocked in respect of S phase entry. Comparable results were obtained with the p16-expressing myotubes. We conclude that progression through S or M phase is not required for generation of mononucleate cells and suggest that such cells may arise by budding from the muscle syncytium.     

On the Limited Potential of Azorean Fleshy Fruits for Oceanic Dispersal

How plants arrived to originally sterile oceanic islands has puzzled naturalists for centuries. Dispersal syndromes (i.e., diaspore traits that promote dispersal by long-distance dispersal vectors), are generally considered to play a determinant role in assisting island colonization. However, the association between diaspore traits and the potential vectors by which diaspores are dispersed is not always obvious. Fleshy fruits, in particular, are considered to have evolved to promote the internal dispersal of seeds by frugivores (endozoochory), however some fleshy fruits can also float in saltwater, and thus be potentially transported by oceanic current (thalassochory). We performed saltwater floatation and viability experiments with fruits of the 14 European fleshy-fruited species that naturally colonized the Azores archipelago (North Atlantic Ocean). We show that only Corema album (a berry) and Juniperus oxycedrus (a fleshy cone) floated for as long as 60 days, the estimated minimum time needed to reach the Azores by oceanic currents. Regardless the floatation potential, exposure to saltwater largely reduced the viability of most seeds of the 14 species (46% of viability decline within 15 days and 77% within 60 days of immersion), including those of Corema album (61%) and Juniperus oxycedrus (83%). Floatability and viability trials suggest that while some fleshy-fruited species might have arrived to the Azores by oceanic currents, such would have required extreme meteorological events that could largely reduce the duration of the trip. Thus, the alternative hypothesis that fleshy-fruited species were mostly dependent on animal dispersers (endozoochory) to colonize these remote islands is reinforced.     

Rapid sodium cyanide depletion in cell culture media: outgassing of hydrogen cyanide at physiological pH

During the course of in vitro studies on cyanide exposure with SH-SY5Y human neuroblastoma cells, we found that sodium cyanide (NaCN) up to a concentration of 10 mM had no significant toxic effect under our culture conditions. Further investigation of this apparent cyanide resistance revealed that the sodium cyanide was being rapidly depleted from the cell culture medium. Cyanide was interacting with constituents of the cell culture medium and was somehow being detoxified or removed from solution. The reaction of cyanide with cell culture media in 96-well culture plates reduced cyanide concentrations rapidly (80-90% in 2 h at 37 degrees C). Running the same reaction in capped tubes significantly reduced cyanide loss from solution. Incubation of cyanide with individual constituents of the cell culture medium in solution showed that glucose, phenol red, and amino acids all acted to detoxify or remove cyanide from solution. When amino acids or buffers were incubated with sodium cyanide in aqueous solution at pH 7.4, hydrogen cyanide (HCN) was found to degas from the solutions. We compared HCN outgassing over a range of pH values. As expected, HCN remained very soluble at high pH, but as the pH was reduced to 7.0, the rate of HCN formation and outgassing increased dramatically. Acid-base reactions involving cyanide and proton donors, such as amino acids and other cell culture media constituents, at physiological pH result in rapid HCN outgassing from solution at 37 degrees C. These results indicate that previous in vitro cyanide toxicity studies done in standard culture media with prolonged incubation times using gas-exchanging culture containers might have to be reevaluated in light of the fact that the effective cyanide concentrations in the culture media were significantly lower than reported.     

增效混剂对神经细胞钠通道的抑制作用

应用膜片钳技术,以MN-9D神经细胞为材料研究了溴氰菊酯及辛硫磷混剂的增效机理。膜片钳实验表明10^-5mol/L辛硫磷对Na⁺通道电流抑制作用很小,并随作用时间延长而逐步恢复。加药1 min Na⁺电流抑制率为6.99%,10 min为3.65%。10^-6 mol/L溴氰菊酯1 min抑制率为20.28%,10 min为21.43%。对蜚蠊中枢神经系统传导的动作电位抑制中时为53 min;10^-6mol/L溴氰菊酯与10^-5 mol/L辛硫磷混剂1 min抑制率为34.15%,10 min为36.69%,动作电位抑制中时为40 min,因此混剂可增强对Na⁺通道电流的抑制作用。通过Na⁺电流数据、尾电流衰减时间常数统计分析表明溴氰菊酯的修饰作用主要发生在关闭和静止状态的Na⁺通道,减缓通道的打开,延长通道关闭或失活状态。     

Growth and fat formation of Aspergillus oryzae and Aspergillus terreus on enzyme-hydrolyzed sweet potatoes

Two fungi, namely,Aspergillus oryzae andAspergillus terreus, have been grown on enzyme-hydrolyzed sweet potato medium either as such or supplemented with an external source of nitrogen in the form of ammonium carbonate, sodium nitrate, or asparagine. Hydrolysis alone promoted carbohydrate uptake, growth as well as fat formation to a remarkable extent in both fungi.Addition of an external supply of nitrogen to the hydrolyzed media increased further carbohydrate absorption which continued until the potato media were almost completely depleted; on unhydrolysed potato media, appreciable amounts of carbohydrates always remained unutilized. It also accelerated the rate of building up so that the dry mycelium increased by more than 4 times in some cases. Fat formation was consequently remarkably enhanced specially in the presence of ammonium carbonate or asparagine.     

Human Pluripotent Stem Cell-Derived Cardiomyocytes: Response to TTX and Lidocain Reveals Strong Cell to Cell Variability

Stem cell derived cardiomyocytes generated either from human embryonic stem cells (hESC-CMs) or human induced pluripotent stem cells (hiPSC-CMs) hold great promise for the investigation of early developmental processes in human cardiomyogenesis and future cell replacement strategies. We have analyzed electrophysiological properties of hESC-CMs (HES2) and hiPSC-CMs, derived from reprogrammed adult foreskin fibroblasts that have previously been found to be highly similar in terms of gene expression. In contrast to the similarity found in the expression profile we found substantial differences in action potentials (APs) and sodium currents at late stage (day 60) of in vitro differentiation with higher sodium currents in hiPSC-CMs. Sensitivity to lidocain was considerably reduced in hESC-CMs as compared to hiPSC-CMs, and the effect could not be explained by differences in beating frequency. In contrast, sensitivity to tetrodotoxin (TTX) was higher in hESC-CMs suggesting different contributions of TTX-sensitive and TTX-resistant sodium channels to AP generation. These data point to physiological differences that are not necessarily detected by genomics. We conclude that novel pharmacological screening-assays using hiPSC-CMs need to be applied with some caution.     

Effect of immersion solutions containing enterocin AS-48 on Listeria monocytogenes in vegetable foods

The effect of immersion solutions containing enterocin AS-48 alone or in combination with chemical preservatives on survival and proliferation of Listeria monocytogenes CECT 4032 inoculated on fresh alfalfa sprouts, soybean sprouts, and green asparagus was tested. Immersion treatments (5 min at room temperature) with AS-48 solutions (25 microg/ml) reduced listeria counts of artificially contaminated alfalfa and soybean sprouts by approximately 2.0 to 2.4 log CFU/g compared to a control immersion treatment in distilled water. The same bacteriocin immersion treatment applied on green asparagus had a very limited effect. During storage of vegetable samples treated with immersion solutions of 12.5 and 25 microg of AS-48/ml, viable listeria counts were reduced below detection limits at days 1 to 7 for alfalfa and soybean sprouts at 6 and 15 degrees C, as well as green asparagus at 15 degrees C. Only a limited inhibition of listeria proliferation was detected during storage of bacteriocin-treated alfalfa sprouts and green asparagus at 22 degrees C. Treatment with solutions containing AS-48 plus lactic acid, sodium lactate, sodium nitrite, sodium nitrate, trisodium phosphate, trisodium trimetaphosphate, sodium thiosulphate, n-propyl p-hydroxybenzoate, p-hydoxybenzoic acid methyl ester, hexadecylpyridinium chloride, peracetic acid, or sodium hypochlorite reduced viable counts of listeria below detection limits (by approximately 2.6 to 2.7 log CFU/g) upon application of the immersion treatment and/or further storage for 24 h, depending of the chemical preservative concentration. Significant increases of antimicrobial activity were also detected for AS-48 plus potassium permanganate and in some combinations with acetic acid, citric acid, sodium propionate, and potassium sorbate.     

The failure of double-half forelimbs to undergo distal transformation following amputation in the axolotl, Ambystoma mexicanum

Although capable of initiating early regenerative responses, axolotl forelimb stumps which are composed of double-half limb tissues fail to undergo the events that normally lead to the replacement of missing parts. In the present study, the posterior halves of right forelimbs were exchanged with the anterior halves of left forelimbs, or the dorsal halves of right forelimbs were exchanged with the ventral halves of left forelimbs. Forelimbs were amputated through the graft region 30 days after grafting. Limb stumps bearing double-dorsal, double-ventral or double-posterior tissues either produced hypomorphic regenerates or failed to form any externally visible outgrowth. When the limb stump bore double-anterior tissues, no externally visible structures were formed. Normal and multiple regenerates were never formed by double-half limbs. These results are discussed in terms of the polar coordinate model and suggest that the regeneration blastema requires a complete circumference of positional values in order to complete distal transformation.     

Long-term changes in neurotrophic factor expression in distal nerve stump following denervation and reinnervation with motor or sensory nerve

Several factors have been proposed to account for poor motor recovery after prolonged denervation, including motor neuron cell death and incomplete or poor regeneration of motor fibers into the muscle. Both may result from failure of the muscle and the distal motor nerve stump to continue expression of neurotrophic factors following delayed muscle reinnervation. This study investigated whether regenerating motor or sensory axons modulate distal nerve neurotrophic factor expression. We found that transected distal tibial nerve up-regulated brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) mRNA, down-regulated neurotrophin-3 and ciliary neurotrophic factor mRNA, and that although these levels returned to normal with regeneration, the chronically denervated distal nerve stump continued to express these neurotrophic factors for at least 6 months following injury. A sensory nerve (the cutaneous saphenous nerve) sutured to distal tibial nerve lowered injury-induced BDNF and GDNF mRNA levels in distal stump, but repair with a mixed nerve (peroneal, containing muscle and cutaneous axons) was more effective. Repair with sensory or mixed nerves did not affect nerve growth factor or neurotrophin-3 expression. Thus, distal nerve contributed to a neurotrophic environment for nerve regeneration for at least 6 months, and sensory nerve repair helped normalize distal nerve neurotrophic factor mRNA expression following denervation. Furthermore, as BDNF and GDNF levels in distal stump increased following denervation and returned to control levels following reinnervation, their levels serve as markers for the status of regeneration by either motor or sensory nerve.     

Voltage-dependent membrane capacitance in rat pituitary nerve terminals due to gating currents

We investigated the voltage dependence of membrane capacitance of pituitary nerve terminals in the whole-terminal patch-clamp configuration using a lock-in amplifier. Under conditions where secretion was abolished and voltage-gated channels were blocked or completely inactivated, changes in membrane potential still produced capacitance changes. In terminals with significant sodium currents, the membrane capacitance showed a bell-shaped dependence on membrane potential with a peak at approximately -40 mV as expected for sodium channel gating currents. The voltage-dependent part of the capacitance showed a strong correlation with the amplitude of voltage-gated Na+ currents and was markedly reduced by dibucaine, which blocks sodium channel current and gating charge movement. The frequency dependence of the voltage-dependent capacitance was consistent with sodium channel kinetics. This is the first demonstration of sodium channel gating currents in single pituitary nerve terminals. The gating currents lead to a voltage- and frequency-dependent capacitance, which can be well resolved by measurements with a lock-in amplifier. The properties of the gating currents are in excellent agreement with the properties of ionic Na+ currents of pituitary nerve terminals.     

Effect of Immersion Solutions Containing Enterocin AS-48 on Listeria monocytogenes in Vegetable Foods

The effect of immersion solutions containing enterocin AS-48 alone or in combination with chemical preservatives on survival and proliferation of Listeria monocytogenes CECT 4032 inoculated on fresh alfalfa sprouts, soybean sprouts, and green asparagus was tested. Immersion treatments (5 min at room temperature) with AS-48 solutions (25 μg/ml) reduced listeria counts of artificially contaminated alfalfa and soybean sprouts by approximately 2.0 to 2.4 log CFU/g compared to a control immersion treatment in distilled water. The same bacteriocin immersion treatment applied on green asparagus had a very limited effect. During storage of vegetable samples treated with immersion solutions of 12.5 and 25 μg of AS-48/ml, viable listeria counts were reduced below detection limits at days 1 to 7 for alfalfa and soybean sprouts at 6 and 15°C, as well as green asparagus at 15°C. Only a limited inhibition of listeria proliferation was detected during storage of bacteriocin-treated alfalfa sprouts and green asparagus at 22°C. Treatment with solutions containing AS-48 plus lactic acid, sodium lactate, sodium nitrite, sodium nitrate, trisodium phosphate, trisodium trimetaphosphate, sodium thiosulphate, n-propyl p-hydroxybenzoate, p-hydoxybenzoic acid methyl ester, hexadecylpyridinium chloride, peracetic acid, or sodium hypochlorite reduced viable counts of listeria below detection limits (by approximately 2.6 to 2.7 log CFU/g) upon application of the immersion treatment and/or further storage for 24 h, depending of the chemical preservative concentration. Significant increases of antimicrobial activity were also detected for AS-48 plus potassium permanganate and in some combinations with acetic acid, citric acid, sodium propionate, and potassium sorbate.     

The cellular and molecular basis of peripheral nerve regeneration

Functional recovery from peripheral nerve injury and repair depends on a multitude of factors, both intrinsic and extrinsic to neurons. Neuronal survival after axotomy is a prerequisite for regeneration and is facilitated by an array of trophic factors from multiple sources, including neurotrophins, neuropoietic cytokines, insulin-like growth factors (IGFs), and glial-cell-line-derived neurotrophic factors (GDNFs). Axotomized neurons must switch from a transmitting mode to a growth mode and express growth-associated proteins, such as GAP-43, tubulin, and actin, as well as an array of novel neuropeptides and cytokines, all of which have the potential to promote axonal regeneration. Axonal sprouts must reach the distal nerve stump at a time when its growth support is optimal. Schwann cells in the distal stump undergo proliferation and phenotypical changes to prepare the local environment to be favorable for axonal regeneration. Schwann cells play an indispensable role in promoting regeneration by increasing their synthesis of surface cell adhesion molecules (CAMs), such asN-CAM, Ng-CAM/L1, N-cadherin, and L2/HNK-1, by elaborating basement membrane that contains many extracellular matrix proteins, such as laminin, fibronectin, and tenascin, and by producing many neurotrophic factors and their receptors. However, the growth support provided by the distal nerve stump and the capacity of the axotomized neurons to regenerate axons may not be sustained indefinitely. Axonal regeneration may be facilitated by new strategies that enhance the growth potential of neurons and optimize the growth support of the distal nerve stump in combination with prompt nerve repair.     

Regeneration in starved planarians depends on TRiC/CCT subunits modulating the unfolded protein response

Planarians are able to stand long periods of starvation by maintaining adult stem cell pools and regenerative capacity. The molecular pathways that are needed for the maintenance of regeneration during starvation are not known. Here, we show that down‐regulation of chaperonin TRiC/CCT subunits abrogates the regeneration capacity of planarians during starvation, but TRiC/CCT subunits are dispensable for regeneration in fed planarians. Under starvation, they are required to maintain mitotic fidelity and for blastema formation. We show that TRiC subunits modulate the unfolded protein response (UPR) and are required to maintain ATP levels in starved planarians. Regenerative defects in starved CCT‐depleted planarians can be rescued by either chemical induction of mild endoplasmic reticulum stress, which leads to induction of the UPR, or by the supplementation of fatty acids. Together, these results indicate that CCT‐dependent UPR induction promotes regeneration of planarians under food restriction.     

Simultaneous modifications of sodium channel gating by two scorpion toxins.

The effects of purified scorpion toxins from two different species on the kinetics of sodium currents were evaluated in amphibian myelinated nerves under voltage clamp. A toxin from Leiurus quinquestriatus slowed and prevented sodium channel inactivation, exclusively, and a toxin from Centruroides sculpturatus Ewing reduced transient sodium currents during a maintained depolarization, and induced a novel inward current that appeared following repolarization, as previously reported by Cahalan (1975, J. Physiol. [Lond.]. 244:511-534) for the crude scorpion venom. Both of these effects were observed in fibers treated with both of these toxins, and the kinetics of the induced current were modified in a way that showed that the same sodium channels were modified simultaneously by both toxins. Although the toxins can act on different sites, the time course of the action of C. sculpturatus toxin was accelerated in the presence of the L. quinquestriatus toxin, indicating some form of interaction between the two toxin binding sites.