Characterization of the oligomer structure of recombinant human mannan-binding lectin

Mannan-binding lectin (MBL) belongs to a family of proteins called the collectins, which show large differences in their ultrastructures. These differences are believed to be determined by different N-terminal disulfide-bonding patterns. So far only the bonding pattern of two of the simple forms (recombinant rat MBL-C and bovine CL-43) have been determined. Recombinant MBL expressed in human cells was purified, and the structure of the N-terminal region was determined. Preliminary results on human plasma-derived MBL revealed high similarity to the recombinant protein. Here we report the structure of the N-terminal part of recombinant human MBL and present a model to explain the oligomerization pattern. Using a strategy of consecutive enzymatic digestions and matrix-assisted laser desorption ionization mass spectrometry, we succeeded in identifying a number of disulfide-linked peptides from the N-terminal cysteine-rich region. Based on these building blocks, we propose a model that can explain the various oligomeric forms found in purified MBL preparations. Furthermore, the model was challenged by the production of cysteine to serine mutants of the three N-terminally situated cysteines. The oligomerization patterns of these mutants support the proposed model. The model indicates that the polypeptide dimer is the basic unit in the oligomerization.     

In vivo and in vitro characterization of novel neuronal plasticity factors identified following spinal cord injury

Following spinal cord injury, there are numerous changes in gene expression that appear to contribute to either neurodegeneration or reparative processes. We utilized high density oligonucleotide microarrays to examine temporal gene profile changes after spinal cord injury in rats with the goal of identifying novel factors involved in neural plasticity. By comparing mRNA changes that were coordinately regulated over time with genes previously implicated in nerve regeneration or plasticity, we found a gene cluster whose members are involved in cell adhesion processes, synaptic plasticity, and/or cytoskeleton remodeling. This group, which included the small GTPase Rab13 and actin-binding protein Coronin 1b, showed significantly increased mRNA expression from 7-28 days after trauma. Overexpression in vitro using PC-12, neuroblastoma, and DRG neurons demonstrated that these genes enhance neurite outgrowth. Moreover, RNAi gene silencing for Coronin 1b or Rab13 in NGF-treated PC-12 cells markedly reduced neurite outgrowth. Coronin 1b and Rab13 proteins were expressed in cultured DRG neurons at the cortical cytoskeleton, and at growth cones along with the pro-plasticity/regeneration protein GAP-43. Finally, Coronin 1b and Rab13 were induced in the injured spinal cord, where they were also co-expressed with GAP-43 in neurons and axons. Modulation of these proteins may provide novel targets for facilitating restorative processes after spinal cord injury.     

Histone H2A ubiquitination does not preclude histone H1 binding, but it facilitates its association with the nucleosome

Histone H2A ubiquitination is a bulky posttranslational modification that occurs at the vicinity of the binding site for linker histones in the nucleosome. Therefore, we took several experimental approaches to investigate the role of ubiquitinated H2A (uH2A) in the binding of linker histones. Our results showed that uH2A was present in situ in histone H1-containing nucleosomes. Notably in vitro experiments using nucleosomes reconstituted onto 167-bp random sequence and 208-bp (5 S rRNA gene) DNA fragments showed that ubiquitination of H2A did not prevent binding of histone H1 but it rather enhanced the binding of this histone to the nucleosome. We also showed that ubiquitination of H2A did not affect the positioning of the histone octamer in the nucleosome in either the absence or the presence of linker histones.     

Binding of antitumor antibiotic daunomycin to histones in chromatin and in solution

Daunomycin is an anticancer drug that is well-known to interact with DNA in chromatin. Using a compositionally defined chicken erythrocyte chromatin fraction, we have obtained conclusive evidence that the drug is also able to interact with chromatin-bound linker histones without any noticeable binding to core histones. The drug can interact in an equal fashion with both histone H1 and H5 and to a greater extent with core histones H3/H4 and H2A/H2B as free proteins in solution. Thus, the binding of daunomycin to linker histones in the chromatin fiber is most likely due to the well-known higher accessibility of these histones to the surrounding environment of the fiber. Binding of daunomycin to linker histones appears to primarily involve the trypsin-resistant (winged-helix) domain of these proteins. The studies described here reveal the occurrence of a previously undisclosed mechanism for the antitumor activity of anthracycline drugs at the chromatin level.     

Association of the DTNBP1 locus with schizophrenia in a U.S. population

Linkage and association studies have recently implicated dystrobrevin-binding protein 1 (DTNBP1) in the etiology of schizophrenia. We analyzed seven previously tested DTNBP1 single-nucleotide polymorphisms (SNPs) in a cohort of 524 individuals with schizophrenia or schizoaffective disorder and 573 control subjects. The minor alleles of three SNPs (P1578, P1763, and P1765) were positively associated with the diagnosis of schizophrenia or schizoaffective disorder in the white subset of the study cohort (258 cases, 467 controls), with P1578 showing the most significant association (odds ratio 1.76, P =.0026). The same three SNPs were also associated in a smaller Hispanic subset (51 cases, 32 controls). No association was observed in the African American subset (215 cases, 74 controls). A stratified analysis of the white and Hispanic subsets showed association with the minor alleles of four SNPs (P1578, P1763, P1320, and P1765). Again, the most significant association was observed for P1578 (P =.0006). Haplotype analysis supported these findings, with a single risk haplotype significantly overrepresented in the white sample (P =.005). Our study provides further evidence for a role of the DTNBP1 gene in the genetic etiology of schizophrenia.     

Geographic variation in patterns of nestedness among local stream fish assemblages in Virginia

Nestedness of faunal assemblages is a multi-scale phenomenon, potentially influenced by a variety of factors. Prior small-scale studies have found freshwater fish species assemblages to be nested along stream courses as a result of either selective colonization or extinction. However, within-stream gradients in temperature and other factors are correlated with the distributions of many fish species and may also contribute to nestedness. At a regional level, strongly nested patterns would require a consistent set of structuring mechanisms across streams, and correlation among species tolerances of the environmental factors that influence distribution. Thus, nestedness should be negatively associated with the spatial extent of the region analyzed and positively associated with elevational gradients (a correlate of temperature and other environmental factors). We examined these relationships for the freshwater fishes of Virginia. Regions were defined within a spatial hierarchy and included whole river drainages, portions of drainages within physiographic provinces, and smaller subdrainages. In most cases, nestedness was significantly stronger in regions of smaller spatial extent and in regions characterized by greater topographic relief. Analysis of hydrologic variability and patterns of faunal turnover provided no evidence that inter-annual colonization/extinction dynamics contributed to elevational differences in nestedness. These results suggest that, at regional scales, nestedness is influenced by interactions between biotic and abiotic factors, and that the strongest nestedness is likely to occur where a small number of organizational processes predominate, i.e., over small spatial extents and regions exhibiting strong environmental gradients.The Virginia Cooperate Fish and Wildlife Research Unit is jointly sponsored by United States Geological Survey, Virginia Polytechnic Institute and State University, and Virginia Department of Game and Inland Fisheries.     

Generation of a tumor vaccine candidate based on conjugation of a MUC1 peptide to polyionic papillomavirus virus-like particles

Virus-like particles (VLPs) are promising vaccine technology due to their safety and ability to elicit strong immune responses. Chimeric VLPs can extend this technology to low immunogenicity foreign antigens. However, insertion of foreign epitopes into the sequence of self-assembling proteins can have unpredictable effects on the assembly process. We aimed to generate chimeric bovine papillomavirus (BPV) VLPs displaying a repetitive array of polyanionic docking sites on their surface. These VLPs can serve as platform for covalent coupling of polycationic fusion proteins. We generated baculoviruses expressing chimeric BPV L1 protein with insertion of a polyglutamic-cysteine residue in the BC, DE, HI loops and the H4 helix. Expression in insect cells yielded assembled VLPs only from insertion in HI loop. Insertion in DE loop and H4 helix resulted in partially formed VLPs and capsomeres, respectively. The polyanionic sites on the surface of VLPs and capsomeres were decorated with a polycationic MUC1 peptide containing a polyarginine-cysteine residue fused to 20 amino acids of the MUC1 tandem repeat through electrostatic interactions and redox-induced disulfide bond formation. MUC1-conjugated fully assembled VLPs induced robust activation of bone marrow-derived dendritic cells, which could then present MUC1 antigen to MUC1-specific T cell hybridomas and primary naïve MUC1-specific T cells obtained from a MUC1-specific TCR transgenic mice. Immunization of human MUC1 transgenic mice, where MUC1 is a self-antigen, with the VLP vaccine induced MUC1-specific CTL, delayed the growth of MUC1 transplanted tumors and elicited complete tumor rejection in some animals.