Real time imaging of calcium-induced localized proteolytic activity after axotomy and its relation to growth cone formation

The emergence of a neuronal growth cone from a transected axon is a necessary step in the sequence of events that leads to successful regeneration. Yet, the molecular mechanisms underlying its formation after axotomy are unknown. In this study, we show by real time imaging of the free intracellular Ca2+ concentration, of proteolytic activity, and of growth cone formation that the activation of localized and transient Ca2+-dependent proteolysis is a necessary step in the cascade of events that leads to growth cone formation. Inhibition of this proteolytic activity by calpeptin, a calpain inhibitor, abolishes growth cone formation. We suggest that calpain plays a central role in the reorganization of the axon's cytoskeleton during its transition from a stable differentiated structure into a dynamically extending growth cone.     

Localized and transient elevations of intracellular Ca2+ induce the dedifferentiation of axonal segments into growth cones

The formation of a growth cone at the tip of a severed axon is a key step in its successful regeneration. This process involves major structural and functional alterations in the formerly differentiated axonal segment. Here we examined the hypothesis that the large, localized, and transient elevation in the free intracellular calcium concentration ([Ca2+]i) that follows axotomy provides a signal sufficient to trigger the dedifferentiation of the axonal segment into a growth cone. Ratiometric fluorescence microscopy and electron microscopy were used to study the relations among spatiotemporal changes in [Ca2+]i, growth cone formation, and ultrastructural alterations in axotomized and intact Aplysia californica neurons in culture. We report that, in neurons primed to grow, a growth cone forms within 10 min of axotomy near the tip of the transected axon. The nascent growth cone extends initially from a region in which peak intracellular Ca2+ concentrations of 300-500 microM are recorded after axotomy. Similar [Ca2+]i transients, produced in intact axons by focal applications of ionomycin, induce the formation of ectopic growth cones and subsequent neuritogenesis. Electron microscopy analysis reveals that the ultrastructural alterations associated with axotomy and ionomycin-induced growth cone formation are practically identical. In both cases, growth cones extend from regions in which sharp transitions are observed between axoplasm with major ultrastructural alterations and axoplasm in which the ultrastructure is unaltered. These findings suggest that transient elevations of [Ca2+]i to 300-500 microM, such as those caused by mechanical injury, may be sufficient to induce the transformation of differentiated axonal segments into growth cones.     

Spinule-type neurite outgrowth from horizontal cells during light adaptation in the carp retina: an actin-dependent process

Dendrites of horizontal cells in the carp retina which invaginate the cone pedicles form numerous spinules during light adaptation. We have analyzed the contribution of cytoskeletal elements to this process. Isolated horizontal cells and frozen sections were screened with phalloidin for the existence of F-actin. F-actin was present in all types of horizontal cells and particularly enriched in the distal parts of the dendrites. Electron microscopical analysis demonstrated that interruption of the F-actin polymerization with cytochalasin B inhibited the formation of spinules during light adaptation. The persistence of spinules was also affected. Cytochalasin B also prevented the light-independent, phorbol ester-induced formation of spinules. Cytochalasin B only affected the morphology of the lateral, spinule-forming dendrites of cone horizontal cells within the cone pedicles, leaving the central, non spinule-forming dendrites of cone horizontal cells and the processes of rod horizontal cells within rod spherules unaffected. Whereas cytochalasin B prevented the protrusion of spinules, the spinule-associated membrane densities were only slightly affected. The two main characteristics of spinules, protrusion and membrane densities are therefore independently regulated processes.     

The Hertzian stress field and formation of cone cracks—I. Theoretical approach

A complete three-dimensional solution has been derived for the Hertzian stress field. The solution was used to define an expression for the largest tensile stress under a spherical indenter. A numerical method was developed to solve the fracture mechanics equation related to cone crack formation, leading to a simple expression for fracture toughness. Examination of the relation between load, cone crack size and stress intensity showed that the critical stress intensity factor is independent of load and crack size. This suggests a new method to determine fracture toughness of brittle materials using Hertzian indentation.     

Interstitial branches develop from active regions of the axon demarcated by the primary growth cone during pausing behaviors

Interstitial branches arise from the axon shaft, sometimes at great distances behind the primary growth cone. After a waiting period that can last for days after extension of the primary growth cone past the target, branches elongate toward their targets. Delayed interstitial branching is an important but little understood mechanism for target innervation in the developing CNS of vertebrates. One possible mechanism of collateral branch formation is that the axon shaft responds to target-derived signals independent of the primary growth cone. Another possibility is that the primary growth cone recognizes the target and demarcates specific regions of the axon for future branching. To address whether behaviors of the primary growth cone and development of interstitial branches are related, we performed high-resolution time-lapse imaging on dissociated sensorimotor cortical neurons that branch interstitially in vivo. Imaging of entire cortical neurons for periods of days revealed that the primary growth cone pauses in regions in which axon branches later develop. Pausing behaviors involve repeated cycles of collapse, retraction, and extension during which growth cones enlarge and reorganize. Remnants of reorganized growth cones are left behind on the axon shaft as active filopodial or lamellar protrusions, and axon branches subsequently emerge from these active regions of the axon shaft. In this study we propose a new model to account for target innervation in vivo by interstitial branching. Our model suggests that delayed interstitial branching results directly from target recognition by the primary growth cone.     

The soluble N-ethylmaleimide-sensitive factor attached protein receptor complex in growth cones: molecular aspects of the axon terminal development

Soluble N-ethylmaleimide-sensitive factor attached protein (SNAP) receptor (SNARE) mechanisms are thought to be involved in two important processes in axonal growth cones: (1) membrane expansion for axonal growth and (2) vesicular membrane fusion for mature synaptic transmission. We investigated the localization and interactions among the proteins involved in SNARE complex formation in isolated growth cone particles (GCP) from forebrain. We demonstrated that the SNARE complex is present in GCPs morphologically without synaptic vesicles (SVs) and associated with growth cone vesicles. However, the apparently SV-free GCP was lacking in the regulatory mechanisms inhibiting SNARE complex formation proposed in SV fusion, i.e., the association of synaptotagmin with the SNARE complex, and vesicle-associated membrane protein (VAMP)-synaptophysin complex formation. The core components of the SNARE complex (syntaxin, SNAP-25, and VAMP) accumulated for several days before postnatal day 7, when SVs first appeared, and preceded the accumulation of marker proteins such as synaptophysin, SV2, and V-ATPase. Our present results suggest that the SNARE mechanism for vesicular transmitter release is not fully functional in growth cones before the appearance of SVs, but the SNARE mechanism is working for membrane expansion in growth cones, which supports our recent report. We concluded that the regulation of the SNARE complex in growth cones is different from that in mature presynaptic terminals and that this switching may be one of the key steps in development from the growth cone to the presynaptic terminal.     

Risk factors for cervical presence of human papillomavirus DNA among women at risk for HIV infection. DIANAIDS Collaborative Study Group

Teleost retinas adapted to light show numerous spinules invaginated in the cone pedicles whereas darkness induces a reduction in the number of spinules. Horizontal cells show nematosomes whose size decreases as the number of spinules increases. We have investigated the involvement of actin filaments in spinule formation, by using cytochalasin D through intraocular injection into an eye. The ultrastructural analysis reveals that cytochalasin D impairs spinule formation and nematosome-size reduction in both treated and contralateral untreated retinas.     

Development of cell markers in subretinal rabbit retinal transplants

Retinas from embryonic rabbits at day E15 were transplanted to the subretinal space in adult rabbits. After survival times between 7 and 193 days, the rabbits were killed, and the transplants were processed for immunohistochemistry. The results show that subretinal transplants from embryonic rabbit retinas develop many, if not all, retinal neuronal types. The cells show approximately normal morphology and express a variety of cell-type-specific markers: photoreceptor cells express visual pigment proteins as identified by antibodies against rhodopsin (R2-15), color-specific cone pigments (COS-1, OS-2) and the cone specific antigen 50-1B11, rod bipolar cells express PKC, horizontal cells HPC-1 antigen and neurofilament 160 kDa, amacrine cells HPC-1 antigen, GABA and neurofilament 160 kDa, and glial cells express vimentin and glial fibrillary acidic protein. The high degree of rosette formation seen in many young grafts, diminishes with time; many transplant cells disappear, and the remaining cells present a less prominent formation of rosettes.     

二辊斜轧延伸大口径P92厚壁管分层缺陷倾向性研究

为研究不同轧辊转速、送进角、轧辊人口锥角等工艺参数对大VI径厚壁P92钢管二辊斜轧延伸过程分层缺陷形成倾向性的影响,借助于商用有限元软件MSC．SuperForm,对不同工艺条件下大口径厚壁P92钢管二辊斜轧延伸过程进行了三维热力耦合模拟;采用Oyane韧性断裂准则分析了轧件损伤场及钢管分层缺陷的倾向性。研究结果表明：钢管内表邻近顶头接触区存在破裂高危带,轧件最大损伤特征值随轧辊转速的降低、送进角和轧辊入口锥角的增大而减小,发生分层缺陷的倾向性降低。此研究为揭示钢管分层缺陷形成机制,确定缺陷发生的敏感工况,制定防止或减轻分层缺陷的有效措施提供科学依据。     

Experimental studies on drop formation at the tip of melting rods

Drop formation at the conical tips of melting rods has been experimentally studied using the transparent wax-alcohol/acetonitrile system. The effects of cone angle, rod diameter, immersion depth, and bath temperature on the detached drop mass have been studied over a wide range, besides recording useful qualitative information based on visual observation. The experimental results suggest that the phenomenon of drop formation at the tip of melting rods has a close parallel with the drop formation at conical tips, at least on a qualitative basis. However, the results could not be quantified owing to difficulties in characterizing the physical properties of the system, despite efforts to minimize them. Formerly Senior Research Fellow with the Department of Metallurgy, Indian Institute of Science, Bangalore 560 012, India     

Median nerve neuropathy from an old lunate dislocation

We performed a histopathologic and immunohistochemical study of eyes obtained at autopsy of an 84-year-old man from a family with X-linked cone degeneration in which affected members have a 6.5-kilobase deletion in the red cone pigment gene. At his most recent ocular examination, at age 71 years, this patient had had a visual acuity of 20/200 OU, fundus changes suggestive of macular degeneration, borderline-normal full-field rod electroretinograms, and profoundly reduced full-field cone electroretinograms. Histopathologic examination demonstrated marked loss of cone and rod photoreceptors and the retinal pigment epithelium in the central macula. The peripheral cone population was reduced, while the peripheral rod population was relatively preserved. Immunohistochemical examination with an antibody to both red and green cone opsin and an antibody to blue cone opsin disclosed a prominent loss of the red and green cone population and preservation of the blue cone population. These findings show that a defect in the red cone pigment gene can result in extensive degeneration of the red and green cone population across the retina.     

Ultrastructure of spermiogenesis and the spermatozoon of Anoplocephaloides dentata (Cestoda, Cyclophyllidea, Anoplocephalidae), an intestinal parasite of Arvicolidae rodents

Spermiogenesis in Anoplocephaloides dentata begins with the formation of a differentiation zone delimited by a ring of arched membranes. This conical area shows 2 parallel centrioles with associated anterior reduced striated roots but without an intercentriolar body. Only 1 of the centrioles develops an axoneme that grows into a cytoplasmic extension. Two crestlike bodies appear when the nucleus initiates its migration along the spermatid body. We describe for the first time at the end of spermiogenesis the formation of an apical cone before the strangulation of the ring of arched membranes. The mature spermatozoon of A. dentata is filiform, tapered at both ends, and lacks mitochondria. Its anterior extremity has an apical cone measuring about 1,400 x 350 nm and 2 crestlike bodies. Cortical microtubules are spiralled at an angle of about 30 degrees to the spermatozoon axis. The axoneme, of the 9+1 pattern of Trepaxonemata (Polycladida, Seriata, "Typhloplanoida," "Dalyellioida," and Neodermata lacks a periaxonemal sheath and does not reach the extremities of the spermatozoon. Numerous granules of electron-dense material are observed in the posterior regions of each spermatozoon. Analysis of ultrastructural features found during spermiogenesis in A. dentata corroborates the presence of striated roots associated with the centrioles in cyclophyllidean species. Moreover, the presence of striated roots is described for the first time in type IV spermiogenesis.     

The photovoltage of macaque cone photoreceptors: adaptation, noise, and kinetics

Whole-cell voltage and current recordings were obtained from red and green cone photoreceptors in isolated retina from macaque monkey. It was demonstrated previously that the cone photovoltage is generated from two sources, phototransduction current in the cone outer segment and photocurrent from neighboring rods. Rod signals are likely transmitted to cones across the gap junctions between rods and cones. In this study, the "pure" cone and rod components of the response were extracted with rod-adapting backgrounds or by subtracting the responses to flashes of different wavelength equated in their excitation of either rods or cones. For dim flashes, the pure cone component was similar in waveform to the cone outer segment current, and the rod component was similar to the photovoltage measured directly in rods. With bright flashes, the high frequencies of the rod signal were filtered out by the rod/cone network. The two components of the cone photovoltage adapted separately to background illumination. The amplitude of the rod component was halved by backgrounds eliciting approximately 100 photoisomerizations sec-1 per rod; the cone component was halved by backgrounds of 8700 photoisomerizations sec-1 per cone. Coupling between rods and cones was not modulated by either dim backgrounds or dopamine. Voltage noise in dark-adapted cones was dominated by elementary events other than photopigment isomerizations. The dark noise was equivalent in magnitude to a steady light eliciting approximately 3800 photoisomerizations sec-1 per cone, a value significantly higher than the psychophysical estimates of cone "dark light."     

Urbanization elicits a more atherogenic lipoprotein profile in carriers of the apolipoprotein A-IV-2 allele than in A-IV-1 homozygotes

PURPOSE: To test the effects of disruption of a conserved cysteine in the green cone opsin molecule on light-activated isomerization, transducin activation, folding, transport, and protein half-life. METHODS: Stable cell lines were established by transfecting 293-EBNA cells with a plasmid containing wild-type or mutant (C203R, C203S, C126S, C126S/C203S) green opsin cDNA molecules. The proteins were induced by culturing the cells in the presence of cadmium chloride and analyzed by spectra, transducin activation, Western blotting, pulse-labeling with immunoprecipitation, and immunocytochemistry. RESULTS: The C203R mutation disrupts the folding and half-life of the green opsin molecule and its abilities to absorb light at the appropriate wavelength and to activate transducin. Similar disruption of folding, half-life, and light activation occurs when Cys203 or its presumed partner for formation of a disulfide bond (Cys126) is replaced by serine residues. CONCLUSIONS: Like rhodopsin, the folding of the cone opsins appears to be dependent on the formation of a disulfide bond between the third transmembrane helix and the second extracellular loop. Disruption of this disulfide bond represents a cause of color vision deficiencies that is unrelated to spectral shifts of the photopigment.     

Suc1-associated neurotrophic factor target (SNT) protein is a major FGF-stimulated tyrosine phosphorylated 90-kDa protein which binds to the SH2 domain of GRB2

Low pH enhances tumor necrosis factor alpha (TNF)-induced cytolysis of cancer cells and TNF-membrane interactions that include binding, insertion, and ion-channel formation. We have also found that TNF increases Na+ influx in cells. Here, we examined the structural features of the TNF-membrane interaction pathway that lead to channel formation. Fluorometric studies link TNF's acid-enhanced membrane interactions to rapid but reversible acquisition of hydrophobic surface properties. Intramembranous photolabeling shows that (i) protonation of TNF promotes membrane insertion, (ii) the physical state of the target bilayer affects the kinetics and efficiency of TNF insertion, and (iii) binding and insertion of TNF are two distinct events. Acidification relaxes the trimeric structure of soluble TNF so that the cryptic carboxyl termini, centrally located at the base of the trimer cone, become susceptible to carboxypeptidase Y. After membrane insertion, TNF exhibits a trimeric configuration in which the carboxyl termini are no longer exposed; however, the proximal salt-bridged Lys-11 residues as well as regional surface amino acids (Glu-23, Arg-32, and Arg-44) are notably more accessible to proteases. The sequenced cleavage products bear the membrane-restricted photoreactive probe, proof that surface-cleaved TNF has an intramembranous disposition. In summary, the trimer's structural plasticity is a major determinant of its channel-forming ability. Channel formation occurs when cracked or partially splayed trimers bind and penetrate the bilayer. Reannealing leads to a slightly relaxed trimeric structure. The directionality of bilayer penetration conforms with x-ray data showing that receptor binding to the monomer interfaces of TNF poises the tip of the trimeric cone directly above the target cell membrane.     

基于尾砂沉降与流变特性的深锥浓密机压耙分析

采用深锥相似模型动态沉降实验及流变参数测定方法研究深锥浓密机压耙原因.结果发现造成深锥浓密机压耙一方面是由全尾砂进料浓度和絮凝剂添加量波动造成全尾砂絮凝沉降效果不佳而引起的;另一方面是间歇式充填排料引起深锥中料浆浓度分布差异性增强,进而导致料浆流变参数突变引起的.通过对深锥压耙机理的研究,为深锥正常运行及事故预测和排除提供理论依据.      

Undetectable S cone electroretinogram b-wave in complete congenital stationary night blindness

AIMS: The short wavelength sensitive (S) cone electroretinograms (ERGs) were examined in two patients with the complete type of congenital stationary night blindness (CSNB). METHODS: Ganzfeld spectral flashes in the presence of strong white adapting fields were used to elicit the S cone ERGs. RESULTS: The S cone ERG b-wave was not detectable to short wavelength stimuli, while the mixed long (L) and middle (M) wavelength sensitive cone responses appeared normal in waveforms with normal amplitude in both patients. Both patients had normal colour vision on the Farnsworth Panel D-15. CONCLUSIONS: These ERG results indicated that the S cone system as well as rod system is more impaired in complete CSNB than the L and M cone system and that normal colour vision may not depend on a normal S cone ERG to full field stimuli.     

HP200型圆锥破碎机定锥总成异常旋转原因分析及改造实践

HP200型圆锥破碎机由于其破碎能力较强、处理能力较大等优点被广泛应用于金属矿山选矿厂、机制砂等生产企业的工艺流程中。针对大溶溪选矿厂在使用HP200型圆锥破碎机进行生产时出现的定锥总成异常顺时针旋转和排料口异常变小等问题,经过定点实地观察、调研总结了异常旋转的原因,提出了解决和预防异常旋转的措施,并对HP200型圆锥破碎机进行了改造。应用实践表明,通过增加锁紧缸的数量、稳定锁紧缸的油压和控制液压油的温度,能够很好地解决HP200型圆锥破碎机定锥总成异常旋转的问题。     

Photoreceptors in a primitive mammal, the South American opossum, Didelphis marsupialis aurita: characterization with anti-opsin immunolabeling

The retinas of placental mammals appear to lack the large number and morphological diversity of cone subtypes found in diurnal reptiles. We have now studied the photoreceptor layer of a South American marsupial (Didelphis marsupialis aurita) by peanut agglutinin labeling of the cone sheath and by labeling of cone outer segments with monoclonal anti-visual pigment antibodies that have been proven to consistently label middle-to-long wavelength (COS-1) and short-wavelength (OS-2) cone subpopulations in placental mammals. Besides a dominant rod population (max. = 400,000/mm2) four subtypes of cones (max. = 3000/mm2) were identified. The outer segments of three cone subtypes were labeled by COS-1: a double cone with a principal cone containing a colorless oil droplet, a single cone with oil droplet, and another single cone. A second group of single cones lacking oil droplets was labeled by OS-2 antibody. The topography of these cone subtypes showed striking anisotropies. The COS-1 labeled single cones without oil droplets were found all over the retina and constituted the dominant population in the area centralis located in the temporal quadrant of the upper, tapetal hemisphere. The population of OS-2 labeled cones was also ubiquitous although slightly higher in the upper hemisphere (200/mm2). The COS-1 labeled cones bearing an oil droplet, including the principal member of double cones, were concentrated (800/mm2) in the inferior, non-tapetal half of the retina. The two spectral types of single cones resemble those of dichromatic photopic systems in most placental mammals. The additional set of COS-1 labeled cones is a distinct marsupial feature. The presence of oil droplets in this cone subpopulation, its absence in the area centralis, and the correlation with the non-tapetal inferior hemisphere suggest a functional specialization, possibly for mesopic conditions. Thus, sauropsid features have been retained but probably with a modified function.     

Functional compartmentalization of the neuronal growth cone: determining calcium's place in signaling cascades

The growth cone is generally regarded as the basic unit of neuronal organization concerned with development of connections within the nervous system. The discussion below illustrates that the growth cone itself can be subdivided into distinct units of organization. It is this functional compartmentalization which enables the growth cone to read the molecular terrain it traverses and to convert this information into precise motor events. Our discussion will focus on the flow of information from the environment to the growth cone. In particular, we will follow signaling events from their remote processing within filopodia to the biological equivalent of a central processing unit in the core of the growth cone.