Bruce E. Kaplan

Lipopeptide epitopes extended by an Nϵ-palmitoyl-lysine moiety increase uptake and maturation of dendritic cells through a Toll-like receptor-2 pathway and trigger a Th1-dependent protective immunity

Lipopeptides, a form of peptide immunogens, are currently under intense investigation as human vaccines for many infectious pathogens and cancers. However, the cellular and molecular mechanisms of lipopeptide immunogenicity are only partially understood. We have investigated the influence of the lipid content on the immunogenicity of lipopeptides using the herpes simplex virus type 1 (HSV‐1) gD1–23 peptide as a model antigen. Totally synthetic lipopeptides were constructed by covalent attachment to the peptide backbone of either Nϵ‐palmitoyl‐lysine (palmitoyl‐lipidated peptide, palmitoyl‐LP) or cholesterol‐lysine (cholesterol‐lipidated peptide, cholesterol‐LP). Immunization of mice with the palmitoyl‐LP, but not with its cholesterol‐LP analog, induced a strong T cell‐dependent protective immunity against lethal HSV‐1 infection. Analysis of cytokine profiles and IgG2a/IgG1 ratios revealed that a dominant Th1‐type immune response was stimulated by the palmitoyl‐LP, as opposed to a Th2 response generated by its cholesterol‐LP analog. The palmitoyl‐LP was efficiently taken up in vitro by immature dendritic cells (DC) in a time‐ and dose‐dependent manner, and induced phenotypic maturation and production of pro‐inflammatory cytokines by DC. Finally, DC stimulated with the palmitoyl‐LP induced antigen‐specific T cell responses through the Toll‐like receptor‐2 pathway. These findings have important implications for the development of effective lipopeptide immunization strategies against infectious pathogens.

On the use of s-t-butylsulphenyl group for protection of cysteine in solid-phase peptide synthesis using fmoc-amino acids

Abstract The utilization of the N α -fluorenylmethoxycarbonyl derivative of S- t -butyl sulphenyl cysteine in solid-phase peptide synthesis for the preparation of several cysteine-containing peptides is described. When this derivative is located in the C-terminal position a side reaction prevents the formation of the desired product. Finally, a peptide containing a disulphide bond has been obtained in excellent yields by selective deprotection of the cysteines and disulfide bond formation on the solid support.

A GH3-like Domain in Reaper Is Required for Mitochondrial Localization and Induction of IAP Degradation*

Reaper is a potent pro-apoptotic protein originally identified in a screen for Drosophila mutants defective in apoptotic induction. Multiple functions have been ascribed to this protein, including inhibition of IAPs (inhibitors of apoptosis); induction of IAP degradation; inhibition of protein translation; and when expressed in vertebrate cells, induction of mitochondrial cytochrome c release. Structure/function analysis of Reaper has identified an extreme N-terminal motif that appears to be sufficient for inhibition of IAP function. We report here that this domain, although required for IAP destabilization, is not sufficient. Moreover, we have identified a small region of Reaper, similar to the GH3 domain of Grim, that is required for localization of Reaper to mitochondria, induction of IAP degradation, and potent cell killing. Although a mutant Reaper protein lacking the GH3 domain was deficient in these properties, these defects could be fully rectified by appending either the C-terminal mitochondrial targeting sequence from Bcl-xL or a homologous region from the pro-apoptotic protein HID. Together, these data strongly suggest that IAP destabilization by Reaper in intact cells requires Reaper localization to mitochondria and that induction of IAP instability by Reaper is important for the potent induction of apoptosis in Drosophila cells.

Ionized and wobble base-pairing for bromouracil-guanine in equilibrium under physiological conditions: A nuclear magnetic resonance study on an oligonucleotide containing a bromouracil-guanine base-pair as a function of pH☆

A one and two-dimensional nuclear magnetic resonance study of a non-selfcomplementary oligonucleotide containing a central 5-bromouracil-guanine pair is reported. For these two bases three types of hydrogen bonding schemes could exist; wobble, rare tautomer and ionized. The two-dimensional spectra of non-exchangeable protons together with one-dimensional spectra recorded in water show that at pH 7.0 the predominant species is a right-handed B-form DNA in which the brU.G pair has wobble geometry. On raising the pH we observe a transition monitored by proton chemical shift changes for the brU.G and adjacent base-pairs. The mid-point of the transition was observed at pH 8.6. Spectra recorded at pH 9.8 show that the helix remains intact with B form conformation. It is shown that this high pH form has an ionized brU.G base-pair now in Watson-Crick geometry. Thus under physiological conditions an equilibrium exists between wobble and ionized structures.

Synthesis of Oligonucleotides Containing the Abasic Site Model Compound 1,4-Anhydro-2-Deoxy-D-Ribitol

Abstract An improved approach for the synthesis of phosphotriester and phosphoramidite derivatives of the 1,4-anhydro-2-deoxy-D-ribitol is presented. The incorporation of these compounds in synthetic DNA and the insertion of purine deoxyribonucleotides opposite the reduced abasic site by DNA polymerase is described.

Characterization of the high pH wobble structure of the 2-aminopurine·cytosine mismatch by N-15 NMR spectroscopy

Transition mutations induced by the base analogue 2-aminopurine arise via the formation of AP.C base pairs during DNA replication. We report here the results of N-15 NMR studies on a duplex oligonucleotide containing N-15 enriched AP and C residues. At high pH (8.6) the AP.C base pair is predominantly wobble. This is the first report on use of a site specifically N-15 enriched oligonucleotide as a probe of aberrant base pairing in DNA.

Structural and dynamic properties of a bromouracil-adenine base pair in DNA studied by proton NMR

We have synthesized and studied by proton NMR a duplex heptaoligonucleotide containing a 5-bromouracil (brU)-adenine base pair. This represents the first structural characterization of a B-form DNA containing brU. The brU.A base pair is Watson-Crick rather than Hoogsteen as seen for the monomers in the crystalline state. From analysis of the NOESY sepctra at very short mixing times evidence is presented that substitution of brU for T induces significant conformational changes from that of a normal B DNA. The helix twist between brU4.A11 and G3.C12 is ca. 15 degrees and for both brU4 and G3 the glycosyl torsion angles are significantly changed. The imino proton of the bru.A base pair shows a pH insensitive line with which shows that the pK of brU in this base pair is very much higher than that of the monomer.

The automated synthesis of oligodeoxyribonucleotides

Abstract Progress in science is a history of the synergistic relationship between technology and theory: growth in theoretical understanding requires and initiates technological change; technological advance opens up new areas of knowledge. The history of molecular genetics is but a single illustration of this process. Four major achievements in understanding DNA have occurred in recent years: they are the use of X-ray diffraction to posit a model for DNA, the discovery of DNA cutting enzymes, the sequencing of DNA, and the chemical synthesis of DNA. This last achievement, the chemical synthesis of DNA, and, more particularly, its automation, will be the focus of this article.