Differential Distribution of Tau Proteins in Developing Cat Cerebral Cortex and Corpus Callosum

During the postnatal development of cat visual cortex and corpus callosum the molecular composition of tau proteins varied with age. In both structures, they changed between postnatal days 19 and 39 from a set of two juvenile forms to a set of at least two adult variants with higher molecular weights. During the first postnatal week, tau proteins were detectable with TAU-1 antibody in axons of corpus callosum and visual cortex, and in some perikarya and dendrites in the visual cortex. At later ages, tau proteins were located exclusively within axons in all cortical layers and in the corpus callosum. Dephosphorylation of postnatal day 11 cortical tissue by alkaline phosphatase strongly increased tau protein immunoreactivity on Western blots and in numerous perikarya and dendrites in all cortical layers, in sections, suggesting that some tau forms had been unmasked. During postnatal development the intensity of this phosphate-dependent somatodendritic staining decreased, but remained in a few neurons in cortical layers II and III. On blots, the immunoreactivity of adult tau to TAU-1 was only marginally increased by dephosphorylation. Other tau antibodies (TAU-2, B19 and BR133) recognized two juvenile and two adult cat tau proteins on blots, and localized tau in axons or perikarya and dendrites in tissue untreated with alkaline phosphatase. Tau proteins in mature tissue were soluble and not associated with detergent-resistant structures. Furthermore, dephosphorylation by alkaline phosphatase resulted in the appearance of more tau proteins in soluble fractions. Therefore tau proteins seem to alter their degree of phosphorylation during development. This could affect microtubule stability as well as influence axonal and dendritic differentiation.     

Inhibition of tubulin polymerization by vitilevuamide, a bicyclic marine peptide, at a site distinct from colchicine, the vinca alkaloids, and dolastatin 10

Vitilevuamide, a bicyclic 13 amino acid peptide, was isolated from two marine ascidians, Didemnum cuculiferum and Polysyncranton lithostrotum. Vitilevuamide was cytotoxic in several human tumor cell lines, with LC(50) values ranging from 6 to 311nM, and analysis in a 25-cell line panel revealed a weak correlation with several taxol analogs. Vitilevuamide was strongly positive in a cell-based screen for inhibitors of tubulin polymerization. Vitilevuamide at 9 microg/mL (5.6 microM) had an effect equivalent to the maximal effect of colchicine at 25 microg/mL (62.5 microM). Vitilevuamide was active in vivo against P388 lymphocytic leukemia, increasing the lifespan of leukemic mice 70% at 30 microg/kg. We hypothesized that at least part of the cytotoxic mechanism of vitilevuamide was due to its inhibition of tubulin polymerization. Vitilevuamide was found to inhibit polymerization of purified tubulin in vitro, with an IC(50) value of approximately 2 microM. Cell cycle analysis showed that vitilevuamide arrested cells in the G(2)/M phase with 78% of treated cells tetraploid after 16hr. Therefore, vitilevuamide was tested for its ability to inhibit binding of known tubulin ligands. Vitilevuamide exhibited non-competitive inhibition of vinblastine binding to tubulin. Colchicine binding to tubulin was stabilized in the presence of vitilevuamide in a fashion similar to vinblastine. Dolastatin 10 binding was unaffected by vitilevuamide at low concentrations, but inhibited at higher ones. GTP binding was also found to be weakly affected by the presence of vitilevuamide. These results suggest the possibility that vitilevuamide inhibits tubulin polymerization via an interaction at a unique site.     

Regional distribution and biochemical characteristics of high molecular weight tau in the nervous system.

The present study examined the distribution of the high molecular weight (HMW) tau protein isoform in the nervous system by immunoblotting and immunohistochemistry. Some of the biochemical properties of this 110 kDa tau protein were explored, including its heat stability, phosphorylation and partitioning with cold/Ca2+ stable vs. soluble microtubules. Qualitative western blot analysis revealed that HMW tau is preferentially expressed in neurons with peripherally projecting axons. For example, this isotype was present in sciatic nerve, ventral and dorsal roots, trigeminal nerve, vagus nerve, dorsal root ganglia (DRG) and spinal cord, but was present in only trace amounts in CNS regions. Another tau isoform of slightly smaller size (90-100 kDa), termed mid-molecular weight (MMW) tau, was present in abundant quantity in optic nerve samples and detectable in several other CNS regions, including hippocampus and cerebellum. The 110 kDa HMW tau as well as MMW tau and the other tau isoforms were found to be heat stable proteins. The HMW and MMW tau isoforms preferentially partitioned with the cold and Ca+2 insoluble tubulin fraction, but the association of HMW tau with stable microtubules was very susceptible to proteolysis. Dephosphorylation of fresh tissue with alkaline phosphatase produced no apparent shift in the mobility of HMW tau on SDS-PAGE but did alter the mobility of other brain tau isoforms, including MMW tau. Immunocytochemical staining with tau-1 antibody in the DRG, which contains HMW tau but no other tau isotypes, showed localization to mainly small neurons and was not altered by dephosphorylation of the histological sections.(ABSTRACT TRUNCATED AT 250 WORDS)     

IQGAP1 activates Tcf signal independent of Rac1 and Cdc42 in injury and repair of bronchial epithelial cells

The process of injury and repair involves spreading, migration and cell proliferation. The functions of Rho GTPases and their effector IQGAP1 are poor known in this process of airway epithelium. In the present study, we employed a widely used in vitro model by scratching a monolayer of BECs. We found that scratching induced decreasing of the GTP-bound Rac1 and Cdc42, but increasing the amounts of IQGAP1 at different time points. Next, we confirmed that IQGAP1 interacted with the constitutively active Rac1 (Rac1^V12) and Cdc42 (Cdc42^V12) rather than the dominant negative Rac1 (Rac1^N17) and Cdc42 (Cdc42^N17). Over-expressions of wild type (WT) IQGAP1 and its mutant (T1050AX2), which was defective to interact with Rho GTPases, induced translocation of β-catenin from the cytoplasm into the nucleus. These results activated Tcf/Lef and increased the expression levels of its target genes of c-myc and cyclin D1. Likewise, the amounts of c-myc and cyclin D1 increased after scratching. Our results suggested that IQGAP1 mediated cell proliferation through activating Tcf in a manner independent of Rac1 and Cdc42 in wound repair of BECs.     

MAP2 Isoforms in Developing Cat Cerebral Cortex and Corpus Callosum

The microtubule-associated protein MAP2 was studied in the developing cat visual cortex and corpus callosum. Biochemically, no MAP2a was detectable in either structure during the first postnatal month; adult cortex revealed small amounts of MAP2a. MAP2b was abundant in cortical tissue during the first postnatal month and decreased in concentration towards adulthood; it was barely detectable in corpus callosum at all ages. MAP2c was present in cortex and corpus callosum at birth; in cortex it consisted of three proteins of similar molecular weights between 65 and 70 kD. The two larger, phosphorylated forms disappeared after postnatal day 28, the smaller form after day 39. In corpus callosum, MAP2c changed from a phosphorylated to an unphosphorylated variant during the first postnatal month and then disappeared. Immunocytochemical experiments revealed MAP2 in cell bodies and dendrites of neurons in all cortical layers, from birth onwards. In corpus callosum, in the first month after birth, a little MAP2, possibly MAP2c, was detectable in axons. The present data indicate that MAP2 isoforms differ in their cellular distribution, temporal appearance and structural association, and that their composition undergoes profound changes during the period of axonal stabilization and dendritic maturation.     

Neuroradiological, neurophysiological and anatomopathological study of a case of locked-in syndrome

We report an anatomoclinical case of locked-in syndrome together with the neuroradiological and neurophysiological findings. MRI confirmed its value in the diagnosis of posterior cranial fossa pathology while the neurophysiological work-up (BAEPs, SEPs, MAPs and TEPs) revealed that the lesion may have broader functional implications than may be gauged from the neuroradiological and pathological evidence. Evoked potentials may therefore be of considerable importance in pinpointing brainstem lesions.

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Sommario Viene riportato un caso anatomoclinico di “Locked in” syndrome e riferiti i risultati delle indagini Neuroradiologiche e Neurofisiologiche. In particolare la RMN conferma la sua utilità diagnostica nella patologia della fossa cranica posteriore; lo studio neurofisiologico (BAEPs, SEPs, MAPs, TEPs) evidenzia che la lesione, sul piano funzionale, può in realtà essere più estesa di quanto dimostrato dagli esami neuroradiologici e dai dati anatomopatologici. I potenziali evocati possono quindi assumere una considerevole importanza per una precisa localizzazione delle lesioni troncali.

     

Recognition and development of traditional medicine in Tanzania

The aim of this paper is to trace developments in Traditional Medicine (TM) and legislation concerning conservation and use of biodiversity in Africa, with Tanzania as a case study. Based on field trips, interviews with different actors, site visits, and literature we explored the history, current status, re-establishment, and development of TM. A summary of laws and regulations concerning forests, access and benefit sharing is presented. During the last decade the Government of Tanzania put forth legislation to address national health needs, traditional knowledge, and the resource base for TM (e.g., practitioners, biodiversity). Our findings indicate that TM is the most common form of health care, and that the HIV pandemic has highlighted the need to work across health sectors. New legislation has facilitated this need. In Tanzania TM is experiencing a renaissance in being formally recognized, integrated into mainstream health care, formal establishment of practitioners, and gaining the interests of different sectors. More studies on bioactivity, safety, domestication, and sustainability of use of medicinal plants are needed. Development of TM can also, other than making a significant contribution to health care and livelihoods, provide income possibilities. It is however yet to be seen if the recent regulations can be made fully operational and implemented.     

Double dimer peptide constructs are immunogenic and protective against Plasmodium falciparum in the experimental Aotus monkey model

Abstract: Multiple antigen peptide constructs (MAPs) have been used to obtain defined multimeric peptide molecules useful in the development of possible synthetic malaria vaccines. In this context, a method was developed, named double dimer constructs (DDCs), involving the direct synthesis of a dimeric peptide with a C‐terminal cysteine. A tetrameric molecule was then obtained by oxidation of sulfhydryl groups. Dimer synthesis was optimized using a Fmoc/tBu strategy, dimers were purified by HPLC, oxidized with DMSO and characterized by HPLC and MALDI‐TOF‐MS. The tetramers or DDCs obtained by this method were used as immunogens in the search for a possible malaria vaccine. It was found that they were immunogenic in the experimental Aotus monkey model, and were able to induce protective immunity when challenged experimentally with a highly infective Plasmodium falciparum malaria strain.     

噻萘普汀对慢性应激大鼠海马CA3区锥体细胞MAP2表达的效应

目的探讨慢性应激大鼠海马CA3区锥体细胞内微管相关蛋白2(MAP2)表达变化及噻萘普汀的效应。方法采用强迫游泳作为应激模型,25只大鼠随机分为对照组、应激组及应激给药组。利用免疫组织化学方法及计算机图像分析技术,定量测定各组大鼠海马CA3区锥体细胞磷酸化MAP2表达水平及阳性细胞数。结果与对照组(149.34±1.81)比较,应激组大鼠海马CA3区锥体细胞磷酸化MAP2表达的平均灰度值(144.99±4.40)明显下降,给药组(148.84±2.73)则明显高于应激组;应激组阳性表达的细胞数(40.36±1.35)明显少于对照组(42.73±1.56),给药组(42.14±1.62)则明显多于应激组。结论噻萘普汀可抑制慢性应激所致的大鼠海马CA3区锥体细胞内磷酸化MAP2表达上调。