Regulation of bile acid synthesis via direct effects on the microsomal membrane

Rats treated with ethinylestradiol (5 mg kg-1 day-1 for 5 days) secrete de novo synthesized bile acids at a markedly reduced rate (-57%). Administration of the nonionic detergent Triton WR-1339 to estradiol-treated rats rapidly restored the rate of secretion of de novo synthesized bile acids to control levels. In contrast, when Triton was administered to control rats, the secretion rate of bile acids was unaffected. The reduction in bile acid synthesis displayed by estradiol-treated rats was similar to the 50% decrease in the activity of hepatic microsomal 7 alpha-hydroxylase. The activity of 7 alpha-hydroxylase was also restored to control levels by the administration of Triton to estradiol-treated rats. We examined the possibility that estradiol acts directly on the hepatic microsomes. Adding increasing amounts of estradiol to microsomes obtained from control rats resulted in decreasing activities of 7 alpha-hydroxylase. The inhibition by estradiol of 7 alpha-hydroxylase obtained in vitro occurred with amounts of estradiol that were found to accumulate in the liver via in vivo treatment. Double-reciprocal analysis showed that at and below 50 micrograms of estradiol/0.5 mg of protein uncompetitive inhibition was displayed. Additional experiments showed that adding Triton to microsomes obtained from estradiol-treated rats increased the activity of 7 alpha-hydroxylase to control levels. In contrast, Triton did not increase the activity of 7 alpha-hydroxylase when it was added to control microsomes. These data show for the first time that the estrogenic steroid estradiol acts directly on the microsomes and inhibits both the activity of 7 alpha-hydroxylase and the rate of bile acid synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue water relations of four co-occurring chaparral shrubs

Summary Chaparral shrubs of California have a suite of morphological and physiological adaptations to withstand the prolonged summer droughts of a mediterranean climate. Not all species of chaparral have the same rooting depth and there is some evidence that those with shallow roots have tissue that is most tolerant to water stress. We tested this notion by comparing the tissue water relations of four co-occurring chaparral shrubs: Quercus durata, Heteromeles arbutifolia, Adenostoma fasciculatum, and Rhamnus californica. We used a pressure-volume technique and a dew-point hygrometer to metsure seasonal changes in osmotic potential when plant tissue was fully hydrated and osmotic potential at predawn, midday, and the turgor loss point. We also calculated seasonal changes in the minimum daily turgor potential, saturated weight/dry weight ratio of leaf tissue, and the bulk modulus of elasticity. We had information on the seasonal water use patterns and apparent rooting depths of these same four shrubs from a previous study (Davis and Mooney 1986). All evidence indicated that Rhamnus had shallow roots and Quercus deep roots. Our results indicated that the tissue water relations of our four co-occurring chaparral shrubs were not alike. Even though Rhamnus had shallow roots, it had the least xerophytic tissue. Seasonal osmotic potential and saturated weight/dry weight ratios were relatively high and bulk modulus of elasticity and minimum daily turgor potentials were low. Furthermore, even though Quercus had deep roots and experienced no seasonal water stress at our study site, its tissue water relations indicated relatively high tolerance to water stress. We conclude that seasonal drought tolerance of stem and leaf tissue of co-occurring chaparral shrubs does not necessarily correspond to rooting depth, to soil moisture resources available to the shrub, or to the degree of seasonal water stress experienced by the shrub.     

Purification of nuclear cAMP-independent protein kinases from rat ventral prostate

Two nuclear cAMP-independent protein kinases (designated PK-N1 and PK-N2) were purified from rat ventral-prostate and liver. The yield of enzyme units was 4-5% and 7-9% for each enzyme from the prostatic nuclei and liver nuclei, respectively. The average fold purification for prostatic nuclear protein kinase N1 and N2 was 1360 and 1833, respectively. The respective average specific activity of the two enzymes towards casein was 81,585 and 110,000 nmol 32P incorporated/hr/mg of enzyme. Protein kinase N1 comprised one polypeptide of Mr 35,000 which underwent phosphorylation in the presence of Mg2+ + ATP. Protein kinase N2 comprised two polypeptides Mr 40,000 and 30,000 of which only the Mr 30,000 polypeptide was autophosphorylated. Both enzymes were active towards casein, phosvitin, dephosphophosvitin, spermine-binding protein, and non-histone proteins in vitro. Little activity was detected towards histones. Both enzymes were stimulated by 150-200 mM NaCl. MgCl2 requirement varied with the protein substrate but was between 2-4 mM for both enzymes. With dephosphophosvitin as substrate, the apparent Km for ATP for N1 protein kinase was 0.01 mM. GTP did not replace ATP in this reaction. Protein kinase N2 was active in the presence of ATP or GTP. The apparent Km was 0.01 mM for ATP, but 0.1 mM for GTP.     

N-acetyl-L-glutamate synthase of Neurospora crassa. Characteristics, localization, regulation, and genetic control

N-Acetylglutamate synthase, an early enzyme of the arginine pathway, provides acetylglutamate for ornithine synthesis in the so-called "acetylglutamate cycle." Because acetylglutamate is regenerated as ornithine is formed, the enzyme has only a catalytic or anaplerotic role in the pathway, maintaining "bound" acetyl groups during growth. We have detected this enzyme in crude extracts of Neurospora crassa and have localized it to the mitochondria along with other ornithine biosynthetic enzymes. The enzyme is bound to the mitochondrial membrane. The enzyme has a pH optimum of 9.0 and Km values for glutamate and CoASAc of 6.3 and 1.6 mM, respectively. It is feedback-inhibited by L-arginine (I0.5 = 0.16 mM), and its specific activity is augmented 2-3-fold by arginine starvation of the mycelium. Mutants of the newly recognized arg-14 locus lack activity for the enzyme. Because these mutants are complete auxotrophs, we conclude that N-acetylglutamate synthase is an indispensible enzyme of arginine biosynthesis in N. crassa. This work completes the assignment of enzymes of the arginine pathway of N. crassa to corresponding genetic loci. The membrane localization of the enzyme suggests a novel mechanism by which feedback inhibition might occur across a semipermeable membrane.     

Cytochalasin D and cationized ferritin as probes for the morphological investigation of blebbing in two human cell lines

Summary The potent fungal metabolite cytochalasin D (CD) and cationized ferritin (CF) are used in combination to test for negative charge distribution on blebs (knobs). Two established human epithelial cell lines, WISH and HeLa, that display blebs in various phases of the cell cycle or under certain culture conditions (37,46) are investigated. CD alone, applied at a low concentration (1.0 μg/ml) and for a short time period (3 min), causes blebs to appear as the prevalent surface feature. These are filled mainly with free ribosomes. Additionally, feltlike mats, presumed to be disorganized, compacted microfilaments, are formed directly beneath the cell membrane. These are especially evident in the cortical cytoplasm below the blebs or bleb clusters. CF (0.345 mg/ml), applied for a 5-min period after CD administration (1.0 μg/ml) for 3 min, appears along the surface of microvilli, at the base of blebs, and in vesicles beneath the bleb clusters. In some cases, microfilaments (6 nm in diameter) are closely related to the vesicles. CF does not preferentially bind to the apical cell membrane of blebs. Above areas of the subplasmalemmal microfilaments, CF membrane binding is apparent, even under circumstances where the filaments are disorganized by cytochalasin treatment. These results seem to show the following: (a) bleb membranes are different from the remainder of the cell and do exhibit a loss of negative charge and (b) surface charge may be dependent on the presence or structural integrity of membrane-related 6-nm microfilaments. The support of this research by a grant from the Baylor College of Dentistry and The Oklahoma College of Osteopathic Medicine and Surgery is gratefully acknowledged. The assistance of Dr. J. H. Martin, Department of Pathology, Baylor University Medical Center, is also greatly appreciated.     

Chain Arrangements in the Gel State and the Transition Temperatures of Phosphatidylcholines

Measures of chain length, inequivalence of chain length, and chain position have been incorporated into a parameter, D, which we call the “relative chain inequivalence.” D has been calculated for a number of saturated phosphatidylcholines (PC) containing one type of chain (homoacid PC), saturated PC containing two different acyl chains (heteroacid PC), and heteroacid PC containing one saturated and one unsaturated chain. The gel to liquid-crystalline transition temperatures and D are related in a regular pattern, which suggests similarity of chain packing in the gel. This pattern may have useful predictive value.     

Analysis of a diffusion-limited hollow fiber reactor for the measurement of effective substrate diffusivities

Mathematical analyses of a diffusion-limited hollow fiber reactor for the measurement of effective substrate diffusivities are presented. An analytical solution to the mathematical model with a first order substrate consumption rate is used to show that the procedure of Webster and Shuler(1) is incorrect. A rigorous analysis that requires numerical solution is also outlined for any form of the substrate consumption rate. These analyses allow for more accurate estimations of effective substrate diffusivities since they should be used in conjunction with integral reactor behavior.     

Microwave-field-driven acoustic modes in DNA.

The direct coupling of a microwave field to selected DNA molecules is demonstrated using standard dielectrometry. The absorption is resonant with a typical lifetime of 300 ps. Such a long lifetime is unexpected for DNA in aqueous solution at room temperature. Resonant absorption at fundamental and harmonic frequencies for both supercoiled circular and linear DNA agrees with an acoustic mode model. Our associated acoustic velocities for linear DNA are very close to the acoustic velocity of the longitudinal acoustic mode independently observed on DNA fibers using Brillouin spectroscopy. The difference in acoustic velocities for supercoiled circular and linear DNA is discussed in terms of solvent shielding of the nonbonded potentials in DNA.     

Histone messenger RNA synthesis and accumulation during early development in the echiuroid worm, Urechis caupo

RNA isolated from Urechis caupo mature oocytes and embryos was analyzed for the presence of histone messenger RNAs (mRNAs) by in vitro translation and by filter blot hybridization to determine the contribution of maternal and newly transcribed histone mRNAs to the pattern of histone synthesis during early development. Histone mRNAs were not detected in mature oocyte RNA which suggests that relatively few if any maternal histone mRNAs are sequestered in the mature oocytes. Histone mRNAs were detected in cleavage-stage RNA and increased in amount from midcleavage through late gastrula stages. The in vitro translation analysis also demonstrated that the amount of H1 histone mRNA in late cleavage- and early blastula-stage embryos exceeds that of the individual core histone mRNAs. The disproportionate accumulation of individual histone mRNAs correlates with the noncoordinate synthesis of H1 and core histones which occurs during early embryogenesis.