Cholesterol alterations in young dystrophic mice

Cholesterol and cholesteryl ester concentrations and cholesteryl ester fatty acid substituents have been measured during the first 10 weeks of life in tissues of normal and dystrophic mice. In normal Swiss and 129ReJ(+/?) mice the concentrations of both cholesterol and cholesteryl esters remain essentially constant in liver, increase in brain and fall sharply in both thigh (mixed fiber type muscles) and chest muscles (predominantly slow oxidative muscles) over this period. In all cases the concentration of free cholesterol exceeds that of esterified cholesterol. In dystrophic mice, similar patterns are found in brain and liver. In both thigh and chest muscles, however, the developmental pattern is significantly different. After an initial decrease the concentrations of cholesterol and cholesteryl esters increase rapidly with the largest increase occurring in the concentration of cholesteryl esters which by 10 weeks of age exceeds the concentration of cholesterol in chest muscle. During the same period the pattern of esterified fatty acids changes gradually in dystrophic tissues towards an increasing ratio of unsaturated/saturated fatty acids. By 10 weeks of age this ratio is significantly higher in dystrophic tissues than normal in all tissues tested.     

Effect of calcium ionophore A23187 on pregnenolone metabolism to progesterone in rat granulosa cells

The effect of calcium ionophore A23187 on the metabolism of pregnenolone to progesterone was examined in rat granulosa cells during a 24-h culture period. Granulosa cells harvested from pregnant mare's serum gonadotropin treated immature rats were incubated in the presence and absence of the divalent cation ionophore A23187. The ionophore induced progesterone synthesis from both endogenous sterol substrate and exogenous pregnenolone in a time- and concentration-dependent manner. Pregnenolone metabolism was examined in the presence of aminoglutethimide phosphate, an inhibitor of endogenous pregnenolone production. Steroid secretion resulting from metabolism of endogenous substrate was more sensitive to A23187 in that a lower concentration of the ionophore was required to induce a significant increase than that noted for exogenous pregnenolone metabolism. In addition, progesterone production from endogenous sterol occurred 6 h earlier than the observed increase in the conversion of pregnenolone to progesterone. These results indicate that A23187 and therefore possibly enhanced calcium influx may play a significant role in the regulation of pregnenolone metabolism in granulosa cells depending on the duration of incubation. The earlier steroidogenic response from endogenous substrate may be a reflection of an acute effect of A23187 on certain steroidogenic steps proximal to pregnenolone production.     

Differential production of steroids by dispersed granulosa and theca interna cells from developing preovulatory follicles of pigs

Dispersed granulosa and theca interna cells were recovered from follicles of prepubertal gilts at 36, 72 and 108 h after treatment with 750 i.u. PMSG, followed 72 h later with 500 i.u. hCG to stimulate follicular growth and ovulation. In the absence of aromatizable substrate, theca interna cells produced substantially more oestrogen than did granulosa cells. Oestrogen production was increased markedly in the presence of androstenedione and testosterone in granulosa cells but only to a limited extent in theca interna cells. The ability of both cellular compartments to produce oestrogen increased up to 72 h with androstenedione being the preferred substrate. Oestrogen production by the two cell types incubated together was greater than the sum produced when incubated alone. Theca interna cells were the principal source of androgen, predominantly androstenedione. Thecal androgen production increased with follicular development and was enhanced by addition of pregnenolone or by LH 36 and 72 h after PMSG treatment. The ability of granulosa and thecal cells to produce progesterone increased with follicular development and addition of pregnenolone. After exposure of developing follicles to hCG in vivo, both cell types lost their ability to produce oestrogen. Thecal cells continued to produce androgen and progesterone but no longer responded to LH in vitro. These studies indicate that several functional changes in the steroidogenic abilities of the granulosa and theca interna compartments occur during follicular maturation.     

Techniques for hand-rearing tree-shrews (Tupaia belangeri) from birth

In captivity, Tupaia belangeri (Thailand tree-shrew) frequently show aberrant patterns of maternal care which result in the death of offspring. In order to maximize the potential of the tree-shrew as an animal resource for experimental studies we have developed a program for hand-rearing tupaiidae from birth. Newborn tree-shrews were removed from mothers with a history of poor parental care to a nursery maintained under conditions of controlled relative humidity (70 ± 10%) and temperature (25 ± 1°C) on a 12 h dark: 12 h light cycle. The young tree-shrews were fed on a liquid formula until the eyes opened (Day 18–23) and for the subsequent 10 days on a transitional diet until they could feed themselves on solid food. Our hand-rearing protocol appears to conform to the natural weaning pattern of tupaiids. Food consumption during the liquid diet phase was linearly related to age and the increase in body weight in both sexes. The initial growth rate of male and female hand-reared tree-shrews was slower than that of maternally reared animals during the liquid diet phase. The growth rate accelerated subsequently and the body weight of hand-reared tree-shrews eventually reached that of maternally reared animals of the same sex. Various developmental changes occurred during the same period in artificially and naturally reared animals. In both hand-reared and maternally reared groups, the growth rate of male tree-shrews exceeded that of females from about Day 40 onward. The accelerated growth rate of male tree-shrews was in apparent association with an increase in androgen secretion at the onset of puberty. The high fecundity of tree-shrews in captivity reinforced with a program for hand-rearing the young make T belangeri a potential alternative to more conventional laboratory species in specific areas of biomedical research.     

Prostaglandin production by dispersed granulosa and theca interna cells from porcine preovulatory follicles

Prepubertal gilts were treated with 750 IU pregnant mares' serum gonadotropin (PMSG) and 72 h later with 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. Dispersed granulosa (GC) and theca interna (TIC) cells were prepared by microdissection and enzymatic digestion from follicles obtained 36, 72 and 108 h after PMSG treatment and incubated for up to 6 h in a chemically defined medium in the presence or absence of arachidonic acid, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and indomethacin. Production of prostaglandin E2 (PGE) and prostaglandin F2 alpha (PGF) was measured by radioimmunoassay. Both GC and TIC had the capacity to produce prostaglandins, with production by each cell type increasing markedly with follicular maturation. PGE was the major prostaglandin produced by both cellular compartments. Only PGE production by GC was consistently enhanced by addition of arachidonic acid to the incubation medium. Neither cell type was responsive to FSH and LH in vitro. Indomethacin inhibited the production of PGE and PGF by both cell types. These results provide convincing evidence for an intrafollicular source of prostaglandins and indicate that both cellular compartments contribute significantly to the increased production of prostaglandins associated with follicular rupture.     

Schistosoma mansoni adult microsomal antigens, a serologic reagent. I. Systematic fractionation, quantitation, and characterization of antigenic components

A systematic and quantitative search was conducted to identify and isolate a serologically pertinent antigen with high specific activity and low cross-reactivity from adult worms of Schistosoma mansoni. Adult worms of S. mansoni quickly thawed from liquid N2 temperatures were disrupted by controlled homogenization in isotonic buffered sucrose. Differential centrifugation of the homogenate yielded three particulate and one soluble fractions: the 480 x G pellet (nuclear), the 7650 x G pellet (mitochondrial), the 360,000 x G pellet (microsomal), and the 360,000 x G supernatant (cytosol). Quantitative analysis indicated a major concentration of specific antigenic activities in the microsomal fraction. Further purifications of the urea-solubilized, n-butanol-treated microsomal particles by gel filtration and ionic-exchange chromatography resulted in a microsomal antigen (MAMA) possessing high specific activity and low cross-reactivity. The final purification post-ionic exchange chromatograph showed a 30-fold increase of specific antigen activity over that of the cytosol fraction. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunotransfer blot by the "Western blot" technique (EITB) indicated specific antigenic activities in association with several different m.w. bands (heterogeneous m.w. by extrapolation = 4.3 to 11.9 x 10(6), 2.0 x 10(5), and 2.0 x 10(4) daltons) of the MAMA fraction. When compared with other reported serologic antigens, MAMA showed substantially higher specific activity and lower cross-reactivity.     

Genomic organization of mouse Dishevelled genes

We report the isolation and characterization of two new genomic loci corresponding to the mouse Dishevelled (Dvl) genes Dvl2 and Dvl3. The Dvl genes are homologs of the Drosophila dsh segment polarity gene, and are involved in the Wnt/wingless signal transduction pathway. Dvl2 and Dvl3 genomic clones were isolated from a mouse 129 strain λFIXII genomic library and have identical exon/intron organization to Dvll. All three Dishevelled genes span 15 exons and 14 introns and have a number of conserved splice junction sites.     

WNT-mediated relocalization of dishevelled proteins

Summary TheWnt family of proto-oncogenes encodes secreted signaling proteins that are required for mouse development. TheDrosophila Wnt homolog, thewingless (Wg) segment polarity gene, mediates a signal transduction pathway in which the downstream elements appear to be conserved through evolution. One such element, thedishevelled gene product, becomes hyperphosphorylated and translocates to the plasma membrane in response to Wg (Yanagawa et al., 1995). We report here that the mouseDishevelle-1 (Dvl-1) andDishevelled-2 genes encode proteins that are differentially localized inWnt-overexpressing PC12 cell lines (PC12/Wnt). WhereasDvl-1 andDvl-2 proteins are limited to the soluble fraction of parental PC12 cells, PC12/Wnt cells display a subset ofDvl-1 protein associated with the membrane andDvl-2 protein with the cytoskeletal fraction. These results suggest a conserved role forDvl inWnt/wg signal transduction.     

Ventilation inhomogeneity in oleic acid-induced pulmonary edema

Tsang, John Y. C., Michael J. Emery, and Michael P. Hlastala. Ventilation inhomogeneity in oleic acid-inducedpulmonary edema. J. Appl. Physiol.82(4): 1040-1045, 1997.Oleic acid causes permeability pulmonaryedema in the lung, resulting in impairment of gas-exchange andventilation-perfusion heterogeneity and mismatch. Previous studies haveshown that by using the multiple-breath helium washout (MBHW)technique, ventilation inhomogeneity (VI) can be quantitativelypartitioned into two components, i.e., convective-dependent inhomogeneity (cdi) and diffusive-convective-dependent inhomogeneity (dcdi). Changes in VI, as represented by the normalized slope of thephase III alveolar plateau, were studied for 120 min in fiveanesthetized mongrel dogs that were ventilated under paralysis by aconstant-flow linear motor ventilator. These animals received oleicacid (0.1 mg/kg) infusion into the right atrium att = 0. MBHWs were done induplicate for 18 breaths every 40 min afterward. Three other dogs thatreceived only normal saline served as controls. The data show that,after oleic acid infusion, dcdi, which represents VI in peripheralairways, is responsible for the increasing total VI as lung wateraccumulates progressively over time. The cdi, which represents VIbetween larger conductive airways, remains relatively constantthroughout. This observation can be explained by increases in theheterogeneity of tissue compliance in the periphery, distal airwayclosure, or by decreases in ventilation through collateral channels.