Reduction of liver macrophage transduction by pseudotyping lentiviral vectors with a fusion envelope from Autographa californica GP64 and Sendai virus F2 domain

Background  

Lentiviral vectors are well suited for gene therapy because they can mediate long-term expression in both dividing and nondividing cells. However, lentiviral vectors seem less suitable for liver gene therapy because systemically administered lentiviral vectors are preferentially sequestered by liver macrophages. This results in a reduction of available virus and might also increase the immune response to the vector and vector products.     

Point mutations of human interleukin-1 with decreased receptor binding affinity

Interleukin-1 (IL-1) is a monocyte-derived polypeptide hormone that interacts with a plasma membrane receptor. We have used oligonucleotide-directed mutagenesis to construct mutant human IL-1 proteins. Three different point mutants in a unique histidine residue (position 30) exhibited varying degrees of reduced IL-1 receptor binding affinity, whereas point mutants at five other residues behaved normally. Structural analysis of these mutant proteins by nuclear magnetic resonance spectroscopy detected no (or only minor) conformational changes relative to wild-type IL-1. These data suggest that the unique histidine residue influences the architecture of the receptor binding site on human IL-1.     

Site directed mutants of human interleukin-1 alpha: a 1H-NMR and receptor binding study

Mutant human interleukin-1 alpha proteins were constructed by oligonucleotide directed mutagenesis. Six different mutants were tested for receptor binding activity and showed no alteration with respect to the wild-type protein. Analysis of these mutants by nuclear magnetic resonance spectroscopy confirmed the structural integrity of the mutant proteins and permitted the sequence specific assignment of the histidine and tryptophan residues.     

Conformation, stability, and folding of interleukin 1 beta

Recombinant human interleukin 1 beta has been studied in solution with respect to its conformation, stability, and characteristics of unfolding and refolding. It is an all-beta-type, stable globular protein with a high cooperativity under conditions where refolding is reversible. The tryptophan residue is approximately 40% exposed to solvent, and the four tyrosines are 50% exposed. The fluorescence of the single tryptophan residue is quenched at pH 7.5 but dequenched by high salt, by titration to lower pH with a pK of 6.59, and by denaturants, resulting in an unusual biphasic change in fluorescence on unfolding. Both histidine and thiol residues have been excluded as being responsible for the pH dependence of fluorescence by site-directed mutagenesis and by chemical modification, respectively. The likely candidate is an aspartate or glutamate.     

Primary antibody-forming cells and secondary B cells are generated from separate precursor cell subpopulations

Two precursor cell subpopulations have been isolated from the spleen cells of nonimmune mice. The major B cell subpopulation binds high levels of the J11D monoclonal antibody and, upon T cell-dependent antigenic stimulation, gives rise to primary antibody-forming cell clones but not secondary B cells. A minority of the 10%-14% of Ia+ precursors that bind low levels of J11D (J11Dlo) also generate antibody-forming cell clones after primary stimulation. However, over 70% of J11Dlo precursors yield no primary antibody-forming cell clones but instead give rise to secondarily responsive B cells. The existence of a distinct precursor cell subpopulation that is responsible for the generation of B cell memory is further evidenced by the distribution of variable region clonotypes among J11Dlo primary precursors, which resembles the clonotype patterns of secondary B cells, and by the accumulation of somatic mutations in their clonal progeny.     

The role of glyceraldehyde 3-phosphate dehydrogenase (GapA-1) in <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> adherence to human cells

Background  

Glyceraldehyde 3-phosphate dehydrogenases (GAPDHs) are cytoplasmic glycolytic enzymes, which although lacking identifiable secretion signals, have also been found localized to the surface of several bacteria (and some eukaryotic organisms); where in some cases they have been shown to contribute to the colonization and invasion of host tissues. Neisseria meningitidis is an obligate human nasopharyngeal commensal which can cause life-threatening infections including septicaemia and meningitis. N. meningitidis has two genes, gapA-1 and gapA-2, encoding GAPDH enzymes. GapA-1 has previously been shown to be up-regulated on bacterial contact with host epithelial cells and is accessible to antibodies on the surface of capsule-permeabilized meningococcal cells. The aims of this study were: 1) to determine whether GapA-1 was expressed across different strains of N. meningitidis; 2) to determine whether GapA-1 surface accessibility to antibodies was dependant on the presence of capsule; 3) to determine whether GapA-1 can influence the interaction of meningococci and host cells, particularly in the key stages of adhesion and invasion.