Mutations to Cu/Zn superoxide dismutase (SOD) linked to familial amyotrophic lateral sclerosis (ALS) enhance an unknown toxic reaction that leads to the selective degeneration of motor neurons. However, the question of how >50 different missense mutations produce a common toxic phenotype remains perplexing. We found that the zinc affinity of four ALS‐associated SOD mutants was decreased up to 30‐fold compared to wild‐type SOD but that both mutants and wild‐type SOD retained copper with similar affinity. Neurofilament‐L (NF‐L), one of the most abundant proteins in motor neurons, bound multiple zinc atoms with sufficient affinity to potentially remove zinc from both wild‐type and mutant SOD while having a lower affinity for copper. The loss of zinc from wild‐type SOD approximately doubled its efficiency for catalyzing peroxynitrite‐mediated tyrosine nitration, suggesting that one gained function by SOD in ALS may be an indirect consequence of zinc loss. Nitration of protein‐bound tyrosines is a permanent modification that can adversely affect protein function. Thus, the toxicity of ALS‐associated SOD mutants may be related to enhanced catalysis of protein nitration subsequent to zinc loss. By acting as a high‐capacity zinc sink, NF‐L could foster the formation of zinc‐deficient SOD within motor neurons.

To gain insight into the mechanisms that regulate the development of the H chain CDR3 (CDR-H3), we used the scheme of Hardy to sort mouse bone marrow B lineage cells into progenitor, immature, and mature B cell fractions, and then performed sequence analysis on VH7183-containing Cμ transcripts. The essential architecture of the CDR-H3 repertoire observed in the mature B cell fraction F was already established in the early pre-B cell fraction C. These architectural features include VH gene segment use preference, DH family usage, JH rank order, predicted structures of the CDR-H3 base and loop, and the amino acid composition and average hydrophobicity of the CDR-H3 loop. With development, the repertoire was focused by eliminating outliers to what appears to be a preferred repertoire in terms of length, amino acid composition, and average hydrophobicity. Unlike humans, the average length of CDR-H3 increased during development. The majority of this increase came from enhanced preservation of JH sequence. This was associated with an increase in the prevalence of tyrosine. With an accompanying increase in glycine, a shift in hydrophobicity was observed in the CDR-H3 loop from near neutral in fraction C (−0.08 ± 0.03) to mild hydrophilic in fraction F (−0.17 ± 0.02). Fundamental constraints on the sequence and structure of CDR-H3 are thus established before surface IgM expression.

Mutations in copper, zinc superoxide dismutase (SOD) have been implicated in the selective death of motor neurons in 2 percent of amyotrophic lateral sclerosis (ALS) patients. The loss of zinc from either wild-type or ALS-mutant SODs was sufficient to induce apoptosis in cultured motor neurons. Toxicity required that copper be bound to SOD and depended on endogenous production of nitric oxide. When replete with zinc, neither ALS-mutant nor wild-type copper, zinc SODs were toxic, and both protected motor neurons from trophic factor withdrawal. Thus, zinc-deficient SOD may participate in both sporadic and familial ALS by an oxidative mechanism involving nitric oxide.

To test the contribution of individual D gene segments to B cell development and function, we used gene targeting to create mice that contain only DFL16.1 in the DH locus. We term this D-limited IgH allele ΔD-DFL. Although the absolute number of IgM+IgD− B cells in the bone marrow was decreased, homozygous ΔD-DFL BALB/c mice contained normal numbers of IgM+IgD+ B cells in bone marrow and spleen and normal numbers of B1a, B1b, and B2 cells in the peritoneal cavity. Bone marrow IgM+IgD+ B cells express a CDR-H3 repertoire similar in length and amino acid composition to the DFL16.1 subset of the wild-type BALB/c repertoire but divergent from sequences that do not contain DFL16.1. This similarity in content is the product of both germline bias and somatic selection, especially in the transition to the mature IgM+IgD+ stage of development. Serum Ig concentrations and the humoral immune response to a T-dependent Ag ([4-hydroxy-3-nitrophenyl]acetyl hapten) were nearly identical to wild-type littermate controls. A greater variance in the immune response to the T-independent Ag (α(1→3)-dextran) was observed in ΔD-DFL homozygotes, with half of the mice exhibiting levels below the range exhibited by controls. Although limited to a repertoire specific to DFL16.1, the presence of a single DH gene segment of normal sequence was sufficient for development of normal numbers of mature B cells and for robust humoral immune function.

The vast initial diversity of the antibody repertoire is generated centrally by means of a complex series of V(D)J gene rearrangement events, variation in the site of gene segment joining, and TdT catalyzed N-region addition. Although the diversity is great, close inspection has revealed distinct and unique characteristics in the antibody repertoires expressed by different B cell developmental subsets. In order to illustrate our approach to repertoire analysis, we present an in-depth comparison of V(D)J gene usage, hydrophobicity, length, D H reading frame, and amino acid usage between heavy chain repertoires expressed by immature, transitional, mature, memory IgD + , memory IgD − , and plasmacytes isolated from the blood of a single individual. Our results support the view that in both human and mouse, the H chain repertoires expressed by individual, developmental B cell subsets appear to differ in sequence content. Sequencing of unsorted B cells from the blood is thus likely to yield an incomplete or compressed view of what is actually happening in the immune response of the individual. Our findings support the view that studies designed to correlate repertoire expression with diseases of immune function will likely require deep sequencing of B cells sorted by subset.