哺乳动物耳蜗外毛细胞的马达蛋白:Prestin

近几年的研究发现,在耳蜗基底膜的外毛细胞膜上有一种新奇的蛋白质:prestin(马达蛋白),它能感受细胞膜电位的变化,进而发生构象改变,引发外毛细胞的形状和表面积的改变.Prestin作为一种独特的马达蛋白,能驱动耳蜗外毛细胞的电能动性(electromotility),产生耳蜗的放大器作用,因而使哺乳动物的听觉具有高度的敏感性,广阔的听觉域,敏锐的频率选择性.这种蛋白质的缺失或基因的突变会导致听觉功能严重受损,对于prestin的深入细致的研究,也许可以使人们进一步认识和理解哺乳动物的听觉调谐机制,通过对这种蛋白质基因的表达的调控,是否能够防治一些与之相关的疾病?这或许将是今后听觉研究领域的一个重要课题.     

衣壳蛋白靶向灭活 一种新型抗病毒策略

衣壳蛋白靶向灭活（CTVI）是一种新型抗病毒策略,它是通过将病毒衣壳蛋白与核酸酶（金黄色葡萄球菌和酸梅、核糖核酸酶Barnase、大肠杆菌RNase HI等）的融合蛋白装配到病毒粒子中,使核酸酶接触并降解病毒核酸,从而达到抑制病毒复制的目的。该策略已经在人免疫缺陷病毒、鼠白血病病毒、乙肝病毒、登革病毒等病毒的抗病毒研究中取得良好效果,展示了广阔的应用前景。     

一种改进的纯化和鉴定牛脑皮层Gs蛋白的方法

用1%胆酸钠和15%饱和硫酸铵相结合的方法,从牛脑皮层细胞膜中抽提得到主要含激活型G-蛋白(G_s)和腺苷酸环化酶(AC)两种蛋白组分的制剂,然后通过Sepharose 6B柱将两者分开.将含G_s高活力的级分用庚胺-Sepharose 4B柱进一步分离,即可获得高活力的G_s,SDS-PAGE显示为分子量45 000和36 000的两条蛋白带.该法具有简便、快速、重复性好、产率高等优点,且可同时获得无G_s污染的AC.用无G_s污染的AC脂酶体测定G_s活力亦简便、可靠、灵敏度高.     

衰老标记蛋白30-一种具有应用前景的新型蛋白

衰老标记蛋白30(SMP-30)是近年来发现的一种新型钙结合蛋白,最初从鼠肝可溶蛋白中分离提出,具有随年龄增加而含量减少的特性以及抗细胞凋亡的作用,并且极有可能是一种新型的肝癌相关抗原.本文从该蛋白的结构、表达、可能的生物功能和机制、与肿瘤的相关特性作一综述.     

利用突变的绿色荧光蛋白基因为标记构建新型克隆载体

将三位点替换突变的绿色荧光蛋白（ＧＦＰ＿Ｓ６５Ｔ、Ｖ６８Ｌ、Ｓ７２Ａ）基因插入到ｐＢｌｕｅｓｃｒｉｐｔＳＫ（＋）的ＸｂａⅠ和ＳａｃⅠ之间,构建为新型的克隆载体ｐＧｒｅｅｎＬＤ。此质粒载体在Ｅ．ｃｏｌｉ中表达后使菌落在日光下呈现黄绿色,而在长波紫外光照射下呈现亮绿荧光,当外源ＤＮＡ片段插入该载体的多克隆位点使ｇｆｐ基因表达受阻时,转化的Ｅ．ｃｏｌｉ菌落为白色。因此可以用Ｅ．ｃｏｌｉ菌落黄绿色／绿色荧光的消失作为检测指标来筛选含有重组质粒的克隆。     

一种亨廷顿舞蹈症体外药物筛选细胞模型的建立

为建立以多聚谷氨酰胺（polyQ）为靶点的亨廷顿舞蹈症体外药物筛选细胞模型,以随机引物PCR法克隆了不同长度的 CAG片段,经序列测定正确后,分别融合到已经建立的氯霉素抗性融合蛋白表达系统pCAR中CAT的N端,重组质粒转化大肠杆菌,并在其中诱导表达,SDS-PAGE 和氯霉素抗性平板试验对目的蛋白可溶性与氯霉素活性进行测定。结果显示长度在40以上的polyQ为不溶性包涵体表达,表现为低水平的氯霉素抗性,40以下的polyQ则以可溶形式表达,表现为高水平氯霉素抗性,从而建立起能够模拟亨廷顿舞蹈症病理过程的体外细胞模型。通过检测模型细胞的氯霉素抗性,可以定性、定量地反映polyQ在体内的折叠状态和可溶性,故可以借助该模型对促溶药物或生物活性物质进行高通量筛选,为亨廷顿舞蹈症预防、诊断、治疗提供了新思路。     

一种新型高糖基化免疫融合蛋白NESP-Fc的研制

新红细胞生成刺激蛋白(NESP),是重组人红细胞生长素(rh EPO)的一种高糖基化类似物,它含有5个N端糖链和比rhEPO高2倍的唾液酸残基,具有较好的代谢稳定性和3倍于rhEPO的半衰期。在新红细胞生成刺激蛋白(NESP)的基础上,通过NESP的cDNA与人IgG2的铰链区与CH2和CH3的cDNA连接,形成了融合蛋白NESP-Fc,来达到提高NESP半衰期的目的。表达载体的构建、融合蛋白的表达纯化和初步的功能性试验等一系列研究证实,所表达的融合蛋白主要以二聚体形式存在;NESP-Fc能明显促进UT-7细胞的生长和小鼠体内网织红细胞的增殖;在大鼠体内的研究发现其半衰期高达56h;小试规模重组蛋白的表达量在1.4g/L左右。这些研究为该融合蛋白最终实现临床应用和产业化打下了良好的基础。     

ＪＩＰ—一种新型的胞浆抑制蛋白

JIP（JNK相互作用蛋白）是JNK的一种特异性胞浆抑制因子,引起卢JNK在胞质中滞留,阻止c-Jun、ATF-2、EIK等转录因子的活化,抑制受JNK调控的下游基因的表达,另外,JIP作为支架蛋白在MAPK信号途径发挥重要作用,现在JIP已作为一种新型的生物分子工具应用于JNK/SAPK/MAPK的研究,在哺乳动物中,发现了JIP的同源基因IB1,JIP参与了Glut2和胰岛素基因的表达,JIP作为肿瘤治疗候选的分子药物,可能在肿瘤治疗中发挥重要作用。     

vMIP-Ⅱ--一种广谱的趋化因子受体拮抗蛋白

人疱疹病毒8型编码的一种趋化因子类似物(vMIP-Ⅱ)能与大量CC类和CXC类细胞因子受体高度亲和性结合,通过拮抗Ca^2 短暂迅速内流引起的信号传导,阻断趋化因子受体的趋化作用。vMiP-Ⅱ对趋化因子受体的广谱拮抗能力为开发广谱的抗人类免疫缺陷病毒药物、在非全身免疫抑制条件下治疗移植免疫排斥反应以及治疗其他炎性疾病引入了新的途径。     

Optimal Electrical Properties of Outer Hair Cells Ensure Cochlear Amplification

The organ of Corti (OC) is the auditory epithelium of the mammalian cochlea comprising sensory hair cells and supporting cells riding on the basilar membrane. The outer hair cells (OHCs) are cellular actuators that amplify small sound-induced vibrations for transmission to the inner hair cells. We developed a finite element model of the OC that incorporates the complex OC geometry and force generation by OHCs originating from active hair bundle motion due to gating of the transducer channels and somatic contractility due to the membrane protein prestin. The model also incorporates realistic OHC electrical properties. It explains the complex vibration modes of the OC and reproduces recent measurements of the phase difference between the top and the bottom surface vibrations of the OC. Simulations of an individual OHC show that the OHC somatic motility lags the hair bundle displacement by ∼90 degrees. Prestin-driven contractions of the OHCs cause the top and bottom surfaces of the OC to move in opposite directions. Combined with the OC mechanics, this results in ∼90 degrees phase difference between the OC top and bottom surface vibration. An appropriate electrical time constant for the OHC membrane is necessary to achieve the phase relationship between OC vibrations and OHC actuations. When the OHC electrical frequency characteristics are too high or too low, the OHCs do not exert force with the correct phase to the OC mechanics so that they cannot amplify. We conclude that the components of OHC forward and reverse transduction are crucial for setting the phase relations needed for amplification.     

Limiting Frequency of the Cochlear Amplifier Based on Electromotility of Outer Hair Cells

Outer hair cells are the critical element for the sensitivity and sharpness of frequency selectivity of the ear. It is believed that fast motility (electromotility) of these cells is essential for this function. Indeed, force produced by outer hair cells follows their membrane potential very closely at least up to 60 kHz. However, it has been pointed out that the cell's receptor potential is attenuated by a low-pass RC circuit inherent to these cells, with the RC roll-off frequencies significantly lower than their operating frequencies. This would render electromotility ineffective in producing force. To address this issue, we assume that multiple degrees of freedom and vibrational modes due to the complex structure of the organ of Corti provide optimal phases for outer hair cells' force to cancel viscous drag. Our derived frequency limit depends on the drag-capacitance product, not directly on the RC time constant. With a reasonable assumption for the viscous drag, the estimated limit is 10–13 kHz, exceeding the RC corner frequency. Our analysis shows that a fast-activating potassium current can substantially extend the frequency limit by counteracting the capacitive current.     

EHD4 and CDH23 Are Interacting Partners in Cochlear Hair Cells

Cadherin 23 (CDH23), a transmembrane protein localized near the tips of hair cell stereocilia in the mammalian inner ear, is important for delivering mechanical signals to the mechano-electric transducer channels. To identify CDH23-interacting proteins, a membrane-based yeast two-hybrid screen of an outer hair cell (OHC) cDNA library was performed. EHD4, a member of the C-terminal EH domain containing a protein family involved in endocytic recycling, was identified as a potential interactor. To confirm the interaction, we first demonstrated the EHD4 mRNA expression in hair cells using in situ hybridization. Next, we showed that EHD4 co-localizes and co-immunoprecipitates with CDH23 in mammalian cells. Interestingly, the co-immunoprecipitation was found to be calcium-sensitive. To investigate the role of EHD4 in hearing, compound action potentials were measured in EHD4 knock-out (KO) mice. Although EHD4 KO mice have normal hearing sensitivity, analysis of mouse cochlear lysates revealed a 2-fold increase in EHD1, but no increase in EHD2 or EHD3, in EHD4 KO cochleae compared with wild type, suggesting that a compensatory increase in EHD1 levels may account for the absence of a hearing defect in EHD4 KO mice. Taken together, these data indicate that EHD4 is a novel CDH23-interacting protein that could regulate CDH23 trafficking/localization in a calcium-sensitive manner.Hair cells located in the mammalian inner ear transform mechanical stimuli into electrical signals that in turn facilitate neurotransmitter release onto auditory neurons. The key element in the transduction process is the mechano-electric transducer (MET)² apparatus located near the top of the stereocilium. CDH23 is a single pass transmembrane protein with 27 extracellular cadherin repeats. It is one of the components of the tip-link (1, 2), which connects the top of a shorter stereocilium to the side of its taller neighbor (3). Vibrations of the basilar membrane of the inner ear ultimately result in deflection of the hair bundles, which modulates tension on the tip-link, thereby controlling the opening probability of cation-selective MET channels (3, 4). Cations, principally K⁺ and Ca²⁺, flow through the MET channels and ultimately change the membrane potential. A mutation in the gene encoding CDH23, the Usher syndrome type 1D factor (USH1D), causes deaf-blindness in humans (5). Several interacting partners of CDH23 have been reported and include another tip-link protein protocadherin 15 (6), a multi-PDZ domain-containing scaffold protein harmonin (7) and a stereociliary scaffolding protein MAGI-1 (8). Protocadherin 15 binds to CDH23 through its extracellular domains (6), whereas the cytoplasmic region of CDH23 interacts with MAGI-1 and harmonin through its PDZ domain-binding interfaces (PBI). Harmonin also associates with other USH1 factors like myosin VIIa, protocadherin 15, and sans (9). These findings indicate that harmonin bridges CDH23 to the cytoskeletal actin core of the stereocilium and is probably essential for the developmental differentiation of stereocilia (10–12). However, it is currently unknown how CDH23 is transported to the tip of stereocilia. To search for additional interacting partners of CDH23, we performed a membrane-based yeast two-hybrid assay, which identified EHD4 as a potential binding partner (13).EHD4 belongs to an evolutionarily conserved EH (Eps 15 homology) domain-containing protein family involved in endocytic trafficking and recycling. Four highly homologous members of this family, EHD1–4, are expressed in mammalian cells. They contain a single C-terminal EH domain, an N-terminal nucleotide-binding loop and a coiled-coil region responsible for oligomerization (14–16). Of the four EHD proteins EHD1 is the best characterized and is involved in regulating the recycling of membrane receptors including the transferrin receptor and the major histocompatibility complex class I (17, 18). EHD1 is also involved in controlling cholesterol recycling and homeostasis (19) and in facilitating endosome to Golgi retrieval (20). EHD3 appears to regulate receptor movements from the early endosome (EE) to the endocytic recycling compartment (ERC) and Golgi (21, 22). EHD2 was isolated from GLUT4-enriched fractions of adipocytes and was shown to regulate insulin-mediated translocation of GLUT4 to the plasma membrane (23, 24). Additionally, EHD2 is involved in the regulation of transferrin receptor internalization (23), recycling (25), and actin cytoskeleton rearrangement (23). EHD4, also called Pincher, was first reported as an extracellular matrix protein (26). Subsequent studies have shown this intracellular protein to be involved in the regulation of neurotrophin receptor TrkA endocytosis in pheochromocytoma (PC12) cells (27). It is also involved in interactions with the cell fate determinant, NUMB, and co-localizes with the small GTP-binding protein, Arf6 (28). Recently, Sharma et al. (29) showed that EHD4 regulates the exit of endocytic cargo from the early endosome toward both the recycling compartment and the late endocytic pathway. They also indicated that EHD4 and EHD1 interact transiently as most of the EHD4 resides on peripheral early endosomes, while EHD1 resides primarily on tubular recycling compartments. This partial overlap/association might be necessary for the transport of proteins through the early endosome to the ERC. Previously, George et al. (25) had also demonstrated that EHD4 interacts with EHD1 and its paralogs, which suggests cooperation and partial overlap of function between EHD4 and EHD1.Unlike other CDH23-binding proteins, EHD4 does not contain a PDZ domain that could bind to the PBI located in the cytoplasmic tail of CDH23. In addition, the cytoplasmic tail of CDH23 lacks an Asn-Pro-Phe (NPF) motif that could mediate an interaction with the EH domain of EHD4. Therefore, we proceeded to characterize the authenticity of interaction between EHD4 and CDH23 identified in yeast and mammalian cells, using both in vitro and in vivo methods. We verified the expression of EHD4 mRNA in mouse cochlea and investigated the physiological role of EHD4 protein in the cochlea using EHD4-KO mice.     

Development and Function of the Voltage-Gated Sodium Current in Immature Mammalian Cochlear Inner Hair Cells

Inner hair cells (IHCs), the primary sensory receptors of the mammalian cochlea, fire spontaneous Ca²⁺ action potentials before the onset of hearing. Although this firing activity is mainly sustained by a depolarizing L-type (Ca_V1.3) Ca²⁺ current (I _Ca), IHCs also transiently express a large Na⁺ current (I _Na). We aimed to investigate the specific contribution of I _Na to the action potentials, the nature of the channels carrying the current and whether the biophysical properties of I _Na differ between low- and high-frequency IHCs. We show that I _Na is highly temperature-dependent and activates at around −60 mV, close to the action potential threshold. Its size was larger in apical than in basal IHCs and between 5% and 20% should be available at around the resting membrane potential (−55 mV/−60 mV). However, in vivo the availability of I _Na could potentially increase to >60% during inhibitory postsynaptic potential activity, which transiently hyperpolarize IHCs down to as far as −70 mV. When IHCs were held at −60 mV and I _Na elicited using a simulated action potential as a voltage command, we found that I _Na contributed to the subthreshold depolarization and upstroke of an action potential. We also found that I _Na is likely to be carried by the TTX-sensitive channel subunits Na_V1.1 and Na_V1.6 in both apical and basal IHCs. The results provide insight into how the biophysical properties of I _Na in mammalian cochlear IHCs could contribute to the spontaneous physiological activity during cochlear maturation in vivo.     

NaHS Protects Cochlear Hair Cells from Gentamicin-Induced Ototoxicity by Inhibiting the Mitochondrial Apoptosis Pathway

Aminoglycoside antibiotics such as gentamicin could cause ototoxicity in mammalians, by inducing oxidative stress and apoptosis in sensory hair cells of the cochlea. Sodium hydrosulfide (NaHS) is reported to alleviate oxidative stress and apoptosis, but its role in protecting aminoglycoside-induced hearing loss is unclear. In this study, we investigated the anti-oxidant and anti-apoptosis effect of NaHS in in vitro cultured House Ear Institute-Organ of Corti 1 (HEI-OC1) cells and isolated mouse cochlea. Results from cultured HEI-OC1 cells and cochlea consistently indicated that NaHS exhibited protective effects from gentamicin-induced ototoxicity, evident by maintained cell viability, hair cell number and cochlear morphology, reduced reactive oxygen species production and mitochondrial depolarization, as well as apoptosis activation of the intrinsic pathway. Moreover, in the isolated cochlear culture, NaHS was also demonstrated to protect the explant from gentamicin-induced mechanotransduction loss. Our study using multiple in vitro models revealed for the first time, the potential of NaHS as a therapeutic agent in protecting against aminoglycoside-induced hearing loss.     

Modulation of Outer Hair Cell Electromotility by Cochlear Supporting Cells and Gap Junctions

Outer hair cell (OHC) or prestin-based electromotility is an active cochlear amplifier in the mammalian inner ear that can increase hearing sensitivity and frequency selectivity. In situ, Deiters supporting cells are well-coupled by gap junctions and constrain OHCs standing on the basilar membrane. Here, we report that both electrical and mechanical stimulations in Deiters cells (DCs) can modulate OHC electromotility. There was no direct electrical conductance between the DCs and the OHCs. However, depolarization in DCs reduced OHC electromotility associated nonlinear capacitance (NLC) and distortion products. Increase in the turgor pressure of DCs also shifted OHC NLC to the negative voltage direction. Destruction of the cytoskeleton in DCs or dissociation of the mechanical-coupling between DCs and OHCs abolished these effects, indicating the modulation through the cytoskeleton activation and DC-OHC mechanical coupling rather than via electric field potentials. We also found that changes in gap junctional coupling between DCs induced large membrane potential and current changes in the DCs and shifted OHC NLC. Uncoupling of gap junctions between DCs shifted NLC to the negative direction. These data indicate that DCs not only provide a physical scaffold to support OHCs but also can directly modulate OHC electromotility through the DC-OHC mechanical coupling. Our findings reveal a new mechanism of cochlear supporting cells and gap junctional coupling to modulate OHC electromotility and eventually hearing sensitivity in the inner ear.     

Microstructures in the Organ of Corti Help Outer Hair Cells Form Traveling Waves along the Cochlear Coil

According to the generally accepted theory of mammalian cochlear mechanics, the fluid in the cochlear scalae interacts with the elastic cochlear partition to generate transversely oscillating displacement waves that propagate along the cochlear coil. Using a computational model of cochlear segments, a different type of propagating wave is reported, an elastic propagating wave that is independent of the fluid-structure interaction. The characteristics of the propagating wave observed in the model, such as the wavelength, speed, and phase lag, are similar to those observed in the living cochlea. Three conditions are required for the existence of the elastic propagating wave in the cochlear partition without fluid-interaction: 1), the stiffness gradient of the cochlear partition; 2), the elastic longitudinal coupling; and 3), the Y-shaped structure in the organ of Corti formed by the outer hair cell, the Deiters cell, and the Deiters cell phalangeal process. The elastic propagating waves in the cochlear partition disappeared without the push-pull action provided by the outer hair cell and Deiters cell phalangeal process. The results suggest that the mechanical feedback of outer hair cells, facilitated by the organ of Corti microstructure, can control the tuning and amplification by modulating the cochlear traveling wave.     

Microstructures in the Organ of Corti Help Outer Hair Cells Form Traveling Waves along the Cochlear Coil

According to the generally accepted theory of mammalian cochlear mechanics, the fluid in the cochlear scalae interacts with the elastic cochlear partition to generate transversely oscillating displacement waves that propagate along the cochlear coil. Using a computational model of cochlear segments, a different type of propagating wave is reported, an elastic propagating wave that is independent of the fluid-structure interaction. The characteristics of the propagating wave observed in the model, such as the wavelength, speed, and phase lag, are similar to those observed in the living cochlea. Three conditions are required for the existence of the elastic propagating wave in the cochlear partition without fluid-interaction: 1), the stiffness gradient of the cochlear partition; 2), the elastic longitudinal coupling; and 3), the Y-shaped structure in the organ of Corti formed by the outer hair cell, the Deiters cell, and the Deiters cell phalangeal process. The elastic propagating waves in the cochlear partition disappeared without the push-pull action provided by the outer hair cell and Deiters cell phalangeal process. The results suggest that the mechanical feedback of outer hair cells, facilitated by the organ of Corti microstructure, can control the tuning and amplification by modulating the cochlear traveling wave.