Rheumatoid arthritis (RA) is a widely prevalent (1-3%) chronic systemic disease thought to have an autoimmune component; both humoral and cellular mechanisms have been implicated. Primary osteoarthritis (OA) is considered to be distinct from rheumatoid arthritis, and here damage is thought to be secondary to cartilage degeneration. In rheumatoid arthritis, immune complexes are present that consist exclusively of immunoglobulin, implying that this is both the 'antibody' (rheumatoid factor [RF]) and the 'antigen' (most commonly IgG). Autoantigenic reactivity has been localized to the constant-region (C gamma 2) domains of IgG. There is no evidence for a polypeptide determinant but carbohydrate changes have been reported. We have therefore conducted a study, simultaneously in Oxford and Tokyo, to compare in detail the N-glycosylation pattern of serum IgG (Fig. 1) isolated from normal individuals and from patients with either primary osteoarthritis or rheumatoid arthritis. The results, which required an evaluation of the primary sequences of approximately 1,400 oligosaccharides from 46 IgG samples, indicate that: (1) IgG isolated from normal individuals, patients with RA and patients with OA contains different distributions of asparagine-linked bi-antennary complex-type oligosaccharide structures, (2) in neither disease is the IgG associated with novel oligosaccharide structures, but the observed differences are due to changes in the relative extent of galactosylation compared with normal individuals. This change results in a 'shift' in the population of IgG molecules towards those carrying complex oligosaccharides, one or both of whose arms terminate in N-acetylglucosamine. These two arthritides may therefore be glycosylation diseases, reflecting changes in the intracellular processing, or post-secretory degradation of N-linked oligosaccharides.

Involvement of Akt/Protein kinase B (PKB), a serine/threonine kinase with a pleckstrin-homology domain, in angiotensin II (ANG II)-induced signal transduction was investigated in cultured vascular smooth muscle cells (VSMC). Stimulation of the cells with ANG II led to a marked increase in the kinase activity of Akt/PKB, which coincided with Ser-473 phosphorylation. ANG II-stimulated Akt/PKB activation was rapid, concentration dependent, and inhibited by the AT1-receptor antagonist CV-11974, but not by pertussis toxin. Akt/PKB activity was stimulated by the Ca2+ ionophore ionomycin, suggesting the possible involvement of Ca2+ in ANG II-stimulated Akt/PKB activation. However, blockade of Ca2+ mobilization by BAPTA-AM only partially inhibited ANG II-stimulated Akt/PKB activation. ANG II-stimulated Akt/PKB activation was inhibited by the tyrosine kinase inhibitors genistein and herbimycin A and by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY-294002. These results indicate that ANG II stimulates Akt/PKB activity via AT1 receptors in VSMC and that the activities of tyrosine kinase and PI3K are required for this activation.

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Cholesterol 7␣-hydroxylase is the rate-limiting enzyme in the degradation of cholesterol to bile salts and plays a central role in regulating cholesterol homeostasis. The mechanisms involved in the transcriptional control of the human gene are largely unknown. HepG2 cells represent an appropriate model system for the study of the regulation of the gene. To identify liverspecific DNA sequences in the promoter of the human CYP7 gene, we first examined the DNase I hypersensitivity in the 5 -region of the gene. An area of hypersensitivity was observed in the region from ؊50 to ؊200 of the human gene in nuclei from transcriptionally active HepG2 cells, but was absent in transcriptionally inactive HeLa cell nuclei or in free DNA. Various 5 -promoter deletion constructs were made and transfected into HepG2 cells. About 300 base pairs of upstream sequence are required for high level promoter activity of the human CYP7 gene in HepG2 cells. DNase I footprinting of the hypersensitive region revealed nine protected sequences. Gel retardation experiments demonstrated binding of HNF-3 to the segment from ؊80 to ؊70 and of hepatocyte nuclear factor HNF-4 (and ARP-1) to the segment from ؊148 to ؊127 of the human CYP7 promoter. Deletion of either of these sites depressed promoter activity in HepG2 cells. A third region from ؊313 to ؊285 is bound by members of the HNF-3 family and acts as an enhancer. Additionally, the segment from ؊197 to ؊173 binds a negative regulatory protein that is present in Chinese hamster ovary cell extracts and in HepG2 cell extracts. These experiments define the key control elements responsible for basal transcription of the human CYP7 gene in HepG2 cells.Cholesterol 7␣-hydroxylase catalyzes the rate-limiting step in the pathway that leads to the catabolism of cholesterol to bile acids (for review, see Ref. 1). Cholesterol 7␣-hydroxylase is a microsomal enzyme member of the cytochrome P-450 family. In human and rat, the major products of this metabolic pathway are cholic acid and chenodeoxycholic acid. Bile acids have an important role in cholesterol homeostasis; their synthesis and excretion cause a decrease in hepatic cholesterol levels, while their presence in the intestine facilitates the solubilization of dietary fats and is required for the absorption of cholesterol and fat-soluble vitamins. Because of the importance of these functions, bile acid synthesis in the liver is carefully regulated to maintain cholesterol homeostasis (1). To date, little is known about the molecular mechanisms that control cholesterol catabolism and bile acid synthesis.The cDNAs and genes for cholesterol 7␣-hydroxylase have been isolated from rat (2-4), human (5, 6), hamster (7), and mouse (8). CYP7 mRNA is found exclusively in the liver (9), making this gene a target for the study of the molecular mechanisms implicated in hepatic-specific gene expression. Work by several groups has demonstrated that CYP7 mRNA levels are modulated in cultured cells by a number of effectors. For example, in cultured rat hepatocytes (10) ...