Ribosomal RNA secondary structure: compensatory mutations and implications for phylogenetic analysis

Using sequence data from the 28S ribosomal RNA (rRNA) genes of selected vertebrates, we investigated the effects that constraints imposed by secondary structure have on the phylogenetic analysis of rRNA sequence data. Our analysis indicates that characters from both base-pairing regions (stems) and non-base-pairing regions (loops) contain phylogenetic information, as judged by the level of support of the phylogenetic results compared with a well-established tree based on both morphological and molecular data. The best results (the greatest level of support of well-accepted nodes) were obtained when the complete data set was used. However, some previously supported nodes were resolved using either the stem or loop bases alone. Stem bases sustain a greater number of compensatory mutations than would be expected at random, but the number is < 40% of that expected under a hypothesis of perfect compensation to maintain secondary structure. Therefore, we suggest that in phylogenetic analyses, the weighting of stem characters be reduced by no more than 20%, relative to that of loop characters. In contrast to previous suggestions, we do not recommend weighting of stem positions by one-half, compared with that of loop positions, because this overcompensates for the constraints that selection imposes on the secondary structure of rRNA.      

Localization of SV40 genes within supercoiled loop domains

Recent studies indicate that eukaryotic DNA is organized into supercoiled loop domains. These loops appear to be anchored at their bases to an insoluble nuclear skeleton or matrix. Most of the DNA in the loops can be released from the matrix by nuclease digestion; the residual DNA remaining with the nuclear matrix represents sequences at the base of the loops, and possibly other sequences which are intimately associated with the nuclear matrix for other reasons. Using a quantitative application of the Southern blotting technique, we have found this residual DNA from SV40 infected 3T3 cells to be enriched in SV40 sequences, indicating that they reside near matrix-DNA attachment points. An enrichment of 3-7 fold relative to total cellular DNA, was found in each of three different lines of SV40 infected 3T3 cells. Control experiments with globin genes showed no such enrichment in this residual matrix DNA. This sequence specificity suggests that the spatial organization of DNA sequences within loops may be related to the functionality of these sequences within the cell.     

An immunodominant MHC class II-restricted tumor antigen is conformation dependent and binds to the endoplasmic reticulum chaperone, calreticulin.

There is accumulating evidence that CD4(+) T cell responses are important in antitumor immunity. Accordingly, we generated CD4(+) T cells against the murine CT26 colon cancer. Three of three independent CT26-specific CD4(+) hybridomas were found to recognize the high m.w. precursor of the env gene product gp90. The CD4(+) response was completely tumor specific in that the same glycoprotein expressed by other tumors was not recognized by the CT26-specific hybridomas. The recognition of gp90 by the hybridomas was strictly dependent on the conformation of gp90. Different procedures that disrupted the conformation of the glycoprotein, such as disulfide bond reduction and thermal denaturation, completely abrogated recognition of gp90 by all three hybridomas. In CT26 cells, but not in other tumor cells tested, a large proportion of gp90 was retained in the endoplasmic reticulum, mostly bound to the endoplasmic reticulum chaperone, calreticulin. Although calreticulin was not essential for the stimulation of the gp90-specific hybridomas, most of the antigenic form of gp90 was bound to it. The antigenicity of gp90 correlated well with calreticulin binding, reflecting the fact that specificity of binding of calreticulin to its substrate required posttranslational modifications that were also necessary for the generation of this tumor-specific CD4(+) epitope.     

Baculovirus-infected insect cells expressing peptide-MHC complexes elicit protective antitumor immunity

Evaluation of T cell responses to tumor- and pathogen-derived peptides in preclinical models is necessary to define the characteristics of efficacious peptide vaccines. We show in this study that vaccination with insect cells infected with baculoviruses expressing MHC class I linked to tumor peptide mimotopes results in expansion of functional peptide-specific CD8+ T cells that protect mice from tumor challenge. Specific peptide mimotopes selected from peptide-MHC libraries encoded by baculoviruses can be tested using this vaccine approach. Unlike other vaccine strategies, this vaccine has the following advantages: peptides that are difficult to solublize can be easily characterized, bona fide peptides without synthesis artifacts are presented, and additional adjuvants are not required to generate peptide-specific responses. Priming of antitumor responses occurs within 3 days of vaccination and is optimal 1 wk after a second injection. After vaccination, the Ag-specific T cell response is similar in animals primed with either soluble or membrane-bound Ag, and CD11c+ dendritic cells increase expression of maturation markers and stimulate proliferation of specific T cells ex vivo. Thus, the mechanism of Ag presentation induced by this vaccine is consistent with cross-priming by dendritic cells. This straightforward approach will facilitate future analyses of T cells elicited by peptide mimotopes.     

Dynamic compression counteracts IL-1β induced inducible nitric oxide synthase and cyclo-oxygenase-2 expression in chondrocyte/agarose constructs

Background  

Nitric oxide and prostaglandin E₂ (PGE₂play pivotal roles in both the pathogenesis of osteoarthritis and catabolic processes in articular cartilage. These mediators are influenced by both IL-1β and mechanical loading, and involve alterations in the inducible nitric oxide synthase (iNOS) and cyclo-oxygenase (COX)-2 enzymes. To identify the specific interactions that are activated by both types of stimuli, we examined the effects of dynamic compression on levels of expression of iNOS and COX-2 and involvement of the p38 mitogen-activated protein kinase (MAPK) pathway.     

Soluble, high-affinity dimers of T-cell receptors and class II major histocompatibility complexes: biochemical probes for analysis and modulation of immune responses

T cell receptors (TCR) and major histocompatibility complex (MHC) molecules are integral membrane proteins that have central roles in cell-mediated immune recognition. Therefore, soluble analogs of these molecules would be useful for analyzing and possibly modulating antigen-specific immune responses. However, due to the intrinsic low-affinity and inherent solubility problems, it has been difficult to produce soluble high-affinity analogs of TCR and class II MHC molecules. This report describes a general approach which solves this intrinsic low-affinity by constructing soluble divalent analogs using IgG as a molecular scaffold. The divalent nature of the complexes increases the avidity of the chimeric molecules for cognate ligands. The generality of this approach was studied by making soluble divalent analogs of two different classes of proteins, a TCR (2C TCR2Ig) and a class II MHC (MCCI-Ek2Ig) molecule. Direct flow cytometry assays demonstrate that the divalent 2C TCR2Ig chimera retained the specificity of the native 2C TCR, while displaying increased avidity for cognate peptide/MHC ligands, resulting in a high-affinity probe capable of detecting interactions that heretofore have only been detected using surface plasmon resonance. TCR2IgG was also used in immunofluorescence studies to show ER localization of intracellular peptide-MHC complexes after peptide feeding. MCCI-Ek2Ig chimeras were able to both stain and activate an MCC-specific T cell hybridoma. Construction and expression of these two diverse heterodimers demonstrate the generality of this approach. Furthermore, the increased avidity of these soluble divalent proteins makes these chimeric molecules potentially useful in clinical settings for probing and modulating in vivo cellular responses.     

Focusing on the genetics of hearing: you ain't heard nothin' yet

The complexity of genetic pathways for hearing is beginning to be amenable to unraveling by systematic functional genomic analysis. Genome-wide mutagenesis studies in the mouse are beginning to shed further light on the structure and regulation of the machinery of hearing.     

Spinning molecular immunology into successful immunotherapy

Until recently, immunotherapies have been of limited success, particularly against cancer. However, recent insights into the cells, molecules and signalling pathways that regulate immune responsiveness are providing new approaches for immunotherapy. In this article, I review some of the most promising molecular and cellular targets for immunotherapy and discuss approaches that use these targets to amplify immune responses and potentially break antigen-specific tolerance. These strategies provide a blueprint for the development of successful immunotherapy over the next decade.