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The rat microphthalmia-associated transcription factor gene (Mitf) maps at 4q34-q41 and is mutated in the mib rats.

The rat gene encoding the microphthalmia-associated transcription factor (Mitf) was assigned to rat Chromosome (Chr) 4q34-q41, as well as the Gata2 and Mem1 genes. Rat Chr 4 is homologous to mouse Chr 6 and human Chr 3, which carry the Mitf (MITF) gene in these species (MMU 6, 40.0 cM, and HSA 3p14.1-p12.3). mib/mib rats, which are characterized by depigmentation, microphtalmy, osteopetrosis, and neurological disorders were shown to bear a deletion covering several kilobases of genomic DNA in the Mitf gene and to lack Mitf mRNA. The Mitf mutation in the mib/mib rats is thus very likely to be a Mitf null mutation, causing a phenotype similar to the one observed in the miVGA-9 mice, but including osteopetrosis as an additional feature.

Animals↗

Antibodies to microtubule-associated protein 2 in patients with neuropsychiatric systemic lupus erythematosus.

OBJECTIVE: Microtubule-associated protein 2 (MAP-2), a cellular protein restricted to neurons, is important in the control of cytoskeletal integrity and other neuronal functions. We undertook this study to examine the presence of autoantibodies to MAP-2 in neuropsychiatric systemic lupus erythematosus (NPSLE). METHODS: Sera from 100 patients with SLE, 74 patients with other neurologic disorders and injuries (including cerebrovascular accidents, brain trauma, brain tumors, and demyelinating disorders), and 60 normal controls were examined both by enzyme immunoassays and by Western immunoblotting for autoantibodies to MAP-2. Sera designated positive for antibodies to MAP-2 were required to be positive in both assays. RESULTS: Seventeen percent of SLE patients had autoantibodies to MAP-2, in contrast to 4% of neurologic injury/disease control patients (P = 0.028) and 1.7% of normal controls. In SLE, anti-MAP-2 positivity in both assays was associated with neuropsychiatric symptoms in 76.5% of patients, whereas the absence of anti-MAP-2 was associated with neuropsychiatric symptoms in 19.7% of patients (P = 0.0002). The neuropsychiatric symptoms in the former group included psychosis, seizure, neuropathy, and cerebritis. CONCLUSION: Autoantibodies to MAP-2, a neuron-restricted cytoskeletal protein, appear to be another immune marker for NPSLE.

Adolescent↗

Characterization of microtubule-associated protein 1-associated protein kinases from rat brain.

The microtubule-associated protein (MAP) 1 preparation, MAP1A and 1B, obtained from rat brain microtubules was associated with protein kinases that were insensitive to cAMP, cGMP, calcium, calcium/calmodulin and calcium/phosphatidylserine. The fractionation of highly purified MAP1 by phosphocellulose chromatography revealed that protein kinase activity to phosphorylate phosvitin was separated into three major peaks (MAP1 kinases A, B and C). MAP1 was recovered in the MAP1 kinase A fraction and phosphorylated by the contained kinase. MAP1 kinase A is a novel protein kinase that is remarkably activated by poly-L-lysine and poly-L-arginine, but very insensitive to heparin among the kinases. Photoaffinity labeling using [alpha-32P]8-azido ATP indicated that the 65 kDa polypeptide is identified as an ATP-binding protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the highly purified MAP1 and MAP1 kinase A fractions. MAP1 kinases B and C may be identified as casein kinase I- and II-like kinases. The present results show that MAP1 is associated with at least three kinases and provide an insight for understanding thoroughly the MAP1-mediated microtubule functions.

Animals↗

Interaction of the Unc-51-like kinase and microtubule-associated protein light chain 3 related proteins in the brain: possible role of vesicular transport in axonal elongation.

We identified two mammalian ULK1 (Unc-51-like kinase involved in neurite extension) binding proteins by yeast two-hybrid screening. Both proteins showed high structural similarity to microtubule-associated protein (MAP) light chain 3 (LC3). One is identical to the Golgi-associated ATPase Enhancer of 16 kDa (GATE-16), an essential factor for intra-Golgi transport [39]. The other is identical to the gamma 2-subunit of GABA-A receptor associated protein (GABARAP) which has a possible role in receptor transport [46]. Using the yeast two-hybrid system and the in vitro GST pull-down assay, we found that the N-terminal proline/serine rich (PS) domain of ULK1 (amino acid 287-416) is required for ULK1-GATE-16 and ULK1-GABARAP protein interactions. However, the kinase activity of ULK1 affected neither ULK1-GATE-16 nor ULK1-GABARAP interaction. Immunohistochemical analysis using ULK1 and GABARAP antibodies showed that the ULK1 and the GABARAP proteins co-localized to many kind of neurons such as pyramidal cells of the hippocampus, mitral cells of the olfactory bulb, and Purkinje cells of the cerebellum. In HeLa cells, endogenous ULK1 and tagged GABARAP showed punctate structures in the cytosol, and were colocalized. These results suggest that the interaction of ULK1 and GABARAP is important to vesicle transport and axonal elongation in mammalian neurons.

Adaptor Proteins, Signal Transducing↗

The neurofilament antibody RT97 recognises a developmentally regulated phosphorylation epitope on microtubule-associated protein 1B.

Microtubules are important for the growth and maintenance of stable neuronal processes and their organisation is controlled partly by microtubule-associated proteins (MAPs). MAP 1B is the first MAP to be expressed in neurons and plays an important role in neurite outgrowth. MAP 1B is phosphorylated at multiple sites and it is believed that the function of the protein is regulated by its phosphorylation state. We have shown that the monoclonal antibody (mAb) RT97, which recognises phosphorylated epitopes on neurofilament proteins, fetal tau, and on Alzheimer's paired helical filament-tau, also recognises a developmentally regulated phosphorylation epitope on MAP 1B. In the rat cerebellum, Western blot analysis shows that mAb RT97 recognises the upper band of the MAP 1B doublet and that the amount of this epitope peaks very early postnatally and decreases with increasing age so that it is absent in the adult, despite the continued expression of MAP 1B in the adult. We confirmed that mAb RT97 binds to MAP 1B by showing that it recognises MAP 1B immunoprecipitated from postnatal rat cerebellum using polyclonal antibodies to recombinant MAP 1B proteins. We established that the RT97 epitope on MAP 1B is phosphorylated by showing that antibody binding was abolished by alkaline phosphatase treatment of immunoblots. Epitope mapping experiments suggest that the mAb RT97 site on MAP 1B is near the N-terminus of the molecule. Despite our immunoblotting data, immunostaining of sections of postnatal rat cerebellum with mAb RT97 shows a staining pattern typical of neurofilaments with no apparent staining of MAP 1B. For instance, basket cell axons and axons in the granule cell layer and white matter stained, whereas parallel fibres did not. These results suggest that the MAP 1B epitope is masked or lost under the immunocytochemical conditions in which the cerebellar sections are prepared. The upper band of the MAP 1B doublet is believed to be predominantly phosphorylated by proline-directed protein kinases (PDPKs). PDPKs are also good candidates for phosphorylating neurofilament proteins and tau and therefore we postulate that the sites recognised by RT97 on these neuronal cytoskeletal proteins may be phosphorylated by similar kinases. Important goals are to determine the precise location of the RT97 epitope on MAP 1B and the kinase responsible.

Animals↗

Three unique 5' untranslated regions are spliced to common coding exons of high- and low-molecular-weight microtubule-associated protein-2.

Three unique 5' untranslated regions (UTRs) have been characterized for human microtubule-associated protein-2 (MAP-2) transcripts. All three UTRs shared a common 171-bp sequence adjacent to the MAP-2 coding region and then diverged upstream. The size of the unique upstream sequence was 281, 146, or 104 bp. PCR of genomic DNA demonstrated that the 5' UTRs span multiple exons. The unique region of the UTRs recognizes a 9.5- and a 6-kb MAP-2 transcript in poly(A)+ mRNA isolated from human MSN cells, and PCR analysis demonstrated that each unique UTR is contained in multiple high- and low-molecular-weight MAP-2 transcripts. Reverse transcription-PCR (RT-PCR) performed on MSN mRNA isolated from polysomes demonstrated that all three of the UTRs contained within multiple MAP-2 transcripts were associated with polysomes and hence translated. RT-PCR from human fetal spinal cord and adult brain mRNA demonstrated that all of the UTRs are expressed at these developmental time points.

Aging↗

Improved understanding of cortical injury by incorporating measures of functional anatomy.

Volume of injury is often used to describe a brain insult. However, this approach assumes cortical equivalency and ignores the special importance that certain cortical regions have in the generation of behaviour. We hypothesized that incorporating knowledge of normal brain functional anatomy into the description of a motor cortex injury would provide an improved framework for understanding consequent behavioural effects. Anatomical scanning was performed in 21 patients with a chronic cortical stroke that involved the sensorimotor cortex. Functional MRI (fMRI) was used to generate separate average activation maps for four tasks including hand, shoulder and face motor tasks in 14 controls. For each task, group average maps for contralateral sensorimotor cortex activation were generated. Injury to these maps was measured by superimposing each patient's infarct. These measurements were then correlated with behavioural assessments. In bivariate analyses, injury to fMRI maps correlated with behavioural assessments more strongly than total infarct volume. For example, performance on the Purdue pegboard test by the stroke-affected hand correlated with the fraction of hand motor map injured (r = -0.79) more strongly than with infarct volume (r = -0.60). In multiple linear regression analyses, measures of functional map injury, but not infarct volume, remained as significant explanatory variables for behavioural assessments. Injury to >37% of the hand motor map was associated with total loss of hand motor function. Hand and shoulder motor maps showed considerable spatial overlap (63%) and similar behavioural consequences of injury to each map, while hand and face motor maps showed limited overlap (10.4%) and disparate behavioural consequences of injury to each map. Lesion effects support current models of broad, rather than focal, sensorimotor cortex somatotopic representation. In the current cross-sectional study, incorporating an understanding of normal tissue function into lesion measurement provided improved insights into the behavioural consequences of focal brain injury.

Adult↗

Cognitive map formation through sequence encoding by theta phase precession.

The rodent hippocampus has been thought to represent the spatial environment as a cognitive map. The associative connections in the hippocampus imply that a neural entity represents the map as a geometrical network of hippocampal cells in terms of a chart. According to recent experimental observations, the cells fire successively relative to the theta oscillation of the local field potential, called theta phase precession, when the animal is running. This observation suggests the learning of temporal sequences with asymmetric connections in the hippocampus, but it also gives rather inconsistent implications on the formation of the chart that should consist of symmetric connections for space coding. In this study, we hypothesize that the chart is generated with theta phase coding through the integration of asymmetric connections. Our computer experiments use a hippocampal network model to demonstrate that a geometrical network is formed through running experiences in a few minutes. Asymmetric connections are found to remain and distribute heterogeneously in the network. The obtained network exhibits the spatial localization of activities at each instance as the chart does and their propagation that represents behavioral motions with multidirectional properties. We conclude that theta phase precession and the Hebbian rule with a time delay can provide the neural principles for learning the cognitive map.

Algorithms↗

Investigating the relationship between hemodynamic and metabolic parameters in portal triade occlusion following hemorrhagic shock.

PURPOSE: To determine whether metabolic and hemodynamic parameters are "mathematically" coupled in rats submitted to portal triad occlusion following controlled hemorrhagic shock state. METHODS: Pearson's coefficient (r-value) analysis was performed. Differences considered significant at p < 0.05 and -0.50 > r > 0.50. RESULTS: It was observed that there is a direct proportional relationship to HCO3- with pCO2 (r = 0.66), base deficit (r = 0.87) and inverse with serum lactate (r = -0.54). pCO2 was directly associated with MAP (r = 0.51), and inversely with pH (r = -0.64). Hematocrit was directly associated with HR (r = 0.72) and CI (r = 0.76), and serum lactated was inversely associated with base deficit (r = -0.61). CONCLUSION: In rats submitted to Pringle's maneuver during 15 minutes following hemorrhagic shock state, there is a mathematical coupling with a very good correlation between several hemodynamic and metabolic variables.

Animals↗

Organization of the adeno-associated virus (AAV) capsid gene: mapping of a minor spliced mRNA coding for virus capsid protein 1.

We have mapped a minor spliced transcript with mRNA properties which is derived from the capsid gene promoter (P40) of the adeno-associated virus (AAV) genome. By S1 nuclease mapping as well as primer extension analysis this mRNA was found to occur by splicing at the same donor site (nucleotide, NT 1907) but at an alternative acceptor site when compared to the major, 2.3-kb spliced P40 transcript which encodes two of the three AAV capsid proteins, namely VP2 and VP3. This minor acceptor site is located at NT 2200, i.e., 27 NT upstream of the acceptor site used to generate the VP2/VP3 mRNA. We have also sequenced the AAV genome in this region and have found one important error in the sequence published by A. Srivastava, E. Lusby, and K.I. Berns (1983, J. Virol., 45, 555-564): residue at NT 2429 has to be deleted from the reported sequence. This correction of the sequence reveals that the open reading frame (ORF) which encodes all three capsid proteins extends from NT 2203 to NT 4321. Furthermore, this entire ORF is contained in the minor but not in the major spliced P40 transcript. We provide evidence that the largest AAV structural protein VP1 is translated from this hitherto undetected spliced transcript by initiation at its first AUG (NT 2203) and termination at NT 4321. The calculated molecular weight of this VP1 polypeptide is 78,247 Da.

Base Sequence↗

MAP, a protein interacting with a tumor suppressor, merlin, through the run domain.

Merlin (or schwannomin) is a tumor suppressor encoded by the neurofibromatosis type 2 gene. Many studies have suggested that merlin is involved in the regulation of cell growth and proliferation through interactions with various cellular proteins. To better understand the function of merlin, we tried to identify the proteins that bind to merlin using the yeast two-hybrid screening. Characterization of the positive clones revealed a protein of 749 amino acids named merlin-associated protein (MAP), which showed wide tissue distribution in Northern blot analysis. Sequence analysis revealed that MAP is a potential homologue of a yeast check-point protein, BUB2, and contains TBC, SH3, and RUN domains, thereby implicating its role in the Ras-like GTPase signal pathways. MAP and merlin were directly associated in vitro and in vivo, and colocalized in NIH3T3 cells. The RUN domain of MAP and the C-terminus of merlin appeared to be responsible for their interaction. MAP decreased the AP-1-dependent promoter activity additively with merlin in NIH3T3 cells. In addition, merlin and MAP synergistically reduced the colony formation of NIH3T3 cells. These results suggest that MAP may play a cooperative role in the merlin-mediated growth suppression of cells.

3T3 Cells↗

Cyclin B interaction with microtubule-associated protein 4 (MAP4) targets p34cdc2 kinase to microtubules and is a potential regulator of M-phase microtubule dynamics.

We previously demonstrated (Ookata et al., 1992, 1993) that the p34cdc2/cyclin B complex associates with microtubules in the mitotic spindle and premeiotic aster in starfish oocytes, and that microtubule-associated proteins (MAPs) might be responsible for this interaction. In this study, we have investigated the mechanism by which p34cdc2 kinase associates with the microtubule cytoskeleton in primate tissue culture cells whose major MAP is known to be MAP4. Double staining of primate cells with anti-cyclin B and anti-MAP4 antibodies demonstrated these two antigens were colocalized on microtubules and copartitioned following two treatments that altered MAP4 distribution. Detergent extraction before fixation removed cyclin B as well as MAP4 from the microtubules. Depolymerization of some of the cellular microtubules with nocodazole preferentially retained the microtubule localization of both cyclin B and MAP4. The association of p34cdc2/cyclin B kinase with microtubules was also shown biochemically to be mediated by MAP4. Cosedimentation of purified p34cdc2/cyclin B with purified microtubule proteins containing MAP4, but not with MAP-free microtubules, as well as binding of MAP4 to GST-cyclin B fusion proteins, demonstrated an interaction between cyclin B and MAP4. Using recombinant MAP4 fragments, we demonstrated that the Pro-rich C-terminal region of MAP4 is sufficient to mediate the cyclin B-MAP4 interaction. Since p34cdc2/cyclin B physically associated with MAP4, we examined the ability of the kinase complex to phosphorylate MAP4. Incubation of a ternary complex of p34cdc2, cyclin B, and the COOH-terminal domain of MAP4, PA4, with ATP resulted in intracomplex phosphorylation of PA4. Finally, we tested the effects of MAP4 phosphorylation on microtubule dynamics. Phosphorylation of MAP4 by p34cdc2 kinase did not prevent its binding to microtubules, but abolished its microtubule stabilizing activity. Thus, the cyclin B/MAP4 interaction we have described may be important in targeting the mitotic kinase to appropriate cytoskeletal substrates, for the regulation of spindle assembly and dynamics.

Amino Acid Sequence↗

DMAP-85: a tau-like protein from Drosophila melanogaster larvae.

Microtubule-associated proteins (MAPs) play major regulatory roles in the organization and integrity of the cytoskeletal network. Our main interest in this study was the identification and the analysis of structural and functional aspects of Drosophila melanogaster MAPs. A novel MAP with a relative molecular mass of 85 kDa from Drosophila larvae was found associated with taxol-polymerized microtubules. In addition, this protein bound to mammalian tubulin in an overlay assay and coassembled with purified bovine brain tubulin in microtubule sedimentation experiments. The estimated stoichiometry of 85-kDa protein versus tubulin in the polymers was 1:5.3 +/- 0.2 mol/mol. It was shown that the 85-kDa protein bound specifically to an affinity column of Sepharose-beta II-(422-434) tubulin peptide, which contains the sequence of the MAP binding domain on beta II-tubulin. Affinity-purified 85-kDa protein enhanced microtubule assembly in a concentration-dependent manner. This effect was significantly decreased by the presence of the beta II-(422-434) peptide in the assembly assays, thus confirming the specificity of the 85-kDa protein interaction with the C-terminal domain on tubulin. Furthermore, this protein also exhibited a strong affinity for calmodulin, based on affinity chromatographic assays. Monoclonal and polyclonal anti-tau antibodies, including sequence-specific probes that recognize repeated microtubule-binding motifs on tau, MAP-2, and MAP-4 and specific N-terminal sequences of tau, cross-reacted with the 85-kDa protein from Drosophila larvae. These results suggest that tau and Drosophila 85-kDa protein share common functional and structural epitopes. We have named this protein as DMAP-85 for Drosophila MAP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Eleven X chromosome breakpoints associated with premature ovarian failure (POF) map to a 15-Mb YAC contig spanning Xq21.

Eleven balanced X-autosome translocations associated with premature ovarian failure (POF) were mapped to a YAC contig spanning most of Xq21 and constructed between the DXS223 and DXS1171 loci. The contig corresponds to a genomic region of about 15 Mb and contains the whole X-Y homologous region. The most proximal and most distal breakpoints associated with POF were mapped 15 Mb apart. The remaining breakpoints were localized along this large region, in the X-specific and in the X-Y homologous region. Four of the YACs contained two breakpoints in the same or in flanking STS intervals. Our results confirm the cytological findings and suggest that a minimum number of eight different genes in Xq21 may be involved with ovary development. Interruption of such loci could be the cause of POF.

Carrier Proteins↗

Catheter ablation of chronic atrial fibrillation with noncontact mapping: are continuous linear lesions associated with ablation success?

Catheter-based, right and left atrial compartmentalization procedure was evaluated using a noncontact mapping (NCM) system. Its usefulness to identify and close discontinuities in linear lesions in both atria was evaluated. The impact of linear lesion continuity on ablation success of chronic AF was also investigated. Nineteen patients with symptomatic, drug refractory chronic AF were studied. Right atrial ablation with three predefined lines was attempted in all patients. In 18 patients, left atrial ablation was performed with four linear lesions. During a follow-up of 12 +/- 3 months, 6 of 19 patients remained in sinus rhythm (SR) without antiarrhythmic agents (AAs). In addition, four patients were maintained in SR with AA. Thirteen of 14 patients with gaps identified during off-line analysis had recurrence of AF. Only one patient with a gap was free of recurrence without AAs. In the remaining five patients without recurrence of AF, no gap was observed during off-line analysis. In all four patients who were free of AF with additional treatment of AAs, two gaps had been identified. In the remaining nine patients with chronic AF recurrence, a mean of 4.9 gaps were identified. Excluding the initial learning period (first five patients) the success rate increased to 43% (6/14 patients) without and to 71% (10/14 patients) with AA. NCM identifies discontinuities in lines of ablation. Successful ablation of chronic AF is associated with continuity of linear lesions and good clinical technique demands a vigilant search for and closure of every gap.

Atrial Fibrillation↗

Possible involvement of 65 kda MAP in elongation growth of azuki bean epicotyls.

Although regulation of the dynamics of plant microtubules (MTs) by microtubule-associated proteins (MAPs) has been suggested, the mechanism has not yet been elucidated. As one candidate, a MAP composed of a 65 kDa polypeptide (65 kDa MAP) has been isolated from tobacco cultured cells [Jiang and Sonobe (1993), J. Cell Sci 105: 8911. To investigate the physiological role of the 65 kDa MAP in situ, we analyzed the changes in content and colocalization of this MAP with cortical MTs in relation to elongation growth, using azuki bean epicotyls (Vigna angularis Ohwi et Ohashi). All apical, intermediate, and basal segments prepared from 6 d seedlings showed high growth activity. In 12 d seedlings, growth activity of intermediate and basal segments was low, although that of apical segments was high. The relationship between the growth activity and the orientation of cortical MTs in the epidermal cells was analyzed. Cells could be classified into four types with respect to orientation of cortical MTs: transverse (T), oblique (O), longitudinal (L) to the vertical axis of cells, and random (R). In rapidly growing segments, three types of cells, T, O, L, were observed at similar ratios. In such segments, significant amounts of the 65 kDa MAP were expressed, and it colocalized well with cortical MTs. In segments showing low growth activity, most of the cells showed oblique and longitudinal orientation of cortical MTs. In such segments, the content of the 65 kDa MAP was low. These results suggested involvement of this 65 kDa MAP in regulation of the elongation growth of this epicotyl.

Fabaceae↗

Expression levels of cytoskeletal proteins indicate pathological aging of S100B transgenic mice: an immunohistochemical study of MAP-2, drebrin and GAP-43.

S100B is a calcium-binding protein found within astroglial cells. When released, S100B has extracellular neurotrophic effects involving the neuronal cytoskeleton. The gene for S100B is located on chromosome 21 and levels of the protein are elevated in Down Syndrome (DS) and Alzheimer's Disease (AD). Thus, overexpression of S100B may be related to the cytoskeletal abnormalities seen in these disorders. Transgenic mice overexpressing human S100B were examined for cytoskeletal changes as young (70 days) and aged (200 days) adults, using immunochemical staining of the dendritic associated protein, MAP-2, the growth-associated protein-43 (GAP-43) and the dendritic spine marker, drebrin. As young adults, the S100B transgenic mice exhibited significantly greater MAP-2-immunoreactivity in the hippocampus, however as older adults, the animals exhibited less staining. In both the CD1 control animals and the S100B animals, the immunoreactivity of drebrin increased with age, however there were no significant between group differences. Finally, the older S100B animals showed more GAP-43 staining than the control animals, suggesting that synaptic remodeling could take place, possibly in response to the loss of MAP-2-ir dendrites. Overall, the data suggest that S100B overexpression leads to changes in cytoskeletal markers. The longitudinal effects of S100B overexpression are discussed with relevance to aging and pathology.

Aging↗

Axonal microtubules of crayfish and spiny lobster nerve cords are decorated with a heat-stable protein of high molecular weight.

Axons of crayfish and spiny lobster ventral nerve cords contain large numbers of microtubules that are decorated with fine filaments. These microtubules can be stabilized in permeabilized axons using buffers that contain either polyethylene glycol or glycerol/dimethyl sulphoxide. In the former, the stabilized microtubules retain their filaments and their normal spacing; in the latter, the filaments are stripped off and the bare microtubules collapse onto one another. This observation has been used as the basis for a method of identifying some of the proteins that make up the filaments. Axons are first permeabilized and stabilized in either buffer and then treated with a microtubule-depolymerizing buffer. The axons treated first with polyethylene glycol release tubulin and significant quantities of microtubule-associated proteins (MAPs), while the axons pre-treated with glycerol release tubulin and only traces of associated proteins. One of the proteins released in largest quantity along with tubulin from polyethylene glycol-treated axons is a high molecular weight, heat-stable MAP that co-electrophoreses with MAP-2 from mammalian brain. This same protein co-purifies with tubulin that is obtained from crayfish nerve cords by two cycles of polymerization and depolymerization. It is concluded that this protein is a component of the filaments that decorate the axonal microtubules of the crayfish and spiny lobster.

Animals↗