DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis.

The availability of sequences of entire genomes has dramatically increased the number of protein targets, many of which will need to be overexpressed in cells other than the original source of DNA. Gene synthesis often provides a fast and economically efficient approach. The synthetic gene can be optimized for expression and constructed for easy mutational manipulation without regard to the parent genome. Yet design and construction of synthetic genes, especially those coding for large proteins, can be a slow, difficult and confusing process. We have written a computer program that automates the design of oligonucleotides for gene synthesis. Our program requires simple input information, i.e. amino acid sequence of the target protein and melting temperature (needed for the gene assembly) of synthetic oligonucleotides. The program outputs a series of oligonucleotide sequences with codons optimized for expression in an organism of choice. Those oligonucleotides are characterized by highly homogeneous melting temperatures and a minimized tendency for hairpin formation. With the help of this program and a two-step PCR method, we have successfully constructed numerous synthetic genes, ranging from 139 to 1042 bp. The approach presented here simplifies the production of proteins from a wide variety of organisms for genomics-based studies.

Many chemokines including CCL20/MIP-3alpha display antimicrobial activity.

Previous studies have demonstrated that beta-defensins exhibit chemotactic activity by sharing the chemokine receptor CCR6 with the CC chemokine ligand CCL20/macrophage-inflammatory protein-3alpha (MIP-3alpha). Structural analysis of CCL20/MIP-3alpha revealed that most of the positively charged residues are concentrated at one area of its topological surface, a characteristic considered to be important for the antimicrobial activity of defensins. Here, we report that similar to defensins, CCL20/MIP-3alpha has antimicrobial effects on Escherichia coli, Pseudomonas aeruginosa, Moraxella catarrhalis, Streptococcus pyogenes, Enterococcus faecium, Staphylococcus aureus, and Candida albicans. Additionally, by screening a total of 30 human chemokines, we have identified an additional 17 human chemokines, which exhibit antimicrobial activity in vitro. Collectively, about two-thirds of the chemokines investigated so far has the capacity to kill microorganisms in vitro, suggesting that antimicrobial activity may be another host-defense function for certain chemokines. Comparison of the structural characteristics between antimicrobial and nonantimicrobial chemokines suggests that topological formation of a large, positively charged electrostatic patch on the surface of the molecule is likely to be a common structural feature of antimicrobial chemokines.

Corpus callosum size is highly heritable in humans, and may reflect distinct genetic influences on ventral and rostral regions.

Anatomical differences in the corpus callosum have been found in various psychiatric disorders, but data on the genetic contributions to these differences have been limited. The current study used morphometric MRI data to assess the heritability of corpus callosum size and the genetic correlations among anatomical sub-regions of the corpus callosum among individuals with and without mood disorders. The corpus callosum (CC) was manually segmented at the mid-sagittal plane in 42 women (healthy, n = 14; major depressive disorder, n = 15; bipolar disorder, n = 13) and their 86 child or adolescent offspring. Four anatomical sub-regions (CC-genu, CC2, CC3 and CC-splenium) and total CC were measured and analyzed. Heritability and genetic correlations were estimated using a variance components method, with adjustment for age, sex, diagnosis, and diagnosis x age, where appropriate. Significant heritability was found for several CC sub-regions (P<0.01), with estimated values ranging from 48% (splenium) to 67% (total CC). There were strong and significant genetic correlations among most sub regions. Correlations between the genu and mid-body, between the genu and total corpus callosum, and between anterior and mid body were all >90%, but no significant genetic correlations were detected between ventral and rostral regions in this sample. Genetic factors play an important role in corpus callosum size among individuals. Distinct genetic factors seem to be involved in caudal and rostral regions, consistent with the divergent functional specialization of these brain areas.

Studies of the biological properties of human beta-defensin 1.

Defensins are small (30-45 amino acid residues) cationic proteins with broad antimicrobial activity against many bacteria and fungi, some enveloped viruses, and other activities such as chemoattraction of a range of different cell types to the sites of inflammation. These proteins represent attractive targets for developing novel antimicrobial agents and modulators of immune responses with therapeutic applicability. In this report, we present the results of functional and structural studies of 26 single-site mutants of human beta-defensin 1 (hBD1). All mutants were assayed for antimicrobial activity against Escherichia coli (ATCC strain 25922) and for chemotactic activity with CCR6-transfected HEK293 cells. To analyze the structural implications of mutagenesis and to verify the correctness of the disulfide connectivity, we used x-ray crystallography to conduct complete structural studies for 10 mutants in which the topology of disulfides was the same as in the native hBD1. Mutations did not induce significant changes of the tertiary structure, suggesting that the observed alterations of biological properties of the mutants were solely associated with changes in the respective side chains. We found that cationic residues located near the C terminus (Arg(29), Lys(31), Lys(33), and Lys(36)) of hBD1 define most of the anti-E. coli in vitro activity of this protein. In turn, nearly all mutations altering the CCR6-mediated chemotaxis are located at one area of the protein, defined by the N-terminal alpha-helical region (Asp(1)... Ser(8)) and a few topologically adjacent residues (Lys(22), Arg(29), and Lys(33)). These experimental results allow for the first time drafting of the CCR6-epitope for a defensin molecule.

Multiple roles of antimicrobial defensins, cathelicidins, and eosinophil-derived neurotoxin in host defense.

Mammals generate a diverse array of antimicrobial proteins, largely represented by defensins or cathelicidins. The direct in vitro microbicidal activity of antimicrobial proteins has long been considered an important innate immune defense, although the in vivo relevance has only very recently been established for certain defensins and cathelicidins. Mammalian defensins and cathelicidins have also been shown to have multiple receptor-mediated effects on immune cells. Beta-defensins interact with CCR6; murine beta-defensin-2 in addition activates TLR4. Cathelicidins act on FPRL1-expressing cells. Furthermore, several defensins have considerable immunoenhancing activity. Thus, it appears that mammalian antimicrobial proteins contribute to both innate and adaptive antimicrobial immunity.

Antimicrobial characterization of human beta-defensin 3 derivatives.

Human beta-defensin 3 (hBD3) is a highly basic 45-amino-acid protein that acts both as an antimicrobial agent and as a chemoattractant molecule. Although the nature of its antimicrobial activity is largely electrostatic, the importance of the molecular structure on this activity is poorly understood. Two isoforms of hBD3 were synthesized: the first with native disulfide linkages and the second with nonnative linkages. In a third synthetic peptide, all cysteine residues were replaced with alpha-aminobutyric acid, creating a completely linear peptide. A series of six small, linear peptides corresponding to regions of hBD3 with net charges ranging from +4 to +8 (at pH 7) and lengths ranging from 9 to 20 amino acids were also synthesized. The linear full-length peptide showed the highest microbicidal activity against Escherichia coli and Staphylococcus aureus, while all three full-length forms showed equal activity against Candida albicans. The linear peptide also showed high activity against Enterococcus faecium and Pseudomonas aeruginosa. Peptides corresponding to the C terminus showed higher activities when tested against E. coli, with the most active peptides being the most basic. However, only the peptide corresponding to the N terminus of hBD3 showed any activity against S. aureus and C. albicans. Further, N-terminal deletion mutants of native hBD3 showed diminished activities against S. aureus. Thus, the antimicrobial properties of hBD3 derivatives are determined by both charge and structure.

Structures of thymus and activation-regulated chemokine (TARC).

Thymus and activation-regulated chemokine (TARC) is a CC chemokine that is mainly expressed in the thymus. TARC interacts primarily with the CCR4 receptor and to a lesser extent with the CCR8 receptor. The structures of TARC have been solved by molecular replacement in two space groups, triclinic (P1) and tetragonal (P4(1)), and refined to resolutions of 1.72 and 2.1 A, respectively, with R factors of 19.8% (R(free) = 24.1%) and 19.8% (R(free) = 27.7%), respectively. The search model originated from the crystal structure of another chemokine, RANTES, and proved to be only modestly similar to the refined structure of TARC. Whereas the tetragonal structure was easily solved using the program AMoRe, solution of the triclinic structure proved to be quite challenging and was obtained by combining the results from four different molecular-replacement programs (AMoRe, CNS, BEAST and EPMR), with subsequent extension of the gathered information. The tertiary structure of TARC is similar to that of other CC chemokines, with a three-stranded antiparallel beta-sheet flanked by a C-terminal helix. Both quaternary structures consist of dimers, which in the triclinic crystals pack further into tetramers. The TARC dimers are similar to those observed previously in the crystal structures of both MCP-1 and RANTES.

Crystallization and preliminary X-ray studies of thymus and activation-regulated chemokine (TARC).

Thymus and activation-regulated chemokine (TARC) is a CC chemokine that is most highly expressed in the thymus. TARC interacts primarily with the CCR4 receptor and to a lesser extent with the CCR8 receptor. Three different crystal forms of synthetically prepared TARC were grown in triclinic, hexagonal and tetragonal systems. The X-ray data for the triclinic crystals (unit-cell parameters a = 56.46, b = 76.48, c = 88.37 A, alpha = 85.8, beta = 72.8, gamma = 70.0 degrees ) extend to 1.85 A on a conventional radiation source. The hexagonal crystals diffracted to 2.2 A at a synchrotron-radiation source and belong to either space group P6(1)22 or P6(5)22, with unit-cell parameters a = 61.8, c = 315. A. The tetragonal crystals diffracted to about 5 A at a synchrotron-radiation source and had approximate unit-cell parameters a = b = 47.7, c = 58.2 A.

Engineering disulfide bridges to dissect antimicrobial and chemotactic activities of human beta-defensin 3.

Human defensins form a family of small, cationic, and Cys-rich antimicrobial proteins that play important roles in innate immunity against invading microbes. They also function as effective immune modulators in adaptive immunity by selectively chemoattracting T lymphocytes and immature dendritic cells. On the basis of sequence homology and the connectivity of six conserved Cys residues, human defensins are classified into alpha and beta families. Structures of several beta-defensins have recently been characterized, confirming the disulfide connectivity conserved within the family, i.e., Cys1-Cys5, Cys2-Cys4, and Cys3-Cys6. We found that human beta-defensin 3 (hBD3), a recently described member of the growing beta family, did not fold preferentially into a native conformation in vitro under various oxidative conditions. Using the orthogonal protection of Cys1-Cys5 and of Cys1-Cys6, we chemically synthesized six topological analogs of hBD3 with predefined disulfide connectivities, including the (presumably) native beta pairing. Unexpectedly, all differently folded hBD3 species exhibited similar antimicrobial activity against Escherichia coli, whereas a wide range of chemotactic activities was observed with these analogs for monocytes and cells transfected by the chemokine receptor CCR6. Furthermore, whereas substitution of all Cys residues by alpha-aminobutyric acid completely abolished the chemotactic activity of hBD3, the bactericidal activity remained unaffected in the absence of any disulfide bridge. Our findings demonstrate that disulfide bonding in hBD3, although required for binding and activation of receptors for chemotaxis, is fully dispensable for its antimicrobial function, thus shedding light on the mechanisms of action for human beta-defensins and the design of novel peptide antibiotics.

The structure of human macrophage inflammatory protein-3alpha /CCL20. Linking antimicrobial and CC chemokine receptor-6-binding activities with human beta-defensins.

Human macrophage inflammatory protein-3alpha (MIP-3alpha; CCL20) is a CC-type chemokine that binds to and activates CC chemokine receptor-6 (CCR6). Although MIP-3alpha does not share the binding site of CCR6 with any other chemokine, human beta-defensin-1 and -2, small cationic antimicrobial peptides, have also been found to bind to and activate CCR6. Conversely, we have found that MIP-3alpha possesses antibacterial activity of greater potency than human beta-defensin-1 and -2 against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, while having no activity against the fungus Candida albicans. There is no clear sequence similarity between beta-defensins and the chemokine MIP-3alpha, beyond an abundance of cationic residues and the presence of disulfide bonds. Nonetheless, there are structural similarities between these three proteins that allow their overlap of chemotactic and antimicrobial activities. In this report, we describe the x-ray crystal structure of human MIP-3alpha refined to a resolution of 1.7 A and compare it with the crystal structures of human beta-defensin-1 and -2. Molecules of MIP-3alpha and the beta-defensins seem to share few structural motifs that are likely associated with their common biological activities.