Sequence and pH effects of LNA-containing triple helix-forming oligonucleotides: physical chemistry, biochemistry, and modeling studies

Triple helix-forming oligonucleotides (TFOs) have been demonstrated to be capable of interfering with gene expression and modifying genomic DNA in a sequence-specific manner. Partial incorporation of 2'-O,4'-C-methylene linked locked nucleic acid (LNA) residues in TFOs has been shown to enhance significantly triple helix formation, whereas the full-length LNA TFO failed to form a stable triplex. This work is aimed at understanding the triple helix-forming properties of LNA-containing TFOs and at optimally designing their sequences. Both DNA thermal melting, gel retardation, and restriction enzyme experiments as well as modeling studies by molecular mechanics were carried out to investigate the base composition/sequence and pH-dependence effects of LNA-containing TFOs, as well as their structural features underlying triple helix formation. Alternating LNA substitution every 2-3 nucleotides in TFOs is mandatory, whereas the use of thymine LNA residues should be favored under neutral pH conditions. A rule for designing optimal LNA-containing TFOs is proposed. In addition, alternative LNA and 2'-O-methyl residues in TFOs do not significantly improve triple helix formation.     

Acoustic detection and satellite-tracking leads to discovery of rare concentration of endangered North Pacific right whales

The North Pacific right whale, Eubalaena japonica, is one of the most endangered species of whale in the world. On 10 August 2004, two right whales were located in the Bering Sea using headings to right whale calls provided by directional sonobuoys. A satellite-monitored radio tag attached to one of these whales functioned for 40 days. Over the 40-day period, this whale moved throughout a large part of the southeast Bering Sea shelf, including areas of the outer-shelf where right whales have not been seen in decades. In September, multiple right whales were acoustically located and subsequently sighted by another survey vessel approaching a near-real-time position from the tag. An analysis of photographs confirmed at least 17 individual whales (not including the tagged whales). Genetic analysis of biopsy samples identified 17 individuals: 10 males and 7 females. The discovery of seven females was significant, as only one female had been identified in the past. Genetics also confirmed the presence of at least two calves. Although the future of this population is highly uncertain, the discovery of additional females and calves gives some hope that this most critically endangered of all whale populations may still possess the capacity to recover.     

Identification of identical TCRs in primary melanoma lesions and tumor free corresponding sentinel lymph nodes

It is generally believed that priming of efficient T-cell responses takes place in peripheral lymphoid tissues. Although this notion has been rigidly proven for infectious diseases, direct evidence for lymph node priming of in vivo T-cell responses against tumors is still lacking. In the present study, we conducted a full and nonbiased comparison of T-cell clonotypes in melanoma lesions and corresponding sentinel lymph nodes. Whereas most tumor lesions comprised a high number of T-cell clonotypes, only a small number of clonally expanded T cells were detected in the draining lymph nodes. Comparative clonotype mapping demonstrated the presence of identical T-cell clonotypes in the tumors and the respective sentinel lymph nodes, only when tumor cells were present in the latter. However, taking advantage of clonotype specific PCR amplification, TCR sequences representing clonally expanded T cells at the tumor site could be detected in the lymph nodes draining the tumors even in the absence of tumor cells. Evidence for the tumor-specific characteristics of these cells was obtained by in situ staining with peptide/HLA class I complexes demonstrating the presence of MART-1/HLA-A2- and MAGE-3/HLA-A2-reactive T cells at the tumor site, as well as in the draining lymph node. Our data indicate that T-cell responses to melanoma are primed in the sentinel lymph node by cross presentation of tumor antigens by dendritic cells.     

Measurement of conformational changes accompanying desensitization in an ionotropic glutamate receptor

The canonical conformational states occupied by most ligand-gated ion channels, and many cell-surface receptors, are the resting, activated, and desensitized states. While the resting and activated states of multiple receptors are well characterized, elaboration of the structural properties of the desensitized state, a state that is by definition inactive, has proven difficult. Here we use electrical, chemical, and crystallographic experiments on the AMPA-sensitive GluR2 receptor, defining the conformational rearrangements of the agonist binding cores that occur upon desensitization of this ligand-gated ion channel. These studies demonstrate that desensitization involves the rupture of an extensive interface between domain 1 of 2-fold related glutamate-binding core subunits, compensating for the ca. 21 degrees of domain closure induced by glutamate binding. The rupture of the domain 1 interface allows the ion channel to close and thereby provides a simple explanation to the long-standing question of how agonist binding is decoupled from ion channel gating upon receptor desensitization.     

Mutations Phe785Leu and Thr618Met in Na+,K+-ATPase, associated with familial rapid-onset dystonia parkinsonism, interfere with Na+ interaction by distinct mechanisms

The Na(+),K(+)-ATPase plays key roles in brain function. Recently, missense mutations in the Na(+),K(+)-ATPase were found associated with familial rapid-onset dystonia parkinsonism (FRDP). Here, we have characterized the functional consequences of FRDP mutations Phe785Leu and Thr618Met. Both mutations lead to functionally altered, but active, Na(+),K(+)-pumps, that display reduced apparent affinity for cytoplasmic Na(+), but the underlying mechanism differs between the mutants. In Phe785Leu, the interaction of the E(1) form with Na(+) is defective, and the E(1)-E(2) equilibrium is not displaced. In Thr618Met, the Na(+) affinity is reduced because of displacement of the conformational equilibrium in favor of the K(+)-occluded E(2)(K(2)) form. In both mutants, K(+) interaction at the external activating sites of the E(2)P phosphoenzyme is normal. The change of cellular Na(+) homeostasis is likely a major factor contributing to the development of FRDP in patients carrying the Phe785Leu or Thr618Met mutation. Phe785Leu moreover interferes with Na(+) interaction on the extracellular side and reduces the affinity for ouabain significantly. Analysis of two additional Phe(785) mutants, Phe785Leu/Leu786Phe and Phe785Tyr, demonstrated that the aromatic function of the side chain, as well as its exact position, is critical for Na(+) and ouabain binding. The effects of substituting Phe(785) could be explained by structural modeling, demonstrating that Phe(785) participates in a hydrophobic network between three transmembrane segments. Thr(618) is located in the cytoplasmic part of the molecule near the catalytic site, and the structural modeling indicates that the Thr618Met mutation interferes with the bonding pattern in the catalytic site in the E(1) form, thereby destabilizing E(1) relative to E(2)(K(2)).     

Versatile interactions of the antimicrobial peptide novispirin with detergents and lipids

Novispirin G-10 is an 18-residue designed cationic peptide derived from the N-terminal part of an antimicrobial peptide from sheep. This derivative is more specific for bacteria than the parent peptide. We have analyzed Novispirin's interactions with various amphipathic molecules and find that a remarkably wide variety of conditions induce alpha-helical structure. Optimal structure induction by lipids occurs when the vesicles contain 40-80% anionic lipid, while pure anionic lipid vesicles induce aggregation. SDS also forms aggregates with Novispirin at submicellar concentrations but induces alpha-helical structures above the cmc. Both types of aggregates contain significant amounts of beta-sheet structure, highlighting the peptide's structural versatility. The cationic detergent LTAC has a relatively strong affinity for the cationic peptide despite the peptide's net positive charge of +7 at physiological pH and total lack of negatively charged side chains. Zwitterionic and nonionic detergents induce alpha-helical structures at several hundred millimolar detergent. We have solved the peptide structure in SDS and LTAB by NMR and find subtle differences compared to the structure in TFE, which we ascribe to the interaction with an amphiphilic environment. Novispirin is largely buried in the SDS-micelle, whereas it does not enter the LTAC-micelle but merely forms a dynamic equilibrium between surface-bound and nonbound Novispirin. Thus, electrostatic repulsion can be overruled by relatively high-detergent concentrations or by deprotonating a single critical side chain, despite the fact that Novispirin's ability to bind to amphiphiles and form alpha-helical structure is sensitive to the electrostatics of the amphiphilic environment. This emphasizes the versatility of cationic antimicrobial peptides' interactions with amphiphiles.     

Affordable biocomputing for everyone: using the Internet,freeware and open-source software

How to build your own complete working biocomputing platform with nothing more than a desktop computer and an Internet connection.     

The specific targeting of immune regulation: T-cell responses against Indoleamine 2,3-dioxygenase

Indoleamine 2,3-dioxygenase (IDO) is an immunoregulatory enzyme that is implicated in suppressing T-cell immunity in many settings including cancer. In recent years, we have described spontaneous CD8(+) as well as CD4(+) T-cell reactivity against IDO in the tumor microenvironment of different cancer patients as well as in the peripheral blood of both cancer patients and to a lesser extent in healthy donors. We have demonstrated that IDO-reactive CD8(+) T cells were peptide-specific, cytotoxic effector cells, which are able to recognize and kill IDO-expressing cells including tumor cells as well as dendritic cells. Consequently, IDO may serve as a widely applicable target for immunotherapeutic strategies with a completely different function as well as expression pattern compared to previously described antigens. IDO constitutes a significant counter-regulatory mechanism induced by pro-inflammatory signals, and IDO-based immunotherapy may consequently be synergistic with additional immunotherapy. In this regard, we have shown that the presence of IDO-specific T cells boosted immunity against CMV and tumor antigens by eliminating IDO(+) suppressive cells and changing the regulatory microenvironment. The current review summarizes current knowledge of IDO as a T-cell antigen, reports the initial results that are suggesting a general function of IDO-specific T cells in immunoregulation, and discusses future opportunities.     

Versatile High Resolution Oligosaccharide Microarrays for Plant Glycobiology and Cell Wall Research

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.