Spider prey-wrapping silk is an α-helical coiled-coil/ß-sheet hybrid nanofiber.

Solid-State NMR results on 13C-Ala/Ser and 13C-Val enriched Argiope argentata prey-wrapping silk show that native, freshly spun aciniform silk nanofibers are dominated by α-helical (∼50% total) and random-coil (∼35% total) secondary structures, with minor ß-sheet nanocrystalline domains (∼15% total). This is the most in-depth study to date characterizing the protein structural conformation of the toughest natural biopolymer: aciniform prey-wrapping silks.

(1)H-(13)C INEPT MAS NMR correlation experiments with (1)H-(1)H mediated magnetization exchange to probe organization in lipid biomembranes.

Two-dimensional (1)H-(13)C INEPT MAS NMR experiments utilizing a (1)H-(1)H magnetization exchange mixing period are presented for characterization of lipid systems. The introduction of the exchange period allows for structural information to be obtained via (1)H-(1)H dipolar couplings but with (13)C chemical shift resolution. It is shown that utilizing a RFDR recoupling sequence with short mixing times in place of the more standard NOE cross-relaxation for magnetization exchange during the mixing period allowed for the identification and separation of close (1)H-(1)H dipolar contacts versus longer-range inter-molecular (1)H-(1)H dipolar cross-relaxation. These 2D INEPT experiments were used to address both intra- and inter-molecular contacts in lipid and lipid/cholesterol mixtures.

The influence of epitope density on the estimation of the affinity of antibody for complex antigens.

The influence of the epitope density of the antigen on antibody affinity values determined by fluid- and solid-phase immunoassays was assessed. The affinity of the interaction of a panel of monoclonal anti-DNP antibodies of different affinities (as determined by equilibrium dialysis) for DNP-protein conjugates of various hapten substitution ratios was used as the test system. The results obtained showed that the epitope density of the antigen markedly influences the observed affinity values obtained by both experimental approaches. However, the monoclonal antibodies were ranked in affinity terms by both assays in a similar order to that given by equilibrium dialysis. It is concluded that provided due care is exercised in choosing an appropriate epitope density for the test antigen, these methods can be used to provide rapid estimations of average antibody affinities.

Distribution effects on 1H double-quantum MAS NMR spectra.

The effect of a distribution in the (1)H-(1)H dipolar coupling on (1)H double-quantum (DQ) magic angle spinning (MAS) nuclear magnetic resonance (NMR) spinning sideband patterns is considered. In disordered or amorphous materials a distribution in the magnitude of the (1)H-(1)H dipolar coupling is a realistic possibility. Simulations of the (1)H DQ MAS NMR spinning sideband spectra were performed with the two-spin approximation. These simulations reveal that a dipolar coupling distribution can greatly affect the DQ spectral shape and behavior of the DQ build-up. The spectral line shapes are quantified by measurement of the relative intensities of the DQ sidebands. These variations in the (1)H DQ NMR spectra are evaluated as a function of the width of the dipolar coupling distribution. As an example, the experimental DQ spinning sideband spectrum for a hydrated polyoxoniobate containing 15 H(2)O molecules per hexaniobate cluster, are better simulated with a distribution of dipolar couplings opposed to a single coupling constant.

Antibody affinity maturation: the role of CD8+ cells.

The cellular control of antibody affinity maturation has been studied in mice genetically selected for their failure to demonstrate an increase in affinity following immunization (low-affinity N/M mice). Depletion of CD8+ cells in low N/M mice by thymectomy and/or anti-CD8 antibody treatment resulted in these animals acquiring the ability to mount an anti-DNP response of progressively increasing affinity. This indicates that CD8+ cells play an important regulatory role in the cellular interactions underlying the process of affinity maturation. In addition, evidence is presented which suggests that the route of immunization may also be critical in determining the affinity of antibody produced.

Interferon-gamma potentiates antibody affinity in mice with a genetically controlled defect in affinity maturation.

Interferon-gamma (IFN-gamma), the tetrapeptide tuftsin and the synthetic nonapeptide from interleukin-1 beta (IL-1 beta) (amino acids 163-171) have previously been shown to act on macrophages and/or T cells and to enhance antibody titres to T cell-dependent antigens. The ability of these immunomodulatory agents to potentiate antibody affinity in addition to antibody titre has been studied in a line of mice that fail to demonstrate normal maturation of antibody affinity (low N/M mice). The results presented here confirm that each of the agents potentiate antibody levels following simultaneous injection with a T cell-dependent antigen but demonstrate that only IFN-gamma is able to enhance antibody affinity in these mice. The observation that IFN-gamma can enhance both antibody affinity and antibody levels suggests that it could be an important adjuvant for vaccine use.

Experimental allergic encephalomyelitis (EAE) in mice selectively bred to produce high affinity (HA) or low affinity (LA) antibody responses.

Induction of experimental allergic encephalomyelitis (EAE) in mice genetically selected to produce either high affinity (HA) or low affinity (LA) antibody responses has revealed significant differences in disease susceptibility between the two lines. HA mice were highly susceptible to EAE following subcutaneous sensitization to mouse central nervous system (CNS) tissue emulsified in Freund's complete adjuvant (FCA). Furthermore, of HA mice surviving acute EAE, up to 93% subsequently developed chronic relapsing disease (CREAE) characterized by variable demyelinating inflammatory changes within the spinal cord. In contrast, LA mice, despite having a major histocompatability complex (MHC) haplotype associated with susceptibility to EAE, were highly resistant to the disease and showed no signs of CREAE when observed for up to 100 days post-sensitization. Antibodies to myelin basic protein (MBP) were detected in both lines but rising titres of high functional affinity antibodies were only seen in HA mice. These HA and LA lines of mice provide a new approach to the study of EAE and, in particular, the role of antibody and antibody affinity in the chronic relapsing form of the disease.