An immunohistochemical study of neuronal microtubule-associated proteins in Hirschsprung's disease

Neuronal microtubule-associated proteins (MAPs) are important components of neurons and are believed to regulate neuronal function and development by controlling the assembly of microtubules and the interaction of microtubules with other cytoplasmic organelles. We studied the immunohistochemical localization of MAPs 1, 2, 5, and tau in the intestinal tissues of five patients with Hirschsprung's disease and in five normal controls using monoclonal antibodies. Microtubule-associated proteins 5 and tau proved to be excellent enteric neuronal markers; they were specifically located in the nerve cell bodies and processes of normal intestine as well as in the abnormal hypertrophied nerve fibers of aganglionic colon. Fine fibrillar structures in the neuroplasm were revealed in greater detail than were those obtained from studies with conventional markers, including neuron-specific enolase, S-100 protein, and neurofilament protein. A slight reduction of MAPs 5 and tau immunoreactivity was observed in the aganglionic colon compared with normal colon. Microtubule-associated proteins 1 and 2 were absent from the nerve fibers in both normal and aganglionic colon. This study suggests that immunostaining for MAPs 5 and tau may be superior to other immunohistochemical methods for diagnosing Hirschsprung's disease; however, in view of its limited retrospective nature these findings need to be corroborated by a large prospective evaluation.     

Improving the flexural rigidity of cold-finished steel

The influence of reduction on the distribution of axial, tangential, and radial residual stress over the cross section of cold-finished steel rods is experimentally studied. Computer modeling of the flexural rigidity of the rods indicates that, with 5–34% reduction, which is the range used in practice, the rigidity of the metal may be almost doubled. It is inexpedient to increase the rigidity of the rods by additional slight reduction, since that would lower the residual tensile stress.     

Protein phosphatase 1 is targeted to microtubules by the microtubule-associated protein Tau

Phosphorylation has been implicated in the regulation of microtubule (MT) stability and function by controlling the interactions between MTs and MT-associated proteins. We found previously that protein phosphatase inhibitors selectively break down stable MTs, suggesting that protein phosphatases may be involved in regulating MT stability. To identify the protein phosphatases involved, we examined purified calf brain MTs and found a protein phosphatase activity that copurified with MTs to constant stoichiometry. Western blot analysis and inhibitor profiles demonstrated that the MT-associated phosphatase was a type 1 protein phosphatase (PP1), which we named PP1MT. Recombinant PP1 catalytic subunit (PP1c) did not bind to MTs, whereas PP1MT did bind, suggesting the presence of proteins that target PP1 to MTs. By Sepharose CL-6B chromatography, the phosphatase activity of PP1MT eluted as a large protein complex of approximately 400 kDa. High salt (2 M NaCl) treatment followed by CL-6B chromatography dissociated PP1MT into PP1c and the MT-targeting subunit(s). The MT-targeting subunit was shown to be the MT-associated protein tau by PP1 blot overlays and other assays. Also, recombinant tau reconstituted the binding of PP1c to MTs. These results identify PP1 as the first tau binding protein and suggest that tau is a novel PP1-targeting subunit.     

Localization of postsynaptic density-93 to dendritic microtubules and interaction with microtubule-associated protein 1A

Postsynaptic density-93 (PSD-93)/Chapsyn-110 is a member of the membrane-associated guanylate kinase (MAGUK) family of PDZ domain-containing proteins. MAGUKs are widely expressed in the brain and are critical elements of the cytoskeleton and of certain synapses. In the ultrastructural studies that are described here, PSD-93 localizes to both postsynaptic densities and dendritic microtubules of cerebellar Purkinje neurons. The microtubule localization is paralleled by a high-affinity in vivo interaction of PSD-93 via its guanylate kinase (GK) domain with microtubule-associated protein 1A (MAP1A). GK domain truncations that mimic genetically identified mutations of a Drosophila MAGUK, discs-large, disrupt the GK/MAP-1A interaction. Additional biochemical experiments demonstrate that intact MAGUKs do not bind to MAP1A as effectively as do isolated GK domains. This appears to be attributable to an intramolecular inhibition of the GK domain by the PDZs, because GK binding activity of full-length MAGUKs is partially restored by a variety of PDZ ligands, including the C termini of NMDA receptor 2B, adenomatous polyposis coli (APC), and CRIPT. Beyond demonstrating a novel cytoskeletal link for PSD-93, these experiments support a model in which intramolecular interactions between the multiple domains of MAGUKs regulate intermolecular associations and thereby may play a role in the proper targeting and function of MAGUK proteins.     

Three-dimensional structure of functional motor proteins on microtubules

BACKGROUND: Kinesins are a superfamily of motor proteins that use ATP hydrolysis to fuel movement along microtubules and participate in many crucial phases of the eukaryotic cell cycle. Usually these motors are heterotetramers of two heavy and two light chains, and have globular motor domains on the two heavy chains. Most kinesins move towards the microtubule 'plus end', but some, such as ncd (nonclaret disjunctional protein), move in the opposite direction. Heavy chain dimers produced by overexpression are viable motors. RESULTS: In order to establish whether the opposite directionality of kinesin and ncd dimers is related to notable conformational differences, we have used electron cryo-microscopy and three-dimensional reconstruction methods to investigate the structure of kinesin and ncd dimers attached to microtubules in the presence of AMP-PNP (5'-adenylylimidodiphosphate), a nonhydrolyzable ATP analogue. Three-dimensional maps of the motor-microtubule complexes show the motors to have one unattached, and one attached head per tubulin dimer. The polarity of the reconstructions was determined for each individual microtubule. Attachment occurs on the crest of a protofilament at the end of the tubulin dimer that points towards the plus end of the microtubule. The attached head extends over the next tubulin molecule along the protofilament. The unattached heads of kinesin and ncd have distinctly different conformations. CONCLUSIONS: The attached heads of kinesin and ncd appear to be similar and to interact with the same region of the plus end-oriented tubulin subunits. The free heads, however, are quite different, which suggests that directionality could be determined by differences in the dimer conformations. Work is in progress to obtain three-dimensional maps in the presence of different nucleotides with the aim of understanding how these motors move along microtubules.     

Modulation of phosphorylation of neuronal cytoskeletal proteins by neuronal depolarization

A carbocyclic analogue of distamycin was obtained, in which the N-methylpyrrole rings were substituted by disubstituted benzene rings. Additionally, N-chloro- or N-bromoacetyl groups, displaying alkylating properties, were introduced. The synthesis, starting from 3,5-dinitrobenzoyl chloride, consisted of five stages.     

Biochemical characterization of mapmodulin, a protein that binds microtubule-associated proteins

Mapmodulin is a 31-kDa protein that stimulates the microtubule- and dynein-dependent localization of Golgi complexes in semi-intact Chinese hamster ovary cells. We have shown previously that it binds the microtubule binding domains of the microtubule-associated proteins, MAP2, MAP4, and tau. We also showed that mapmodulin is identical to a protein named PHAPI (Vaesen, M., Barnikol-Watanabe, S. , G?tz, H., Awni, L.A., Cole, T., Zimmermann, B., Kratzin, H.D. and Hilschmann, N. (1994) Biol. Chem. Hoppe-Seyler 375, 113-126). We report here that mapmodulin is a phosphoprotein that is predominantly cytosolic but is also found peripherally associated with endoplasmic reticulum and Golgi membranes in mammalian cells. The protein occurs as a trimer in cytosol, and phosphorylation is required for its microtubule-associated protein-binding activity. Heat treatment of nonphosphorylated mapmodulin can render it competent for binding to microtubule-associated proteins, suggesting that phosphorylation induces a conformational change in mapmodulin. Finally, despite identity in polypeptide sequence with a protein reported to act as an inhibitor of protein phosphatase 2A, native mapmodulin was not able to inhibit protein phosphatase 2A in Chinese hamster ovary cell cytosol.     

Tau proteins bind to kinesin and modulate its activation by microtubules

Relationships with family and friends by 439 heroin addicts during the first 3 months of drug abuse treatment were examined in relation to behavioral improvements of clients. Family conflict and peer deviance were significant predictors of injection frequency and illegal activity during treatment, and reductions in family conflict were associated with lower drug use, injection frequency, and illegal activity during treatment. These results provide support for treatment emphasis on helping clients reduce conflict among family members, improve dysfunctional relationships with peers, and replace deviant friendships with others that encourage treatment participation and conformance to social norms.     

Quantitation of microvascular plasma perfusion and neuronal microtubule-associated protein in ischemic mouse brain by laser-scanning confocal microscopy

In an exposition of the technique of calculating distribution volumes from laser-scanning confocal microscopic (LSCM) data, three-dimensional images of the distribution of one or two fluorescent markers in mouse brain specimens were generated by LSCM and processed by a system developed for morphometric analysis of fixed and stained serial brain histologic samples. To determine the volume of perfused cerebral capillaries, one of two fluorescent plasma markers, either fluorescein isothiocyanate (FITC)-dextran or Evans blue, was intravenously administered to mice subjected to 1 hour of embolic middle cerebral artery (MCA) occlusion (n = 9) and to mice that were not operated on (n = 3); after 1 minute of circulation, brains were removed, immersion-fixed, and processed for LSCM. In some of these animals (n = 5), the volume of endogenous microtubule-associated protein-2 (MAP2) fluorescence was also determined using immunohistochemical staining. For mice that were not operated on, this methodology yielded highly localized volumes of (1) microvascular plasma, which agree with those determined for rodents by other techniques, and (2) MAP2 expression, which appears physiologically and morphologically reasonable. After 1 hour of MCA occlusion, the MAP2 volumes of distribution were less than 10% of normal in the ipsilateral hemisphere in which plasma perfusion essentially ceased. In conclusion, precise colocalization and quantitation of early ischemic neuronal damage and cerebral plasma perfusion deficit can be done with this three-dimensional, microphysiologic and microanatomic methodology.     

Changes in mRNA abundance of microtubule-associated proteins in the rat brain following electroconvulsive shock

STUDY DESIGN: Case report and review of the literature. OBJECTIVE: To describe a 72-year-old man with thoracic spinal angiomyolipoma in the ventral aspect of the epidural space and extracanal extension to the posterior mediastinum, to discuss the clinical and radiologic features and unique biologic behavior of this entity, and to review of the literature on angiolipoma and angiomyolipoma. SUMMARY OF BACKGROUND DATA: Spinal angiolipoma and angiomyolipoma are rare tumors, which are localized almost exclusively in the dorsal epidural space of the thoracic spine. Most reported cases have no tendency to involve the surrounding tissue. METHODS: The authors describe the radiologic, surgical, and pathologic findings of this patient and review the findings from other reported cases. RESULTS: Anterior decompression was performed using a right transthoracic incision, and the neurologic symptoms improved immediately. There were no signs of recurrence of the tumor or neurologic deficit within a 2-year follow-up period. CONCLUSION: Results of a literature survey of these tumors support management by prompt and radical surgical intervention for long-term cure, even in cases in which the infiltrating nature is recognized.     

Differences in the regulation of microtubule dynamics by microtubule-associated proteins MAP1B and MAP2

The regulation of microtubule dynamics in vitro by microtubule-associated proteins (MAPs) was examined, using purified porcine MAP1B and MAP2. MAP1B has a significantly smaller effect on the observed critical concentration for microtubule assembly than MAP2. Assembly is faster in the presence of either MAP, and the resulting microtubules are shorter, indicating that nucleation is substantially promoted by the MAPs. Both MAPs stabilise the microtubule lattice as observed from podophyllotoxin-induced disassembly, but the effect of MAP1B is weaker than the effect of MAP2. At steady-state of assembly MAP1B still allows microtubule dynamic instability to occur as inferred from microtubule length changes. The comparison of the effects of MAP1B and MAP2 indicates that the reduction of the observed critical concentration is attributable to the reduction of the depolymerisation rate and correlates with the extent of suppression of dynamic instability. Numerical simulations illustrate that microtubule dynamics are strongly influenced by relatively small changes in the strength of a limited subset of subunit interactions in the lattice. The observed characteristic differences between the MAPs may be important for the regulation of distinct populations of microtubules which coexist in the same cell, where differences in stability and dynamics may be essential for their different spatial roles as, for example, in developing neurons.     

Association of neurofilament proteins with neuronal Cdk5 activator

Cdk5 exists in brain extracts in multiple forms, one of which is a macromolecular protein complex comprising Cdk5, neuron-specific Cdk5 activator p35nck5a and other protein components (Lee, K.-Y., Rosales, J. L., Tang, D., and Wang, J.H. (1996) J. Biol. Chem. 271, 1538-1543). The yeast two-hybrid system was employed to identify p35nck5a-interacting proteins from a human brain cDNA library. One of the isolated clones encodes a fragment of glial fibrillary acidic protein, which is a glial-specific protein. Sequence alignment revealed significant homology between the p35nck5a-binding fragment of glial fibrillary acidic protein and corresponding regions in neurofilaments. The association between p35nck5a and neurofilament medium molecular weight subunit (NF-M) was confirmed by both the yeast two-hybrid assay and direct binding of the bacteria-expressed proteins. The p35nck5a binding site on NF-M was mapped to a carboxyl-terminal region of the rod domain, in close proximity to the putative Cdk5 phosphorylation sites in NF-M. A region immediately amino-terminal to the kinase-activating domain in p35nck5a is required for its binding with NF-M. In in vitro binding assays, NF-M binds both monomeric p35nck5a and the Cdk5/p35nck5a complex. The binding of NF-M has no effect on the kinase activity of Cdk5/p35nck5a.     

14-3-3 proteins in neuronal development and function

The 14-3-3 proteins are small, cytosolic, evolutionarily conserved proteins expressed abundantly in the nervous system. Although they were discovered more than 30 yr ago, their function in the nervous system has remained enigmatic. Several recent studies have helped to clarify their biological function. Crystallographic investigations have revealed that 14-3-3 proteins exist as dimers and that they contain a specific region for binding to other proteins. The interacting proteins, in turn, contain a 14-3-3 binding motif; proteins that interact with 14-3-3 dimers include PKC and Raf, protein kinases with critical roles in neuronal signaling. These proteins are capable of activating Raf in vitro, and this role has been verified by in vivo studies in Drosophila. Most interestingly, mutations in the Drosophila 14-3-3 genes disrupt neuronal differentiation, synaptic plasticity, and behavioral plasticity, establishing a role for these proteins in the development and function of the nervous system.     

Regulation of cytoskeletal proteins by thyroid hormone during neuronal maturation and differentiation

Nicotine is known to have multiple effects on neuroendocrine, autonomic, and behavioral responses. Its neuroendocrine effect on the stress-responsive hormone, ACTH, depends on central pathways that act on corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus of the hypothalamus (PVN). Other CRH neurons throughout the brain also are involved in coordinating aspects of the stress response, but very little is known about the effect of nicotine on CRH neurons in extrahypothalamic regions that are involved in the autonomic and behavioral responses to stress. The current study sought to determine the extent of nicotinic activation of extrahypothalamic CRH neurons, since these neurons may be involved in mediating the central effects of nicotine. Freely moving rats were pretreated with a low dose of colchicine, infused with nicotine (0.045 mg/kg/30 s or 0.135 mg/kg/90 s, i.v.), and cardiac perfused 1 h later. Double-label immunocytochemistry identified the activated (positive for cFos protein) CRH neurons in limbic structures (bed nucleus of the stria terminalis [BNST] and central nucleus of the amygdala [CNA]), the dorsal raphe (DR), and Barrington's nucleus (BN); comparisons were made to the PVN. In all of these areas, nicotine activated CRH neurons in a dose-dependent manner, showing differential sensitivity and efficacy with respect to region. CNA CRH neurons were most responsive and were maximally stimulated by the low dose of nicotine (62% of CRH neurons were cFos+, compared to 10-27% of the CRH population in other regions, including the PVN). Although the BNST also was activated by the low dose, only the non-CRH+ neurons were involved; in contrast, 41% of the BNST CRH neurons responded to the higher dose. Nicotinic activation of DR neurons was dose-dependent, with 22% of the CRH neurons activated by the high dose. Few BN neurons were activated by the low dose of nicotine, but 26% of the CRH population responded to the higher dose. These results indicate that the effect(s) of nicotine on the brain may be mediated, in part, by the selective activation of specific extrahypothalamic regions containing CRH neurons that also are involved in autonomic and behavioral responses to stress. The large fraction of CRH neurons responding to the low dose of nicotine in the CNA suggests that this limbic region may be particularly important in mediating these CNS effects of nicotine.     

Morphological transformation of liposomes caused by assembly of encapsulated tubulin and determination of shape by microtubule-associated proteins (MAPs)

To examine the role of cytoskeletons in cellular morphogenesis, we generated liposomes encapsulating tubulin, with or without microtubule-associated proteins (MAPs), and observed their transformation using dark-field microscopy. When tubulin was polymerized with MAPs in liposomes, liposomes were transformed into a "bipolar" shape with a central sphere and two tubular membrane protrusions that aligned in a straight line. On the other hand, when pure tubulin was polymerized in liposomes without MAPs, they initially transformed into a bipolar shape but subsequently re-transformed into a "monopolar" shape, i.e. a sphere with only one straight tubular portion. This re-transformation occurred in two ways: first, by shortening of one of the tubular portions due to microtubule disassembly; or second, by fluctuation of the central sphere toward one of the ends without shortening of the tube portion. MAPs prevented this re-transformation, and their role in stabilizing the shape of transformed liposomes was studied by the co-sedimentation method. The results show that MAPs, particularly MAP1 and MAP2, mediate binding between microtubules and the liposomal membrane. However, MAP2 by itself did not bind to liposomes, but was able to stabilize bipolar liposomes. This stabilization is caused not only by direct links between microtubules and liposomes, but also by prevention of Brownian motion of microtubules through an increase in friction.     

Sulphonate buffers affect the recovery of microtubule-associated proteins MAP1 and MAP2: evidence that MAP1A promotes microtubule assembly

The influence of two commonly used sulphonate buffers, PIPES and MES, on the in vitro assembly of bovine brain microtubule protein was examined. Microtubule assembly was monitored by turbimetry and, after centrifugation, the polymerised protein was analysed by SDS-PAGE and western blotting. Assembly in MES when compared with PIPES resulted in a higher recovery of microtubule proteins at both pH 6.4 and pH 6.9 and in an altered protein composition. The buffer pH affected the total amount of protein polymerised but did not significantly affect the protein composition. At both pH conditions the recovery of HMW-MAPs was markedly increased in MES buffer and this increase was mostly due to an increase in the amount of MAP1.     

Problem-solving rigidity and decision time.

"The present study failed to reveal any significant relationship between rigidity and decision time… . The present results suggest that attempts to explain behavioral rigidity may have to resort to more complex concepts than impulsivity and caution." (PsycINFO Database Record (c) 2010 APA, all rights reserved)