Rearrangement of microtubule polarity orientation during conversion of dendrites to axons in cultured pyramidal neurons

Axons and dendrites of neurons differ in the polarity orientation of their microtubules. Whereas the polarity orientation of microtubules in axons is uniform, with all plus ends distal, that in dendrites is nonuniform. The mechanisms responsible for establishment and maintenance of microtubule polarity orientation in neuronal processes remain unclear, however. We previously described a culture system in which dendrites of rat cortical neurons convert to axons. In the present study, we examined changes in microtubule polarity orientation in such dendrites. With the use of the hooking procedure and electron microscopy, we found that microtubule polarity orientation changed from nonuniform to uniform, with a plus end-distal arrangement, in dendrites that gave rise to axons during culture of neurons for 24 h. Microtubule polarity orientation remained nonuniform in dendrites that did not elongate. Axon regeneration at the dendritic tip thus triggered the disappearance of minus end-distal microtubules from dendrites. These minus end-distal microtubules also disappeared from dendrites during axon regeneration in the presence of inhibitors of actin polymerization, suggesting that actin-dependent transport of microtubules is not required for this process and implicating a previously unidentified mechanism in the establishment and maintenance of microtubule polarity orientation in neuronal processes.     

Role for the pleckstrin homology domain-containing protein CKIP-1 in phosphatidylinositol 3-kinase-regulated muscle differentiation

In this work, we report the implication of the pleckstrin homology (PH) domain-containing protein CKIP-1 in phosphatidylinositol 3-kinase (PI3-K)-regulated muscle differentiation. CKIP-1 is upregulated during muscle differentiation in C2C12 cells. We show that CKIP-1 binds to phosphatidylinositol 3-phosphate through its PH domain and localizes to the plasma membrane in a PI3-K-dependent manner. Activation of PI3-K by insulin or expression of an active form of PI3-K p110 induces a rapid translocation of CKIP-1 to the plasma membrane. Conversely, expression of the 3-phosphoinositide phosphatase myotubularin or PI3-K inhibition by LY294002, wortmannin, or mutant p85 abolishes CKIP-1 binding to the membrane. Upon induction of differentiation in low-serum medium, CKIP-1 overexpression in C2C12 myoblasts first promotes proliferation and then stimulates the expression of myogenin and cell fusion in a manner reminiscent of the dual positive effect of insulin-like growth factors on muscle cells. Interference with the PI3-K pathway impedes the effect of CKIP-1 on C2C12 cell differentiation. Finally, silencing of CKIP-1 by RNA interference abolishes proliferation and delays myogenin expression. Altogether, these data strongly implicate CKIP-1 as a new component of PI3-K signaling in muscle differentiation.     

An essential role for katanin in severing microtubules in the neuron

Several lines of evidence suggest that microtubules are nucleated at the neuronal centrosome, and then released for transport into axons and dendrites. Here we sought to determine whether the microtubule-severing protein known as katanin mediates microtubule release from the neuronal centrosome. Immunomicroscopic analyses on cultured sympathetic neurons show that katanin is present at the centrosome, but is also widely distributed throughout the neuron. Microinjection of an antibody that inactivates katanin results in a dramatic accumulation of microtubules at the centrosome, indicating that katanin is indeed required for microtubule release from the centrosome. However, the antibody also causes an inhibition of axon outgrowth that is more immediate than expected on this basis alone. It may be that katanin severs microtubules throughout the cell body to keep them sufficiently short to be efficiently transported into developing processes. Consistent with this idea, there were significantly fewer free ends of microtubules in the cell bodies of neurons that had been injected with the katanin antibody compared with controls. These results indicate that microtubule-severing by katanin is essential for releasing microtubules from the neuronal centrosome, and also for regulating the length of the microtubules after their release.     

Autosomal recessive inheritance of goiter in Dutch goats

The inheritance of congenital goiter due to a thyroglobulin synthesis defect in a strain of Dutch goats has been studied by Mendelian and biochemical methods. Mendelian analysis of 301 matings, resulting in 591 kids, showed an autosomal recessive mode of inheritance. A restriction fragment length polymorphism (RFLP) in the thyroglobulin gene also was used to confirm the recessive mode of inheritance of the defect. In a pedigree consisting of 27 goats, spanning four generations, the genotype determined by RFLP study was in accordance with the observed phenotype and the autosomal inheritance of the defect. Although phenotypically no differences were detected between normal and heterozygous animals, the use of RFLPs allowed the diagnosis of the three genotypes.     

Human immunodeficiency virus seroconversion presenting with acute inflammatory demyelinating polyneuropathy: a case report

Introduction

Electrocardiogram (ECG) abnormalities in patients with blunt chest trauma are diverse and non-specific, but may be indicative of potentially life-threatening conditions.

Case presentation

We report a rare case of pneumopericardium with extreme ECG abnormalities after blunt chest trauma in a 22-year-old male. The diagnosis was confirmed using computed tomography (CT) scanning. The case is discussed, together with its differential diagnosis and the aetiology of pneumopericardium and tension pneumopericardium.

Conclusion

Pneumopericardium should be distinguished from other pathologies such as myocardial contusion and myocardial infarction because of the possible development of tension pneumopericardium. Early CT scanning is important in the evaluation of blunt chest trauma.     

Oxygen depletion during and after mTHPC-mediated photodynamic therapy in RIF1 and H-MESO1 tumors

During photodynamic therapy (PDT), low oxygenation levels, induced both by oxygen consumption and by vascular occlusion, can lead to an inefficient photochemical reaction that may compromise the efficacy of PDT. In the present studies, tumor oxygenation was measured before, during and after meta-tetrahydroxyphenylchlorin (mTHPC)-mediated PDT of murine RIF1 tumors and human mesothelioma xenografts (H-MESO1). Tumor pO2 was measured in real time with Eppendorf polarography, and the extent of relative hypoxia at specific times was measured by immunohistochemical staining. Significant decreases in median pO2 values, as well as an increase in the number of values below 2.5 mmHg, were seen during and after PDT in RIF1 tumors, although there was a large intertumoral variation. Tumor pO2 values did not change significantly in H-MESO1 tumors. Staining with antibodies against the hypoxia marker EF3 showed significant increases in relative hypoxia after PDT in both tumor types compared with separate groups of untreated controls. Our results are consistent with PDT-induced oxygen depletion (reduced pO2) leading to an increase in relative hypoxia in RIF1 tumors. Extensive necrosis in the H-MESO1 tumors may have prevented the detection of PDT-induced hypoxia using the Eppendorf polarographic needle, whereas immunohistochemistry did reveal increases in relative hypoxia.     

Leaf anatomy of Ficus subsection Urostigma (Moraceae)

Species of Ficus subsection Urostigma show much overlapping variation in vegetative morphology, which often precludes correct identification of the species. The aim of this study was to describe the leaf anatomical characters and their variation and to check their suitability for identification. Included were 41 samples belonging to 25 species of subsection Urostigma, four samples belonging to two species of section Leucogyne and one specimen of Ficus glaberrima subsp. siamensis of subsection Conosycea. Transverse sections of lamina, midrib and petiole and cuticular macerations were used, and the observed anatomical characters are described for each species. On the basis of a limited number of studied samples, leaf anatomy shows little variation within each species and each species has a unique combination of character states, facilitating identification. Ficus arnottiana shows some leaf anatomical characters that are quite different from those of other members of subsection Urostigma, including a multiple epidermis and enlarged lithocysts on both sides of the leaf. Both characters are generally considered as typical for Ficus subsection Conosycea. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 175 , 259–281.     

Identification of a Microtubule-associated Motor Protein Essential for Dendritic Differentiation

The quintessential feature of the dendritic microtubule array is its nonuniform pattern of polarity orientation. During the development of the dendrite, a population of plus end–distal microtubules first appears, and these microtubules are subsequently joined by a population of oppositely oriented microtubules. Studies from our laboratory indicate that the latter microtubules are intercalated within the microtubule array by their specific transport from the cell body of the neuron during a critical stage in development (Sharp, D.J., W. Yu, and P.W. Baas. 1995. J. Cell Biol. 130:93– 104). In addition, we have established that the mitotic motor protein termed CHO1/MKLP1 has the appropriate properties to transport microtubules in this manner (Sharp, D.J., R. Kuriyama, and P.W. Baas. 1996. J. Neurosci. 16:4370–4375). In the present study we have sought to determine whether CHO1/MKLP1 continues to be expressed in terminally postmitotic neurons and whether it is required for the establishment of the dendritic microtubule array. In situ hybridization analyses reveal that CHO1/MKLP1 is expressed in postmitotic cultured rat sympathetic and hippocampal neurons. Immunofluorescence analyses indicate that the motor is absent from axons but is enriched in developing dendrites, where it appears as discrete patches associated with the microtubule array. Treatment of the neurons with antisense oligonucleotides to CHO1/MKLP1 suppresses dendritic differentiation, presumably by inhibiting the establishment of their nonuniform microtubule polarity pattern. We conclude that CHO1/MKLP1 transports microtubules from the cell body into the developing dendrite with their minus ends leading, thereby establishing the nonuniform microtubule polarity pattern of the dendrite.     

Characterization of bone-implant fixation using modal analysis: Application to a press-fit implant model

Orthopaedic implant fixation is strongly dependant upon the effective mechanical properties of newly formed tissue. In this study, we evaluated the potential of modal analysis to derive viscoelastic properties of periprosthetic tissue. We hypothesized that Young's modulus and loss factor could be obtained by a combined theoretical, computational and experimental modal analysis approach. This procedure was applied to ex vivo specimens from a cylindrical experimental implant placed in cancellous bone in an unloaded press-fit configuration, obtained after a four week observation period. Four sections each from seven textured titanium implants were investigated. The first resonant frequency and loss factor were measured. Average experimentally determined loss factor was 2% (SD 0.4%) and average first resonant frequency was 2.1 KHz (SD: 50). A 2D axisymmetric finite element (FE) model identified effective Young's modulus of tissue using experimental resonant frequencies as input. Average value was 42 MPa (SD: 2.4) and no significant difference between specimens was observed.In this pilot study, the non-destructive method allowed accurate measure of dynamic loss factor and resonant frequency and derivation of effective Young's modulus. Prior to implementing this dynamic protocol for broader mechanical evaluation of experimental implant fixation, further work is needed to determine if this affects results from subsequent destructive shear push-out tests.     

Effects of dynactin disruption and dynein depletion on axonal microtubules

We investigated potential roles of cytoplasmic dynein in organizing axonal microtubules either by depleting dynein heavy chain from cultured neurons or by experimentally disrupting dynactin. The former was accomplished by siRNA while the latter was accomplished by overexpressing P50-dynamitin. Both methods resulted in a persistent reduction in the frequency of transport of short microtubules. To determine if the long microtubules in the axon also undergo dynein-dependent transport, we ascertained the rates of EGFP-EB3 "comets" observed at the tips of microtubules during assembly. The rates of the comets, in theory, should reflect a combination of the assembly rate and any potential transport of the microtubule. Comets were initially slowed during P50-dynamitin overexpression, but this effect did not persist beyond the first day and was never observed in dynein-depleted axons. In fact, the rates of the comets were slightly faster in dynein-depleted axons. We conclude that the transient effect of P50-dynamitin overexpression reflects a reduction in microtubule polymerization rates. Interestingly, after prolonged dynein depletion, the long microtubules were noticeably misaligned in the distal regions of axons and failed to enter the filopodia of growth cones. These results suggest that the forces generated by cytoplasmic dynein do not transport long microtubules, but may serve to align them with one another and also permit them to invade filopodia.