Variants in the gene encoding C3 are associated with asthma and related phenotypes among African Caribbean families.

Proinflammatory and immunoregulatory products from C3 play a major role in phagocytosis, respiratory burst, and airways inflammation. C3 is critical in adaptive immunity; studies in mice deficient in C3 demonstrate that features of asthma are significantly attenuated in the absence of C3. To test the hypothesis that the C3 gene on chromosome 19p13.3-p13.2 contains variants associated with asthma and related phenotypes, we genotyped 25 single nucleotide polymorphism (SNP) markers distributed at intervals of approximately 1.9 kb within the C3 gene in 852 African Caribbean subjects from 125 nuclear and extended pedigrees. We used the multiallelic test in the family-based association test program to examine sliding windows comprised of 2-6 SNPs. A five-SNP window between markers rs10402876 and rs366510 provided strongest evidence for linkage in the presence of linkage disequilibrium for asthma, high log[total IgE], and high log[IL-13]/[log[IFN-gamma] in terms of global P-values (P = 0.00027, 0.00013, and 0.003, respectively). A three-SNP haplotype GGC for the first three of these markers showed best overall significance for the three phenotypes (P = 0.003, 0.007, 0.018, respectively) considering haplotype-specific tests. Taken together, these results implicate the C3 gene as a priority candidate controlling risk for asthma and allergic disease in this population of African descent.

Direct sequencing of bacterial and P1 artificial chromosome-nested deletions for identifying position-specific single-nucleotide polymorphisms.

A loxP-transposon retrofitting strategy for generating large nested deletions from one end of the insert DNA in bacterial artificial chromosomes and P1 artificial chromosomes was described recently [Chatterjee, P. K. & Coren, J. S. (1997) Nucleic Acids Res. 25, 2205-2212]. In this report, we combine this procedure with direct sequencing of nested-deletion templates by using primers located in the transposon end to illustrate its value for position-specific single-nucleotide polymorphism (SNP) discovery from chosen regions of large insert clones. A simple ampicillin sensitivity screen was developed to facilitate identification and recovery of deletion clones free of transduced transposon plasmid. This directed approach requires minimal DNA sequencing, and no in vitro subclone library generation; positionally oriented SNPs are a consequence of the method. The procedure is used to discover new SNPs as well as physically map those identified from random subcloned libraries or sequence databases. The deletion templates, positioned SNPs, and markers are also used to orient large insert clones into a contig. The deletion clone can serve as a ready resource for future functional genomic studies because each carries a mammalian cell-specific antibiotic resistance gene from the transposon. Furthermore, the technique should be especially applicable to the analysis of genomes for which a full genome sequence or radiation hybrid cell lines are unavailable.

Genomic organization, fine-mapping, and expression of the human islet-brain 1 (IB1)/c-Jun-amino-terminal kinase interacting protein-1 (JIP-1) gene.

Islet-brain 1 (IB1), a regulator of the pancreatic beta-cell function in the rat, is homologous to JIP-1, a murine inhibitor of c-Jun amino-terminal kinase (JNK). Whether IB1 and JIP-1 are present in humans was not known. We report the sequence of the 2133-bp human IB1 cDNA, the expression, structure, and fine-mapping of the human IB1 gene, and the characterization of an IB1 pseudogene. Human IB1 is 94% identical to rat IB1. The tissue-specific expression of IB1 in human is similar to that observed in rodent. The IB1 gene contains 12 exons and maps to chromosome 11 (11p11.2-p12), a region that is deleted in DEFECT-11 syndrome. Apart from an IB1 pseudogene on chromosome 17 (17q21), no additional IB1-related gene was found in the human genome. Our data indicate that the sequence and expression pattern of IB1 are highly conserved between rodent and human and provide the necessary tools to investigate whether IB1 is involved in human diseases.

Genomic characterization of the coding region of the human type II 5'-deiodinase gene.

Type II 5'-Deiodinase (5'DII) is a key element in the maintenance of peripheral thyroid hormone homeostasis through the regulation of local T4 to T3 conversion in pituitary, brain, brown adipose tissue and placenta. The cDNA containing the coding region of the human 5'DII (HDII) has been recently cloned from infant brain. In the present paper we report the genomic structure, chromosomal localization and restriction map of the coding region of HDII. The presence of a single intron located at codon 75 was demonstrated using a PCR-based strategy; the exon-intron junctions were then cloned and partially sequenced. Chromosomal localization was performed by radiation hybrid mapping. This study demonstrated that the entire coding region of the HDII gene is contained in two exons spliced at codon 75 by a 7.4 Kb intron and that the HDII chromosomal location is 14q24.3. These data will allow further studies of the role of HDII in the pathophysiology of thyroid homeostasis.

Identification of Rad's effector-binding domain, intracellular localization, and analysis of expression in Pima Indians.

In order to characterize the endogenous gene product for rad (ras-related protein associated with diabetes), we prepared antibodies to synthetic peptides that correspond to amino acids (109-121, 178-195, 254-271) within the protein. These antibodies were used to analyze the expression, structure, and function of rad. Western analysis with these antibodies revealed that rad was a 46 kDa protein which was expressed during myotube formation. Further, immunolocalization studies showed that rad localized to thin filamentous regions in skeletal muscle. Interestingly, when muscle biopsies from diabetic and control Pima Indians were compared, no differences in rad protein or mRNA expression were observed. Similarly, no differences were observed in protein expression in diabetic and control Zucker diabetic fatty (ZDF) rats. Functional analysis of muscle rad revealed that its GTP-binding activity was inhibited by the addition of N-ethylmaliemide, GTP, GTP gamma S, and GDP beta S but not ATP or dithiothreitol. Moreover, cytosol-dependent rad-GTPase activity was stimulated by the peptide corresponding to amino acids 109-121. Antibodies corresponding to this epitope inhibited cytosol-dependent rad-GTPase activity. Taken together, the results indicate that 1) rad is a 46 kDa GTP-binding protein localized to thin filaments in muscle and its expression increases during myoblast fusion, 2) expression of rad in Pima Indians and ZDF rats does not correlate with diabetes, and 3) the amino acids (109-121) may be involved in regulating rad-GTPase activity, perhaps by interacting with a cytosolic factor(s) regulating nucleotide exchange and/or hydrolysis.

Chromosomal localization and partial genomic structure of the human peroxisome proliferator activated receptor-gamma (hPPAR gamma) gene.

We determined the chromosomal localization and partial genomic structure of the coding region of the human PPAR gamma gene (hPPAR gamma), a nuclear receptor important for adipocyte differentiation and function. Sequence analysis and long PCR of human genomic DNA with primers that span putative introns revealed that intron positions and sizes of hPPAR gamma are similar to those previously determined for the mouse PPAR gamma gene[13]. Fluorescent in situ hybridization localized hPPAR gamma to chromosome 3, band 3p25. Radiation hybrid mapping with two independent primer pairs was consistent with hPPAR gamma being within 1.5 Mb of marker D3S1263 on 3p25-p24.2. These sequences of the intron/exon junctions of the 6 coding exons shared by hPPAR gamma 1 and hPPAR gamma 2 will facilitate screening for possible mutations. Furthermore, D3S1263 is a suitable polymorphic marker for linkage analysis to evaluate PPAR gamma's potential contribution to genetic susceptibility to obesity, lipoatrophy, insulin resistance, and diabetes.

Muscle Rad expression and human metabolism: potential role of the novel Ras-related GTPase in energy expenditure and body composition.

Ras associated with diabetes (Rad), a new ras-related GTPase, was recently identified by subtractive cloning as an mRNA in skeletal muscle that is overexpressed in NIDDM. To better understand its metabolic significance, we measured skeletal muscle Rad expression in well-characterized insulin sensitive (IS) and insulin resistant (IR) subjects with normal glucose tolerance and in untreated NIDDM patients. We found no differences in expression of Rad mRNA levels among IS, IR, and NIDDM groups using a ribonuclease protection assay (0.22 +/- 0.06, 0.13 +/- 0.01, and 0.16 +/- 0.02 relative units, respectively; NS) and no differences in Rad protein expression using a specific anti-peptide Rad antibody (1.05 +/- 0.18, 1.14 +/- 0.08, and 1.08 +/- 0.21 units/mg protein, respectively; NS). However, Rad protein levels were positively correlated with BMI (r = 0.43, P = 0.03) and percentage body fat (r = 0.55, P < 0.005), two independent measures of obesity, and negatively correlated with resting metabolic rate (r = 0.49, P = 0.01). In multiple regression analyses, percentage body fat and resting metabolic rate independently accounted for 30 and 10% of individual variability in muscle Rad protein expression. In conclusion, Rad expression in skeletal muscle is not altered as a function of insulin resistance or NIDDM in humans. However, these data, for the first time, implicate a role for Rad in regulating body composition and energy expenditure and provide a framework for studies designed to elucidate Rad's cellular functions.

Molecular scanning of the human peroxisome proliferator activated receptor gamma (hPPAR gamma) gene in diabetic Caucasians: identification of a Pro12Ala PPAR gamma 2 missense mutation.

Peroxisome proliferator activated receptor-gamma (PPAR gamma) is a nuclear receptor that regulates adipocyte differentiation, and possibly lipid metabolism and insulin sensitivity. As such, PPAR gamma is a promising candidate gene for several human disorders including obesity and type 2 diabetes mellitus. Screening for mutations in the entire coding region of the PPAR gamma gene (both gamma 1 and gamma 2 isoforms) was performed with DNA of 26 diabetic Caucasians with or without obesity. Two base substitutions were identified: a silent mutation at nucleotide 1431 (CACHis-->CATHis) and a missense mutation (CCGPro-->GCGAla) at codon 12 of PPAR gamma 2. The allele frequency of the Pro12Ala PPAR gamma 2 variant was 0.12 in Caucasian Americans, 0.10 in Mexican Americans, 0.08 in Samoans, 0.03 in African Americans, 0.02 in Nauruans, and 0.01 in Chinese. We conclude that the Pro12Ala PPAR gamma 2 gene variant is present in diverse populations. Further studies of the Pro12Ala variant will determine its relevance to obesity, insulin resistance, and type 2 diabetes.

Anti-inflammatory activity of phosphodiesterase (PDE)-IV inhibitors in acute and chronic models of inflammation.

Inhibitors of cyclic nucleotide phosphodiesterases are known to suppress lipopolysaccharide (LPS)-induced tumour necrosis factor-alpha (TNF-alpha) production in vitro in human monocytes. The most potent of these have selectivity for type IV PDEs, suggesting that this class of PDE is the major type involved in the regulation of human TNF-alpha production. Using compounds of two distinct chemical structural classes, a quinazolinedione (CP-77059) and a 4 arylpyrrolidinone (rolipram), we show here that PDE-IV-specific inhibitors are also potent in suppressing LPS-induced TNF-alpha production in vitro in sodium periodate-elicited murine macrophages (IC50s of 1 and 33, respectively). We then report the in vivo anti-inflammatory effect of PDE-IV inhibition in five murine models of inflammation: (i) elevation of serum TNF-alpha induced by a sublethal LPS injection; (ii) LPS-induced endotoxic shock; (iii) LPS/galactosamine-induced endotoxic shock; (iv) carrageenan-induced paw oedema; and (v) adjuvant arthritis. Following a sublethal (5 micrograms/mouse) injection of LPS, serum TNF-alpha levels in mice peaked sharply, reaching concentrations of 3-12 ng/ml 90 min after injection. In this sublethal LPS assay, CP-77059 was about 30 times more potent than rolipram, with a minimum effective dose of 0.1 mg/kg versus 3 mg/kg for rolipram. This rank order is in keeping with the relative in vitro IC50s for CP-77059 and rolipram, as well as their relative Ki against the human PDE-IV enzyme (46 nM and 220 nM, respectively). In LPS-induced endotoxic shock, rolipram and CP-77059 at relatively high doses of 30 and 10 mg/kg, respectively, significantly reduced serum TNF-alpha levels, and also inhibited mortality 66%. In the LPS/galactosamine shock model, in which mice are rendered exquisitely sensitive to LPS by co-injection with galactosamine, only 0.1 microgram of LPS/mouse is necessary for serum TNF-alpha elevation and death. Both rolipram and the CP-77059 caused dose-dependent reduction of serum TNF-alpha and lethality. In the carrageenan-induced paw oedema model, in which there is a pronounced local TNF-alpha response (without a serum TNF-alpha elevation), rolipram significantly inhibited paw swelling as well as localized TNF-alpha levels in the paw. In the adjuvant arthritis model, a chronic model of inflammation also possessing localized TNF-alpha elevation in the inflamed paw, rolipram and CP-77059 suppressed ankle swelling and radiological evidence of joint damage. These data are consistent with a major role for PDE-IV in regulation of TNF-alpha production and inflammatory responses in murine systems.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential regulation of human monocyte-derived TNF alpha and IL-1 beta by type IV cAMP-phosphodiesterase (cAMP-PDE) inhibitors.

Elevation of cAMP downregulates certain functions of inflammatory cells, including the release of TNF alpha and IL-1 beta by macrophages. Intracellular cAMP levels can be modulated pharmacologically by adding cell-permeable cAMP analogs, by stimulating adenylate cyclase or by inhibiting degradation of cAMP by cAMP-phosphodiesterases (cAMP-PDE). Multiple forms of cAMP-PDEs have been identified in various tissues and cells using both biochemical characterization and selective inhibitors. Therefore, we wanted to determine which of these different PDE isoforms was present in human monocytes and whether this isoform could regulate cytokine release from human monocytes by a mechanism similar to that seen with dbcAMP or PGE1. Our results demonstrate that selective inhibitors of type IV cAMP-PDE, such as rolipram and Ro20-1724, are clearly the most effective compounds at enhancing cAMP levels and inhibiting the release of TNF alpha and IL-1 beta in these cells. The type III cAMP-PDE-selective inhibitors C1930 and cilostamide and the nonselective PDE inhibitors IBMX and pentoxifylline were significantly less potent. In agreement with these data, cAMP-PDE activity in cytosolic extracts from human monocytes was also much more sensitive to inhibition by rolipram than by cilostamide. Additionally, rolipram dramatically reduced TNF alpha mRNA accumulation, which supports previous findings that cAMP regulates TNF alpha at the transcriptional level. Surprisingly, rolipram, rolipram, dbcAMP or PGE1 increased IL-1 beta was reduced, which indicates that cAMP can have both positive and negative effects on the regulation of IL-1 beta.(ABSTRACT TRUNCATED AT 250 WORDS)

Elevated levels of TNF in the joints of adjuvant arthritic rats.

The primary purpose of this study was to determine whether local levels of tumor necrosis factor (TNF) were elevated in chronically inflamed joints in rats with adjuvant-induced arthritis (AA). We also wished to develop methodology for the quantitative measurement of joint TNF, and to examine the effects of known anti-inflammatory agents on joint TNF levels. TNF levels were measured in joints from AA rats taken during the systemic phase (day 20) of arthritic disease. Using the L929 bioassay, joint extracts from AA rats had significantly greater TNF levels (1054 +/- 147 pg/g tissue) than joint extracts from normal rats (110 +/- 42 pg/g tissue). Administration of ibuprofen failed to significantly inhibit TNF levels in the joint at a time point when paw swelling was significantly reduced. The immunomodulating agents, methotrexate, cyclosporin A (CSA) and HWA486 profoundly inhibited both joint TNF levels and paw swelling. The specificity of this assay for TNF was supported by studies with a polyclonal rabbit anti-mouse TNF antibody which neutralized 78-87% of the TNF activity in the joint extracts. Our studies demonstrate a quantitative increase in local TNF expression in adjuvant arthritis and support a role for TNF in chronic inflammation.

Effect of acetylation on arthropathic activity of group A streptococcal peptidoglycan-polysaccharide fragments.

Purified group A streptococcal peptidoglycan-polysaccharide (PG-PS) fragments were either de-O-acylated, or acetylated and then de-O-acylated to yield N-acetylated PG-PS. Native PG-PS was poorly degraded, N-acetylated PG-PS was extensively degraded, and de-O-acylated PG-PS was only slightly degraded by hen egg white lysozyme. N-acetylated PG-PS was also extensively degraded by human lysozyme and partially degraded by rat serum or rat liver extract. After a single intraperitoneal injection of rats with a sterile, aqueous suspension, all PG-PS preparations induced acute arthritis. The acute arthritis induced by N-acetylated PG-PS was significantly more severe than that induced by native PG-PS; that induced by de-O-acylated PG-PS was of intermediate severity. After the acute reaction, rats injected with native PG-PS developed chronic relapsing erosive synovitis which remained severe for the duration of the experiment (83 days). In contrast, joint inflammation induced by N-acetylated PG-PS resolved within 6 weeks with little evidence of recurrent disease. Chronic arthritis induced by de-O-acylated PG-PS was of intermediate severity. In another assay of arthropathic activity, the arthritis in all rat ankle joints, which had been injected directly with native PG-PS, could be reactivated 3 weeks later by the intravenous injection of a small dose of PG. In contrast, only 50% of the joints initially injected with de-O-acylated PG-PS and none of the joints injected with N-acetylated PG-PS could be reactivated. These studies support the concepts that the resistance of PG-PS to muralytic digestion is crucial for chronic arthropathic activity and that the nature and degree of PG acetylation are important molecular determinants of the phlogistic activities of PG-PS polymers.

Restriction in the lytic efficiency of complement of different erythrocyte targets: a re-examination of the activities of horse C8 and C9.

Differences in the lytic efficiency of different complement sources have frequently been observed. This effect has been shown to be related to both the species of the target erythrocyte and the species composition of terminal complement components within the 5b-9 membrane attack complex. The majority of studies have indicated that the source of C9 is critical in controlling the range of erythrocyte species that can be lysed efficiently. One exception to this general finding was the report by Lachmann et al., 1973 (Immunology 24, 135-145), using horse serum as a complement source. In that study, horse C8 rather than C9 was implicated as the critical component. In this study, we have re-examined this observation and find that the restricted hemolytic potential of horse complement correlates absolutely with the presence of horse C9. The reason for the differences between our findings and those of the earlier study are discussed.