Citations

The major histocompatibility complex class II genes play an important role in the genetic predisposition to many autoimmune diseases. In the case of rheumatoid arthritis (RA), the human leukocyte antigen (HLA)-DRB1 locus has been implicated in the disease predisposition. The "shared epitope" hypothesis predicts that similar motifs within the third hypervariable (HV3) regions of some HLA-DRB1 alleles are responsible for the class II-associated predisposition to RA. Using a line of transgenic mice expressing the DQB1*0302/DQA1*0301 (DQ8) genes in the absence of endogenous mouse class II molecules, we have analyzed the antigenicity of peptides covering the HV3 regions of RA-associated and nonassociated DRB1 molecules. Our results show that a correlation exists between proliferative response to peptides derived from the HV3 regions of DRB1 chains and nonassociation of the corresponding alleles with RA predisposition. While HV3 peptides derived from nonassociated DRB1 molecules are highly immunogenic in DQ8 transgenic mice, all the HV3 peptides derived from RA-associated DRB1 alleles fail to induce a DQ8-restricted T-cell response. These data suggest that the role of the "shared epitope" in RA predisposition may be through the shaping of the T-cell repertoire.

Among the four subtypes of Hodgkin disease (HD), lymphocyte-predominant (LP) HD is now generally considered as a separate entity. The B cell nature of the typical Hodgkin and Reed–Sternberg (HRS) cells and their variants (L and H, lymphocytic and histiocytic cells) in LP HD has long been suspected, but the question of whether these cells represent a true tumor clone is unclear. We previously demonstrated clonal Ig gene rearrangements in one case of LP HD. In the present study, five cases of LP HD were analyzed by micromanipulation of single HRS cells from frozen tissue sections and DNA amplification of rearranged Ig heavy chain genes from those cells. Clonal V gene rearrangements harboring somatic mutations were detected in each case. In three cases ongoing somatic mutation was evident. This shows that HRS cells in LP HD are a clonal tumor population derived from germinal center B cells. The pattern of somatic mutation indicates that HRS cells in LP HD are selected for antibody expression. This, and the presence of ongoing mutation discriminates LP from classical HD.

The completion of sequencing projects for various organisms has already advanced our insight into the evolution of entire genomes and the role of gene duplications. One multigene family that has served as a paradigm for the study of gene duplications and molecular evolution is the family of homeodomain-encoding genes. I present here an analysis of the homeodomain repertoire of an entire genome, that of the nematode Caenorhabditis elegans , in relation to our current knowledge of these genes in plants, arthropods, and mammals. A methodological framework is developed that proposes approaches for the analysis of homeodomain repertoires and multigene families in general.

The immune system is equipped with an extremely large spectrum of structurally diverse receptors to recognize all potential antigens. This fundamental principle of receptor diversity is no longer upheld in patients with rheumatoid arthritis (RA), who have a marked contraction of the T cell receptor repertoire. In this study, the ability of RA patients to produce T cells and to maintain T cell homeostasis was examined. CD4 T cells containing T cell receptor rearrangement excision circles (TREC) were substantially reduced in RA patients; TREC levels in young adult patients matched those of controls 20 years older. Increased self-replication of T cells in RA was indicated by age-inappropriate erosion of telomeres in circulating T cells with almost complete attrition of telomeric reserves in patients 20–30 yr of age. The degree of telomere loss was not related to disease duration or the use of disease-modifying medication and was most pronounced in CD4 + CD45RO null (naive) T cells. The loss of TREC-positive T cells could be a consequence of a primary defect in peripheral T cell homeostasis. Alternatively, RA patients may have impaired thymic function with the increased turnover of peripheral T cells being a secondary compensatory event.

Cross-sectional positron emission tomography (PET) studies find that cognitively normal carriers of the apolipoprotein E (APOE) ɛ4 allele, a common Alzheimer's susceptibility gene, have abnormally low measurements of the cerebral metabolic rate for glucose (CMRgl) in the same regions as patients with Alzheimer's dementia. In this article, we characterize longitudinal CMRgl declines in cognitively normal ɛ4 heterozygotes, estimate the power of PET to test the efficacy of treatments to attenuate these declines in 2 years, and consider how this paradigm could be used to efficiently test the potential of candidate therapies for the prevention of Alzheimer's disease. We studied 10 cognitively normal ɛ4 heterozygotes and 15 ɛ4 noncarriers 50–63 years of age with a reported family history of Alzheimer's dementia before and after an interval of approximately 2 years. The ɛ4 heterozygotes had significant CMRgl declines in the vicinity of temporal, posterior cingulate, and prefrontal cortex, basal forebrain, parahippocampal gyrus, and thalamus, and these declines were significantly greater than those in the ɛ4 noncarriers. In testing candidate primary prevention therapies, we estimate that between 50 and 115 cognitively normal ɛ4 heterozygotes are needed per active and placebo treatment group to detect a 25% attenuation in these CMRgl declines with 80% power and P = 0.005 in 2 years. Assuming these CMRgl declines are related to the predisposition to Alzheimer's dementia, this study provides a paradigm for testing the potential of treatments to prevent the disorder without having to study thousands of research subjects or wait many years to determine whether or when treated individuals develop symptoms.

The NUP98 gene encodes precursor proteins that generate two nucleoplasmically oriented nucleoporins, NUP98 and NUP96. By using gene targeting, we have selectively disrupted the murine NUP98 protein, leaving intact the expression and localization of NUP96. We show that NUP98 is essential for mouse gastrulation, a developmental stage that is associated with rapid cell proliferation, but dispensable for basal cell growth. NUP98−/− cells had an intact nuclear envelope with a normal number of embedded nuclear pore complexes. Typically, NUP98-deficient cells contained on average approximately 5-fold more cytoplasmic annulate lamellae than control cells. We found that a set of cytoplasmically oriented nucleoporins, including NUP358, NUP214, NUP88, and p62, assembled inefficiently into nuclear pores of NUP98−/− cells. Instead, these nucleoporins were prominently associated with the annulate lamellae. By contrast, a group of nucleoplasmically oriented nucleoporins, including NUP153, NUP50, NUP96, and NUP93, had no affinity for annulate lamellae and assembled normally into nuclear pores. Mutant pores were significantly impaired in transport receptor-mediated docking of proteins with a nuclear localization signal or M9 import signal and showed weak nuclear import of such substrates. In contrast, the ability of mutant pores to import ribosomal protein L23a and spliceosome protein U1A appeared intact. These observations show that NUP98 disruption selectively impairs discrete protein import pathways and support the idea that transport of distinct import complexes through the nuclear pore complex is mediated by specific subsets of nucleoporins.

Proteolytic cleavage of the six known insulin-like growth factor binding proteins (IGFBPs) is a powerful means of rapid structure and function modification of these important growth-regulatory proteins. Intact IGFBP-4 is a potent inhibitor of IGF action in vitro, and cleavage of IGFBP-4 has been shown to abolish its ability to inhibit IGF stimulatory effects in a variety of systems, suggesting that IGFBP-4 proteolysis acts as a positive regulator of IGF bioavailability. Here we report the isolation of an IGF-dependent IGFBP-4-specific protease from human fibroblast-conditioned media and its identification by mass spectrometry microsequencing as pregnancy-associated plasma protein-A (PAPP-A), a protein of unknown function found in high concentrations in the maternal circulation during pregnancy. Antibodies raised against PAPP-A both inhibited and immunodepleted IGFBP-4 protease activity in human fibroblast-conditioned media. Moreover, PAPP-A purified from pregnancy sera had IGF-dependent IGFBP-4 protease activity. PAPP-A mRNA was expressed by the human fibroblasts and osteoblasts, and PAPP-A protein was secreted into the culture medium. In conclusion, we have identified an IGF-dependent IGFBP protease and at the same time assigned a function to PAPP-A. This represents an unanticipated union of two areas of research that were not linked in any way before this report.

In skeletal muscle, acetylcholinesterase (AChE) exists in homomeric globular forms of type T catalytic subunits (ACHE T ) and heteromeric asymmetric forms composed of 1, 2, or 3 tetrameric ACHE T attached to a collagenic tail (ColQ). Asymmetric AChE is concentrated at the endplate (EP), where its collagenic tail anchors it into the basal lamina. The ACHE T gene has been cloned in humans; COLQ cDNA has been cloned in Torpedo and rodents but not in humans. In a disabling congenital myasthenic syndrome, EP AChE deficiency (EAD), the normal asymmetric species of AChE are absent from muscle. EAD could stem from a defect that prevents binding of ColQ to ACHE T or the insertion of ColQ into the basal lamina. In six EAD patients, we found no mutations in ACHE T . We therefore cloned human COLQ cDNA, determined the genomic structure and chromosomal localization of COLQ , and then searched for mutations in this gene. We identified six recessive truncation mutations of COLQ in six patients. Coexpression of each COLQ mutant with wild-type ACHE T in SV40-transformed monkey kidney fibroblast (COS) cells reveals that a mutation proximal to the ColQ attachment domain for ACHE T prevents association of ColQ with ACHE T ; mutations distal to the attachment domain generate a mutant ≈10.5S species of AChE composed of one ACHE T tetramer and a truncated ColQ strand. The ≈10.5S species lack part of the collagen domain and the entire C-terminal domain of ColQ, or they lack only the C-terminal domain, which is required for formation of the triple collagen helix, and this likely prevents their insertion into the basal lamina.

Two putative ribonucleases have been isolated from the secondary granules of mouse eosinophils. Degenerate oligonucleotide primers inferred from peptide sequence data were used in reverse transcriptase-PCR reactions of bone marrow-derived cDNA. The resulting PCR product was used to screen a C57BL/6J bone marrow cDNA library, and comparisons of representative clones showed that these genes and encoded proteins are highly homologous (96% identity at the nucleotide level; 92/94% identical/similar at the amino acid level). The mouse proteins are only weakly homologous (approximately 50% amino acid identity) with the human eosinophil-associated ribonucleases (i.e., eosinophil-derived neurotoxin and eosinophil cationic protein) and show no sequence bias toward either human protein. Phylogenetic analyses established that the human and mouse loci shared an ancestral gene, but that independent duplication events have occurred since the divergence of primates and rodents. The duplication event generating the mouse genes was estimated to have occurred < 5 x 10(6) years ago (versus 30 to 40 x 10(6) years ago in primates). The identification of independent duplication events in two extant mammalian orders suggests a selective advantage to having multiple eosinophil granule ribonucleases. Southern blot analyses in the mouse demonstrated the existence of three additional highly homologous genes (i.e., five genes total) as well as several more divergent family members. The potential significance of this observation is the implication of a larger gene subfamily in primates (i.e., humans).