Endotoxin-induced cytokine gene transcription in monocytes and macrophages is regulated in part by NF-B. We have previously shown that the p38 mitogenactivated protein (MAP) kinase is necessary for endotoxin-induced cytokine gene transcription. Due to the fact that most cytokine promoter sequences have active NF-B sites, we hypothesized that the p38 MAP kinase was necessary for NF-B-dependent gene expression. We found that NF-B-dependent gene expression was reduced to near control levels with either SB 203580 or a dominant-negative p38 MAP kinase expression vector. Inhibition of the p38 MAP kinase did not alter NF-B activation at any level, but it significantly reduced the DNA binding of TATA-binding protein (TBP) to the TATA box. The dominant-negative p38 MAP kinase expression vector interfered with the direct interaction of native TFIID (TBP) with a co-transfected p65 fusion protein. Likewise, this dominant-negative plasmid also interfered with the direct interaction of a co-transfected TBP fusion protein with the native p65 subunit. The p38 kinase also phosphorylated TFIID (TBP) in vitro, and SB 203580 inhibited phosphorylation of TFIID (TBP) in vivo. Thus, the p38 MAP kinase regulates NF-B-dependent gene transcription, in part, by modulating activation of TFIID (TBP). Cytokine gene expression in endotoxin (LPS)1 -stimulated macrophages is regulated, at least in part, at the level of transcription. A transcription factor that is necessary for the transcription of many cytokine genes is nuclear factor B (NF-B) (1-4). In addition to others, we have previously shown that NF-B binds to specific cytokine promoter sequences (1-8). NF-B is composed of heterodimers (most commonly p50 and p65) of members of the Rel family of transcription factors. In quiescent cells the heterodimers are kept in the cytoplasm by an inhibitor protein, IB (9 -11). NF-B translocation and DNA binding is dependent on IB kinase (IKK), which phosphorylates IB on serines within the amino-terminal domain (12)(13)(14)(15)(16)(17). This phosphorylation results in IB degradation, thus allowing NF-B translocation to the nucleus. Other factors, however, have been shown to be essential for NF-B transcriptional activation, especially phosphorylation of the p65 subunit of NF-B in one of two of its transactivation domains (18). In addition, the association of the carboxyl terminus of p65 with basal transcription factors, such as transcription factor IIB (TFIIB) and TATA-binding protein (TBP), is known to be important for transcriptional regulation of .One family of kinases that is essential for transferring signals from the cell surface to the nucleus is the mitogen-activated protein (MAP) kinases. We and others have shown that the p38 MAP kinase is critical for LPS-induced cytokine gene expression (23)(24)(25)(26).Some of these studies showed that LPS, interleukin 1, and osmotic stress activate p38 MAP kinases, and inhibition with SB 203580, a competitive inhibitor of the p38 MAP kinases, reduced cytokine release but did not affect cytokine mRNA accumu...

Infertility and spontaneous pregnancy losses are an enduring problem to women's health. The establishment of pregnancy depends on successful implantation, where a complex series of interactions occurs between the heterogeneous cell types of the uterus and blastocyst. Although a number of genes are implicated in embryo-uterine interactions during implantation, genetic evidence suggests that only a small number of them are critical to this process. To obtain a global view and identify novel pathways of implantation, we used a dual screening strategy to analyze the expression of nearly 10,000 mouse genes by microarray analysis. Comparison of implantation and interimplantation sites by a conservative statistical approach revealed 36 up-regulated genes and 27 down-regulated genes at the implantation site. We also compared the uterine gene expression profile of progesterone-treated, delayed implanting mice to that of mice in which delayed implantation was terminated by estrogen. The results show up-regulation of 128 genes and down-regulation of 101 genes after termination of the delayed implantation. A combined analysis of these experiments showed specific up-regulation of 27 genes both at the implantation site and during uterine activation, representing a broad diversity of molecular functions. In contrast, the majority of genes that were decreased in the combined analysis were related to host immunity or the immune response, suggesting the importance of these genes in regulating the uterine environment for the implanting blastocyst. Collectively, we identified genes with recognized roles in implantation, genes with potential roles in this process, and genes whose functions have yet to be defined in this event. The identification of unique genetic markers for the onset of implantation signifies that genome-wide analysis coupled with functional assays is a promising approach to resolve the molecular pathways required for successful implantation.

We used expression quantitative trait locus mapping in the laboratory rat (Rattus norvegicus) to gain a broad perspective of gene regulation in the mammalian eye and to identify genetic variation relevant to human eye disease. Of >31,000 gene probes represented on an Affymetrix expression microarray, 18,976 exhibited sufficient signal for reliable analysis and at least 2-fold variation in expression among 120 F2 rats generated from an SR͞JrHsd ؋ SHRSP intercross. Genome-wide linkage analysis with 399 genetic markers revealed significant linkage with at least one marker for 1,300 probes (␣ ‫؍‬ 0.001; estimated empirical false discovery rate ‫؍‬ 2%). Both contiguous and noncontiguous loci were found to be important in regulating mammalian eye gene expression. We investigated one locus of each type in greater detail and identified putative transcription-altering variations in both cases. We found an inserted cREL binding sequence in the 5 flanking sequence of the Abca4 gene associated with an increased expression level of that gene, and we found a mutation of the gene encoding thyroid hormone receptor ␤2 associated with a decreased expression level of the gene encoding short-wavelength sensitive opsin (Opn1sw). In addition to these positional studies, we performed a pairwise analysis of gene expression to identify genes that are regulated in a coordinated manner and used this approach to validate two previously undescribed genes involved in the human disease Bardet-Biedl syndrome. These data and analytical approaches can be used to facilitate the discovery of additional genes and regulatory elements involved in human eye disease.bioinformatics ͉ expression quantitative trait locus analysis ͉ gene regulation ͉ ophthalmology

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Our studies suggest that the phase-on genotypes of licA and igaB are important for early human nasopharynx colonization. lex2A showed a trend from phase off to phase on, suggesting a potentially important role in the colonization process.

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SummaryMycoplasma arthritidis, an agent of chronic proliferative arthritis of rodents, secretes a potent soluble superantigen, MAM, that is active for both murine and human T and B lymphocytes. We now report the complete nucleotide and amino acid sequence of MAM and show it to be distinct from other proteins and not closely related phylogeneticaUy to other superantigens. Two functional domains on MAM are identified based on the ability ofpeptides encompassing these regions to inhibit lymphocyte proliferation by the intact MAM molecule. One of these domains shares short sequences or epitopes with other microbial superantigens. The second domain contains the consensus legume lectin motif-B, which is important for T cell activation by concanavalin (Con) A. MAM and Con A peptides containing this motif are functionally cross reactive, suggesting a novel secondary pathway for T cell activation by MAM. S uperantigens are potent immunomodulatory moleculesthat are produced by a number of pathogenic bacteria (1, 2), endogenous murine retroviruses (3), and may be associated with HIV-1 (4, 5). Superantigens have been hypothesized to play a role in human diseases including Kawasaki's disease, toxic shock syndrome, rheumatoid arthritis, and diabetes (6-8). The Mycoplasma arthritidis mitogen (MAM) ~ (9) is a particularly interesting superantigen as it is produced by an organism that causes spontaneous chronic arthritis in rodents (10). Furthermore, evidence suggests that MAM triggers autoimmune arthritis in collagen-injected mice by activating the specific T cells that drive the inflammatory response (11).MAM is a typical superantigen in that it is presented to murine and human T cells by direct binding to MHC molecules present on accessory cell surfaces and is recognized by specific V~ chain segments of the TCR without MHC restriction (9). Although MAM is much less potent in activating human T cells than are the Staphylococcus aureusderived superantigens, unlike the latter it promotes a strong polyclonal B cell activation in human PBL. This property suggests that MAM may be an ideal model for the study of 1Abbreviations used in this paper: MAM, Mycoplasma arthtitidis mitogen; SEA, SEB, SEC, staphylococcal enterotoxin A, B, and C, respectively. the role of superantigens in the pathogenesis of human autoimmune disease characterized by hypergammaglobulinemia (12). We show here that MAM is a unique protein, phylogenetically distinct from other superantigens, but which contains cross-reactive motifs found in other T cell mitogens. Materials and MethodsCloning and Sequencing of MAM. Degenerate oligonucleotide primers were designed corresponding to the NH 2-and COOHterminal ends of the previously sequenced peptide (13), based upon the codon usage ofMycoplasma capricolum (14). These primers were used to amplify, by PCtL, chromosomal DNA from M. arthritidis strain PG6. The PCIL product was labeled with 32p and used to screen an M. arthtitidis PG6 genomic library constructed in EMBL3 using previously described methods (15). Three clone...

Abstract-The mouse is useful in studies of vascular biology because of its well-defined genetics and because the mouse genome can be manipulated. However, because only small amounts of mRNA can be extracted from blood vessels, the quantification of gene expression in individual mice is difficult. Endothelial NO synthase (eNOS) plays a major role in the regulation of vascular tone and growth. In addition, there appear to be sex differences in the production of NO under basal conditions in mouse aortas. The goals of this study were to develop a real-time polymerase chain reaction (PCR) method to quantify eNOS mRNA in blood vessels from mice and to examine eNOS mRNA levels in vessels from male and female mice. Blood vessels were isolated from C57BL/6 mice. Total RNA from individual mice was isolated and reverse-transcribed. The number of molecules of eNOS mRNA (after reverse transcription) was determined against cDNA standards, with 18S rRNA used as a control for RNA input and reverse-transcription efficiency. When expressed as copy numbers per nanogram of total RNA or as the ratio of eNOS to 18S rRNA, eNOS mRNA was lower in the aortas of female mice than in those of male mice at 7 to 9 months of age. In contrast, no difference in eNOS mRNA was found in the aortas of 2-month-old mice. In addition, eNOS mRNA levels were similar in the carotid, cerebral, and coronary arteries. These findings provide the first quantitative measurements of eNOS mRNA by using real-time PCR in the vessels of mice and suggest age-and sex-related differences in the basal levels of eNOS mRNA in mice. In addition, the eNOS region that was used for real-time PCR was amplified and sequenced for monkeys and other species. Key Words: endothelial NO synthase Ⅲ real-time polymerase chain reaction Ⅲ mice Ⅲ aortas Ⅲ sex E ndothelial NO synthase (eNOS) plays a major role in the regulation of vascular tone and growth in large arteries and in the microcirculation. 1,2 In addition to mechanisms that regulate enzyme activity acutely, eNOS is regulated at mRNA and protein levels. 3,4 Steady-state levels of eNOS mRNA are increased by shear stress, 5 estrogen, 6 and exercise, 7 but these levels are decreased during heart failure 8 and possibly other disease states. A sensitive and reproducible method to quantify eNOS expression is of great importance in studies of vascular physiology and pathophysiology.Quantification of relative eNOS mRNA levels has been accomplished previously by using Northern blotting, 5,7,8 RNase protection assay, 6 and competitive reverse transcription (RT)-polymerase chain reaction (PCR). 9 Real-time PCR (after RT) is a new, sensitive, and accurate method for the quantification of mRNA levels 10 and has had limited application in vascular studies. 11 It requires a small amount of starting RNA and uses the initial (linear) rate of PCR (C T ) as the measure of the original amount of cDNA. With at least 5 orders of linear dynamic range, there is no need to analyze dilutions of each sample, and this, coupled with no need for post-PCR pro...