Single human B cell-derived monoclonal anti-Candida antibodies enhance phagocytosis and protect against disseminated candidiasis.

The high global burden of over one million annual lethal fungal infections reflects a lack of protective vaccines, late diagnosis and inadequate chemotherapy. Here, we have generated a unique set of fully human anti-Candida monoclonal antibodies (mAbs) with diagnostic and therapeutic potential by expressing recombinant antibodies from genes cloned from the B cells of patients suffering from candidiasis. Single class switched memory B cells isolated from donors serum-positive for anti-Candida IgG were differentiated in vitro and screened against recombinant Candida albicans Hyr1 cell wall protein and whole fungal cell wall preparations. Antibody genes from Candida-reactive B cell cultures were cloned and expressed in Expi293F human embryonic kidney cells to generate a panel of human recombinant anti-Candida mAbs that demonstrate morphology-specific, high avidity binding to the cell wall. The species-specific and pan-Candida mAbs generated through this technology display favourable properties for diagnostics, strong opsono-phagocytic activity of macrophages in vitro, and protection in a murine model of disseminated candidiasis.

Stabilization of RNA oligomers through reverse micelle encapsulation.

The cellular milieu is rich in diversity of both simple and complex molecules and is also quite crowded. By contrast, typical sample concentrations employed for in vitro investigation of biophysics and structural biology make use of purified macromolecules in simple buffer systems at concentrations that range from micromolar to millimolar. Although this formulation has proven to be compatible with a wide range of biological and structural studies, it is quite different from the relatively crowded conditions typically found within cells. The importance of these crowding effects for proteins has been recognized for some time, but the equivalent analysis is underexplored in nucleic acids. Encapsulation with surfactant-based reverse micelles has emerged as an effective biophysical tool, allowing study of the influence of ionic strength, pH, hydration, and crowding on biologically active macromolecules over a wide range of conditions. We have encapsulated an oligonucleotide model of TAR RNA from HIV and the 5' stem loop oligonucleotide of the U4 snRNA. Observation of imino (1)H resonances is an established method for evaluating the stability of nucleic acid oligonucleotides, implying the presence of stacked, hydrogen bonded base pairs. Inspection of (1)H NMR spectra of the RNA molecules reveals that the intensity of several of the imino resonances increases upon encapsulation. Additional resonances not observed in spectra of the oligonucleotides free in solution support the suggestion that the molecules have gained stability. These results indicate that RNA oligonucleotides may acquire significant stability in the presence of cellular levels of crowding.

Assignment of 1H, 13C, and 15N resonances of the RNA binding protein Snu13p from Saccharomyces cerevisiae.

Snu13p is a highly conserved RNA binding protein from Saccharomyces cerevisiae required for both eukaryotic pre-mRNA splicing and pre-rRNA processing. The 1H, 13C, and 15N assignments were determined from multidimensional, multinuclear NMR experiments conducted at 25 degrees C.

Recognition and removal of oxidized guanines in duplex DNA by the base excision repair enzymes hOGG1, yOGG1, and yOGG2.

8-Oxo-7,8-dihydroguanine (OG) is susceptible to further oxidation in vitro to form two secondary oxidation products, guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp). Previous work from this laboratory has shown that OG, Gh, and Sp are recognized and excised from duplex DNA substrates by the Escherichia coli DNA repair enzyme Fpg. In this report, we extend these studies to the functionally related eukaryotic OG glycosylases (OGG) from yeast and humans: yOGG1, yOGG2, and hOGG1. The hOGG1 enzyme was active only toward the removal of 8-oxoguanine, exhibiting a 1000-fold faster rate of removal of 8-oxoguanine from OG.C-containing duplexes relative to their OG.A counterparts. Duplexes containing Gh or Sp opposite any of the four natural bases were not substrates for the hOGG1 enzyme. In contrast, both yOGG1 and yOGG2 enzymes removed Gh and Sp in a relatively efficient manner from an 18 bp duplex. No significant difference was observed in the rate of reaction of Gh- and Sp-containing duplexes with yOGG1. However, yOGG2 removed Sp at a faster rate than Gh. Both yOGG enzymes exhibit a negligible dependence on the base opposite the lesion, suggesting that the activity of these enzymes may be promutagenic. Surprisingly, in the 18 bp sequence context, both yOGG enzymes did not exhibit OG removal activity. However, both removed OG in a 30 bp duplex with a different sequence surrounding the OG. The wide range of repair efficiencies observed by these enzymes with different substrates in vitro suggests that this could greatly affect the mutagenicity of these lesions in vivo. Indeed, the greater efficiency of the yOGG proteins for removal of the further oxidized products, Gh and Sp, over their 8-oxoguanine parent, suggests that these lesions may be the preferred substrates in vivo.