Human esterase D gene: complete cDNA sequence,genomic structure,and application in the genetic diagnosis of human retinoblastoma

Summary The gene encoding human esterase D (EsD), a member of the nonspecific esterase family, is a useful genetic marker for retinoblastoma (RB) and Wilson's disease. Previously we identified a cDNA clone from this gene and determined its chromosomal location. In this report, we present the complete cDNA sequence of the human EsD gene. A long open reading frame encoded a predicted protein of 282 amino acids with molecular weight of 30 kD. A computer-assisted search of a protein sequence data base revealed homology with two other esterases, acetylcholinesterase of Torpedo and esterase-6 of Drosophila. Homologous region were centered around presumptive active sites, suggesting that the catalytic domains of the esterases are conserved during evolution. Three genomic clones of this gene were also isolated and characterized by restriction mapping. At least ten exons were distributed over a 35-kb (kilobase pair) region; each exon contained an average of 100 basepairs (bp). A polymorphic site for Apa I, located within an intron of the esterase D gene, can be used to identify chromosome 13 carrying defective RB alleles within retinoblastoma families.     

Studies on the submicrosomal fractions of bovine oligodendroglia: Lipid composition and glycolipid biosynthesis

Oligodendroglia were isolated from bovine brain, and a crude, microsomal fraction obtained from cell homogenates was subfractionated into myelin (MP), plasma membranes (PM), Golgi (GF), smooth (SER) and rough (RER) endoplasmic membranes using discontinuous-sucrose gradient centrifugation. The submicrosomal fractions were characterized by ultrastructural examination and analysis of the specific organelle markers. The myelin and plasma membrane rich fractions contained characteristically the highest amounts of the lipid with lower mole percentages of total phospholipids and phosphatidylcholine, and higher concentrations of phosphatidylethanolamine (+plasmalogens), cholesterol and galactolipids. Considerable amounts of the typical myelin galactolipids (galacto-cerebrosides, sulfatides and monogalactosyl diglycerides) were also found in the Golgi fraction (GF). The GF fraction had the greatest enrichment of glycolipid-forming galactosyltransferases, and the distribution of these enzymes correlated well with that of the Golgi marker enzymes. The results give evidence that intracellular Golgi apparatus of oligodendroglia is rich in the myelin-specific lipids, and suggest its involvement in the synthesis and processing of myelin lipids.     

On the validity of the steady state assumption of enzyme kinetics

By estimating relevant time scales, a simple new condition can be found that ensures the validity of the steady state assumption for a standard enzyme-substrate reaction. The generality of the approach is demonstrated by applying it to the determination of validity criteria for the steady state assumption applied to an enzyme-substrate-inhibitor system.     

Magnetic properties of tunicate blood cells. I. Ascidia nigra

The magnetic properties of intact and freeze-dried blood cells of the tunicate Ascidia nigra and of model vanadium(III) and (IV) compounds as polycrystalline solids and in aqueous solution have been measured up to 50 kOe with a SQUID susceptometer. Corrections for the samples' diamagnetism were extracted from the temperature dependence of the data without any further assumptions. For vanadium(IV), measured values of the magnetic moment at different values of the applied magnetic field over the temperature range 2-100 K obey a Brillouin function with spin 1/2. For vanadium(III), the magnetic moment data did not obey a Brillouin function and were analyzed in terms of a spin Hamiltonian with S = 1. Measurements on both whole and freeze-dried blood samples give consistent results with vanadium(III) the predominant species. These results are discussed in terms of the mechanisms of vanadium accumulation and the use of vanadium oxidation states as criteria of ascidian taxonomy.     

Effect of dietary protein on cholesterol homeostasis in diabetic rats

Normal and streptozotocin (STZ)-diabetic rats were studied in order to examine the effects of altering the type of dietary protein on cholesterol homeostasis. Rats were fed a non-purified or a purified diet containing either casein or soybean protein. The results obtained on the specific aspects of lipid metabolism were remarkably similar in control rats fed the non-purified (Purina Lab Chow) diet or the purified diet with the soybean protein. However, most of the findings obtained with the above two groups were different from those obtained with rats fed the purified diet containing casein. In the latter group, plasma cholesterol was elevated following a 15-day feeding period as compared to the other two dietary groups. The excess plasma cholesterol in the casein-fed group was found in two lipoprotein fractions with densities of 1.023-1.045 g/ml and 1.045-1.086 g/ml, respectively. The latter lipoprotein fraction was also enriched with apolipoprotein E. The casein-fed animals also showed a lower fractional rate of plasma cholesterol esterification and an abnormal accumulation of cholesterol in the body despite inhibition of cholesterol synthesis in the liver and in the intestines. Twelve to 15 days after the induction of diabetes, plasma cholesterol increased to a similar extent in the rats on all three diets. However, the distribution of cholesterol among the lipoprotein fractions was markedly different. The percentage of cholesterol in fractions of d less than 1.086 g/ml was increased while that carried in the fraction of d 1.086-1.161 g/ml decreased in the rats fed the nonpurified diet and the casein diet. In contrast, there was no change in the distribution of lipoprotein cholesterol between the diabetic and the control rats fed the soybean protein diet. The hepatic synthesis of cholesterol was unaltered in diabetic rats fed the nonpurified diet and the purified diet with soybean protein, but was increased 2.4-fold in diabetic rats fed casein. Intestinal cholesterol synthesis was increased in all three dietary groups. The increase was highest in the rats fed casein and lowest in rats fed soybean protein. The rate of sterol synthesis in the kidneys was not significantly affected by the diet or diabetes. In all three dietary groups diabetes led to an abnormal accumulation of cholesterol in the body. This accumulation was highest in the casein-fed rats and lowest in those fed the soybean protein diet. The cholesterol content of the kidneys was markedly increased by dietary casein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differences in the gastrointestinal microbiota of specific pathogen free mice: an often unknown variable in biomedical research

Large differences were found in the numbers of facultatively anaerobic Gram-negative bacteria in the gastrointestinal tract of mice from 3 major specific pathogen free (SPF) units in Australia. The species isolated also differed between mouse colonies. In one unit the presence of Enterobacter cloacae was found to dramatically influence the survival of mice following total body irradiation. This finding conforms with previous studies which have shown the influence of variation in gastrointestinal microbiota on the immune system and on susceptibility to infection. Given that the presence or absence of Enterobacteriaceae in the intestines of mice under investigation may influence experimental results, researchers using SPF rodents are encouraged to determine the baseline loading of these bacteria in their animals. Where results of immunological or irradiation studies from different colonies are likely to be compared, the enterobacterial status of the colony being used should be reported.     

Mutagenic activities of selected nitrofluoranthene derivatives in Salmonella typhimurium strains TA98, TA98NR and TA98/1,8-DNP6

The mutagenic activities of novel nitrofluoranthene derivatives in Salmonella strains TA98, TA98NR and TA98/1,8-DNP6 (with and without S9 addition) are given. These derivatives were produced from the reactions of fluoranthene (FL) and its directly mutagenic 2- and 3-nitro derivatives with covalent dinitrogen pentoxide (N2O5) in CCl4 solution at ambient temperature. The influence of the addition of a nitro group on the observed activity of the resulting di- and tri-nitrofluoranthenes is discussed.     

Estradiol enhances binding to cultured human keratinocytes of antibodies specific for SS-A/Ro and SS-B/La. Another possible mechanism for estradiol influence of lupus erythematosus

A strong association between anti-SS-A/Ro and anti-SS-B/La antibodies and skin lesions has been well documented in subacute cutaneous lupus erythematosus and neonatal lupus erythematosis in which 70 to 80% of patients are female. In order to better understand the mechanisms of the influence of sex hormones on cutaneous lupus, we designed immunopathological in vitro experiments to evaluate the effects of estradiol and other sex steroids on the binding of SS-A/Ro- and SS-B/La-specific antibodies to cultured human keratinocytes from neonates. Cultured human keratinocytes incubated with antisera specific for SS-A/Ro or SS-B/La Ag were fixed with either acetone or paraformaldehyde and then analyzed in indirect immunofluorescent assays or by FACS analysis to detect cell surface IgG binding as an indirect measure of SS-A/Ro and SS-B/La Ag expression on the cell surface of keratinocytes. Estradiol (10(-5) to 10(-7) M) augmented binding of antiserum probes on the surface of cultured keratinocytes, with 10(-7) M estradiol showing the highest induction of cell surface binding of antisera specific for SS-A/Ro plus SS-B/La Ag (24.5% of cells were positive). In contrast, dihydrotestosterone, testosterone, and progesterone showed no augmentation. The augmentation by estradiol was partially inhibited by the antiestrogen nafoxidine. Estradiol augmented the relative incidence and absolute number of small or cuboidal cells binding antibodies specific for SS-A/Ro and SS-B/La Ag, whereas the number and incidence of larger differentiated cells binding anti-SS-A/Ro and anti-SS-B/La decreased significantly in cell cultures stimulated with estradiol. Flow cytometric analysis utilizing monospecific anti-SS-A/Ro or anti-SS-B/La sera showed that estradiol induced binding of anti-SS-A/Ro in 13.1% of cultured keratinocytes, of anti-SS-A/La in 14.4%, and of sera specific for both Ag in 21.4%. This direct association between estradiol and the augmentation of binding to the cell surface of human keratinocytes of IgG from antisera specific for SS-A/Ro and SS-B/La Ag may be a trigger factor of immunologic damage in lupus and may be important in the different sex rates observed in skin manifestation of subacute cutaneous and neonatal lupus erythematosis.     

Anion transport during maturation of erythroblastic cells

Bromide uptake was measured in single maturing erythroblastic cells of rabbits by means of X-ray microanalysis. Increase in bromide uptake as the cells matured was observed. The order of cells from low to high bromide uptake was: early erythroblast less than late erythroblast less than marrow red cells less than peripheral red blood cells. The transition from low to high bromide uptake is correlated to the accumulation of iron which begins in the late erythroblast. A decrease in rubidium uptake also occurs as iron accumulates in the cell. These results indicate that the anion and cation transport changes during maturation are parallel in time course but opposite in direction. In addition, the increase in bromide uptake can be accounted for by the increase in surface-to-volume ratios of the cells. Surface-to-volume ratios were estimated by morphometric techniques.     

Synergism of cortisol and thyrotropin-releasing hormone in lung maturation in fetal sheep

The effects of fetal infusions of cortisol and thyrotropin-releasing hormone (TRH) singly and together on pressure-volume relationships and saturated phosphatidylcholine (SPC) concentrations in the lungs were studied in 28 fetal sheep delivered at 128 days of gestation. Four groups each of 7 fetuses were infused with either saline (for 156 h), TRH (25 micrograms/h in 60-s pulses for 156 h), TRH (for 156 h) combined with cortisol (1 mg/h for 84 h), or cortisol (for 84 h). Cortisol had no effect on SPC concentrations, whereas both TRH and cortisol plus TRH increased the concentration of SPC in lavage fluid but not lung tissue. Neither cortisol nor TRH significantly affected lung distensibility [V40; 0.64 +/- 0.04 and 0.57 +/- 0.10 (SE) ml/g, respectively, vs. 0.41 +/- 0.03 ml/g in controls] or stability (V5; 0.24 +/- 0.01 and 0.35 +/- 0.07 ml/g vs. 0.24 +/- 0.03 ml/g), whereas treatment with a combination of the two hormones was associated with a fourfold increase in V40 (1.70 +/- 0.16 ml/g) and V5 (1.03 +/- 0.15 ml/g). Since raised concentrations of cortisol, triiodothyronine, and estradiol-17 beta (treatment with cortisol) had no effect on V40 and V5, whereas similar hormonal changes associated with elevated prolactin levels (treatment with cortisol plus TRH) had marked effects, we conclude that prolactin plays an essential part in the synergism of cortisol and TRH.