Identification of a zinc finger protein that binds to the sterol regulatory element.

Cholesterol balance in mammalian cells is maintained in part by sterol-mediated repression of gene transcription for the low density lipoprotein receptor and enzymes in the cholesterol biosynthetic pathway. A promoter sequence termed the sterol regulatory element (SRE) is essential for this repression. With the use of an oligonucleotide containing the SRE to screen a human hepatoma complementary DNA expression library, a clone for a DNA binding protein was isolated that binds to the conserved SRE octanucleotide in both a sequence-specific and a single-strand--specific manner. This protein contains seven highly conserved zinc finger repeats that exhibit striking sequence similarity to retroviral nucleic acid binding proteins (NBPs). We have designated the protein "cellular NBP" (CNBP). CNBP is expressed in a wide variety of tissues, is up regulated by sterols, and exhibits binding specificity that correlates with in vivo function. These properties are consistent with a role in sterol-mediated control of transcription.

Hormone-sensitive lipase: sequence, expression, and chromosomal localization to 19 cent-q13.3.

Hormone-sensitive lipase, a key enzyme in fatty acid mobilization, overall energy homeostasis, and possibly steroidogenesis, is acutely controlled through reversible phosphorylation by catecholamines and insulin. The 757-amino acid sequence predicted from a cloned rat adipocyte complementary DNA showed no homology with any other known lipase or protein. The activity-controlling phosphorylation site was localized to Ser563 in a markedly hydrophilic domain, and a lipid-binding consensus site was tentatively identified. One or several messenger RNA species (3.3, 3.5, or 3.9 kilobases) were expressed in adipose and steroidogenic tissues and heart and skeletal muscle. The human hormone-sensitive lipase gene mapped to chromosome 19 cent-q13.3.

Gender- and compartment-specific bone loss in C57BL/6J mice: correlation to season?

Seasonal variation in bone mineral density (BMD) has been documented in humans, and has been attributed to changes in 25-hydroxyvitamin D [25(OH)D] synthesis. To test the hypothesis that seasonal changes in bone mass occur in laboratory mice, we measured body composition, femoral bone phenotypes, and serum bone markers in 16-wk-old male and female C57BL/6 (B6) mice during the summer (June-August) and winter (December-February) months at The Jackson Laboratory in Bar Harbor, Maine. Both male and female B6 mice had higher volumetric BMD in the summer than winter. Females showed reduced trabecular bone, whereas males showed changes in bone volume. Males, but not females, had higher insulin-like growth factor 1 in summer than in winter, and only males showed an increase in body weight during the winter. No seasonal differences in serum TRAP5b, osteocalcin, or 25(OH)D were noted for either sex. We conclude that seasonal variation in skeletal and body composition parameters in B6 mice is significant and must be considered when performing longitudinal phenotyping of the skeleton. Further studies are needed to determine the environmental factors that cue seasonal changes in body composition and the mechanisms that produce these changes.

Coincidence of genetic loci for plasma cholesterol levels and obesity in a multifactorial mouse model.

We have examined backcross progeny derived from a cross of Mus spretus with C57BL/6J, that range from 1 to 50% carcass lipid (n = 215), and from 22 to 130 mg/dl plasma total cholesterol (n = 238). Statistical analysis revealed that distal mouse chromosome 7 exhibits significant linkage both to plasma total cholesterol (likelihood of the odds [LOD] 5.8) and to carcass lipid (LOD 3.8). A locus on chromosome 6 also shows significant linkage to plasma total cholesterol (LOD 5.6), but no linkage to carcass lipid. Neither chromosomal region contains any previously mapped genes likely to influence lipoprotein metabolism, indicating that novel genetic factors contributing to plasma lipoprotein levels have been identified.

Identification of four chromosomal loci determining obesity in a multifactorial mouse model.

We previously described a new mouse model for multigenic obesity, designated BSB. We now report the use of a complete linkage map approach to identify loci contributing to body fat and other traits associated with obesity in this model. Four loci exhibiting linkage with body fat, or with the weights of four different fat depots, residing on mouse chromosomes 6, 7, 12, and 15, were identified and confirmed by analysis of additional BSB mice. Each of the four loci differed with respect to their effects on the percent of body fat, specific fat depots and plasma lipoproteins. The loci exhibited allele-specific, non-additive interactions. A locus for hepatic lipase activity was co-incident with the body fat and total cholesterol loci on chromosome 7, providing a possible mechanism linking plasma lipoproteins and obesity. The chromosome 7 locus affecting body fat, total cholesterol and hepatic lipase activity was isolated in congenic strains whose donor strain regions overlap with the chromosome 7 BSB locus. These results provide candidate genes and candidate loci for the analysis of human obesity.

Molecular cloning and sequence of a cholesterol-repressible enzyme related to prenyltransferase in the isoprene biosynthetic pathway.

Differential hybridization and molecular cloning have been used to isolate CR39, a cDNA which hybridizes to a 1.2-kilobase (kb) mRNA in rat liver. The level of CR39 mRNA was increased seven- to ninefold over normal levels by dietary cholestyramine and mevinolin and decreased about fourfold compared with normal levels by cholesterol feeding or administration of mevalonate. Similar changes in the mRNA levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and HMG-CoA synthase were observed under the various conditions. In vitro translation of either CR39 hybrid selected RNA or 1.2-kb CR39 RNA generated by an SP6 in vitro transcription system produced a polypeptide of 39,000 daltons. As deduced from the nucleotide sequence of a full-length CR39 cDNA, the rat CR39 polypeptide contained 344 amino acids and had a molecular weight of 39,615. The predicted amino acid composition and submit molecular weight of the rat CR39 were very similar to those of prenyltransferases isolated from chicken, pig, and human. The sequence of amino acid residues 173 through 203 in the rat CR39 polypeptide showed that 17 out of 30 matched an active-site peptide of avian liver prenyltransferase. Thus, alterations in the rate of cholesterogenesis resulted in the coordinate regulation of three mRNAs encoding HMG-CoA reductase, HMG-CoA synthase, and CR39, the latter being tentatively identified as prenyltransferase.

Renal interstitial fibrosis and vascular changes. Occurrence in patients with autoimmune diseases treated with cyclosporine.

Histologic examinations of kidney biopsy specimens from six patients with relapsing polychondritis (n = 1), Behçet's syndrome (n = 3), and chronic uveitis (n = 2) were performed after four to 36 months of treatment with cyclosporine. Five of the patients had a variable degree of focal interstitial fibrosis and tubular atrophy, with and without minimal interstitial inflammation. Arteriolar hyalinization was noted in four and glomerular sclerosis in two patients. These renal lesions could not be attributed to underlying autoimmune disease or previous drug therapy but were similar to those recently reported in kidney and heart recipients receiving long-term cyclosporine. The initial cyclosporine doses were 15 mg/kg body weight in one patient and 10 mg/kg in the others. The maintenance cyclosporine doses ranged from 2.5 to 7.5 mg/kg with appropriate trough cyclosporine plasma levels (60 to 130 ng/mL). A rough correspondence between the extent of the histologic renal changes and the cumulative cyclosporine was seen, whereas serum creatinine increase or the development of hypertension during treatment did not predict the degree of interstitial fibrosis or the presence of arteriolar changes. Neither did a rapid fall in the serum creatinine level after withdrawal of cyclosporine exclude focal irreversible renal lesions. Since the histopathologic changes found in the kidneys are potentially progressive, we believe that, until more is known, long-term cyclosporine treatment should be reserved for situations where more established immunosuppression has failed to control an autoimmune process threatening the function of vital organs.

Serum lipoprotein in active rheumatoid arthritis and other chronic inflammatory arthritides. I. Relativity to inflammatory activity.

Lipoprotein metabolism was investigated in 69 patients with untreated active rheumatoid arthritis (n = 48) and in seronegative spondylarthropathies (n = 21). The patients had high inflammatory activity as measured by erythrocyte sedimentation rate and C-reactive protein (CRP). Serum cholesterol and cholesterol levels in the very-low-density lipoprotein (VLDL), low-density lipoprotein, and high-density lipoprotein fractions were reduced by 20% to 30% compared with healthy controls; and triglyceride levels in VLDL and high-density lipoprotein were reduced by 10% to 30%. There were significant correlations between the inflammatory activity and certain lipoprotein lipids, ie, between CRP and VLDL triglycerides, VLDL cholesterol, and serum triglycerides. The fractional elimination rate (K2) measured by an intravenous fat tolerance test was 30% higher in the patients than in the controls despite reduced tissue lipoprotein lipase activities. There was correlation between CRP and the K2 value. These findings suggest that it is the degree of inflammatory activity that governs the altered lipoprotein metabolism in untreated active chronic inflammatory arthritides. The relationships between CRP and VLDL and between CRP and K2 suggest that the VLDL particles may be altered by inflammatory process, and that the increased elimination may take place through the "scavenger pathway."

Serum lipoprotein in active rheumatoid arthritis and other chronic inflammatory arthritides. II. Effects of anti-inflammatory and disease-modifying drug treatment.

Serum lipids and lipoprotein patterns were prospectively analyzed in 33 previously untreated patients with active chronic inflammatory arthritides during different anti-inflammatory and disease-modifying drug regimens. Before treatment the lipoprotein pattern was characterized by low cholesterol concentrations in all lipoprotein fractions and low triglyceride concentrations in the very-low-density lipoprotein fraction as well as in the high-density lipoprotein fraction. During treatment with prednisolone combined with azathioprine or cyclophosphamide (n = 10), a reduction of the disease activity was achieved and the lipoprotein pattern was normalized; similar results were noted in a small group of patients (n = 4) treated with prednisolone alone while nonsteroidal anti-inflammatory drug therapy (n = 9) neither significantly affected the lipoprotein pattern nor the inflammatory activity measured by the acute-phase reactants. The long-term treatment with penicillamine (n = 4) and chloroquine (n = 6) induced both a clinical remission of the disease and a reduction of the inflammatory activity. The lipoprotein concentrations started to reverse to the normal values during penicillamine treatment. In contrast, in the chloroquine-treated group the alterations in lipoprotein lipid concentrations were further pronounced, ie, the cholesterol and triglyceride concentrations in serum and the very-low-density lipoprotein fraction decreased.

Quantitative trait loci mapping for cholesterol gallstones in AKR/J and C57L/J strains of mice.

Quantitative trait locus (QTL) mapping was used to locate genes that determine the difference in cholesterol gallstone disease between the gallstone-susceptible strain C57L/J and the gallstone-resistant strain AKR/J. Gallstone weight was determined in 231 male (AKR x C57L) F(1) x AKR backcross mice fed a lithogenic diet containing 1% cholesterol, 0.5% cholic acid, and 15% butterfat for 8 wk. Mice having no stones and mice having the largest stones were genotyped at approximately 20-cM intervals to find the loci determining cholesterol gallstone formation. The major locus, Lith1, mapped near D2Mit56 and was confirmed by constructing a congenic strain, AK. L-Lith1(s). Another locus, Lith2, mapped near D19Mit58 and was also confirmed by constructing a congenic strain AK.L-Lith2(s). Other suggestive, but not statistically significant, loci mapped to chromosomes 6, 7, 8, 10, and X. The identification of these Lith genes will elucidate the pathophysiology of cholesterol gallstone formation.

Regulation of chicken apolipoprotein B: cloning, tissue distribution, and estrogen induction of mRNA.

Apolipoprotein (apo) B is a major protein component of plasma very low-density and low-density lipoproteins (VLDL and LDL, respectively) and serves as a recognition signal for the cellular binding and internalization of LDL by the apoB/E receptor. In contrast to the situation in mammals, avian apoB is also a component of specialized VLDL particles that are produced by the liver in response to estrogen. These particles transport cholesterol and triglyceride from the liver to the ovary for deposition in egg yolk. We report here the identification and characterization of cDNA clones for chicken apoB and their use in examining the tissue distribution and hormonal regulation of chicken apoB mRNA. The cDNA clones were identified by immunological screening of a phage lambda gt11 library constructed with hen liver mRNA and their identity was supported by sequence comparisons with mammalian apoB. The chicken apoB mRNA is approximately the same size as mammalian apoB mRNA (14 kb), and, as occurs in mammals, is present at high levels in liver and small intestine. Unlike mammals, the chicken apoB mRNA is also found at high levels in the kidney, consistent with previous protein biosynthetic studies. A DNA-excess solution-hybridization assay was used to quantitate apoB mRNA in these tissues and to examine its hormonal regulation. In control roosters the liver and kidney contained 65% and 10%, respectively, as much apoB mRNA as the small intestine. Within 24 h after estradiol administration, apoB mRNA was increased five- to seven-fold in liver but was unchanged in intestine and kidney. The increase in apoB mRNA content and the kinetics of induction parallel hepatic apoB synthesis, indicating that estrogen regulates apoB production through changes in the cellular abundance of apoB mRNA. The apoB mRNA increased rapidly following hormone treatment while the mRNA for another VLDL protein (apoII) showed a lag or slow phase of several hours before significant mRNA accumulation occurred. These data indicate that the liver can respond immediately to estrogen to increase apoB mRNA accumulation, while apoII mRNA accumulation appears to involve additional events or signals which occur slowly and are specific to this gene.

Impaired glucose handling in active rheumatoid arthritis: relationship to peripheral insulin resistance.

Glucose metabolism was studied after an intravenous glucose loading in normal-weighted, previously untreated patients (n = 14) with active rheumatoid arthritis (RA). The patients displayed an enhanced insulin response and impaired glucose handling compared with healthy controls (P less than .001). The insulin sensitivity, measured as the glucose utilization rate during steady state of euglycemia (M) was significantly decreased (P less than .01) among the patients compared to the controls (5.5 +/- 1.9 mg/kg BW/min [mean +/- SD] and 7.2 +/- 1.2, respectively). The corresponding values for the metabolic clearance rate (MCR) were 5.8 +/- 0.6 mL/kg BW/min and 8.2 +/- 0.4, respectively (P less than .01). In the patient group the k value correlated with the peripheral insulin sensitivity (P less than .01), which, in turn, was inversely related to the acute phase reaction (P less than .05). During 1 week of potent anti-inflammatory treatment with corticosteroids (prednisolone 20 mg daily) the k value improved P less than .001), the insulin sensitivity tended to improve and the insulin response increased (P less than .001) after an intravenous glucose loading. Five patients who had a remission of their disease on sulphasalazine as antirheumatic therapy were reexamined. A normalization of the inflammatory activity as well as the glucose handling and insulin sensitivity was achieved. The data obtained indicate that impaired glucose handling in active RA is related to insulin resistance. The linkage between inflammatory indices and glucose metabolism might reflect a special consequence of inflammation, but the influence of nonspecific disease manifestations, ie, malnutrition, inactivity, and myopenia, has to be considered.

Impaired glucose handling in active rheumatoid arthritis: effects of corticosteroids and antirheumatic treatment.

Forty-two patients with active rheumatoid arthritis were studied serially with respect to glucose metabolism after the institution of different anti-inflammatory and antirheumatic therapies. Sixteen patients received 20 mg of prednisolone daily. After 1 week of treatment the mean k value in glucose tolerance tests increased from 1.0 +/- 0.1 (SEM) to 1.6 +/- 0.1 (P less than .001). The corticosteroid therapy thus restored the glucose tolerance to normal and significantly enhanced the insulin response (P less than .01). Corticosteroids also normalized the growth hormone response to glucose infusion but had no effect on plasma glucagon. Treatment with nonsteroidal anti-inflammatory drugs did not affect the k values nor the hormonal pattern either after short-term treatment or after three months of therapy, except for causing a minor increase in the plasma glucagon levels both before and after glucose infusion. The long-term effects of treatment with penicillamine (n = 4), chloroquine (n = 7), and immunosuppressive agents [corticosteroids combined with azathioprine or cyclophosphamide (n = 7)], were an improvement of the clinical state, a reduction of the inflammatory activity, and a reversal of the glucose handling to normal.

Impaired glucose handling in active rheumatoid arthritis: relationship to the secretion of insulin and counter-regulatory hormones.

An intravenous glucose tolerance test was performed in 45 untreated patients with active inflammatory rheumatoid arthritis and in age- and sex-matched healthy subjects. The mean k value in the patients, which correlated to the inflammatory activity, was 1.0 +/- 0.05 (SEM), which was significantly lower (P less than .001) than in the controls (1.8 +/- 0.09). The basal serum insulin concentration and the maximum insulin response to glucose loading were significantly higher (P less than .001 and P less than .01, respectively) in the patient group. The patients had a normal basal concentration of growth hormone in the serum, but during glucose infusion the concentration increased. The plasma glucagon level was significantly lower than in the controls (P less than .001). The urinary output of cortisol and catecholamines was normal. It is concluded that impaired glucose handling in active chronic inflammatory disease cannot be explained as a stress reaction but may be due to peripheral insulin resistance mediated by the inflammatory process. A paradoxical increase in growth hormone secretion during glucose infusion may suggest that this hormone is one factor that influences glucose handling in chronic inflammation. The pathophysiologic relevance of altered glucose metabolism and enhanced insulin secretion is uncertain but may reflect a possible link with the proposedly increased risk of atherosclerotic cardiovascular disease in rheumatoid arthritis.

Reduced zinc in peripheral blood cells from patients with inflammatory connective tissue diseases.

By the use of the nuclear microprobe technique, the concentrations of zinc in isolated erythrocytes, platelets, and granulocytes were measured in patients with rheumatoid arthritis, other inflammatory arthritides, and scleroderma. Markedly reduced cellular zinc values were found compared to those measured in healthy subjects. No relation was found to inflammatory activity or disease duration. Plasma zinc was reduced in the majority of the patients and was negatively correlated to the inflammatory activity estimated by ESR and serum orosomucoid. No relation was found between total zinc values in plasma or cells or disease duration. Corticosteroid therapy was instituted in a number of the patients with inflammatory arthritides and induced a significant elevation of total zinc in all cell types, although normalization was not achieved. Plasma zinc values remained unchanged during the treatment.

Tubular proteinuria during treatment with cyclosporin A--a case report.

A patient with relapsing polychondritis developed a progressive destruction of tracheal cartilage despite treatment with immunosuppressive and cytotoxic agents. Cyclosporin A therapy was instituted and has been continued for more than two years, concomitant with a steady improvement and remission of the disease. During the treatment period an increased urinary excretion of beta 2-microglobulin was measured, indicating renal tubular damage. The tubular proteinuria preceded an elevation of serum creatinine and a drop in creatinine-clearance. Thus, beta 2-microglobulin might be a sensitive indicator of nephrotoxicity and of value for the evaluation of the long-term side effects of Cyclosporin A particularly in patients with extrarenal disease.

Physiological effects of housing density on C57BL/6J mice over a 9-month period.

The NRC has consistently recommended floor space for animals used in science and agriculture. For mice, the recommended floor space is 77.4 cm(2) (12 in(2)) for a 15- to 25-g mouse. The NRC noted that its recommendations were based on "best professional judgment" and encouraged alternatives that were data driven. As part of a continual effort of The Jackson Laboratory to ensure the health and well-being of production and research mice, while promoting cost-effective, state-of-the-art research, several density-driven studies have been conducted by lab researchers. The objectives of this study were to determine the effect of housing density on variables related to mouse physiology and air quality in cages and assess the value of specific measured variables in such studies. In the present study, we monitored C57BL/6J mice in individually ventilated cages from weaning until 9 mo of age. Housing densities were equivalent to 66.4 or 36.8 cm(2) per mouse (10.3 or 5.7 in(2)). Clinical physiological variables representing general health and well-being were measured. Hematological traits, plasma lipids, and glucose, growth, bone mineral density, and percent body fat did not differ between housing densities. In the more densely housed mice, however, adrenal glands were significantly smaller, heart rates were significantly less, and food consumption was less. Cage air microenvironment was evaluated for ammonia, carbon dioxide, temperature, and humidity in cages changed weekly or every 2 wk. The cage microenvironment remained within acceptable limits at the higher density of mice at both cage-changing frequencies. The results suggest that mice housed for as long as 9 mo at up to twice the density currently recommended by NRC show no measurable adverse effects. Continued re-evaluation of the recommendation by measuring additional relevant variables of health and general well-being, and studying additional strains of mice is warranted.

Abnormal calcium and magnesium stores in erythrocytes and granulocytes from patients with inflammatory connective tissue diseases. Relationship to inflammatory activity and effect of corticosteroid therapy.

The mass fraction of Ca and Mg in isolated erythrocytes and granulocytes was measured using the nuclear microprobe technique. Conspicuous abnormalities were observed in cells from patients with rheumatoid arthritis and other inflammatory arthritides. Compared with the normal cellular content, total Ca was increased an average of 3 times in erythrocytes and 5 times in granulocytes. Total granulocyte Mg was increased about 3 times, whereas erythrocyte Mg was reduced to as much as 60% of normal. These abnormalities were less prominent or were absent in scleroderma patients, except for levels of granulocyte Ca, which were increased more than 3 times beyond normal in this patient group. A significant positive correlation was found between serum haptoglobin and erythrocyte or granulocyte Ca content among these patients, but not between haptoglobin and erythrocyte or granulocyte Mg values. During corticosteroid treatment, a significant increase in erythrocyte Mg and a significant reduction in erythrocyte Ca were noted, but normalization of these levels was not achieved. Granulocyte Ca was also significantly reduced, while granulocyte Mg remained unaltered. Serum levels of Ca and Mg were within normal ranges and were not influenced by corticosteroid therapy. The results indicate that at least Ca abnormalities in erythrocytes and granulocytes are associated with the intensity of the inflammatory process and that the amounts of Ca and Mg in these cells are influenced by potent antiinflammatory therapy.

Eosinophil involvement in rheumatoid arthritis as reflected by elevated serum levels of eosinophil cationic protein.

Circulating levels of eosinophil cationic protein (ECP), an eosinophil specific granule protein, and numbers of peripheral eosinophils were determined in 42 patients with rheumatoid arthritis. At the time of the investigation the patients were without drug treatment. They had normal blood counts of eosinophils but on average a five-fold increase of the serum ECP values compared with healthy subjects. The intracellular content of ECP in eosinophils isolated from 14 patients was normal. High serum levels of ECP were particularly observed in patients with a disease of rather short duration but with a more aggressive course. Other factors associated with high ECP values were blood eosinophil counts in the upper normal range, high rheumatoid factor titre and increased inflammatory activity as defined by elevated serum haptoglobin and blood platelet counts. No relation was found between serum ECP and circulating immune complexes or serum total IgE. Synovial fluids obtained from 14 patients with rheumatoid arthritis contained very high concentration of ECP; on average nine times higher than those in the circulation of the patients. During corticosteroid but not NSAID therapy serum ECP decreased on average about 50% compared with pre-treatment values. Although eosinophils are not a notable feature of the synovial membrane infiltrate or cellular joint exudate, data obtained indirectly indicates their participation in the inflammatory reaction in RA.

Elevated granulocyte manganese in rheumatoid arthritis and other connective tissue diseases.

Higher than normal amounts (p less than 0.001) of manganese were found in granulocytes isolated from patients with rheumatoid arthritis, other inflammatory arthritides, and scleroderma, while erythrocytes and platelets contained normal amounts of manganese. The cause of manganese accumulation is not known but evidently is linked to the intensity of the inflammatory process since significant positive correlations (p less than 0.001) were seen between erythrocyte sedimentation rate or serum haptoglobin and granulocyte manganese. During corticosteroid therapy granulocyte manganese started to return to normal levels but the relative changes were modest. Strong positive correlations (p less than 0.001) were found between granulocyte manganese and granulocyte stores of calcium and iron, suggesting a close relationship in the cellular regulation of these elements in chronic inflammatory conditions.