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.

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In the bone marrow, the passage of developing B cells through critical checkpoints of differentiation is associated with a reduction of specific categories of CDR3 of the Ig heavy chain (CDR-H3), particularly those with excessive hydrophobic or charged amino acids and those with a length of eight or fewer residues. To gain insight into the role of CDR-H3 content in the development of B cells in the spleen, we compared the sequences of V H 7183DJCl transcripts from sorted transitional T1, marginal zone, and follicular B cell subsets to those expressed by immature IgM + IgD -and mature IgM lo IgD hi B cells in the bone marrow. Although differences in V H utilization were noted, the T1 CDR-H3 repertoire showed extensive similarity to that of immature bone marrow B cells, and the follicular CDR-H3 repertoire most resembled that of mature bone marrow B cells. Unlike the splenic follicular and bone marrow mature B cell CDR-H3 repertoires, the marginal zone B cell CDR-H3 repertoire retained both short and highly charged amino acid motifs, including those with two arginines. Our findings suggest that antigen binding sites containing specific categories of CDR-H3 sequence content may inhibit, permit, or even facilitate passage of the host B cell through critical checkpoints in peripheral as well as central development.

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.

Stimulation of Na+/H' exchange by growth factors has been implicated as a mechanism allowing quiescent cells to resume growth because of a predicted elevation of intracellular pH (pHi). We tested this prediction in NR6 cells by using a further development of our technique for pHi measurement, based on introduction of the fluorescent pH indicator 4',5'-dimethylfluorescein (pKa = 6.75) coupled to dextran into the cytoplasm. Addition of the potent mitogens platelet-derived growth factor (PDGF) or serum to NR6 cells stimulates an amiloride-sensitive 'Na' uptake and causes an elevation of pH,. The PDGF-dependent pHi increase follows a lag period of a2 min, reaches a maximal level within 10 min (ApHi 0.1 at an external pH of 7.18), and remains at this level for at least 1 hr. Serum addition initially produces a large elevation of pHi, which later declines to a level similar to that obtained with PDGF. The effects of PDGF and serum are partially additive (ApHi 0.14). The magnitude of pHi elevation by PDGF decreases with increasing extracellular pH. Serum-and PDGF-dependent elevations of pHi are inhibited by amiloride and by eliminating Na+ from the medium. Under conditions in which Na+/H+ exchange is inhibited, PDGF and serum induce an initial cytoplasmic acidification that does not show a lag period. The results show that a single purified growth factor, as well as serum, can promote a sustained elevation of pHi by stimulating Na+/H+ exchange. The extent of pHi elevation may be modulated by the concomitant stimulation by the growth factor of a process generating H+ within the cell.Addition of growth factors to quiescent mammalian cells triggers emergence of the cells from the Go/G1 phase of the cell cycle and subsequent progression along a defined pathway culminating in DNA synthesis and mitosis. Recently, particular interest has focused on the hypothesis that elevation of intracellular pH (pHi) may act as a mediator of growth factor action. Both serum and pure growth factors have been found to rapidly stimulate an electroneutral and amiloride-sensitive Na+ uptake, suggesting a stimulation of Na+/H+ exchange (1-4). Since under physiological conditions there is a large Na+ concentration gradient directed inward, stimulation of Na+/H+ exchange may lead to cytoplasmic alkalinization. This has been shown to occur in the sea urchin egg (5), in which an increase in pHi brought about by stimulation of Na+/H+ exchange on fertilization appears to provide a crucial stimulus for egg development.Assessing cytoplasmic pH in cells that depend on growth factors for proliferation has been hampered by lack of suitable methods for the determination of pHi in cells grown in monolayer cultures. Schuldiner and Rozengurt (6) recently used the distribution of weak acids across the cell membrane to demonstrate cytoplasmic alkalinization by -0. 15 pH unit on addition of a combination of mitogens to Swiss 3T3 cells. However, the weak acid distribution method has disadvantages because of limited temporal resolution and compartmentaliza...