Cross-hemispheric collaboration and segregation associated with task difficulty as revealed by structural and functional connectivity.

Although it is known that brain regions in one hemisphere may interact very closely with their corresponding contralateral regions (collaboration) or operate relatively independent of them (segregation), the specific brain regions (where) and conditions (how) associated with collaboration or segregation are largely unknown. We investigated these issues using a split field-matching task in which participants matched the meaning of words or the visual features of faces presented to the same (unilateral) or to different (bilateral) visual fields. Matching difficulty was manipulated by varying the semantic similarity of words or the visual similarity of faces. We assessed the white matter using the fractional anisotropy (FA) measure provided by diffusion tensor imaging (DTI) and cross-hemispheric communication in terms of fMRI-based connectivity between homotopic pairs of cortical regions. For both perceptual and semantic matching, bilateral trials became faster than unilateral trials as difficulty increased (bilateral processing advantage, BPA). The study yielded three novel findings. First, whereas FA in anterior corpus callosum (genu) correlated with word-matching BPA, FA in posterior corpus callosum (splenium-occipital) correlated with face-matching BPA. Second, as matching difficulty intensified, cross-hemispheric functional connectivity (CFC) increased in domain-general frontopolar cortex (for both word and face matching) but decreased in domain-specific ventral temporal lobe regions (temporal pole for word matching and fusiform gyrus for face matching). Last, a mediation analysis linking DTI and fMRI data showed that CFC mediated the effect of callosal FA on BPA. These findings clarify the mechanisms by which the hemispheres interact to perform complex cognitive tasks.

Detection of amino-terminal extracellular domain of somatostatin receptor 2 by specific monoclonal antibodies and quantification of receptor density in medulloblastoma.

Somatostatin receptor 2 (SSTR2) is expressed by most medulloblastomas (MEDs). We isolated monoclonal antibodies (MAbs) to the 12-mer (33)QTEPYYDLTSNA(44), which resides in the extracellular domain of the SSTR2 amino terminus, screened the peptide-bound MAbs by fluorescence microassay on D341 and D283 MED cells, and demonstrated homogeneous cell-surface binding, indicating that all cells expressed cell surface-detectable epitopes. Five radiolabeled MAbs were tested for immunoreactive fraction (IRF), affinity (KA) (Scatchard analysis vs. D341 MED cells), and internalization by MED cells. One IgG(3) MAb exhibited a 50-100% IRF, but low KA. Four IgG(2a) MAbs had 46-94% IRFs and modest KAs versus intact cells (0.21-1.2 x 10(8) M(-1)). Following binding of radiolabeled MAbs to D341 MED at 4 degrees C, no significant internalization was observed, which is consistent with results obtained in the absence of ligand. However, all MAbs exhibited long-term association with the cells; binding at 37 degrees C after 2 h was 65-66%, and after 24 h, 52-64%. In tests with MAbs C10 and H5, the number of cell surface receptors per cell, estimated by Scatchard and quantitative FACS analyses, was 3.9 x 10(4) for the "glial" phenotype DAOY MED cell line and 0.6-8.8 x 10(5) for four neuronal phenotype MED cell lines. Our results indicate a potential immunotherapeutic application for these MAbs.

HDAC6 and Ubp-M BUZ domains recognize specific C-terminal sequences of proteins.

The BUZ/Znf-UBP domain is a protein module found in the cytoplasmic deacetylase HDAC6, E3 ubiquitin ligase BRAP2/IMP, and a subfamily of ubiquitin-specific proteases. Although several BUZ domains have been shown to bind ubiquitin with high affinity by recognizing its C-terminal sequence (RLRGG-COOH), it is currently unknown whether the interaction is sequence-specific or whether the BUZ domains are capable of binding to proteins other than ubiquitin. In this work, the BUZ domains of HDAC6 and Ubp-M were subjected to screening against a one-bead-one-compound (OBOC) peptide library that exhibited random peptide sequences with free C-termini. Sequence analysis of the selected binding peptides as well as alanine scanning studies revealed that the BUZ domains require a C-terminal Gly-Gly motif for binding. At the more N-terminal positions, the two BUZ domains have distinct sequence specificities, allowing them to bind to different peptides and/or proteins. A database search of the human proteome on the basis of the BUZ domain specificities identified 11 and 24 potential partner proteins for Ubp-M and HDAC6 BUZ domains, respectively. Peptides corresponding to the C-terminal sequences of four of the predicted binding partners (FBXO11, histone H4, PTOV1, and FAT10) were synthesized and tested for binding to the BUZ domains by fluorescence polarization. All four peptides bound to the HDAC6 BUZ domain with low micromolar K(D) values and less tightly to the Ubp-M BUZ domain. Finally, in vitro pull-down assays showed that the Ubp-M BUZ domain was capable of binding to the histone H3-histone H4 tetramer protein complex. Our results suggest that BUZ domains are sequence-specific protein-binding modules, with each BUZ domain potentially binding to a different subset of proteins.

The Macrophage-Specific Promoter mfap4 Allows Live, Long-Term Analysis of Macrophage Behavior during Mycobacterial Infection in Zebrafish.

Transgenic labeling of innate immune cell lineages within the larval zebrafish allows for real-time, in vivo analyses of microbial pathogenesis within a vertebrate host. To date, labeling of zebrafish macrophages has been relatively limited, with the most specific expression coming from the mpeg1 promoter. However, mpeg1 transcription at both endogenous and transgenic loci becomes attenuated in the presence of intracellular pathogens, including Salmonella typhimurium and Mycobacterium marinum. Here, we describe mfap4 as a macrophage-specific promoter capable of producing transgenic lines in which transgene expression within larval macrophages remains stable throughout several days of infection. Additionally, we have developed a novel macrophage-specific Cre transgenic line under the control of mfap4, enabling macrophage-specific expression using existing floxed transgenic lines. These tools enrich the repertoire of transgenic lines and promoters available for studying zebrafish macrophage dynamics during infection and inflammation and add flexibility to the design of future macrophage-specific transgenic lines.