Distinct labelling of fusion events in rat lactotrophs by FM 1-43 and FM 4-64 is associated with conformational differences

AIM: Conformational analysis of fluorescent styryl dyes FM 1-43 and FM 4-64 was undertaken to clarify if distinct activity-dependent labelling of single lactotrophs vesicles and plasma membrane by two dyes is associated with their structural differences. METHODS: The activity-dependent labelling of single vesicles and plasma membrane by FM 1-43 and FM 4-64 was studied using confocal microscopy. The fluorescence intensity of vesicles fused with the plasma membrane, and the plasma membrane alone was measured; the ratio of their respective peak amplitudes was calculated. The conformational analysis of FM 1-43 and FM 4-64 was further undertaken by employing the Monte Carlo approach to search the conformational space of these molecules. RESULTS: In FM 1-43 staining of vesicles and plasma membrane, the ratio of the fluorescence peak amplitudes (vesicle vs. plasma membrane) was 2.6 times higher in comparison with FM 4-64 staining. In FM 4-64 molecule the low-energy conformations are distributed in three conformational states (consisting of 3, 4 and 2 conformers respectively) in which the proportion of the molecules residing in a given state is 62%, 28% and 9% respectively. In FM 1-43 the conformation distribution is limited to just one conformational state with three approximately equally populated conformers what can be explained by greater intrinsic rigidity of the molecule. CONCLUSIONS: The observed structural characteristics of FM 1-43 molecules may account for a higher increase in quantum yield and/or binding affinity upon incorporation of the dye into the vesicle matrix and therefore stronger fluorescence emission in comparison with FM 4-64.     

Developmental changes in pulpal sensory innervation of rat incisors and molars shown on a single injection of the fluorescent dye AM1-43

Developmental changes in pulpal innervation of rat incisors and molars were examined using the fluorescent styryl dye AM1-43, which labels sensory cells and nerves in vivo. From 2 to 40 days after birth, the animals were injected once with a small amount of AM1-43 solution (2 microg/g bodyweight). One day after the injection, the animals were killed and examined. In 3-day-old rats, neither incisors nor molars were innervated. In 7-day-old rats, the pulp of incisors and molars was innervated as indicated by fine intensely stained varicose sensory fibers and thicker intensely stained fibers running mostly along the blood vessels. In 15-, 27-, and 41-day-old rats, sensory nerve fibers neither passed through the odontoblast layer nor penetrated into the dentin in incisors, whereas the sensory nerve fibers penetrated into the coronal dentin through the odontoblast layers in molars. These results suggest that innervation of dental pulp is composed of two phases: (i) linear penetration of nerve fibers along blood vessels into the pulp space; and (ii) sprouting and extension of nerve fibers into coronal dentin. Innervation of the incisor pulp may stop at the first phase.     

Discrimination of live and early apoptotic mononuclear cells by the fluorescent SYTO 16 vital dye

Accurate detection of apoptotic cells is important for the determination of cell viability. The aim of this study was to compare the sensitivity of the cell permeant SYTO 16 fluorescent dye for detecting early apoptotic mononuclear cells (MNCs) in normal donor blood with other apoptosis assays [i.e. Annexin-V, light scatter/7-amino-actinomycin-D (7-AAD) and chloromethyl-X-rosamine (CMXRos)] and to identify critical parameters for optimal SYTO 16 staining. Apoptosis was induced in normal human leukocytes from adult peripheral blood or cord blood, or the Jurkat T-lymphocytic cell line and assessed by fluorescence microscopy and flow cytometry. Dual labelling showed that SYTO 16 detected more apoptotic MNCs compared to Annexin-V. SYTO 16 staining intensity was consistent with the light scatter profiles expected of live, apoptotic and necrotic MNCs and was more objective than light scatter/7-AAD. CMXRos staining required considerable care and may not be a robust marker of apoptotic primary MNCs. For SYTO 16 flow cytometric analysis, the optimal conditions for staining 1x10(6) leukocytes were 4 nM SYTO 16 in the presence of 30 muM verapamil for 25-45 min at 37 degrees C in media containing calcium/magnesium supplemented with protein. A P-glycoprotein inhibitor, such as verapamil, and calcium/magnesium are essential for optimal loading of SYTO 16 into live MNCs and discrimination of apoptotic MNCs in normal blood samples. SYTO 16 is a sensitive, simple, inexpensive 'live cell' method for the discrimination of live, apoptotic and necrotic normal blood MNCs and is more sensitive for detecting apoptosis in these cells than Annexin-V or light scatter/7-AAD.     

Vlgr1 is required for proper stereocilia maturation of cochlear hair cells

Very large G-protein coupled receptor (Vlgr1b) is the largest known G-protein coupled receptor. Its function is unknown, although mice with deletion of Vlgr1 (Vlgr1b together with other splicing variants, Vlgr1c, Vlgr1d and Vlgr1e) are known to exhibit audiogenic seizure susceptibility and VLGR1 is reported to be the gene responsible for Usher type 2C syndrome. We demonstrated here that Vlgr1-mutated mice suffered from a hearing defect because of inner ear dysfunction, as indicated by auditory brainstem response (ABR) and distortion product oto-acoustic emissions (DPOAE). The expression of Vlgr1 was identified in the developing hair cells perinatally, and the translated products were seen to be localized in the base of stereocilia on hair cells using confocal microscopy. This Vlgr1 localization was limited to the base of stereocilia within approximately 200-400 nm from the apical surface of hair cells, as shown by immunoelectron microscopy. The Vlgr1-mutated mice exhibited malformation of the stereocilia; the cochlear hair bundles were apparently normal at birth but then became disarranged at postnatal day 8. Furthermore, the stereocilia in the mutant mice became slanted and disarranged thereafter. These results indicate that loss of Vlgr1 resulted in abnormal development of stereocilia formation.     

电离辐射对大鼠耳蜗外毛细胞Prestin蛋白表达影响的实验研究

目的探讨电离辐射下大鼠耳蜗外毛细胞Prestin蛋白表达的改变及意义。方法建立大鼠耳放射损伤模型,检测其听力复合动作电位（CAP）阈值及畸变产物耳声发射（DPOAEs）幅值,验证其电离辐射晚期感音神经性耳聋（SNHL）的发生。提取大鼠耳蜗组织mRNA及蛋白,荧光实时定量PCR检测耳蜗Prestin蛋白mRNA水平的表达,以及Western杂交检测Prestin蛋白水平的表达。结果成功建立了电离辐射晚期SNHL大鼠模型,其耳蜗Prestin蛋白无论是mRNA表达水平还是蛋白表达水平均较未照射组明显降低。结论放疗晚期感音神经性耳聋的发生机制可能与电离辐射导致内耳外毛细胞Prestin蛋白的表达异常有关。     

大鼠触须毛囊干细胞的分离培养和鉴定

背景：毛囊干细胞在体内膀胱及周围组织环境作用下有可能向尿路上皮细胞和平滑肌细胞分化,成为目前研究较新的干细胞之一。 目的：建立一种高效简单的分离培养和鉴定毛囊干细胞的方法。 方法：取大鼠触须部皮肤组织,体式显微镜下分离出毛囊组织,中性蛋白酶Ⅱ与胰蛋白酶和乙二胺四乙酸混合液“两步酶法”消化;所得细胞悬液中添加含体积分数10%胎牛血清的角质细胞无血清培养基,Ⅳ型胶原差速贴壁法筛选毛囊干细胞,20 min以内贴壁的细胞作为实验组,未贴壁的细胞作为对照组;待筛选后的细胞生长至70%~80%汇合后,进行传代培养。 结果与结论：筛选后的细胞形态均匀一致,折光性强,呈典型的“铺路石状”。透射电镜显示细胞处于原始状态。流式细胞仪检测实验组CD34和β1整合素的表达分别为(39.52±19.57)%和(93.46±4.73)%,对照组相应为(19.20±11.53)%和(63.57± 14.42)%,两组间差异有显著性意义(P < 0.05)。结果表明联用显微分离技术、二步酶法及差速贴壁法筛选可以获得纯度较高的毛囊干细胞。CD34和β1整合素表达是较理想的鉴定方法。     

大鼠原代肝细胞的培养及鉴定

目的:探讨体外大鼠肝细胞的分离培养及纯化的方法。方法:采用胶原酶Ⅳ消化组织块法分离新生大鼠肝细胞,对大鼠肝细胞进行纯化、培养,并采用免疫组织化学方法对其进行鉴定。结果:培养24 h,有部分细胞贴壁,48 h有大量细胞贴壁,但含有较多杂细胞。经过4~5次传代后,细胞形态一致,可见许多双核细胞和多核细胞。采用抗大鼠细胞角蛋白(ck-18)的特异抗体进行免疫细胞化学鉴定,经SP染色DAB显色后,阳性着色部位在胞浆呈棕黄色颗粒,阴性对照未见着色。结论:成功地体外培养了大鼠肝细胞并对其进行了鉴定,为进一步的实验研究奠定了基础。     

Forces involved in length changes of cochlear outer hair cells

Motion or force generation of outer hair cells may contribute to active modulation of cochlear mechanics. In order to determine the force involved in length changes of outer hair cells, a new in vitro method was used. In the first series of experiments, apical and basolateral extracellular spaces of outer hair cells of the guinea-pig cochlea were separated. Changes of the voltage between the two extracellular spaces induced reversible, proportional changes of the cell length of 4.4 nm/ mV if the cell had a length of 80 m. In the second series of experiments, cell elongations in response to negative pressure applied to the basal end of the cells were measured and corrected for frictional effects. From these data, the compliance of the longitudinal axis of the hair cells was calculated. It was 220±130 m/N (n =25) and 240±170 m/N /(n = 24) for cells of the third and fourth cochlear turns, respectively, if the water permeability of the cell membrane was neglected. If the water permeability was taken into account, the compliance was probably around 5 km/N. Thus, a mechanism that changes the cell length by 1 m must generate a static force of at least around 200 pN in an outer hair cell of the organ of Corti. Electromotility of outer hair cells, induced by changes of the electrical potential difference across the outer hair cell, is a mechanism that generates this force.     

Immunohistochemistry of connexin 43 throughout anterior pituitary gland in a transgenic rat with green fluorescent protein-expressing folliculo-stellate cells

Folliculo-stellate (FS) cells in the anterior pituitary gland have been speculated to possess multifunctional properties. Because gap junctions (GJ) have been identified between FS cells, FS cells may be interconnected electrophysiologically by GJ and serve as signal transmission networks to modulate hormone release in the anterior pituitary gland. But whether GJ are localized among FS cells from the pars tuberalis through the pars distalis is unclear. The S100b-GFP transgenic rat has recently been generated, which expresses green fluorescent protein (GFP) specifically in FS cells in the anterior pituitary. This model is expected to be a powerful tool for studies of FS cells. The purpose of the present paper was therefore to examine the localization of GJ on connexin 43 immunohistochemistry throughout the anterior pituitary gland of S100b-GFP rats under confocal laser microscopy. The localization patterns of FS cells was also observed in primary culture of anteiror pituitary cells and the question of whether GJ between FS cells are reconstructed in vitro was investigated. In vivo studies showed that GJ were present specifically between FS cells from the pars tuberalis to the pars distalis in the anterior pituitary gland. The appearance of FS cells was distinguished into two types, with localization of GJ differing between types. In vitro, it was observed for the first time that FS cells in primary culture could be categorized into two types. In vivo localization of GJ between FS cells was reconstructed in vitro. These morphological observations are consistent with the hypothesis that FS cells form an electrophysiological network throughout the anterior pituitary for signal transmission.     

The mechanism of pneumolysin-induced cochlear hair cell death in the rat

Streptoccocus pneumoniae infection can result in local and systemic diseases such as otitis media, pneumonia and meningitis. Sensorineural hearing loss associated with this infection is mediated by the release of an exotoxin, pneumolysin. The goal of the present study was to characterize the mechanisms of pneumolysin toxicity in cochlear hair cells in vitro . Pneumolysin induced severe damage in cochlear hair cells, ranging from stereocilia disorganization to total cell loss. Surprisingly, pneumolysin-induced cell death preferentially targeted inner hair cells. Pneumolysin triggered in vitro cell death by an influx of calcium. Extracellular calcium appeared to enter the cell through a pore formed by the toxin. Buffering intracellular calcium with BAPTA improved hair cell survival. The mitochondrial apoptotic pathway involved in pneumolysin-induced cell death was demonstrated by the use of bongkrekic acid. Binding of pneumolysin to the hair cell plasma membrane was required to induce cell death. Increasing external calcium reduced cell toxicity by preventing the binding of pneumolysin to hair cell membranes. These results showed the significant role of calcium both in triggering pneumolysin-induced hair cell apoptosis and in preventing the toxin from binding to its cellular target.     

FM1-43在显示豚鼠食道肌间神经节内板状末梢中的应用

目的: 用苯乙烯基染料FM1-43结合牵拉刺激显示豚鼠食道IGLEs (intraganglionic laminar endings) 的特异结构，与neurobiotin神经顺行标记技术相结合，以FM1-43 染色从功能及形态两方面验证IGLEs 是否是迷走神经感受机械刺激的受体。 方法: 比较给予豚鼠食道组织牵拉刺激前后FM1-43 染色显示的IGLEs的数目与形态； Neurobiotin神经顺行标记技术与牵拉刺激后FM1-43染色相结合，比较两者显示的IGLEs形态是否一致。 结果: 牵拉刺激后FM1-43 染色显示的IGLEs的数目明显高于未牵拉组FM1-43染色(P<0.01)； Neurobiotin神经顺行标记技术与牵拉刺激后FM1-43染色显示的IGLEs形态完全一致。 结论: FM1-43 染色及neurobiotin神经顺行标记技术均可以特异地显示IGLEs的结构， 而FM1-43 更能从功能上证实IGLEs 是迷走神经感受机械刺激的受体。     

Fast adaptation of mechanoelectrical transducer channels in mammalian cochlear hair cells

Outer hair cells are centrally involved in the amplification and frequency tuning of the mammalian cochlea, but evidence about their transducing properties in animals with fully developed hearing is lacking. Here we describe measurements of mechanoelectrical transducer currents in outer hair cells of rats between postnatal days 5 and 18, before and after the onset of hearing. Deflection of hair bundles using a new rapid piezoelectric stimulator evoked transducer currents with ultra-fast activation and adaptation kinetics. Fast adaptation resembled the same process in turtle hair cells, where it is regulated by changes in stereociliary calcium. It is argued that sub-millisecond transducer adaptation can operate in outer hair cells under the ionic, driving force and temperature conditions that prevail in the intact mammalian cochlea.     

Large releasable pool of synaptic vesicles in chick cochlear hair cells

Hearing requires the hair cell synapse to maintain notable temporal fidelity (< or =1 ms) while sustaining neurotransmitter release for prolonged periods of time (minutes). Here we probed the properties and possible anatomical substrate of prolonged neurotransmitter release by using electrical measures of cell surface area as a proxy for neurotransmitter release to study hair cell exocytosis evoked by repetitive stimuli. We observed marked depression of exocytosis by chick tall hair cells. This exocytic depression cannot be explained by calcium current inactivation, presynaptic autoinhibition by metabotropic glutamate receptors, or postsynaptic receptor desensitization. Rather, cochlear hair cell exocytic depression resulted from the exhaustion of a functional vesicle pool. This releasable vesicle pool is large, totaling approximately 8,000 vesicles, and is nearly 10 times greater than the number of vesicles tethered to synaptic ribbons. Such a large functional pool suggests the recruitment of cytoplasmic vesicles to sustain exocytosis, important for maintaining prolonged, high rates of neural activity needed to encode sound.     

The function of connexin 43 on the differentiation of rat bone marrow cells in culture

Connexin (Cx) 43-mediated gap-junctional intercellular communication (GJC) mainly regulates the osteoblastic differentiation, but much of the function of Cx43 on the differentiation of bone marrow cells is unclear. This study is aimed to clarify relationship between the differentiation of rat bone marrow cells and the function of Cx43. Bone marrow cells derived from four-week-old Wistar strain rats were grown in the presence and absence of 18-alpha-glycyrrhetinic acid (AGA, 100 muM) to inhibit Cx43-mediated GJC. Expression of Cx43 gene and protein, and the level of intracellular cyclic adenosine monophosphate (cAMP) were determined as the assessment of the function in Cx43-mediated GJC, and alkaline phosphatase (ALP) activity and mineralization were measured as the assessment of osteoblastic differentiation. The Cx43 gene expression was first observed at 2 days, but under the condition in which rat bone marrow cells were treated with AGA, there was no significant effect on the Cx43 gene expression. By administrating AGA to rat bone marrow cells, all parameters of maturation but the Cx43 gene expression significantly decreased. The results of this experiment suggest that Cx43-mediated GJC plays a critical role in rat bone marrow cells, progress toward maturation.