Interferon regulatory factor-5 is genetically associated with systemic lupus erythematosus in African Americans.

Increased expression of interferon (IFN)-inducible genes is implicated in the pathogenesis of systemic lupus erythematosus (SLE). One transcription factor responsible for regulating IFN, interferon regulatory factor-5 (IRF5), has been associated with SLE in genetic studies of Asian, Caucasian and Hispanic populations. We genotyped up to seven polymorphic loci in or near IRF5 in a total of 4870 African-American and Caucasian subjects (1829 SLE sporadic cases and 3041 controls) from two independent studies. Population-based case-control comparisons were performed using the Pearson's chi(2)-test statistics and haplotypes were inferred using HaploView. We observed significant novel associations with the IRF5 variants rs2004640 and rs3807306 in African Americans and replicated previously reported associations in Caucasians. While we identified risk haplotypes, the majority of haplotypic effects were accounted for by one SNP (rs3807306) in conditional analyses. We conclude that genetic variants of IRF5 associate with SLE in multiple populations, providing evidence that IRF5 is likely to be a crucial component in SLE pathogenesis among multiple ethnic groups.

Conservation of FcepsilonRI gamma chain coding region in normals and in SLE patients.

High-frequency single nucleotide polymorphism (SNP) alleles are useful in mapping genes responsible for disease susceptibility. Functionally, Fcgamma receptors (FcgammaR) have been implicated in autoimmune disease, and the gene encoding the signaling element for several FcgammaR, Fc-epsilon-receptor gamma-chain (FcepsilonRIgamma), has several SNPs in the immunoreceptor tyrosine activation motif (ITAM) recorded in GenBank. Direct sequencing of the FcepsilonRIgamma coding region found potentially polymorphic sites in the 5'-->3' direction in control donors, which were not confirmed in the reverse direction (n = 66), and further exploration of 80 SLE patients revealed no non-synonymous SNPs. One normal donor was heterozygous for a non-synonymous SNP at nt 38 which changed the fifth codon from valine (GTG) to methionine (ATG). Although the EST databases suggest candidate SNPs, insertions and deletions, these appear to be artifacts, most probably due to secondary structure. The coding region of FcepsilonRIgamma shows a remarkable absence of nucleotide diversity. Either as yet unidentified regulatory elements of FcepsilonRIgamma or other genes in the region of human chromosome 1q23 are likely to be systemic lupus erythematosus disease susceptibility and severity genes.

Interleukin 10 polymorphisms as predictors of sustained response in antiviral therapy for chronic hepatitis C infection.

Host genetic factors have been reported to influence the natural history of hepatitis C virus (HCV) infection. We examined whether variation in interleukin 10 (IL-10) and tumor necrosis factor alpha (TNF-alpha) genes would predict the likelihood of sustained response to antiviral therapy. Single nucleotide polymorphisms (SNPs) and microsatellites at two loci encoding the cytokines IL-10 and TNF-alpha were determined by polymerase chain reaction (PCR)-based techniques. Their relationship to the outcome of antiviral therapy for chronic HCV infection was studied in 49 white patients who had a virologically sustained response (SR) and in 55 white nonresponders (NR) to a combination of interferon alfa-2b and ribavirin (IFN + R). Several IL-10 variants were more frequent among SRs compared with NRs. Carriage of the -592A or the -819T SNP was associated with SR (odds ratio [OR] = 2.2; P =.016). The -592A/A and the exclusively linked -819T/T genotypes were also associated with SR (OR = 16.6; P =.013 for either). The haplotype consisting of the 108-bp IL-10.R microsatellite and -3575T, -2763C, -1082A, -819T, -592A was also associated with SR (OR = 2.65; P =.01). Stratification for viral genotype, baseline viral RNA concentration, and histologic status identified homozygosity for the haplotype as the principal determinant: all 5 homozygous individuals achieved SR (OR(crude) = 13.7; P =.025; stratified ORs = 1.9-7.0), whereas heterozygotes differed only slightly from wild-type carriers. In contrast, TNF alleles defined by promoter sequences -238G/A and -308G/A were approximately equally distributed among SR and NR. In conclusion, homozygosity for -592A, -819T or the extended haplotype (108bp) - (-2575T) - (-2763C) - (-1082A) - (-819T) - (-592A) is associated with SR to IFN + R.

Novel single nucleotide polymorphisms in the distal IL-10 promoter affect IL-10 production and enhance the risk of systemic lupus erythematosus.

Family studies of first-degree relatives and analysis of twins indicate that as much as 75% of the differences in quantitative IL-10 production in man derive from heritable genetic factors. Studies of single nucleotide polymorphisms (SNP) in the proximal 1.0 kb of the IL-10 promoter have yielded inconsistent association with IL-10 production and variable results in promoter-reporter studies. However, in normal donors, an association of quantitative production with certain alleles of the IL-10.R short tandem repeat polymorphism at -4.0 kb suggested that SNPs in the more distal promoter might be informative. We have identified seven novel SNP sites in the genomic sequence of the first 4 kb of the IL-10 promoter region 5' to the ATG start site from Caucasian individuals with either a high or a low IL-10 production phenotype. We have also identified eight SNP haplotypes in the distal promoter that segregate with significant differences in quantitative IL-10 production in normal donors. These SNPs are significantly associated with systemic lupus erythematosus in African-Americans and may define one component of the genetic susceptibility to systemic lupus erythematosus in this group.

Functional domain analysis of the Saccharomyces MAL-activator.

MAL63 of the MAL6 locus and its homologues at the other MAL loci encode transcription activators required for the maltose-inducible expression of the MAL structural genes. We carried out a deletion analysis of LexA-MAL63 gene fusions to localize the functional domains of the Mal63 MAL-activator protein. Our results indicate that the sequence-specific DNA-binding domain of Mal63p is contained in residues 1-100; that residues 60-283 constitute a functional core region including the transactivation domain; that residues 251-299 are required to inhibit the activation function of Mal63p; and that the rest of the C-terminal region of the protein contains a maltose-responsive domain that acts to relieve the inhibitory effect on the activation function. Abundant overproduction of Mal63p does not overcome the negative regulation of MAL gene expression in the absence of maltose, suggesting that a titratable MAL-specific repressor similar to Gal80p is not involved in the negative regulation of the MAL-activator. A model for maltose-inducible autoregulation of the MAL-activator is presented.

Single-nucleotide polymorphisms of T cell receptor zeta chain in patients with systemic lupus erythematosus.

OBJECTIVE: Signaling molecules from the T cell receptor zeta/Fcepsilon receptor gamma (TCRzeta/FcRgamma) family play a critical role in the function of Fcgamma receptors and the TCR and are located on human chromosome 1, where lupus susceptibility genes are located. This study was undertaken to investigate the possibility of polymorphisms and/or mutations of TCRzeta in systemic lupus erythematosus (SLE). METHODS: We amplified the whole coding region of TCRzeta by reverse transcriptase-polymerase chain reaction (PCR) and directly sequenced the PCR products with a dye primer technique to facilitate heterozygote detection. RESULTS: An alternative splicing form of TCRzeta, with a CAG codon (glutamine) inserted at the splice junction of exons 4 and 5, was found both in SLE and in non-SLE subjects. Both splice isoforms of TCRzeta occurred in human mixed peripheral blood mononuclear cells, natural killer cells, and Jurkat T cells. In TCRzeta, 2 silent and 2 missense mutations were found, but neither coding change occurred in the immunoreceptor tyrosine-activation motif. No unique mutations were found in Caucasian, African American, Hispanic, Chinese, or Japanese SLE patients living in North America. CONCLUSION: The uncommon and equal occurrence of novel single-nucleotide polymorphisms in both SLE patients and normal subjects makes it improbable that they play important roles in genetic susceptibility to SLE.

High levels of gelatinase-B and active gelatinase-A in metastatic glioblastoma.

PURPOSE: Extra-neural metastases from glioblastoma multiforme (GBM) are rare. Because gelatinases-A and -B have been implicated in tumor invasion/metastasis in non-neural tumors, we compared the expression of gelatinase-A and -B in 2 patients (both had a prior craniotomy performed) with extraneural metastases from GBM to expression levels in 24 other gliomas; 15 non-metastatic GBMs, 9 other lower grade gliomas, and 7 normal brain tissues. METHODS: The intracerebral tumor from both patients, patient # 1's extraneural metastases, 24 other gliomas, 1 sample of reactive astrocytes and 7 normal brain tissues were studied using gelatin zymography. The active form of gelatinases was confirmed by co-migration after activation with APMA. RESULTS: Expression of the latent form of gelatinase-A correlated with glioma grade (r = 0.486; p = 0.0053). Active gelatinase-A was found only in the 2 GBMs with extraneural metastases and patient # 1's cervical metastases. In contrast, latent gelatinase-B levels correlated more strongly with histologic grade (r = 0.577; p = 0.0009) (higher levels with higher grades). Very high levels of gelatinase-B were seen in both GBMs with extraneural metastases, a cervical extraneural metastases, and 2 GBMs without metastases. CONCLUSIONS: We observed that gelatinases-A and -B are present in most gliomas but we found active gelatinase-A only in the GBMs with extraneural metastases suggesting that the active form of this enzyme may determine the metastatic potential of GBMs. We propose that high levels of gelatinolytic activities are associated with intracerebral invasion and rarely, metastases of GBMs.

Constitutive mutations of the Saccharomyces cerevisiae MAL-activator genes MAL23, MAL43, MAL63, and mal64.

We report the sequence of several MAL-activator genes, including inducible, constitutive, and noninducible alleles of MAL23, MAL43, MAL63, and mal64. Constitutive alleles of MAL23 and MAL43 vary considerably from inducible alleles in their C-terminal domain, with many of the alterations clustered and common to both alleles. The 27 alterations from residues 238-461 of Mal43-C protein are sufficient for constitutivity, but the minimal number of alterations needed for the constitutive phenotype could not be determined. The sequence of mal64, a nonfunctional homologue of MAL63, revealed that Mal64p is 85% identical to Mal63p. Two mutations that activate mal64 and cause constitutivity are nonsense mutations resulting in truncated proteins of 306 and 282 residues. We conclude that the C-terminal region of the MAL-activator, from residues 283-470, contains a maltose-responsive negative regulatory domain, and that extensive mutation or deletion of the entire region causes loss of the negative regulatory function. Additionally, certain sequence elements in the region appear to be necessary for efficient induction of the full-length Mal63 activator protein. These studies highlight the role of ectopic recombination as an important mechanism of mutagenesis of the telomere-associated family of MAL loci.

Apoptosis induced by c-myc overexpression is dependent on growth conditions.

Recent reports have suggested a causal link between the expression of the c-myc gene and ensuing cell death by apoptosis, particularly in cells prevented from dividing or after withdrawal of growth factors. In contrast, other studies have suggested that cells constitutively expressing c-myc are actually more resistant to cell death induced by some chemotherapeutic drugs that block cell division. We have examined the frequency of cell death in several Chinese hamster ovary cell lines that contain 20 to 30 copies of the human c-myc gene and that express high levels of human c-myc mRNA and protein. We found that constitutive c-myc expression in cells incubated at low density in medium containing 0.1% serum correlates with increased cell death due to apoptosis, as indicated by oligonucleosomal DNA fragmentation and a requirement for ongoing protein synthesis. However, apoptosis was not enhanced in these cells when they were blocked in cell division in the presence of serum, nor when grown at moderate to high density under low-serum conditions, despite their continued accumulation of high levels of c-Myc protein. Our results show that overexpression of c-Myc protein can promote cell death under some but not all conditions that block cell division and further suggest that c-Myc may accelerate but does not necessarily initiate apoptosis.

c-Myc antigens in the mammalian enteric nervous system.

We have investigated the presence of c-Myc-like antigens in the enteric nervous system of the guinea-pig, rat, dog, sheep, monkey and human. c-Myc-like immunoreactivity was demonstrated by immunohistochemistry in the enteric nervous system of all animals tested, with one or more monoclonal antibodies raised against peptide sequences found in the human c-Myc protein. While in most cases the labelling was nuclear, cytoplasmic labelling was also observed. In the guinea-pig enteric nervous system, c-Myc-like immunoreactivity detected by two different antibodies remained detectable for up to 4 h in the presence of cycloheximide. The size and density of labelled nuclei in the ileal submucous plexus were consistent with exclusive neuronal labelling by one antibody and neuronal plus glial labelling by the other. Double-labelling with antiserum directed against vasoactive intestinal peptide revealed a subset of c-Myc-immunoreactive neurons that also contain this neuropeptide. Anti-c-Myc antibodies specifically immunoprecipitated proteins from guinea-pig myenteric plexus-longitudinal muscle preparations whose sizes were consistent with previous observations for c-Myc antigens and whose distribution was consistent with synthesis in the myenteric plexus. We conclude that c-Myc proteins are expressed in mammalian enteric neurons and that they have characteristics similar to those of c-Myc proteins in other nonproliferative cells.

A possible role for c-Myc oncoproteins in post-transcriptional regulation of ribosomal RNA.

Overexpression of members of the myc family of oncogenes contributes to the development of many vertebrate malignancies. Although several functions for myc gene products have been proposed, the targets for Myc activity during oncogenesis or normal development remain to be identified. Oocytes of Xenopus laevis, which are non-dividing cells that accumulate abundant c-Myc protein, provide an unusual opportunity to examine c-Myc function. We have investigated whether the accumulation of massive amounts of ribosomal RNA (rRNA) by Xenopus laevis oocytes may be related to their elevated expression of myc proto-oncogenes. Our data show that anti-Myc antibodies and some (but not all) c-Myc peptides from conserved regions of the c-Myc protein enhance the turnover of rRNA synthesized in homogenates of oocyte nuclei. These results suggest that one or more members of the Myc protein family may be involved in post-transcriptional regulation of rRNA metabolism. The regulation by c-Myc of rRNA could account, in part, for the generalized stimulation of cells during tumorigenesis.

Multiple antigens recognized by anti-c-myc antibodies in human cells and Xenopus oocytes.

We have investigated the localization, solubility, serum regulation, and phosphorylation of MYC antigens from Colo 320 cells, a human transformed cell line with an amplified c-myc gene, and from Xenopus oocytes, which express high levels of c-myc mRNA. Although MYC proteins are often reported to range from 60 to 68 kilodaltons, our panel of anti-MYC monoclonal antibodies recognized a number of higher and lower molecular mass antigens, in addition to proteins within this range. Based upon various criteria, including cross-recognition by several anti-MYC antibodies, we suggest that some of these antigens are bona fide MYC family proteins. Our results, as well as those of others reported previously, suggest that several MYC antigens may be simultaneously present in cells. The apparent diversity among members of the MYC family of antigens raises the possibility of multiple cellular functions and regulatory roles for these proteins.

Migratory and invasive behavior of pigment cells in normal and animalized sea urchin embryos.

Pigment cell precursors in the vegetal plate of late mesenchyme blastulae of the sea urchin Strongylocentrotus purpuratus begin to express a cell surface epitope recognized by the monoclonal antibody SP-1/20.3.1. When one-quarter gastrulae are dissociated into ectodermal and mesenchymal fractions, most SP-1/20.3.1 immunoreactive cells separate into the mesenchymal fraction, whereas at the full gastrula and all later stages almost all epitope-bearing cells are in the ectodermal fraction. Exposure of embryos to sulfate-free seawater p-nitrophenyl beta-D-xyloside, and tunicamycin, all of which prevent primary mesenchyme migration, does not inhibit SP-1/20.3.1 immunoreactive cells from distributing similarly to those in controls, although pigment synthesis is completely inhibited in sulfate-free conditions. Time-lapse video sequences reveal that pigment cells, and a small set of rapidly migrating, SP-1/20.3.1 immunoreactive amoeboid cells that appear in the pluteus, remain closely associated with the ectodermal epithelium during most of larval development. Transmission electron microscopy observations of plutei show pigment cells tightly apposed to the ectodermal epithelium at discontinuities in the basal lamina and sandwiched between the basal lamina and the epithelial cells. It is concluded that SP-1/20.3.1 immunoreactive mesenchymal cells invade the ectodermal epithelium and may use migratory substrates other than those used by primary mesenchymal cells.

The origin of pigment cells in embryos of the sea urchin Strongylocentrotus purpuratus.

A monoclonal antibody (SP1/20.3.1) that recognizes a cell surface epitope expressed by pigment cells in the pluteus larva of Strongylocentrotus purpuratus has been produced. Using indirect immunofluorescence, the epitope is first detected in nonpigmented cells of the vegetal plate after primary mesenchyme ingression. Between the beginning of gastrulation, and when the archenteron is one-third the distance across the blastocoel, SP1/20.3.1-positive cells are free within the blastocoel, at the tip of the archenteron, and dispersed within the blastoderm. Cells at the tip of the archenteron, and mesenchyme near the tip in later stages of gastrulation (secondary mesenchyme), do not express the SP1/20.3.1 antigen. By the completion of gastrulation all SP1/20.3.1-positive cells are dispersed throughout the epidermis. It has been concluded that in S. purpuratus pigment cell precursors are released from the vegetal plate during the initial phase of gastrulation. The cells migrate first to the vegetal ectoderm, and subsequently disperse throughout the ectoderm and develop pigment granules.