We found that the dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, can be applied as a fluorescent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry (J. Cell. Biol. 1985. 100: 965-973). To understand the selectivity of the staining, we examined the fluorescence properties of nile red in the presence of organic solvents and model lipid systems. Nile red was found to be both very soluble and strongly fluorescent in organic solvents. The excitation and emission spectra of nile red shifted to shorter wavelengths with decreasing solvent polarity. However, the fluorescence of nile red was quenched in aqueous medium. Nile red was observed to fluoresce intensely in the presence of aqueous suspensions of phosphatidylcholine vesicles (excitation maximum: 549 nm; emission maximum: 628 nm). When neutral lipids such as triacylglycerols or cholesteryl esters were incorporated with phosphatidylcholine to form microemulsions, nile red fluorescence emission maxima shifted to shorter wavelengths. Serum lipoproteins also induced nile red fluorescence and produced spectral blue shifts. Nile red fluorescence was not observed in the presence of either immunoglobulin G or gelatin. These results demonstrate that nile red fluorescence accompanied by a spectral blue shift reflects the presence of nile red in a hydrophobic lipid environment and account for the selective detection of neutral lipid by the dye. Nile red thus serves as an excellent fluorescent lipid probe.

We have found that the myc oncogene has been modified by abortive recombination with the alpha heavy-chain immunoglobulin constant-region (C alpha) gene in five different mouse plasmacytoma lines. Recombination occurred approximately 0.8-2.0 kb to the 5' side of two distinct coding regions, defined by sequence homology between the chicken cellular and plasmacytoma myc genes. The myc and C alpha genes were always in opposite transcriptional orientation, with the recombination site within the C alpha switch region sequences. DNA recombination was found to correlate with the production of a novel 2.1 kb species of myc RNA that was 0.4 kb shorter than the normal cellular transcript. No elevated levels of myc RNA were evident, suggesting that DNA rearrangements have altered the myc oncogene product. This oncogene activation corresponds to the chromosomal translocations found in nearly all plasmacytomas.

Expression of the interleukin-2 receptor (IL-2R alpha) gene is activated by the transcriptional activator protein, Tax (previously referred to as the tat gene product), encoded by the human T-cell leukemia virus (HTLV-I). Multiple protein binding sites for specific DNA-protein interactions were identified over the upstream IL-2R alpha transcriptional regulatory sequences. However, only one region, which includes the sequence motif GGGGAATCTCCC, was required for activation by both the tax gene product and mitogenic stimulation. Remarkably, this sequence also bound the nuclear factor NF kappa B, which is important for induction of kappa-immunoglobulin gene expression. A model is presented whereby regulation of cellular gene expression by the HTLV-I tax gene product occurs via an indirect mechanism that may involve a post-translational modification of preexistent cellular transcription factors.

Mice lacking the gene encoding poly(ADP-ribosyl) transferase (PARP or ADPRT) display no phenotypic abnormalities, although aged mice are susceptible to epidermal hyperplasia and obesity in a mixed genetic background. Whereas embryonic fibroblasts lacking PARP exhibit normal DNA excision repair, they grow more slowly in vitro. Here we investigated the putative roles of PARP in cell proliferation, cell death, radiosensitivity, and DNA recombination, as well as chromosomal stability. We show that the proliferation deficiency in vitro and in vivo is most likely caused by a hypersensitive response to environmental stress. Although PARP is specifically cleaved during apoptosis, cells lacking this molecule apoptosed normally in response to treatment with anti-Fas, tumor neurosis factor alpha, gamma-irradiation, and dexamethasone, indicating that PARP is dispensable in apoptosis and that PARP-/- thymocytes are not hypersensitive to ionizing radiation. Furthermore, the capacity of mutant cells to carry out immunoglobulin class switching and V(D)J recombination is normal. Finally, primary PARP mutant fibroblasts and splenocytes exhibited an elevated frequency of spontaneous sister chromatid exchanges and elevated micronuclei formation after treatment with genotoxic agents, establishing an important role for PARP in the maintenance of genomic integrity.

Alpha/beta interferon (IFN) and gamma IFN exert widely overlapping biological effects. Still, mice with individually inactivated alpha/beta or gamma receptors exhibit variably severely reduced resistance to infection and altered immune responses. To investigate to what extent the two IFN systems are functionally redundant, we generated mice with a combined receptor defect (AG129 mice). Like mice with individual mutations, AG129 mice had no apparent anomalies, confirming that in the mouse the IFN system is not essential for normal development. These mice showed an additive phenotype with respect to antiviral defense and exhibited an increased susceptibility to lymphocytic choriomeningitis virus (LCMV) and notably vaccinia virus infection. Because of unlimited replication and subsequent rapid exhaustion of cytotoxic T lymphocyte (CTL) precursors, these mice were unable to mount a CTL response to LCMV. CD8(+)-mediated immunopathology was absent in LCMV-infected mice, and virus persisted. Vaccinia virus replicated much faster in AG129 mice, and a 10(4)-fold lower dose of vaccinia virus was sufficient to prime these mice. With the normal priming dose of 10(6) PFU, cytopathic effects and overwhelming infection possibly causing partial exhaustion of CTL interfered with the anti-vaccinia virus response. Even though global antiviral immunoglobulin G (IgG) titers were within normal ranges, the IgG subclass distribution was heavily biased toward IgG1.

Neutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Here, we characterized polyclonal immunoglobulin Gs (IgGs) and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/neutralization of IgGs over Fabs. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1^A and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4 Å cryo-electron microscopy (cryo-EM) structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses, and characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.

Keywords: COVID-19; ELISA; Fab; IgG; MERS-CoV; SARS-CoV; SARS-CoV-2; convalescent plasma; coronavirus; electron microscopy.

CD28 is a homodimeric glycoprotein expressed on the surface of a major subset of human T cells that has recently been identified as a member of the immunoglobulin supergene family. The binding of monoclonal antibodies to the CD28 antigen on purified T cells does not result in proliferation; however, previous studies have shown that the combination of CD28 stimulation and protein kinase C activation by phorbol myristate acetate (PMA) results in T-cell proliferation that is independent of both accessory cells and activation of the T-cell receptor-CD3 complex. In the present study, effects of stimulation by anti-CD28 on cell cycle progression and on the interleukin 2 (IL-2) and IL-2 receptor system have been investigated on primary cultures of purified peripheral-blood CD28+ T cells. There was no measurable effect on cell size or on DNA synthesis after stimulation of resting (G0) cells by CD28 alone. After 3 h of activation of T cells by PMA alone, a slight (8%) increase in cell volume occurred that did not progress to DNA synthesis. In contrast, T-cell stimulation by CD28 in combination with PMA resulted in a progressive increase in cell volume in approximately 100% of cells at 12 to 14 h after stimulation. Northern blot (RNA blot) analysis revealed that CD28 stimulation alone failed to cause expression of the alpha chain of the IL-2 receptor or of IL-2 mRNA, and in accord with previous studies, stimulation by PMA alone resulted in the accumulation of IL-2 receptor transcripts but no detectable IL-2 mRNA. In contrast, T-cell stimulation by the combination of CD28 and PMA resulted in the appearance of IL-2 transcripts and enhanced expression of IL-2 receptor mRNA. Functional studies revealed that the proliferation induced by CD28 and PMA stimulation was entirely resistant to cyclosporine, in contrast to T-cell activation induced by the CD3-T-cell receptor complex. Cyclosporine was found not to affect the accumulation of IL-2 mRNA after CD28 plus PMA stimulation, although there was no detectable IL-2 mRNA after stimulation by CD3 in the presence of the drug. Furthermore, stimulation by CD28 in combination with immobilized CD3 antibodies caused a striking enhancement of IL-2 mRNA expression that was, in part, resistant to the effects of cyclosporine. These studies indicate that the CD28 molecule synergizes with protein kinase C activation to induce IL-2 gene expression and demonstrate that stimulation by the CD28 pathway can cause vigorous T-cell proliferation even in the presence of cyclosporine and that cyclosporine does not prevent transcription of 16-2 mRNA, as has been suggested previously. Moreover, these findings suggest that a potential role for the CD28 molecule in vivo may be to augment IL-2 production after stimulation of the CD3-T-cell receptor molecular complex and thereby to amplify an antigen-specific immune response. Finally, these results provide further evidence that the CD28 molecule triggers T-cell proliferation in a manner that differs biochemically from CD3-T-cell receptor-induced proliferation.

BACKGROUND

The CD19 protein forms a complex with CD21, CD81, and CD225 in the membrane of mature B cells. Together with the B-cell antigen receptor, this complex signals the B cell to decrease its threshold for activation by the antigen.

METHODS

We evaluated four patients from two unrelated families who had increased susceptibility to infection, hypogammaglobulinemia, and normal numbers of mature B cells in blood. We found a mutation in the CD19 gene in all four patients. The CD19 gene in the patients and their first-degree relatives was sequenced, and flow-cytometric immunophenotyping of B cells, immunohistochemical staining of lymphoid tissues, and DNA and messenger RNA analysis were performed. B-cell responses on the triggering of the B-cell receptor were investigated by in vitro stimulation; the antibody response after vaccination with rabies vaccine was also studied.

RESULTS

All four patients had homozygous mutations in the CD19 gene. Levels of CD19 were undetectable in one patient and substantially decreased in the other three. Levels of CD21 were decreased, whereas levels of CD81 and CD225 were normal, in all four patients. The composition of the precursor B-cell compartment in bone marrow and the total numbers of B cells in blood were normal. However, the numbers of CD27+ memory B cells and CD5+ B cells were decreased. Secondary follicles in lymphoid tissues were small to normal in size and had a normal cellular composition. The few B cells that showed molecular signs of switching from one immunoglobulin class to another contained V(H)-C(alpha) and V(H)-C(gamma) transcripts with somatic mutations. The response of the patients' B cells to in vitro stimulation through the B-cell receptor was impaired, and in all four patients, the antibody response to rabies vaccination was poor.

CONCLUSIONS

Mutation of the CD19 gene causes a type of hypogammaglobulinemia in which the response of mature B cells to antigenic stimulation is defective.

The intestinal mucosa contains the largest population of antibody-secreting plasma cells in the body, and in humans several grams of secretory immunoglobulin A (SIgA) are released into the intestine each day. In the gut lumen, SIgA serves as a first-line barrier that protects the epithelium from pathogens and toxins. Recently, next-generation sequencing has revolutionized our understanding of the nature of the intestinal microbiota and has also shed new light on the important roles of SIgA in the regulation of host-commensal homeostasis. Here, I discuss pathways of IgA induction in the context of SIgA specificity and function.

Spectrin repeats fold into triple helical coiled-coils comprising approximately 106 amino acid residues. Using an AFM-related technique we measured the force required to mechanically unfold these repeats to be 25 to 35 pN. Under tension, individual spectrin repeats unfold independently and in an all-or-none process. The dependence of the unfolding forces on the pulling speed reveals that the corresponding unfolding potential is shallow with an estimated width of 1.5 nm. When the unfolded polypeptide strand is relaxed, several domains refold within less than a second. The unfolding forces of the alpha-helical spectrin domains are five to ten times lower than those found in domains with beta-fold, like immunoglobulin or fibronectin Ill domains, where the tertiary structure is stabilized by hydrogen bonds between adjacent strands. This shows that the forces stabilizing the coiled-coil lead to a mechanically much weaker structure than multiple hydrogen-bonded beta-sheets.

The development and maturation of the immune system in zebrafish was investigated using immune-related gene expression profiling by quantitative real-time polymerase chain reaction, in situ hybridization (ISH), immunoglobulin (Ig) detection by immuno-affinity purification and Western blotting as well as immersion immunization experiments. Ikaros expression was first detected at 1 day post-fertilization (dpf) and thereafter increased gradually to more than two-fold between 28 and 42dpf before decreasing to less than the initial 1dpf expression level in adult fish (aged 105dpf). Recombination activating gene-1 (Rag-1) expression levels increased rapidly (by 10-fold) between 3 and 17dpf, reaching a maximum between 21 and 28dpf before decreasing gradually. However, in adult fish aged 105dpf, the expression level of Rag-1 had dropped markedly, and was equivalent to the expression level at 3dpf. T-cell receptor alpha constant region and immunoglobulin light chain constant region (IgLC) isotype-1, 2 and 3 mRNAs were detected at low levels by 3dpf and their expression levels increased steadily to the adult range between 4 and 6 weeks post-fertilization (wpf). Using tissue-section ISH, Rag-1 expression was detected in head kidney by 2wpf while IgLC-1, 2 and 3 were detected in the head kidney and the thymus by 3wpf onwards. Secreted Ig was only detectable using immuno-affinity purification and Western blotting by 4wpf. Humoral response to T-independent antigen (formalin-killed Aeromonas hydrophila) and T-dependent antigen (human gamma globulin) was observed in zebrafish immunized at 4 and 6wpf, respectively, indicating that immunocompetence was achieved. The findings reveal that the zebrafish immune system is morphologically and functionally mature by 4-6wpf.

Infections, no matter how mild, have adverse effects on nutritional status. The significance of these effects depends on the previous nutritional status of the individual, the nature and duration of the infection, and the diet during the recovery period. Conversely, almost any nutrient deficiency, if sufficiently severe, will impair resistance to infection. Iron deficiency and protein-energy malnutrition, both highly prevalent, have the greatest public health importance in this regard. Remarkable advances in immunology of recent decades have increased insights into the mechanisms responsible for the effects of infection. These include impaired antibody formation; loss of delayed cutaneous hypersensitivity; reduced immunoglobulin concentrations; decreased thymic and splenic lymphocytes; reduced complement formation, secretory immunoglobulin A, and interferon; and lower T cells and T cells subsets (helper, suppressor-cytotoxic, and natural killer cells) and interleukin 2 receptors. The effects observed with single or multiple nutrient deficiencies are due to some combination of these responses. In general, cell-mediated and nonspecific immunity are more sensitive than humoral immunity.

The generation of B-lymphocytes from hematopoietic stem cells is controlled by multiple transcription factors regulating distinct developmental aspects. Ikaros and PU.1 act in parallel pathways to control the development of lymphoid progenitors in part by regulating the expression of essential signaling receptors (Flt3, c-Kit, and IL-7R alpha). The generation of the earliest B cell progenitors depends on E2A and EBF, which coordinately activate the B cell gene expression program and immunoglobulin heavy-chain gene rearrangements at the onset of B-lymphopoiesis. Pax5 restricts the developmental options of lymphoid progenitors to the B cell lineage by repressing the transcription of lineage-inappropriate genes and simultaneously activating the expression of B-lymphoid signaling molecules. LEF1 and Sox4 contribute to the survival and proliferation of pro-B cells in response to extracellular signals. Finally, IRF4 and IRF8 together control the termination of pre-B cell receptor signaling and thus promote differentiation to small pre-B cells undergoing light-chain gene rearrangements.

Immunoglobulin (Ig) genes are hypermutated in B lymphocytes that are the precursors to memory B cells. The mutations are linked to transcription initiation, but non-Ig promoters are permissible for the mutation process; thus, other genes expressed in mutating B cells may also be subject to somatic hypermutation. Significant mutations were not observed in c-MYC, S14, or alpha-fetoprotein (AFP) genes, but BCL-6 was highly mutated in a large proportion of memory B cells of normal individuals. The mutation pattern was similar to that of Ig genes.

Examination of the subclass distribution of murine antibodies directed against groups A and C streptococcal carbohydrate, alpha-(1 leads to 3) dextran and phosphocholine yields the surprising observation that these carbohydrate antigens stimulate IgG responses largely restricted to the rare IgG3 subclass. This subclass restriction is particularly impressive in light of the low circulating levels of IgG3 in nonimmune mouse serum and the failure of a variety of other antigens including proteins and aromatic haptens to stimulate IgG3 antibody production. Attempts to alter the subclass restriction of antibodies with carbohydrate specificity by immunization with carbohydrate-coupled protein have been unsuccessful and indicate that immunoregulation of subclass expression probably occurs at the level of the antibody forming (B) cell. It is therefore conceivable that VH regions of murine immunoglobulins may be restricted to particular IgG subclasses. A similar type of subclass restriction has been reported in human and rat anti-carbohydrate antibodies. This recruitment of a minor immunoglobulin isotype by carbohydrate antigens in several species further supports the concept of immunoregulation at the level of subclass, and suggests that these and other mammals may share a structurally similar isotype with perhaps a common evolutionary origin.

OBJECTIVE

Steroid-refractory Crohn's disease responds to therapy with the chimeric anti-tumor necrosis factor (TNF)-alpha antibody infliximab. Etanercept, a recombinant TNF receptor/immunoglobulin G fusion protein, is highly effective in rheumatoid arthritis but not in Crohn's disease. Because both infliximab and etanercept are TNF-alpha-neutralizing drugs, we investigated the differences in TNF-alpha-neutralizing capacity and human lymphocyte binding and apoptosis-inducing capacity of both molecules.

METHODS

We used a nuclear factor kappaB reporter assay and a cytotoxicity bioassay to study TNF-alpha neutralization by infliximab and etanercept. Lymphocyte binding and apoptosis-inducing capacity was investigated using fluorescence-activated cell sorter analysis, annexin V staining, and cleaved caspase-3 immunoblotting using mixed lymphocyte reaction-stimulated peripheral blood lymphocytes (PBL) from healthy volunteers and lamina propria T cells from patients with Crohn's disease.

RESULTS

Both infliximab and etanercept neutralized TNF-alpha effectively. Infliximab bound to activated PBL and lamina propria T cells, whereas binding of etanercept was equal to a nonspecific control antibody. Infliximab but not etanercept induced peripheral and lamina propria lymphocyte apoptosis when compared with a control antibody. Infliximab activated caspase 3 in a time-dependent manner, whereas etanercept did not.

CONCLUSIONS

Although both infliximab and etanercept showed powerful TNF-alpha neutralization, only infliximab was able to bind to PBL and lamina propria T cells and subsequently to induce apoptosis of activated lymphocytes. These data may provide a biological basis for the difference in efficacy of the 2 TNF-alpha-neutralizing drugs.

Trichothecene mycotoxins are a group of structurally similar fungal metabolites that are capable of producing a wide range of toxic effects. Deoxynivalenol (DON, vomitoxin), a trichothecene, is prevalent worldwide in crops used for food and feed production, including in Canada and the United States. Although DON is one of the least acutely toxic trichothecenes, it should be treated as an important food safety issue because it is a very common contaminant of grain. This review focuses on the ability of DON to induce toxicologic and immunotoxic effects in a variety of cell systems and animal species. At the cellular level, the main toxic effect is inhibition of protein synthesis via binding to the ribosome. In animals, moderate to low ingestion of toxin can cause a number of as yet poorly defined effects associated with reduced performance and immune function. The main overt effect at low dietary concentrations appears to be a reduction in food consumption (anorexia), while higher doses induce vomiting (emesis). DON is known to alter brain neurochemicals. The serotoninergic system appears to play a role in mediation of the feeding behavior and emetic response. Animals fed low to moderate doses are able to recover from initial weight losses, while higher doses induce more long-term changes in feeding behavior. At low dosages of DON, hematological, clinical, and immunological changes are also transitory and decrease as compensatory/adaptation mechanisms are established. Swine are more sensitive to DON than mice, poultry, and ruminants, in part because of differences in metabolism of DON, with males being more sensitive than females. The capacity of DON to alter normal immune function has been of particular interest. There is extensive evidence that DON can be immunosuppressive or immunostimulatory, depending upon the dose and duration of exposure. While immunosuppression can be explained by the inhibition of translation, immunostimulation can be related to interference with normal regulatory mechanisms. In vivo, DON suppresses normal immune response to pathogens and simultaneously induces autoimmune-like effects which are similar to human immunoglobulin A (IgA) nephropathy. Other effects include superinduction of cytokine production by T helper cells (in vitro) and activation of macrophages and T cells to produce a proinflammatory cytokine wave that is analogous to that found in lipopolysaccharide-induced shock (in vivo). To what extent the elevation of cytokines contributes to metabolic effects such as decreased feed intake remains to be established. Although these effects have been largely characterized in the mouse, several investigations with DON suggest that immunotoxic effects are also likely in domestic animals. Further toxicology studies and an assessment of the potential of DON to be an etiologic agent in human disease are warranted.

Interleukin-1 alpha and -1 beta (IL-1 alpha and IL-1 beta) are cytokines that participate in the regulation of immune responses, inflammatory reactions, and hematopoiesis. A direct expression strategy was used to clone the receptor for IL-1 from mouse T cells. The product of the cloned complementary DNA binds both IL-1 alpha and IL-1 beta in a manner indistinguishable from that of the native T cell IL-1 receptor. The extracellular, IL-1 binding portion of the receptor is 319 amino acids in length and is composed of three immunoglobulin-like domains. The cytoplasmic portion of the receptor is 217 amino acids long.

To determine the extent of airway infection and inflammation in adolescents and adults with cystic fibrosis (CF) who have mild lung disease and are without symptoms of active infection, we performed bronchoalveolar lavage (BAL) on 18 CF patients>> or = 12 yr of age who were stable, appeared clinically well, and had mean (+/- SEM) FEV1 of 79 +/- 4% of predicted. We quantitated the bacteria, inflammatory cells, immunoglobulins, and mediators of inflammatory tissue damage in the epithelial lining fluid (ELF) of these patients and in 23 healthy control subjects. All CF patients were found to be infected with Pseudomonas aeruginosa, Staphylococcus aureus, and/or Haemophilus influenzae; no organisms were isolated from the control subjects. The mean number of cells in the ELF was 14 times greater in the CF patients than in the control subjects. Neutrophils constituted 57% of the recovered cells in the CF patients versus 3% in the control subjects, and their concentration was 380 times greater in the CF patients versus the control subjects. IgG, IgA, and IgM were 2.5 to 6 times greater in CF ELF versus that of control subjects. Abundant active elastase was present in the ELF of the CF patients (2.3 +/- 0.9 microM) despite threefold elevated levels of alpha 1-protease inhibitor (alpha 1-PI). No active elastase was detectable in the control subjects. alpha 1-PI was functional in CF as demonstrated by elevated elastase:alpha 1-PI complex (0.045 microM in CF versus 0.002 microM in control subjects). This active elastase caused proteolytic destruction of surface complement receptors on airway neutrophils in situ.(ABSTRACT TRUNCATED AT 250 WORDS)

Receptors for the Fc domain of IgG (Fc gamma R) provide a critical link between specific humoral responses and the cellular branch of the immune system. When hFc gamma R interact with immunoglobulin, a variety of biological responses are triggered. These include phagocytosis, endocytosis, antibody-dependent cellular cytotoxicity (ADCC), release of inflammatory mediators, and enhancement of antigen presentation. In the last few years our understanding of the Fc gamma receptor structure has increased dramatically, due to the availability of monoclonal antibodies (mAb) and cDNA probes. Fc gamma R are members of the immunoglobulin superfamily and three main classes, hFc gamma RI, hFc gamma RII, and hFc gamma RIII are recognized in man generating at least 12 different isoforms. A further level of complexity is introduced by various genetic polymorphisms and, importantly, recent evidence points at the relevance of this Fc gamma R heterogeneity.

Teleost fish are the most primitive bony vertebrates that contain immunoglobulins. In contrast to mammals and birds, these species are devoid of immunoglobulin A (IgA) or a functional equivalent. This observation suggests that specialization of immunoglobulin isotypes into mucosal and systemic responses took place during tetrapod evolution. Challenging that paradigm, here we show that IgT, an immunoglobulin isotype of unknown function, acts like a mucosal antibody. We detected responses of rainbow trout IgT to an intestinal parasite only in the gut, whereas IgM responses were confined to the serum. IgT coated most intestinal bacteria. As IgT and IgA are phylogenetically distant immunoglobulins, their specialization into mucosal responses probably occurred independently by a process of convergent evolution.

In addition to the two previously identified genes rearranged and expressed in a cytotoxic T-lymphocyte clone, we have identified a third gene that is also rearranged and expressed in the same clone. This new gene shows clonal diversity, codes for a polypeptide chain that contains immunoglobulin-like variable and constant domains, carries potential N-glycosylation sites and is a particularly attractive candidate for the gene that encodes the alpha-subunit of the heterodimeric antigen receptor of this T-cell clone.

Ku70, Ku80, and DNA-PKcs are subunits of the DNA-dependent protein kinase (DNA-PK), an enzyme implicated in DNA double-stranded break repair and V(D)J recombination. Our Ku70-deficient mice were about 50% the size of control littermates, and their fibroblasts were ionizing radiation sensitive and displayed premature senescence associated with the accumulation of nondividing cells. Ku70-deficient mice lacked mature B cells or serum immunoglobulin but, unexpectedly, reproducibly developed small populations of thymic and peripheral alpha/beta T lineage cells and had a significant incidence of thymic lymphomas. In association with B and T cell developmental defects, Ku70-deficient cells were severely impaired for joining of V(D)J coding and recombination signal sequences. These unanticipated features of the Ku70-deficient phenotype with respect to lymphocyte development and V(D)J recombination may reflect differential functions of the three DNA-PK components.

Here, we provide an analysis of molecular evolution of five of the most populated protein folds: immunoglobulin fold, oligonucleotide-binding fold, Rossman fold, alpha/beta plait, and TIM barrels. In order to distinguish between "historic", functional and structural reasons for amino acid conservations, we consider proteins that acquire the same fold and have no evident sequence homology. For each fold we identify positions that are conserved within each individual family and coincide when non-homologous proteins are structurally superimposed. As a baseline for statistical assessment we use the conservatism expected based on the solvent accessibility. The analysis is based on a new concept of "conservatism-of-conservatism". This approach allows us to identify the structural features that are stabilized in all proteins having a given fold, despite the fact that actual interactions that provide such stabilization may vary from protein to protein. Comparison with experimental data on thermodynamics, folding kinetics and function of the proteins reveals that such universally conserved clusters correspond to either: (i) super-sites (common location of active site in proteins having common tertiary structures but not function) or (ii) folding nuclei whose stability is an important determinant of folding rate, or both (in the case of Rossman fold). The analysis also helps to clarify the relation between folding and function that is apparent for some folds.