Two chitin synthase genes, designated chsA and chsB, were isolated from Aspergillus nidulans with the Saccharomyces cerevisiae CHS2 gene as the hybridization probe. Nucleotide sequencing showed that chsA and chsB encoded polypeptides consisting of 1013 and 916 amino acid residues, respectively; the hydropathy profiles of the enzymes were similar to those of other fungal chitin synthases. Northern analysis indicated that both genes were transcribed, suggesting that cellular chitin in A. nidulans is synthesized by at least two chitin synthases. For examination of the roles of the chitin synthase genes in cell growth, gene disruption experiments were done. The chsA disruptant grew as well as the wild-type strain, but the chsB disruptant had severe growth defects that could not be overcome by the addition of 1.2M sorbitol as an osmotic stabilizer. These findings suggested that chsB but not chsA is essential for hyphal growth.

Nine (15%) of 60 patients with repaired myelomeningocele exhibited late deterioration of neurological function with a tethered cord syndrome. Dense adhesions at the lowest laminae and at the site of previous repair were the most common findings at surgery. Postoperatively, 71% of the patients improved. Magnetic resonance (MR) imaging was performed in 29 of the 60 patients. Eight of these 29 patients exhibited a tethered cord syndrome. The MR images in all patients showed a low-lying conus fixed at the site of previous repair, irrespective of the presence or absence of a tethered cord syndrome. The MR images were classified into two groups depending upon the site of adhesions: Group A had potential sites of tethering at the ventral aspect of the last laminae and at the site of previous repair, and Group B showed the adhesion point only at the site of previous repair. Most patients with a tethered cord syndrome were found to be in Group A; conversely, most patients without the syndrome were in Group B. An enlarged low conus was seen in symptomatic patients more commonly than in those without this syndrome. It is concluded that the presence of adhesions specifically at the last laminae as well as a widened low-lying conus may be the cause of tethered cord syndrome in patients with repaired myelomeningoceles. A clear understanding of the tethering process and preoperative evaluation of potential sites of tethering, based on the MR findings, are very important for planning surgery. The release of adhesions at the lowest laminae by laminectomy appeared essential for improvement.

Cholesterol homeostasis is maintained by coordinate regulation of cholesterol synthesis and its conversion to bile acids in the liver. The excretion of cholesterol from liver and intestine is regulated by ATP-binding cassette half-transporters ABCG5 and ABCG8. The genes for these two proteins are closely linked and divergently transcribed from a common intergenic promoter region. Here, we identified a binding site for hepatocyte nuclear factor 4␣ (HNF4␣) in the ABCG5/ABCG8 intergenic promoter, through which HNF4␣ strongly activated the expression of a reporter gene in both directions. The HNF4␣-responsive element is flanked by two conserved GATA boxes that were also required for stimulation by HNF4␣. GATA4 and GATA6 bind to the GATA boxes, coexpression of GATA4 and HNF4␣ leads to a striking synergistic activation of both the ABCG5 and the ABCG8 promoters, and binding sites for HNF4␣ and GATA were essential for maximal synergism. We also show that HNF4␣, GATA4, and GATA6 colocalize in the nuclei of HepG2 cells and that a physical interaction between HNF4␣ and GATA4 is critical for the synergistic response. This is the first demonstration that HNF4␣ acts synergistically with GATA factors to activate gene expression in a bidirectional fashion.Cholesterol homeostasis is maintained by a series of regulatory pathways that control the synthesis of endogenous cholesterol, the absorption of dietary sterol, and the elimination of cholesterol and its catabolic end products, bile acids. Transcriptional control of many genes vital to these processes can be attributed to two classes of transcription factors: sterol regulatory element-binding proteins (SREBPs), especially SREBP-2, which control the production of key enzymes in cholesterol biosynthesis (11,36,38,39), and the nuclear hormone receptor family, including liver X receptor (LXR), farnesoid X receptor, small heterodimer partner, liver receptor homolog1 (LRH-1), and hepatocyte nuclear factor 4␣ (HNF4␣), which control the expression of genes involved in cholesterol efflux, catabolism, and elimination (3, 27).HNF4␣ is the most abundant nuclear orphan receptor expressed in the liver, and it is involved in early liver development (22). HNF4␣ is also expressed in kidney, intestine, and pancreas and is required for expression of many tissue-specific traits in all of these organs. Transcriptional activation by HNF4␣ is mediated by its binding as a homodimer to a DNA sequence composed of two direct repeats (DRs) of the hexanucleotide motif AGGTCA separated by 1 base, referred to as an HNF4␣ response element of the DR-1 type. Like other nuclear receptors, HNF4␣ exhibits a modular structure with six distinct domains (A to F). The N-terminal A/B domain is highly variable among nuclear receptors and contains a ligandindependent activation function 1 (AF-1) domain. The highly conserved C domain encodes the DNA binding domain of nuclear receptors and confers sequence-specific DNA recognition. By linking the highly structured C and E domains, the hinge D region may allow for flexibil...

In obesity-related insulin resistance, pancreatic islets compensate for insulin resistance by increasing secretory capacity. Here, we report the identification of sex-determining region Y-box 6 (SOX6), a member of the high mobility group box superfamily of transcription factors, as a co-repressor for pancreatic-duodenal homeobox factor-1 (PDX1). SOX6 mRNA levels were profoundly reduced by both a long term high fat feeding protocol in normal mice and in genetically obese ob/ob mice on a normal chow diet. Interestingly, we show that SOX6 is expressed in adult pancreatic insulin-producing ␤-cells and that overexpression of SOX6 decreased glucosestimulated insulin secretion, which was accompanied by decreased ATP/ADP ratio, Ca 2؉ mobilization, proinsulin content, and insulin gene expression. In a complementary fashion, depletion of SOX6 by small interfering RNAs augmented glucose-stimulated insulin secretion in insulinoma mouse MIN6 and rat INS-1E cells. These effects can be explained by our mechanistic studies that show SOX6 acts to suppress PDX1 stimulation of the insulin II promoter through a direct protein/protein interaction. Furthermore, SOX6 retroviral expression decreased acetylation of histones H3 and H4 in chromatin from the promoter for the insulin II gene, suggesting that SOX6 may decrease PDX1 stimulation through changes in chromatin structure at specific promoters. These results suggest that perturbations in transcriptional regulation that are coordinated through SOX6 and PDX1 in ␤-cells may contribute to the ␤-cell adaptation in obesity-related insulin resistance.

Lipoprotein lipase (LPL) is a key enzyme in the hydrolysis of TG-rich lipoproteins. To elucidate the physiological roles of LPL in lipid and lipoprotein metabolism, we generated transgenic rabbits expressing human LPL. In postheparinized plasma of transgenic rabbits, the human LPL protein levels were about 650 ng/ml, and LPL enzymatic activity was found at levels up to 4-fold greater than that in nontransgenic littermates. Increased LPL activity in transgenic rabbits was associated with as much as an 80% decrease in plasma triglycerides and a 59% decrease in high density lipoprotein-cholesterol. Analysis of the lipoprotein density fractions revealed that increased expression of the LPL transgene resulted in a remarkable reduction in the level of very low density lipoproteins as well as in the level of intermediate density lipoproteins. In addition, LDL cholesterol levels in transgenic rabbits were significantly increased. When transgenic rabbits were fed a cholesterol-rich diet, the development of hypercholesterolemia and aortic atherosclerosis was dramatically suppressed in transgenic rabbits. These results demonstrate that systemically increased LPL activity functions in the metabolism of all classes of lipoproteins, thereby playing a crucial role in plasma triglyceride hydrolysis and lipoprotein conversion, and that overexpression of LPL protects against diet-induced hypercholesterolemia and atherosclerosis. Lipoprotein lipase (LPL)1 plays a crucial role in lipid metabolism and transport by catalyzing the hydrolysis of triglyceriderich (TG-rich) lipoproteins such as chylomicrons and very low density lipoproteins (VLDL). Through the hydrolysis of TG in these particles, LPL converts these lipoproteins to denser lipoproteins such as chylomicron remnants, intermediate density lipoprotein (IDL), and low density lipoproteins (LDL) (1-3). This process generates free fatty acids (FFA), which are taken up and used for metabolic energy or stored as TG after reesterification and also results in the generation of surface remnants, which give rise to high density lipoproteins (HDL). It has been suggested that LPL influences not only plasma TG levels but also plasma HDL levels (4).LPL is mainly produced by mesenchymal cells such as adipose and muscle cells and then transported to the luminal surface of the vascular endothelium, where it is bound to heparan sulfate proteoglycans (HSPG). Small amounts of LPL are also present in other types of tissues, including the adrenals, brain, lung, and spleen (5). Furthermore, LPL is also expressed by macrophages and smooth muscle cells in atherosclerotic lesions (6, 7), suggesting that LPL modulates vascular functions and may be involved in atherogenesis. Elucidation of the precise roles of LPL in atherosclerosis has been compounded by the fact that LPL has multiple functions in lipoprotein metabolism through its catalytic properties and acts as a ligand for the LDL receptor-related protein (8) or a bridge between lipoproteins and HSPG (9). In humans, it has been found that familial L...