Function of the upstream hypersensitive sites of the chicken beta-globin gene cluster in mice.

We have shown previously that the chicken beta A-globin gene, with its 3' enhancer, is expressed in a copy number-dependent manner in transgenic mice. The expression level was low but increased approximately 6-fold upon inclusion of 11 kb of upstream DNA containing four DNase I hypersensitive sites. To study the effect of the individual upstream hypersensitive sites on transgene expression, we produced lines of mice in which the individual upstream sites were linked to the beta A gene and enhancer. RNA levels were measured in blood from adult animals. With each of these four constructs, the level of transgene RNA per DNA copy varied over a > 20-fold range. These data suggest that addition of a hypersensitive site to the beta A-globin/enhancer region abrogates its position independent expression. The average beta A-globin expression per copy in the lines carrying an upstream site was comparable with that in lines without an upstream site. Thus, no single upstream hypersensitive site accounts for the higher level of beta A-globin expression seen in mice containing the complete upstream region. We had shown previously that control of the chicken beta-globin cluster is distributed between at least two regions, the beta A/epsilon enhancer and the upstream region. Our current results suggest that the control mediated by the upstream DNA is itself distributed and is not due to a single hypersensitive site.     

Evaluation of factors responsible for the inability of insulin to antagonize lipolysis due to high concentrations of catecholamines

Previous studies using rat adipocytes have shown that the ability of insulin to antagonize lipolysis induced by physiological concentrations of catecholamines is diminished at high concentrations of these hormones. Since such high concentrations of catecholamines cause an accumulation of free fatty acids, a decrease in cellular ATP level and a ‘short lived’ increase in cAMP (that is many fold higher than required to activate lipolysis maximally), we studied which of these modulates the antilipolytic activity of insulin. We found that inhibition of adenylate cyclase by virazole (2 mM), which lowers the initial cyclic AMP burst by about 70%, enables insulin to antagonize lipolysis at high isoproterenol concentrations. In contrast, reduction of cellular ATP level by 40% and 70%, using cyanide ion, or increasing free fatty acids in the medium to a level that suppresses the effects of insulin on glucose metabolism, failed to compromise the antilipolytic activity of the hormone. These data indicate that the inability of insulin to antagonize lipolysis induced by high isoproterenol concentrations is the direct consequence of the initial, larger burst of cyclic AMP.     

Demonstration of the heterozygous state for I-cell disease and pseudo-Hurler polydystrophy by assay of N-acetylglucosaminylphosphotransferase in white blood cells and fibroblasts

The biochemical abnormalities of I-cell disease (mucolipidosis II) and pseudo-Hurler polydystrophy (mucolipidosis III) can be explained by a deficiency of the enzyme UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. We demonstrate here that obligate heterozygotes for these autosomal recessive diseases have intermediate levels of this enzymatic activity in homogenates of peripheral blood white cells and in extracts from cultured fibroblasts. This finding provides further evidence that the enzyme deficiency is the primary genetic defect in these diseases. In addition, the previous observation that obligate heterozygotes for mucolipidosis III have elevations of total serum beta-hexosaminidase outside the range of normal was confirmed. In studies of three pedigrees of patients with mucolipidosis III, these techniques were used to score individuals at risk for the carrier state.     

Growth, adipose, brain, and skin alterations resulting from targeted disruption of the mouse peroxisome proliferator-activated receptor beta(delta)

To determine the physiological roles of peroxisome proliferator-activated receptor beta (PPARbeta), null mice were constructed by targeted disruption of the ligand binding domain of the murine PPARbeta gene. Homozygous PPARbeta-null term fetuses were smaller than controls, and this phenotype persisted postnatally. Gonadal adipose stores were smaller, and constitutive mRNA levels of CD36 were higher, in PPARbeta-null mice than in controls. In the brain, myelination of the corpus callosum was altered in PPARbeta-null mice. PPARbeta was not required for induction of mRNAs involved in epidermal differentiation induced by O-tetradecanoylphorbol-13-acetate (TPA). The hyperplastic response observed in the epidermis after TPA application was significantly greater in the PPARbeta-null mice than in controls. Inflammation induced by TPA in the skin was lower in wild-type mice fed sulindac than in similarly treated PPARbeta-null mice. These results are the first to provide in vivo evidence of significant roles for PPARbeta in development, myelination of the corpus callosum, lipid metabolism, and epidermal cell proliferation.     

Search for an Endogenous Bombesin-Like Receptor 3 (BRS-3) Ligand Using Parabiotic Mice

Bombesin-like receptor 3 (BRS-3) is an X-linked G protein-coupled receptor involved in the regulation of energy homeostasis. Brs3 null (Brs3 ^-/y) mice become obese. To date, no high affinity endogenous ligand has been identified. In an effort to detect a circulating endogenous BRS-3 ligand, we generated parabiotic pairs of mice between Brs3 ^-/y and wild type (WT) mice or between WT controls. Successful parabiosis was demonstrated by circulatory dye exchange. The Brs3 ^-/y-WT and WT-WT pairs lost similar weight immediately after surgery. After 9 weeks on a high fat diet, the Brs3 ^-/y-WT pairs weighed more than the WT-WT pairs. Within the Brs3 ^-/y-WT pairs, the Brs3 ^-/y mice had greater adiposity than the WT mice, but comparable lean and liver weights. Compared to WT mice in WT-WT pairs, Brs3 ^-/y and WT mice in Brs3 ^-/y-WT pairs each had greater lean mass, and the Brs3 ^-/y mice also had greater adiposity. These results contrast to those reported for parabiotic pairs of leptin receptor null (Lepr ^db/db) and WT mice, where high leptin levels in the Lepr ^db/db mice cause the WT parabiotic partners to lose weight. Our data demonstrate that a circulating endogenous BRS-3 ligand, if present, is not sufficient to reduce adiposity in parabiotic partners of Brs3 ^-/y mice.     

Characterization of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> Beijing isolates from the Mediterranean area

Background  

The Beijing lineage of Mycobacterium tuberculosis is causing concern due to its global distribution and its involvement in severe outbreaks. Studies focused on this lineage are mainly restricted to geographical settings where its prevalence is high, whereas those in other areas are scarce. In this study, we analyze Beijing isolates in the Mediterranean area, where this lineage is not prevalent and is mainly associated with immigrant cases.     

The relation between cartilage biomarkers (C2C,C1,2C,CS846, and CPII) and the long-term outcome of rheumatoid arthritis patients within the CAMERA trial

Introduction  

The aim of this study was to investigate whether serum biomarker levels of C2C, C1,2C, CS846, and CPII can predict the long-term course of disease activity and radiographic progression early in the disease course of rheumatoid arthritis (RA).