Colocalization of ��-actinin and Synaptopodin in the Pyramidal Cell Axon Initial Segment

The cisternal organelle that resides in the axon initial segment (AIS) of neocortical and hippocampal pyramidal cells is thought to be involved in regulating the Ca(2+) available to maintain AIS scaffolding proteins, thereby preserving normal AIS structure and function. Through immunocytochemistry and correlative light and electron microscopy, we show here that the actin-binding protein ?-actinin is present in the typical cistenal organelle of rodent pyramidal neurons as well as in a large structure in the AIS of a subpopulation of layer V pyramidal cells that we have called the "giant saccular organelle." Indeed, this localization of ?-actinin in the AIS is dependent on the integrity of the actin cytoskeleton. Moreover, in the cisternal organelle of cultured hippocampal neurons, ?-actinin colocalizes extensively with synaptopodin, a protein that interacts with both actin and ?-actinin, and they appear concomitantly during the development of these neurons. Together, these results indicate that ?-actinin and the actin cytoskeleton are important components of the cisternal organelle that are probably required to stabilize the AIS.

Rammed earth walls in the late middle age castles in the actual province of Soria, Spain

This paper analyzed the building techniques based in the use of rammed earth in the Late Medieval fortifications in the province of Soria, Spain. The manorial castles of Ser��n de N��gima and Yanguas were built completely with rammed earth. However, these techinques are different. Through the study of the constructive signals, we can reconstruct the constructive process and made an hypothesis of the auxiliary scaffolding necessary for the construction. After, other four cases in which there is presence of rammed earth are described: ��greda, Arcos de Jal��n, Caracena and the tower of Mart��n Gonz��lez or castle of La Raya (the Border). Rammed earth is used in some secondary architectural elements and also as a filling of the masonry walls, but somtimes there are ancient rammed earth walls overlaid with stone masonry.

Doubling the width of the platform of the San Pedro bridge in Spain

The San Pedro Bridge has six spans and is 750 m (2460 ft) long, 88 m (290 ft) high, 12 m (39 ft) wide, and curved with a radius of 700 m (2300 ft). It was built in 1993 using the cantilever method. Its super - structure is a prestressed concrete box girder with main spans of 150 m (490 ft). In 2008 and 2009, the width of the platform was enlarged to 23 m (75 ft) using five movable sets of scaffolding. The bridge remained open to traffic during construction. The original platform was widened 6 m (20 ft) on each side by connecting a new lightweight concrete cantilever to the original upper slab. These cantilevers were supported by steelstruts. The tie into the upper slab was made with new transverse post-tensioned tendons. The original superstructure was strengthened to resist the additional dead load of the expansion and live loads of the extra traffic. An additional new central web and a composite concrete-steel section were constructed and connected to the concrete box and central web using vertical high-strength post-tensioning bars. Also, external post-tensioning cables were implemented. It was also necessary to strengthen the connection of the original concrete box section to the piers. Detailed calculations were performed to evaluate the load distribution transmitted to the piers by the webs and by the original inclined concrete walls of the box girder. Finally, a detailed second-order-analysis of the complete structure was made to guarantee the resistance of the piers compared with actual loads

Spinophilin, a novel protein phosphatase 1 binding protein localized to dendritic���spines

Dendritic spines receive the vast majority of excitatory synaptic contacts in the mammalian brain and are presumed to contain machinery for the integration of various signal transduction pathways. Protein phosphatase 1 (PP1) is greatly enriched in dendritic spines and has been implicated in both the regulation of ionic conductances and long-term synaptic plasticity. The molecular mechanism whereby PP1 is localized to spines is unknown. We have now characterized a novel protein that forms a complex with the catalytic subunit of PP1 and is a potent modulator of PP1 enzymatic activity in vitro. Within the brain this protein displays a remarkably distinct localization to the heads of dendritic spines and has therefore been named spinophilin. Spinophilin has the properties expected of a scaffolding protein localized to the cell membrane and contains a single consensus sequence in PSD95/DLG/zo-1, which implies cross-linking of PP1 to transmembrane protein complexes. We propose that spinophilin represents a novel targeting subunit for PP1, which directs the enzyme to those substrates in the dendritic spine compartment, e.g., neurotransmitter receptors, which mediate the regulation of synaptic function by PP1.

Recruitment of SMRT/N-CoR-mSin3A-HDAC-repressing complexes is not a general mechanism for BTB/POZ transcriptional repressors: The case of HIC-1 and ��FBP-B

Hypermethylated in cancer (HIC-1), a new candidate tumor suppressor gene located in 17p13.3, encodes a protein with five C2H2 zinc fingers and an N-terminal broad complex, tramtrack, and bric �� brac/poxviruses and zinc-finger (BTB/POZ) domain found in actin binding proteins or transcriptional regulators involved in chromatin modeling. In the human B cell lymphoma (BCL-6) and promyelocityc leukemia (PLZF) oncoproteins, this domain mediates transcriptional repression through its ability to recruit a silencing mediator of retinoid and thyroid hormone receptor (SMRT)/nuclear receptor corepressor (N-CoR)-mSin3A-histone deacetylase (HDAC) complex, a mechanism shared with numerous transcription factors. HIC-1 appears unique because it contains a 13-aa insertion acquired late in evolution, because it is not found in its avian homologue, ��F1-binding protein isoform B (��FBP-B), a transcriptional repressor of the ��F-crystallin gene. This insertion, located in a conserved region involved in the dimerization and scaffolding of the BTB/POZ domain, mainly affects slightly the ability of the HIC-1 and ��FBP-B BTB/POZ domains to homo- and heterodimerize in vivo, as shown by mammalian two-hybrid experiments. Both the HIC-1 and ��FBP-B BTB/POZ domains behave as autonomous transcriptional repression domains. However, in striking contrast with BCL-6 and PLZF, both HIC-1 and ��FBP-B similarly fail to interact with members of the HDAC complexes (SMRT/N-CoR, mSin3A or HDAC-1) in vivo and in vitro. In addition, a general and specific inhibitor of HDACs, trichostatin A, did not alleviate the HIC-1- and ��FBP-B-mediated transcriptional repression, as previously shown for BCL-6. Taken together, our studies show that the recruitment onto target promoters of an HDAC complex is not a general property of transcriptional repressors containing a conserved BTB/POZ domain.

Epstein���Barr virus nuclear protein 2 interacts with p300, CBP, and PCAF histone acetyltransferases in activation of the LMP1 promoter

The Epstein���Barr virus (EBV) nuclear protein 2 (EBNA2) and herpes simplex virion protein 16 (VP16) acidic domains that mediate transcriptional activation now are found to have affinity for p300, CBP, and PCAF histone acetyltransferases (HATs). Transcriptionally inactive point mutations in these domains lack affinity for p300, CBP, or PCAF. P300 and CBP copurify with the principal HAT activities that bind to EBNA2 or VP16 acidic domains through velocity sedimentation and anion-exchange chromatography. EBNA2 binds to both the N- and C-terminal domains of p300 and coimmune-precipitates from transfected 293T cells with p300. In EBV-infected Akata Burkitt's tumor cells that do not express the EBV encoded oncoproteins EBNA2 or LMP1, p300 expression enhances the ability of EBNA2 to up-regulate LMP1 expression. Through its intrinsic HAT activity, PCAF can further potentiate the p300 effect. In 293 T cells, P300 and CBP (but not PCAF) can also coactivate transcription mediated by the EBNA2 or VP16 acidic domains and HAT-negative mutants of p300 have partial activity. Thus, the EBNA2 and VP16 acidic domains can utilize the intrinsic HAT or scaffolding properties of p300 to activate transcription.

CMS: An adapter molecule involved in cytoskeletal rearrangements

Cas ligand with multiple Src homology (SH) 3 domains (CMS) is an ubiquitously expressed signal transduction molecule that interacts with the focal adhesion protein p130Cas. CMS contains three SH3 in its NH2 terminus and proline-rich sequences in its center region. The latter sequences mediate the binding to the SH3 domains of p130Cas, Src-family kinases, p85 subunit of phosphatidylinositol 3-kinase, and Grb2. The COOH-terminal region contains putative actin binding sites and a coiled-coil domain that mediates homodimerization of CMS. CMS is a cytoplasmic protein that colocalizes with F-actin and p130Cas to membrane ruffles and leading edges of cells. Ectopic expression of CMS in COS-7 cells resulted in alteration in arrangement of the actin cytoskeleton. We observed a diffuse distribution of actin in small dots and less actin fiber formation. Altogether, these features suggest that CMS functions as a scaffolding molecule with a specialized role in regulation of the actin cytoskeleton.

Cadherin Sequences That Inhibit ��-Catenin Signaling: A Study in Yeast and Mammalian Cells

Drosophila Armadillo and its mammalian homologue ��-catenin are scaffolding proteins involved in the assembly of multiprotein complexes with diverse biological roles. They mediate adherens junction assembly, thus determining tissue architecture, and also transduce Wnt/Wingless intercellular signals, which regulate embryonic cell fates and, if inappropriately activated, contribute to tumorigenesis. To learn more about Armadillo/��-catenin's scaffolding function, we examined in detail its interaction with one of its protein targets, cadherin. We utilized two assay systems: the yeast two-hybrid system to study cadherin binding in the absence of Armadillo/��-catenin's other protein partners, and mammalian cells where interactions were assessed in their presence. We found that segments of the cadherin cytoplasmic tail as small as 23 amino acids bind Armadillo or ��-catenin in yeast, whereas a slightly longer region is required for binding in mammalian cells. We used mutagenesis to identify critical amino acids required for cadherin interaction with Armadillo/��-catenin. Expression of such short cadherin sequences in mammalian cells did not affect adherens junctions but effectively inhibited ��-catenin���mediated signaling. This suggests that the interaction between ��-catenin and T cell factor family transcription factors is a sensitive target for disruption, making the use of analogues of these cadherin derivatives a potentially useful means to suppress tumor progression.

RNA is a structural element in retrovirus particles

A single retroviral protein, Gag, is sufficient for virus particle assembly. While Gag is capable of specifically packaging the genomic RNA into the particle, this RNA species is unnecessary for particle assembly in vivo. In vitro, nucleic acids profoundly enhance the efficiency of assembly by recombinant Gag proteins, apparently by acting as ���scaffolding��� in the particle. To address the participation of RNA in retrovirus assembly in vivo, we analyzed murine leukemia virus particles that lack genomic RNA because of a deletion in the packaging signal of the viral RNA. We found that these particles contain cellular mRNA in place of genomic RNA. This result was particularly evident when Gag was expressed by using a Semliki Forest virus-derived vector: under these conditions, the Semliki Forest virus vector-directed mRNA became very abundant in the cells and was readily identified in the retroviral virus-like particles. Furthermore, we found that the retroviral cores were disrupted by treatment with RNase. Taken together, the data strongly suggest that RNA is a structural element in retrovirus particles.

The effects of practice on the functional anatomy of task���performance

The effects of practice on the functional anatomy observed in two different tasks, a verbal and a motor task, are reviewed in this paper. In the first, people practiced a verbal production task, generating an appropriate verb in response to a visually presented noun. Both practiced and unpracticed conditions utilized common regions such as visual and motor cortex. However, there was a set of regions that was affected by practice. Practice produced a shift in activity from left frontal, anterior cingulate, and right cerebellar hemisphere to activity in Sylvian-insular cortex. Similar changes were also observed in the second task, a task in a very different domain, namely the tracing of a maze. Some areas were significantly more activated during initial unskilled performance (right premotor and parietal cortex and left cerebellar hemisphere); a different region (medial frontal cortex, ���supplementary motor area���) showed greater activity during skilled performance conditions. Activations were also found in regions that most likely control movement execution irrespective of skill level (e.g., primary motor cortex was related to velocity of movement). One way of interpreting these results is in a ���scaffolding-storage��� framework. For unskilled, effortful performance, a scaffolding set of regions is used to cope with novel task demands. Following practice, a different set of regions is used, possibly representing storage of particular associations or capabilities that allow for skilled performance. The specific regions used for scaffolding and storage appear to be task dependent.

Oligomeric structure of caveolin: implications for caveolae membrane organization.

A 22-kDa protein, caveolin, is localized to the cytoplasmic surface of plasma membrane specializations called caveolae. We have proposed that caveolin may function as a scaffolding protein to organize and concentrate signaling molecules within caveolae. Here, we show that caveolin interacts with itself to form homooligomers. Electron microscopic visualization of these purified caveolin homooligomers demonstrates that they appear as individual spherical particles. By using recombinant expression of caveolin as a glutathione S-transferase fusion protein, we have defined a region of caveolin's cytoplasmic N-terminal domain that mediates these caveolin-caveolin interactions. We suggest that caveolin homooligomers may function to concentrate caveolin-interacting molecules within caveolae. In this regard, it may be useful to think of caveolin homooligomers as "fishing lures" with multiple "hooks" or attachment sites for caveolin-interacting molecules.

Parent-Child Interaction and Children's Self-Concept

This study is designed to investigate the relationships between marital communication, the quality of parents' ability to assist their children in joint problem-solving, and children's independent mastery attempts and perceived competence at problem-solving, and behavioral indicators of self-esteem. Couples' skill at regulating their own and their children's negative affect within the marital and parent-child family subsystems is hypothesized to predict the quality of their assistance, or scaffolding behavior, to their children during joint problem-solving. Further, the quality of parental scaffolding behavior is expected to predict children's independent mastery attempts, levels of perceived competence at problemsolving, and behavioral indicators of self-esteem. Families for the study will be those with children between 3 1/2 to six years of age recruited from subjects participating in a longitudinal study of communication in marriage being conducted at the Denver Center for Marital and Family Studies. Families will participate in three interaction tasks designed to tap parental scaffolding behavior during problemsolving with their children. Children will be administered self-report measures to tap their perceived competence at such problem-solving as those in the interaction tasks and parents will complete a questionnaire tapping the behavioral indicators of their child's self-esteem. Family interaction data will be coded with the use of a microanalytic coding system developed by this study, the Parent-Child Interaction Coding System. Marital communication data at three time points, premaritally, during the transition to parenthood , and concurrently, will be obtained from couples' interactions from the longitudinal study. The clinical significance of this study includes implications for training couples how to effectively regulate negative affect and offer their children sensitive assistance during joint problem-solving.

Towards a model-driven engineering approach of data mining

Nowadays, data mining is based on low-level specications of the employed techniques typically bounded to a specic analysis platform. Therefore, data mining lacks a modelling architecture that allows analysts to consider it as a truly software-engineering process. Here, we propose a model-driven approach based on (i) a conceptual modelling framework for data mining, and (ii) a set of model transformations to automatically generate both the data under analysis (via data-warehousing technology) and the analysis models for data mining (tailored to a specic platform). Thus, analysts can concentrate on the analysis problem via conceptual data-mining models instead of low-level programming tasks related to the underlying-platform technical details. These tasks are now entrusted to the model-transformations scaffolding.

Integrating the development of data mining and data warehouses via model-driven engineering

Data mining is one of the most important analysis techniques to automatically extract knowledge from large amount of data. Nowadays, data mining is based on low-level specifications of the employed techniques typically bounded to a specific analysis platform. Therefore, data mining lacks a modelling architecture that allows analysts to consider it as a truly software-engineering process. Bearing in mind this situation, we propose a model-driven approach which is based on (i) a conceptual modelling framework for data mining, and (ii) a set of model transformations to automatically generate both the data under analysis (that is deployed via data-warehousing technology) and the analysis models for data mining (tailored to a specific platform). Thus, analysts can concentrate on understanding the analysis problem via conceptual data-mining models instead of wasting efforts on low-level programming tasks related to the underlying-platform technical details. These time consuming tasks are now entrusted to the model-transformations scaffolding. The feasibility of our approach is shown by means of a hypothetical data-mining scenario where a time series analysis is required.

Race at the Interface: Rendering Blackness on WorldStarHipHop.com

WorldStarHipHop.com (WSHH) is an online video aggregating website that describes itself as ���the premiere online hip hop destination��� and a home for ���urban media.��� Yet, browsing through the site provides little clarity on what constitutes a hip-hop video or urban Internet space because of the disparate video content, the actual racial diversity of the performers, and the website���s generic design. As a result, WSHH���s taglines make a strange claim about the current state of the black musical tradition. Through close readings of the site, this article considers the architecture of this space of interracial exchange and identifies the interface as an example of Modernist architectural simplicity. I argue WSHH���s modular design is flexible enough to include non-black bodies, while remaining a black ���urban��� space. Thus, the site���s straightforward architecture paradoxically becomes the scaffolding of a much more complex, de-corporealized, and ���shareable��� blackness.