Human amnion membrane serves as a substratum for growing axons in vitro and in vivo

The epithelial cell layer of human amnion membrane can be removed while the basement membrane and stromal surfaces remain morphologically intact. Such a preparation has been used as a substratum for the in vitro culture of dissociated neurons. Embryonic motor neurons from chick ciliary ganglion attached to both surfaces but grew extensive neurites only on the basement membrane. On cross sections of rolled amnion membranes, regenerating axons of cultured neurons were guided along pathways of basement membrane that were immunoreactive with an antibody to laminin. In addition, when rolled amnion membranes were implanted into a lesion cavity between the rat septum and hippocampus, cholinergic neurons extended axons through the longitudinally oriented implant into the hippocampus. Thus, this amnion preparation can serve as a bridge to promote axonal regeneration in vivo in damaged adult brain.     

Nonvisual photoreception in the chick iris

The embryonic chicken iris constricts to light ex vivo, but with characteristics atypical of visual phototransduction. The chick iris was most sensitive to short-wavelength light, demonstrating an action spectrum consistent with cryptochrome rather than with opsin pigments. Pupillary responses did not attenuate after saturating light exposure, but showed paradoxical potentiation. Iris photosensitivity was not affected by retinoid depletion or inhibitors of visual phototransduction. Knockdown of cryptochrome expression, but not of melanopsin expression, decreased iris photosensitivity. These data characterize a non-opsin photoreception mechanism in a vertebrate eye and suggest a conserved photoreceptive role for cryptochromes in vertebrates.     

Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway

The failure of axons to regenerate is a major obstacle for functional recovery after central nervous system (CNS) injury. Removing extracellular inhibitory molecules results in limited axon regeneration in vivo. To test for the role of intrinsic impediments to axon regrowth, we analyzed cell growth control genes using a virus-assisted in vivo conditional knockout approach. Deletion of PTEN (phosphatase and tensin homolog), a negative regulator of the mammalian target of rapamycin (mTOR) pathway, in adult retinal ganglion cells (RGCs) promotes robust axon regeneration after optic nerve injury. In wild-type adult mice, the mTOR activity was suppressed and new protein synthesis was impaired in axotomized RGCs, which may contribute to the regeneration failure. Reactivating this pathway by conditional knockout of tuberous sclerosis complex 1, another negative regulator of the mTOR pathway, also leads to axon regeneration. Thus, our results suggest the manipulation of intrinsic growth control pathways as a therapeutic approach to promote axon regeneration after CNS injury.     

Reinnervated eye muscles do not respond to impulses in foreign nerves

Normal movements return to carp eyes after section and regeneration of the IIIrd and IVth nerve trunks. Two months after reinnervation, records of impulses in the inferior oblique nerve during tilting of the body show activity of the normal motoneurons to that muscle, together with discharge patterns characteristic of the antagonistic superior oblique and some of the rectus muscles. These axons must have found their way into the inferior oblique trunk during sprouting at the lesion and must be maintained after reinnervation. Impulses from foreign axons are without detectable effect on eye movement and therefore must be blocked at their termination in the muscle. Previous study of cross-innervated and doubly innervated fish eye muscles revealed only structurally normal neuromuscular junctions. Transmission from foreign junctions in multiply innervated muscle is blocked by competitive molecular recognition and control mechanisms that do not cause degeneration.     

印度谷螟成虫复眼的外部形态及显微结构观察

利用扫描电镜和石蜡切片技术观察研究了印度谷螟[Plodia interpunctella(Hübener)]成虫复眼的外部形态和明暗适应对其内部显微结构的影响.结果显示:(1)该蛾复眼半球形,位于头部两侧,略成“八”字形排列,单个复眼的小眼数约为1 950;(2)每个小眼主要由角膜、晶锥、小网膜细胞柱、视杆和基膜组成,其中角膜、晶锥、小网膜细胞周围环绕着由初级虹膜色素细胞分泌的初级虹膜色素颗粒和由6个次级虹膜色素细胞分泌的次级虹膜色素颗粒,基膜处分布有基膜色素颗粒,且密集的气管穿过基膜进入网膜区;(3)明适应时,大部分次级虹膜色素颗粒向视杆远心端移动,覆盖整个小网膜细胞柱区域;暗适应时,次级虹膜色素颗粒逐渐向晶锥方向聚集,分布在各晶锥周围和小网膜细胞柱的远心端.结果表明:印度谷螟成虫复眼为典型的重叠像眼,其主要通过色素颗粒发生纵向位移来调节对光的吸收,以适应外界光环境的变化.     

Myelin-associated glycoprotein as a functional ligand for the Nogo-66 receptor

Axonal regeneration in the adult central nervous system (CNS) is limited by two proteins in myelin, Nogo and myelin-associated glycoprotein (MAG). The receptor for Nogo (NgR) has been identified as an axonal glycosyl-phosphatidyl-inositol (GPI)-anchored protein, whereas the MAG receptor has remained elusive. Here, we show that MAG binds directly, with high affinity, to NgR. Cleavage of GPI-linked proteins from axons protects growth cones from MAG-induced collapse, and dominant-negative NgR eliminates MAG inhibition of neurite outgrowth. MAG-resistant embryonic neurons are rendered MAG-sensitive by expression of NgR. MAG and Nogo-66 activate NgR independently and serve as redundant NgR ligands that may limit axonal regeneration after CNS injury.     

PirB is a functional receptor for myelin inhibitors of axonal regeneration

A major barrier to regenerating axons after injury in the mammalian central nervous system is an unfavorable milieu. Three proteins found in myelin--Nogo, MAG, and OMgp--inhibit axon regeneration in vitro and bind to the glycosylphosphatidylinositol-anchored Nogo receptor (NgR). However, genetic deletion of NgR has only a modest disinhibitory effect, suggesting that other binding receptors for these molecules probably exist. With the use of expression cloning, we have found that paired immunoglobulin-like receptor B (PirB), which has been implicated in nervous system plasticity, is a high-affinity receptor for Nogo, MAG, and OMgp. Interfering with PirB activity, either with antibodies or genetically, partially rescues neurite inhibition by Nogo66, MAG, OMgp, and myelin in cultured neurons. Blocking both PirB and NgR activities leads to near-complete release from myelin inhibition. Our results implicate PirB in mediating regeneration block, identify PirB as a potential target for axon regeneration therapies, and provide an explanation for the similar enhancements of visual system plasticity in PirB and NgR knockout mice.     

Control of axon branch dynamics by correlated activity in vivo

To determine how patterned visual activity regulates the development of axonal projections, we collected in vivo time-lapse images of retinal axons from albino Xenopus tadpoles in which binocular innervation of the optic tectum was induced. Axons added branch tips with nearly equal probability in all territories, but eliminated them preferentially from territory dominated by the opposite eye. This selective branch elimination was abolished by blockade of N-methyl-D-aspartate receptors. These results describe a correlation-based mechanism by which visual experience directly governs axon branch dynamics that contribute to the development of topographic maps.     

虹膜诊断研究述评

虹膜诊断为目前国内外研究热点,是遵循眼是人体的缩影、眼可以反映出人体全息的原理,观察虹膜组织形态学的动态变化,利用照虹膜分区定位图来诊断疾病的技术。虹膜诊断属于中医目诊范畴,虹膜诊断能进一步丰富中医目诊内涵,并在望目辨证中提供辨证依据。目前研究表明,虹膜特征性改变与全身疾病具有相关性,虹膜的结构受全身疾病的影响,现将虹膜诊断的研究进展进行述评。     

鸡腔上囊传出神经元的分布——用CB—HRP法研究

将CB—HRP注入鸡腔上囊壁内,支配腔上囊的神经元被标记。支配腔上囊的长轴突型交感节前神经元在胸7—腰荐3(简称T_7—LS_3)髓节的Terni氏柱内,主要位于LS_1—LS_2髓节。在LS_8—LS_(11)髓节中央管背侧和背外侧区有大量标记细胞,主要集中于LS_8髓节,这些标记细胞是支配腔上囊的副交感节前神经元,其轴突大部分行经同侧盆神经到达腔上囊,少数行经对侧的盆神经到达腔上囊。腔上囊的交感节后神经元位于T_6—L_(13)交感干神经节和肾上腺神经节,交感干的标记细胞集中位于LS_9—LS_(11)和LS_2—LS_3。副交感节后神经元位于盆神经和泄殖腔神经节内。在肠神经内有大量的标记细胞。     

传染性喉气管炎病毒gD蛋白多克隆抗体制备与运用

鸡传染性喉气管炎(Infectious laryngotracheitis,ILT)是由传染性喉气管炎病毒(Infectious laryngotracheitis virus,ILTV)引起的一种急性接触性上部呼吸道病。ILTV表面糖蛋白g D能刺激机体产生良好体液和细胞免疫应答,编码该蛋白的g D基因是ILTV主要抗原性基因之一。以ILTV-LJS09株基因组为模板,PCR扩增g D基因主要抗原区域(54~344 aa)及g D基因,分别亚克隆至p GEX-6P-1和p CAGGS载体,构建原核表达载体p GEX-6P-1-g D163/1032和真核表达载体p CAGGS-g D。利用纯化ILTV、原核表达纯化获得重组蛋白r-g D和真核质粒p CAGGS-g D,对新西兰大白兔进行免疫与加强免疫,制备兔抗ILTV g D多克隆抗体。通过间接免疫荧光试验确定ILTV g D多克隆抗体和ILTV感染的鸡肝癌细胞表达蛋白发生反应,表明制备的ILTV g D多克隆抗体能识别ILTV天然抗原表位,最佳稀释度为11 000;用ILTV感染的LMH细胞培养物进行Western Blot分析,在40 ku左右出现一条特异性条带,与预期ILTV g D蛋白大小相符。激光共聚焦定位感染鸡肝癌细胞中ILTV g D蛋白,发现ILTV g D蛋白主要表达于感染细胞胞浆及融合细胞交界处。     

EGFR activation mediates inhibition of axon regeneration by myelin and chondroitin sulfate proteoglycans

Inhibitory molecules associated with myelin and the glial scar limit axon regeneration in the adult central nervous system (CNS), but the underlying signaling mechanisms of regeneration inhibition are not fully understood. Here, we show that suppressing the kinase function of the epidermal growth factor receptor (EGFR) blocks the activities of both myelin inhibitors and chondroitin sulfate proteoglycans in inhibiting neurite outgrowth. In addition, regeneration inhibitors trigger the phosphorylation of EGFR in a calcium-dependent manner. Local administration of EGFR inhibitors promotes significant regeneration of injured optic nerve fibers, pointing to a promising therapeutic avenue for enhancing axon regeneration after CNS injury.     

Photoelectric technique for measuring eye movements

By the system described, the movement of a stimulus and the correlated tracking movements of the eye are recorded simultaneously. The technique for measuring the eye movements consists of detecting and amplifying by photomultiplication the total amount of light passing through a small slit upon which is imaged a small portion of the light-dark field represented by the iris and sclera of the eye. This total amount of light varies directly with the angular position of the eye.     

Nerve growth factor: stimulation of regenerative growth of central noradrenergic neurons

The growth of new axonal sprouts was studied from transected, ascending noradrenergic axons into transplants of iris tissue in the caudal hypothalamus of the rat. A single intraventricular injection of nerve growth factor, given at the time of axonal damage, resulted in an increased formation and growth of new noradrenaline sprouts 7 days later. The effect seemed to be proportional to the administered dose of nerve growth factor.     

飞蝗几丁质酶5-1抗体制备及表达特性分析

【目的】通过制备飞蝗（Locusta migratoria）几丁质酶5-1（LmCht5-1）的多克隆抗体,建立飞蝗体内LmCht5-1蛋白水平检测方法,分析LmCht5-1在4龄飞蝗的表达特性及组织定位。【方法】采用MEGA 软件对LmCht5-1和LmCht5-2氨基酸序列进行比对,利用Expression网站对LmCht5-1氨基酸序列进行抗原表位预测分析,获得LmCht5-1抗原结构区域;以飞蝗cDNA为模板,通过PCR扩增LmCht5-1的抗原片段;同时通过酶切连接构建含有鸡血清白蛋白OVA的载体pET32a-OVA;然后通过酶切连接将LmCht5-1插入到pET32a-OVA载体构建pET32a-OVA-LmCht5-1;将pET32a-OVA-LmCht5-1转入表达菌株BL21（DE3）中,IPTG诱导表达重组蛋白OVA-LmCht5-1;利用Ni-NTA纯化后免疫新西兰白兔,制备多克隆抗体。通过ELISA方法检测抗体效价;利用Western blot检测抗体特异性。提取4龄飞蝗不同日龄表皮,采用Western blot分析LmCht5-1蛋白表达模式。对4龄飞蝗注射dsLmCht5-1,检测蛋白水平LmCht5-1表达量,并通过免疫组化方法对LmCht5-1进行定位及功能分析。【结果】通过LmCht5-1和LmCht5-2序列比对和抗原表位预测分析,获得LmCht5-1的471-533AA可作为抗原区域;通过酶切连接分别获得pET32a-OVA和pET32a-OVA-LmCht5-1。经诱导表达和纯化后获得重组蛋白OVA-LmCht5-1,分子量为71.34 kD;免疫后制备多克隆抗体OVA-LmCht5-1,ELISA检测效价为1﹕102 400。多克隆抗体OVA-LmCht5-1用于Western blot检测时可特异性识别LmCht5-1,与LmCht5-2蛋白无交叉反应。利用Western blot方法检测4龄若虫各日龄表皮中LmCht5-1蛋白的表达,发现LmCht5-1蛋白随发育日龄其表达量逐渐增加,在蜕皮当天达到峰值,蜕皮后快速降至最低点。对飞蝗若虫N4D2注射dsLmCht5-1,检测到在N4D5时,LmCht5-1的转录水平和蛋白水平均被显著抑制,抑制率分别为70.0%和73.6%。选取注射dsLmCht5-1和dsGFP的4龄飞蝗表皮,进行免疫组化实验显示LmCht5-1定位于表皮细胞和旧表皮中,其功能失活引起旧表皮中几丁质不降解。【结论】获得飞蝗LmCht5-1多克隆抗体可特异性识别LmCht5-1,可用于Western blot和免疫组化检测。明确LmCht5-1在飞蝗4龄若虫蜕皮当天表达最高,定位于表皮细胞和旧表皮中。dsLmCht5-1可减少表皮细胞和旧表皮中LmCht5-1的表达,阻碍旧表皮中几丁质的降解。     

禽流感病毒感染与疫苗免疫鉴别诊断试纸条的研制及应用

 【目的】结合胶体金免疫层析技术建立一种适合养鸡业基层生产人员使用的便捷的快速诊断禽流感病毒感染鸡群的方法。【方法】将含禽流感病毒非结构基因ns1的表达载体KG-NS1转化入BL21（DE3）感受态细胞,筛选阳性克隆进行诱导表达,表达产物用GST亲和层析柱进行纯化和Western-blot分析;以NS1重组蛋白为诊断抗原,抗鸡IgFc的单抗标记的胶体金为示踪物,结合免疫层析技术,组装禽流感病毒NS1抗体的免疫胶体金鉴别诊断试纸条,评价其灵敏度和特异性,并对试验感染和临床采集的血样进行检测。【结果】NS1重组蛋白分子量约为52 kD,具有免疫学活性。在25 min内用肉眼观察到禽流感病毒感染鸡血清和NS1蛋白免疫鸡血清在试纸条的检测线处出现明显的棕红色,呈明显的阳性反应,而其它病原的血清在试纸条的检测线处不出现任何颜色,呈阴性反应。而且感染鸡群的NS1抗体在感染后3 d即可检测到,感染后1～2周为高峰期,维持时间约1周,检测阳性率为80%左右。临床样品的阳性率为9.1%。【结论】试纸条使用方便,操作简单,25 min内可以用肉眼判断结果,可区分禽流感疫苗免疫和野毒感染家禽,具有很大的推广应用价值。     

Transport of protein by goldfish optic nerve fibers

After tritiated leucine was injected into the eye of goldfish, radio-active protein synthesized by the ganglion cell bodies moved down the optic axons at an average rate of 0.4 mm per day. Radioautograms of the optic tectum in which these axons end show that, as early as 24 hours after the injection, before the radioactivity in the tectal layer containing the optic axons had risen above background level, the layer containing the axon terminals was already heavily labeled. The radioactivity in the terminals reached a maximum about 48 hours after the injection and remained approximately constant for at least 23 days thereafter, whereas the radioactivity in the fiber layer increased significantly during the same interval, as the slowly moving protein component entered it. Thus there appears to be a special mechanism for rapid transport of protein from the cell body to the synaptic terminals, as well as a slower movement of protein down the axon.     

锚定NDV HN蛋白重组干酪乳杆菌诱导雏鸡黏膜免疫应答

以干酪乳杆菌作为呈递抗原活载体,表达新城疫病毒保护性抗原HN蛋白。重组干酪乳杆菌表达外源蛋白后,经SDS-PAGE、Western blot及间接免疫荧光分析,表明目的蛋白表达并展示到乳酸菌表面。将重组菌及空质粒菌株分别滴鼻、点眼免疫雏鸡,于不同时间检测血清样品中特异性IgG;于三免后不同时间采集雏鸡的泪液、气管洗液、胆汁和肠洗液样品,采用间接ELISA方法检测样品的特异性sIgA;用MTT法检测免疫鸡脾淋巴细胞增殖情况,结果显示重组干酪乳杆菌表达系统能刺激动物黏膜免疫反应和系统免疫反应。     

The nucleocapsid of hepatitis B virus is both a T-cell-independent and a T-cell-dependent antigen

One characteristic of the immune response during hepatitis B virus (HBV) infection in humans is the vigorous production and subsequent persistence of antibodies of immunoglobulin (Ig) classes M and G to the nucleocapsid antigen (HBcAg). In this study HBcAg was shown to be similarly immunogenic in mice. When injected into athymic (nude) B10.BR and athymic BALB/c mice, HBcAg induced IgM and IgG class antibodies to HBc in spite of the absence of T cells in nude mice. In euthymic mice, HBcAg efficiently stimulated T-cell proliferation in vitro and helper T-cell function in vivo. The dual functions of HBcAg as a T-cell-independent and a T-cell-dependent antigen may explain its enhanced immunogenicity. Denaturation of HBcAg yields a nonparticulate antigen designated HBeAg; when HBeAg was used as the immunogen, antibody production required helper T-cell function. Although HBcAg and HBeAg are serologically distinct, they are structurally related, and in these experiments were highly cross-reactive at the T-cell level. These results suggest that the elevated levels of IgM antibodies to HBc and the enhanced immunogenicity of HBcAg during HBV infection in humans reflect the ability of HBcAg to directly activate B cells to produce antibodies to HBc in the presence or absence of HBcAg- or HBeAg-sensitized T cells.     

Geometrical differences among homologous neurons in mammals

The dendritic arbors of sympathetic neurons in different species of mammals vary systematically: the superior cervical ganglion cells of smaller mammals have fewer and less extensive dendrites than the homologous neurons in larger animals. This difference in dendritic complexity according to body size is reflected in the convergence of ganglionic innervation; the ganglion cells of progressively larger mammals are innervated by progressively more axons. These relations have implications both for the function of homologous neural systems in animals of different sizes and for the regulation of neuronal geometry during development.