Electroporation as a technique for the transfer of macromolecules into mammalian cell lines

Summary Numerous methods have been devised to facilitate the introduction of exogenous compounds into cells. The technique of electroporation allows the direct physical transfer of numerous kinds of molecules into essentially any cell, but the major application has been for transfection of DNA and this emphasis is recapitulated here. However, the conditions for transfer of DNA or other macromolecules are sufficiently similar that the same protocol is followed regardless. In addition, as electroporation involves a mechanism distinct from that of most other methods of transfection, it has distinct advantages and disadvantages relative to other transfection techniques. This review is designed to allow one to simplify the processes of determining whether electroporation is appropriate to a given experimental design, to indicate how to minimize the disadvantages, and to simplify the requisite process of parameter optimization required to evaluate and apply electroporation to the system of choice. Practical aspects are highlighted but the theoretical bases are discussed when relevant for application of the technique.     

Ethanol modulates the ionic permeability of sodium channels in rat sensory neurons

The effects of ethanol on tetrodotoxin-sensitive (TTX_s) and tetrodotoxin-resistant (TTX_r) sodium channels in rat spinal ganglia were studied using a patch-clamp method. Application of ethanol (10 and 100 mM) to both sides of membranes resulted in decreases in the reversion potentials of both types of sodium channels. In the case of TTX_r channels, ethanol decreased their selectivity in relation to Na ions and altered the sequence of ion selectivity of these channels for different cations from row X to row XI of the Eisenman selectivity classification. It is suggested that this change in ion selectivity is associated with ethanol-induced disruption of hydrogen bonds which stabilize the spatial structure of ion channel macromolecules, whith may lead to changes in the steric parameters of the pores formed by these channels. Translated from Rossiskii Fiziologisheskii Zhurnal imeni I. M. Sechenova, Vol. 85, No. 1, pp. 110–118, January, 1999.     

Regional and temporal expression of sodium channel messenger RNAs in the rat brain during development

Summary The distribution of mRNA expression for three types of voltage gated neuronal sodium-channels was studied in the rat brain at different developmental stages (embryonal day E18, postnatal day P5 and adult). With the in-situ hybridization technique, using synthetic DNA-oligomer probes, pronounced regional and temporal variations in the expression levels of the different channel subtypes could be detected. In comparison with types I and III, sodium channel II mRNA was the most abundant subtype at all developmental stages. Maximal expression of sodium channel II mRNA was seen at P5 in virtually all parts of the grey matter, except for the cerebellum. In adult rat brain in contrast, sodium channel II mRNA levels were maximal in the granular layer of the cerebellum, whereas in all other regions expression had decreased to roughly 50% of postnatal levels. Na channel I expression was virtually absent at E18 and showed highest levels at P5, with maxima in the caudate nucleus and hippocampus. In the adult brain, expression of Na-channel I was nearly absent in the neocortex, but well detectable in the cerebellum and, at lower levels in the striatum and thalamus. Sodium channel III was mainly expressed at the embryonal stage and showed a decrease to very low levels with little regional preferences in the adult.Supported by Deutsche Forschungsgemeinschaft grant no.: Cr 30/16     

Functional expression of a large-conductance Ca-activated K channel in mouse substantia nigra pars compacta dopaminergic neurons

The existence of large-conductance Ca²⁺-activated K⁺ (BK) channels in substantia nigra pars compacta (SNc) has been a matter of debate. Using the patch-clamp technique in the inside-out configuration, we have recorded BK channel currents in SNc dopaminergic neurons. The channel has a conductance of 301 pS with a slight inward rectification and is both voltage- and calcium-dependent. Paxilline, a specific BK channel blocker, can completely block the channel, while tetraethylammonium (TEA), a nonspecific blocker of voltage-gated potassium channels, reduces its conductance and a high concentration of TEA (30 mM) inhibits its activity. ATP and GTP reduce the channel activity, while ADP is less potent, and AMP has no effect. The channel is also sensitive to changes in intracellular pH. Our results indicate that functional BK channels are expressed in SNc and suggest the possibility that the BK channel may be involved in the response of SNc dopaminergic neurons to metabolic stress.     

The amygdala of the common shrew,guinea pig,rabbit, fox and pig: five flavours of the mammalian amygdala as a consequence of clade-specific mosaic-like evolution

The amygdala is a part of neural networks that contribute to the regulation of emotional behaviours and emotional learning, stress response, and olfactory, pheromonal and reproductive functions. All these various functions are processed by the three main functional systems, frontotemporal, autonomic and olfactory, which are derived from different telencephalic sources (claustrum, striatum and olfactory cortex) and are represented, respectively, by the basolateral complex (BLC), the central complex (CC) and corticomedial complex (CMC) of the amygdala. The question arises of how these three functional systems evolved during mammalian phylogeny to fit the amygdala to specific needs of various animals. In the present study, we provide quantitative information regarding the individual volumes and neuron numbers in the BLC, CC and CMC of the common shrew, guinea pig, rabbit, fox and pig, a series of animals arranged according to increasing size and complexity of the brain. The results show that, in this series of animals, the BLC underwent a gradual size increase in volume and number of neurons, whereas the CMC was gradually reduced with regard to both these measures. The CC was more or less conserved across studied species. For example, the volume of the amygdala in pigs is ~250 times larger than that in shrews and it also has almost 26 times as many neurons as the amygdala of shrews. However, the volumes of the BLC, CC and CMC were ~380, 208 and 148 times larger, respectively, in pigs than in shrews. The number of neurons in these three regions was ~38, 23 and 20 times greater, respectively, in pigs than in shrews. The results also show striking morphometric similarities of the amygdala in the guinea pig and rabbit as well as fox and pig. For example, the percentages of neurons in the fox and pig are 42.23% and 42.78%, respectively, for the BLC, 16.64% and 16.58%, respectively, for the CC, and 41.12% and 40.64%, respectively, for the CMC. In conclusion, our results indicate that the amygdala does not evolve as a single unit but, instead, the three main functional systems evolved independently, which suggests that brain structures with major functional links evolve together independently of evolutionary changes in other unrelated structures. The size progression of the BLC parallels the size progression of the neocortex with which it is strongly functionally linked, whereas the CMC is strongly connected to olfactory regions, and all these structures follow the same regression course. Remarkable morphometric similarity of the amygdala in the guinea pig and rabbit as well as in the fox and pig, however, suggest that there must also be another mechanism shaping the morphology of the amygdala and the brain during evolution. The gradual nature of size changes in the BLC and CMC support this hypothesis as well.     

Functional expression and properties of the human skeletal muscle sodium channel

Full-length deoxyribonucleic acid, complementary (cDNA) constructs encoding the-subunit of the adult human skeletal muscle Na⁺ channel, hSkM1, were prepared. Functional expression was studied by electrophysiological recordings from cRNA-injectedXenopus oocytes and from transiently transfected tsA201 cells. The Na⁺ currents of hSkM1 had abnormally slow inactivation kinetics in oocytes, but relatively normal kinetics when expressed in the mammalian cell line. The inactivation kinetics of Na⁺ currents in oocytes, during a depolarization, were fitted by a weighted sum of two decaying exponentials. The time constant of the fast component was comparable to that of the single component observed in mammalian cells. The block of hSkM1 Na⁺ currents by the extracellular toxins tetrodotoxin (TTX) and -conotoxin (CTX) was measured. The IC₅₀ values were 25 nM (TTX) and 1.2 M (CTX) in oocytes. The potency of TTX is similar to that observed for the rat homolog rSkM1, but the potency of CTX is 22-fold lower in hSkM1, primarily due to a higher rate of toxin dissociation in hSkM1. Single-channel recordings were obtained from outside-out patches of oocytes expressing hSkM1. The single-channel conductance, 24.9 pS, is similar to that observed for rSkM1 expressed in oocytes.     

Expression of the American cockroach Per a 1 allergen in mammalian cells

BACKGROUND: Cockroach allergens are one of the major etiologic risk factors for developing IgE-mediated allergic respiratory illness throughout the world. Per a 1 is a cross-reactive allergen of American and German cockroaches. This study aimed to investigate the expression of a recombinant American cockroach (Periplaneta americana) Per a 1, C42, allergen in mammalian COS-1 cells. METHODS: The COS-1 cells and Escherichia coli were used to express the P. americana C42 allergen. Recombinant proteins were purified with hydroxylapatite and DE52 chromatography. Biologic reactivities of recombinant proteins were examined by direct IgE binding and IgE inhibition studies with the enzyme-linked immunosorbent assay (ELISA). RESULTS: C42 was successfully expressed in the mammalian COS-1 cell as a 50-kDa secreted protein, and purified from the culture medium. The specific human IgE antibodies against recombinant C42 from either E. coli (C42-E. coli) or COS-1 (C42-COS-1) were compared by ELISA with 12 sera from Per a 1 and C42 skin-test-positive patients. All atopic sera contained specific IgE antibodies to C42 from either E. coli or COS-1. Moreover, recombinant C42-COS-1 bound higher levels of serum IgE than recombinant C42-E. coli among C42-sensitive atopic patients, and a statistically significant difference (P<0.01) was found between them. In addition, recombinant C42-COS-1 as an inhibitor revealed higher inhibition of IgE binding to natural Per a 1 than recombinant C42-E. coli. CONCLUSIONS: The biologically highly reactive recombinant C42 produced in the COS-1 cell provides an alternative expression system and will facilitate studies on the immune response of asthma patients to cockroach allergens.     

Effects of static magnetic fields on the voltage-gated potassium channel currents in trigeminal root ganglion neurons

To evaluated the effects of moderate-intensity static magnetic fields (SMF) on two types of voltage-gated potassium channel (VGPC) currents: I(K,A) and I(K,V), whole-cell patch-clamp experiments were conducted on acute dissociated rat trigeminal root ganglion (TRG) neurons. The results demonstrated that 125 mT SMF could influence the inactivation kinetics of these two VGPC currents by altering the inactivation rate and velocity. No significant change was observed in the activation properties. These findings supported the hypothesis that biological membrane would be deformed in moderate-intensity SMF and the physiological characteristics of ion channels on the membrane would be influenced. The mechanism underlying the different effects of SMF on the I(K,A) and I(K,V) inactivation was also discussed.     

From virus to vaccine: recombinant mammalian cell lines as substrates for the production of herpes simplex virus vaccines

The use of mammalian cells as substrates for the production of various vaccines is a time-honored procedure. With the advent of recombinant DNA technology, various investigators soon realized that these techniques could be applied to mammalian cells to convert them to reliable, safe producers of a variety of vaccines. While none of these types of recombinant vaccines have, as yet, been widely utilized, many individuals believe that these technologies are the wave of the future in vaccine production. In this paper our work on the use of mammalian cells for the development of subunit vaccines for the prevention of infection by herpes simplex virus (HSV) types 1 and 2 is reviewed. A surface glycoprotein of these viruses, gD, was chosen as a likely candidate for a subunit vaccine based upon its primary sequence conservation between the two viral types as well as upon data suggesting that antibodies against this glycoprotein were highly efficient at neutralizing virus infectivity. Stable mammalian cell lines were constructed that expressed secreted forms of this protein, and the protein could be purified from cell-conditioned supernatants to near-homogeneity. Vaccination of mice with this glycoprotein was found to protect them from a lethal intraperitoneal infection by either the type 1 or type 2 forms of this virus. In a more realistic study, guinea pigs vaccinated with either the type 1 or type 2 form of gD were found to be protected from a genital form of HSV 2 infection.(ABSTRACT TRUNCATED AT 250 WORDS)     

分泌性腹泻对小鼠钙激活Cl~-通道基因表达的影响

目的探讨肠道组织钙激活Cl~-通道(CLCAs)基因表达与分泌性腹泻发生的关系。方法选取昆明小鼠24只,雌雄各半,随机分为3组:对照组、实验1h和8h组,每组8只。对照组小鼠经腹腔注射0.2ml生理盐水,实验组小鼠经腹腔注射脂多糖(LPS,6mg/kg)分别作用1h和8h,于注射后观察小鼠活动特征及肠道组织形态,以判定分泌性腹泻模型的建立,利用实时光定量PCR法检测各段肠道组织CLCAs基因的表达。结果 LPS成功诱导小鼠发生了分泌性腹泻;小鼠十二指肠、空肠、回肠和结肠中均有CLCAs基因的转录;注射LPS后,十二指肠、空肠、回肠和结肠的CLCA1、CLCA2、CLCA3、CLCA4、CLCA6基因转录水平均发生上调,与1h组相比,8h组各基因转录水平显著上调。注射LPS后,十二指肠未检测到CLCA5的表达,但空肠、回肠CLCA5的表达均上调。结论 LPS引起的分泌性腹泻与各肠段CLCAs基因的差异表达有关。     

Effects of divalent cations on the activation of a calcium-dependent potassium channel in hippocampal neurons

The activation of a calcium-dependent K⁺ channel K(Ca) in cultured hippocampal neurons has been studied after the addition, to the internal solution, of the divalent cations magnesium (Mg²⁺), strontium (Sr²⁺) or barium (Ba²⁺). With physiological K⁺ across inside-out patches and Ca²⁺ present at 0.2 mM in the bath solution, a 90-pS channel was activated with an open probability in excess of 75%. When the internal Ca²⁺ was reduced to levels near 5 M, the channel-open probability was significantly diminished. However, if 0.2 mM concentrations of either Mg²⁺ or Sr²⁺ were added to the internal solution, the open probability was increased to a value close to original level. In the presence of internal Ca²⁺ at 0.1 M, the K(Ca) channel was not active and was not activated with the addition of 0.2 mM Mg²⁺ or Sr²⁺ to the internal solution. Thus, Mg²⁺ or Sr²⁺ were not able to active K(Ca) in the absence of Ca²⁺; however, both of these divalent cations could potentiate the Ca²⁺-induced activation of K(Ca) if internal Ca²⁺ was near 5 M. The results indicate that Mg²⁺ could have a role as an internal modulator of K(Ca) in the Ca²⁺-dependent regulation of excitability in nerve membrane. Replacement of Ca²⁺ with Ba²⁺, or addition of Ba²⁺ to Ca-containing solutions, caused significant decreases in the channel-open probability for K(Ca). The action of Ba²⁺ was primarily mediated by a decrease in the frequency of channel opening. At a concentration of 5 M, Ba²⁺ diminished the channel-open probability by one-half.     

CD63 expression on basophils as a tool for the diagnosis of pollen-associated food allergy: sensitivity and specificity

BACKGROUND: Basophil activation is associated with the expression of CD63. Because allergens can induce basophil activation by cross-linking specific IgE, increased CD63 expression has been proposed as a novel in vitro test for immediate type allergy. OBJECTIVE: We compared the CD63-based basophil activation test (BAT) in the diagnosis of allergy to carrot, celery and hazelnut with skin prick tests (SPT) and measurement of allergen-specific IgE. METHODS: Twenty-nine patients with a history of an oral allergy syndrome induced by carrot, celery or hazelnut (n = 20 for each allergen) and 20 controls were studied. SPT were performed with standardized and native carrot, celery and hazelnut extracts. Allergen-specific IgE was determined by the CAP FEIA method and basophil activation was determined by flow cytometry upon double staining with anti-IgE/anti-CD63 mAb. RESULTS: SPT with native carrot, celery and hazelnut showed sensitivities of 100%, 100% and 90%, and specificities of 80%, 80% and 90%. SPT with commercial extracts of the same allergens gave sensitivities of 85%, 80% and 85%, and specificities of 80%, 80% and 90%. Sensitivity of allergen-specific IgE and the BAT for carrot, celery and hazelnut was 80% vs. 85%, 70% vs. 85%, and 80% vs. 90%, with corresponding specificities of 80% vs. 85%, 80% vs. 80%, and 95% vs. 90%. The cut-off for a positive BAT was 10% CD63+ basophils. Moreover, there was a positive correlation between IgE reactivity and the number of CD63+ basophils for all food allergens (carrot: r = 0.69, celery: r = 0.67, hazelnut: r = 0.66). CONCLUSIONS: Quantification of basophil activation by CD63 expression is a valuable new in vitro method for diagnosis of immediate type food sensitization. Although double-blind placebo-controlled food challenges remain the gold standard, the CD63-based BAT may supplement routine diagnostic tests such as SPT or allergen-specific IgE in the future.     

手阳明大肠经和手厥阴心包经相关的脊髓前角运动神经元投射支配特性

目的绘制支配手阳明大肠经和手厥阴心包经相关的脊髓运动神经元在脊髓的空间定位和树突投射的特异性分布。方法利用霍乱毒素B单位结合辣根过氧化物酶(CB-HRP)可被外周神经末梢吸收和运输的特性,在大鼠前肢手阳明大肠经和手厥阴心包经路径上的肌肉的每一穴位点分别注射5μL的CB-HRP(1%)溶液,经过3~4 d的药物吸收后,通过灌流固定-切片-TMB组织化学反应程序,显示出支配这些肌肉的运动神经元胞体及突起,并观察手阳明大肠经和手厥阴心包经相关的运动神经元在脊髓的空间的分布特性。结果大鼠手阳明大肠经以及手厥阴心包经相关的运动神经元在颈3-颈7节段脊髓腹角分别形成首尾走向、定位明确的运动神经元柱状联结;同一条经对应的运动神经元之间存在丰富的树突-树突和树突-胞体定向投射,形成一个由运动神经元胞体及其树突构成的细胞柱状实体。结论同经相关的运动神经元之间构成一个紧密连接的神经元柱,柱间的神经元的树突-树突和树突-胞体定向投射可能是循经感传反射活动的结构基础。     

Proton binding sites involved in the activation of acid-sensing ion channel ASIC2a

Most acid-sensing ion channel (ASIC) subunits are activated by protons, but ASIC2b (a splice variant of ASIC2a) is acid-insensitive. Differences in protonatable residues between the extracellular loop regions of ASIC2a and ASIC2b may explain this difference. Site-directed mutagenesis, combined with immunocytochemistry and whole-cell patch clamp, demonstrated that mutating any one of five ASIC2a sites produces channels that traffic normally to the cell surface membrane but are insensitive to protons. One of the mutants forms functional heteromers with ASIC1a and ASIC2a, demonstrating that ion transport is intact in this mutant. These five sites may be involved in the activation of ASIC2a by protons.     

Linkage of malignant hyperthermia and hyperkalemic periodic paralysis to the adult skeletal muscle sodium channel (SCN4A) gene in a large pedigree

Hyperkalemic periodic paralysis (HPP) is caused by mutations of the adult skeletal muscle sodium channel (SCN4A) gene on chromosome 17. Malignant hyperthermia (MH) is a genetically heterogeneous autosomal-dominant disorder occurring in association with various neuromuscular diseases or without other apparent abnormalities. In some families, MH is associated with mutations of a calcium release channel (RYR1) gene on chromosome 19. In other families, linkage of this disorder to the SCN4A gene on chromosome 17 has been suggested. We report on linkage analysis in a family in which both HPP and MH are inherited as autosomal-dominant traits. Two polymorphisms within the SCN4A locus, an RFLP and a (C-A)_n repeat, were typed on multiple family members. The findings were consistent with linkage of the polymorphic markers within the SCN4A gene to both HPP (Z_max = 6.79 at θ = 0.0) and MH (Z_max = 1.76 at θ = 0) in this family. Our data provide further evidence that MH is linked to the SCN4A locus in some families. Am. J. Med. Genet. 76:21–27, 1998. © 1998 Wiley-Liss, Inc.     

Drosophila as a model to study the role of glia in neurodegeneration

Neurons and glia interact reciprocally. Glia perform many important functions in the development and proper functioning of the nervous system throughout different stages of life. Neurons also affect the development and function of glia. Neurodegenerative diseases are usually late-onset, progressive, and affect specific parts of the nervous system. Many neurodegenerative disorders have been extensively studied. However, the majority of the studies have focused on the events that occur in neurons. The events that occur in glia, and whether and how glia participate in the pathogenesis of these diseases, have not been as well studied. In this review, we will focus on how the fruit fly Drosophila melanogaster has been used as a model to study neuron–glia interactions in neurodegenerative disorders. We discuss how glia are affected in these models of human neurodegenerative disorders and how glia contribute to their pathogenesis. These studies have provided important insight into the mechanisms of diverse neurodegenerative disorders, and have suggested possibilities for early diagnosis.     

Stimulation of protein phosphatases as a mechanism of the muscarinic-receptor-mediated inhibition of cardiac L-type Ca2+ channels

Acetylcholine decreases currents through cardiac L-type Ca²⁺ channels after stimulation with agents which elevate levels of cyclic adenosine monophosphate, such as isoproterenol, but there is still a controversy over the mechnisms of this muscarinic effect. We tested the hypothesis of whether, after isoproterenol stimulation, protein phosphatases are activated by acetylcholine. Whole-cell currents were recorded from guinea-pig ventricular myocytes. The effect of 10^–5 M acetylcholine on currents induced by 10^–8 M isoproterenol was studied in the absence or presence of protein phosphatase inhibitors. Three agents reduced the acetylcholine response: okadaic acid (3 or 9 · 10^–6 M) and cantharidin (3 · 10^–6 M) added to the pipette solution, and bath-applied fluoride (3 mM). In contrast, pipette application of other phosphatase inhibitors, namely the inhibitor PPI₂ (1000 U/ml), ciclosporin (10^–5 M), or calyculin A (10^–6 M) did not significantly diminish the acetylcholine effect. Interestingly, there was no correlation between the effects of the compounds on basal Ca²⁺ current and their interference with the muscarinic response. An activation of type 2A phosphatases by acetylcholine would explain these findings. Indeed, okadaic acid is 3 orders of magnitude more potent in vitro in its inhibition of this isoform (purified from cardiac myocytes) than is calyculin A, while type-1 phosphatases are inhibited equally. The data support the attractive possibility that stimulation of protein phosphatases is part of the signal transduction cascade of Ca²⁺ channel inhibition by acetyl-choline.     

Age-related downregulation of the CaV3.1 T-type calcium channel as a mediator of amyloid beta production

Alzheimer's is a crippling neurodegenerative disease that largely affects aged individuals. Decades of research have highlighted age-related changes in calcium homeostasis that occur before and throughout the duration of the disease, and the contributions of such dysregulation to Alzheimer's disease pathogenesis. We report an age-related decrease in expression of the CaV_3.1 T-type calcium channel at the level of messenger RNA and protein in both humans and mice that is exacerbated with the presence of Alzheimer's disease. Downregulating T-type calcium channels in N2a cells and the 3xTg-AD mouse model of Alzheimer's disease, by way of pharmacologic inhibition with NNC-55-0396, results in a rapid increase in amyloid beta production via reductions in non-amyloidogenic processing, whereas genetic overexpression of the channel in human embryonic kidney cells expressing amyloid precursor protein produces complementary effects. The age-related decline in CaV_3.1 expression may therefore contribute to a pro-amyloidogenic environment in the aging brain and represents a novel opportunity to intervene in the course of Alzheimer's disease pathogenesis.     

Long term exposure to the chemokine CCL2 activates the nigrostriatal dopamine system: a novel mechanism for the control of dopamine release

Accumulating evidence show that chemokines can modulate the activity of neurons through various mechanisms. Recently, we demonstrated that CCR2, the main receptor for the chemokine CCL2, is constitutively expressed in dopamine neurons in the rat substantia nigra. Here we show that unilateral intranigral injections of CCL2 (50 ng) in freely moving rats increase extracellular concentrations of dopamine and its metabolites and decrease dopamine content in the ipsilateral dorsal striatum. Furthermore, these CCL2 injections are responsible for an increase in locomotor activity resulting in contralateral circling behavior. Using patch-clamp recordings of dopaminergic neurons in slices of the rat substantia nigra, we observed that a prolonged exposure (>8 min) to 10 nM CCL2 significantly increases the membrane resistance of dopaminergic neurons by closure of background channels mainly selective to potassium ions. This leads to an enhancement of dopaminergic neuron discharge in pacemaker or burst mode necessary for dopamine release. We provide here the first evidence that application of CCL2 on dopaminergic neurons increases their excitability, dopamine release and related locomotor activity.