Interleukin-1β Induces Prostaglandin G/H Synthase-2 (Cyclooxygenase-2) in Primary Murine Astrocyte Cultures

Abstract: Activation of glial cells and the consequent release of cytokines, proteins, and other intercellular signaling molecules is a well-recognized phenomenon in brain injury and neurodegenerative disease. We and others have previously described an inducible prostaglandin G/H synthase, known as PGHS-2 or cyclooxygenase-2, that is up-regulated in many cell systems by cytokines and growth factors and down-regulated by glucocorticoid hormones. In cultured mouse astrocytes we observed increased production of prostaglandin E₂ (PGE₂) after stimulation with either interleukin-1β (IL-1β) or the protein kinase C activator phorbol 12-myristate 13-acetate (TPA). This increase in PGE₂ content was blocked by pretreatment with dexamethasone and correlated with increases in cyclooxygenase activity measured at 4 h. Northern blots revealed concomitant increases in PGHS-2 mRNA levels that peaked at 2 h and were dependent on the dosage of IL-1β. Dexamethasone inhibited this induction of PGHS-2 mRNA by IL-1β. TPA, basic fibroblast growth factor, and the proinflammatory factors tumor necrosis factor α and lipopolysaccharide, but not interleukin-6, also stimulated PGHS-2 mRNA expression. Relative to IL-1β, the greater increases in PGE₂ production and cyclooxygenase activity caused by TPA correlated with a greater induction of PGHS-2 mRNA. Furthermore, NS-398, a specific inhibitor of cyclooxygenase-2, blocked >80% of the cyclooxygenase activity in TPA-treated astrocytes. These findings indicate that increased expression of PGHS-2 contributes to prostaglandin production in cultured astrocytes exposed to cytokines and other factors.     

Zn(II)-113Cd(II) and Zn(II)-Mg(II) hybrids of alkaline phosphatase. 31P and 113Cd NMR

Methods have been developed for the addition of different metal ion species to the three distinct pairs of metal sites (A, B, and C) found in the dimer of apoalkaline phosphatase. This allows the preparation of hybrid alkaline phosphatases in which A and B sites of each monomer contain two different species of metal ion or the A and B sites of one monomer contain the same species of metal ion, while the adjacent monomer contains a second species. The following hybrids have been characterized in detail: (Zn(II)ACd(II)B)2 alkaline phosphatase, (Zn(II)AMg(II)B)2 alkaline phosphatase, (Cd(II)AZn(II)B)2 alkaline phosphatase, and (Zn(II)AZn(II]B)(Cd(II)ACd(II)B) alkaline phosphatase. 31P and, where appropriate, 113Cd NMR have been used to monitor the behavior of the covalent (E-P) and noncovalent (E X P) phosphointermediates and of the A and B metal ions. From the pH dependencies of the E-P in equilibrium E X P in equilibrium E + Pi equilibria, it is clear that A site metal is the dominant influence in dephosphorylation of E-P and may have a coordinated water molecule, which ionizes to ZnOH- at a low pH providing the nucleophile for dephosphorylation. A site metal also serves to coordinate phosphate in the E X P complex. B site metal has a much smaller effect on dephosphorylation rates, although it does dramatically alter the Pi dissociation rate, which is the rate-limiting step for the native enzyme at alkaline pH, and is probably important in neutralizing the charge on the phosphoseryl residue, thus potentiating the nucleophilic attack of the OH- bound at A site. Phosphate dissociation is slowed markedly by replacement of B site zinc by cadmium. There is clear evidence for long range effects of subunit-subunit interactions, since metal ion and phosphate binding at one active center alters the environments of A and B site metal ions and phosphoserine at the other active site.     

Effects of repeated removal of large ovarian follicles and treatment with progestin on ovarian function in the ewe

Preovulatory follicles were removed from ewes during estrus to determine hormonal, ovarian and behavioral responses. In Experiment 1, new follicles were recruited and ovulated within 4 days, and a second estrous period was observed in most ewes. In Experiment 2, follicles were removed at Day 0 (estrus), Day 3.5 and Day 7.0 to determine responses to repeated follicular removal in the absence of a corpus luteum (CL). Ewes in two groups were given exogenous progestin at the time of first or second surgery. Each follicular removal was followed by a surge of follicle-stimulating hormone (FSH) and follicular growth, and in many cases, behavioral estrus and/or a surge of luteinizing hormone (LH) was detected around the time of the next follicular removal. Although not necessary for display of estrus, treatment with progestin during follicular maturation increased the number of ewes showing estrus. When the newly developing follicles were allowed to ovulate, resulting corpora lutea produced low levels of progesterone or had a short life span.     

Analysis of myogenesis by somatic cell hybridization: III. Myogenic competence of hybrids derived from rat L6 myoblasts and mouse primary fibroblasts and myoblasts

Hybrid cells derived from rat L6 myoblasts and mouse primary fibroblasts (M x F hybrids), as well as those derived from rat L6 myoblasts and mouse primary myoblasts (M x M hybrids), were examined for their ability to engage in myogenesis as judged by muscle fiber formation plus the expression of skeletal muscle myosin and creatine kinase (CK). Of 172 primary hybrid colonies scored, 59% were myogenic in the M x F fusion and 97% exhibited muscle fiber formation in the M x M fusion. Individual hybrid clones from each cross were isolated, expanded and analyzed for myogenic capabilities as well. All three M x M and all ten M x F isolated clones exhibited preferential elimination of mouse chromosomes. Nonetheless, all were capable of fusing spontaneously and of elaborating skeletal muscle myosin and CK. The three M x M hybrids expressed only MM-CK whereas nine out of ten M x F hybrids produced all three CK isoenzymes (MM, MB, BB). These results suggest that M X M hybrids express CK patterns reminiscent of the rat L6 parental cells while M X F hybrids apparently mimic mouse muscle fiber CK patterns. Various models are discussed which address these phenomena.     

Hormonal regulation of plasminogen activator in rat hepatoma cells

Plasminogen activators are membrane-associated, arginine-specific serine proteases which convert the inactive plasma zymogen plasminogen to plasmin, an active, broad-spectrum serine protease. Plasmin, the major fibrinolytic enzyme in blood, also participates in a number of physiologic functions involving protein processing and tissue remodelling, and may play an important role in tumor invasion and metastasis. In HTC rat hepatoma cells in tissue culture, glucocorticoids rapidly decrease plasminogen activator (PA) activity. We have shown that this decrease is mediated by induction of a soluble inhibitor of PA activity rather than modulation of the amount of PA. The hormonally-induced inhibitor is a cellular product which specifically inhibits PA but not plasmin. We have isolated variant lines of HTC cells which are selectively resistant to the glucocorticoid inhibition of PA but retain other glucocorticoid responses. These variants lack the hormonally-induced inhibitor; PA from these variants is fully sensitive to inhibition by inhibitor from steroid-treated wild-type cells. Cyclic nucleotides dramatically stimulate PA activity in HTC cells in a time- and concentration-dependent manner. Paradoxically, glucocorticoids further enhance this stimulation. Thus glucocorticoids exert two separate and opposite effects on PA activity. The availability of glucocorticoid-resistant variant cell lines, together with the unique regulatory interactions of steroids and cyclic nucleotides, make HTC cells a useful experimental system in which to study the multihormonal regulation of plasminogen activator.     

Neutral lipid storage disease: a possible functional defect in phospholipid- linked triacylglycerol metabolism

Neutral lipid storage disease (NLSD) (Chanarin-Dorfman Syndrome) is an autosomal recessive disorder of multisystem triacylglycerol (TAG) storage. Previous work has pointed to a defect in intracellular TAG metabolism. In the studies reported here, the lipid metabolism of three lines of NLSD fibroblasts were compared to normal skin fibroblasts. When pulsed with [3H]oleic acid, the earliest observed abnormality in NLSD cell lines was increased incorporation into phosphatidylethanolamine, followed by accumulation of radiolabel in TAG. Activities of several glycerolipid synthetic enzymes were comparable in NLSD and normal fibroblast lines, excluding oversynthesis of glycerolipid. The proportion of plasmalogen and neutral ether lipid synthesized was normal and alkylglycerols did not accumulate, excluding a defect in ether lipid metabolism. Activities of both acid lipase and Mn2(+)-sensitive lipase within the particulate fractions of NLSD and normal fibroblasts were comparable. These studies are most consistent with functional deficiency of a TAG lipase with activity against a pool of TAG that are normally utilized for phospholipid biosynthesis.     

The influence of conditions at the time of mating on reproduction of commercial pigs

The objective of this study was to compare a new mating system, called the Detection-Mating Area (DM Area), and a conventional mating system on the long-term reproductive performance of commercial pigs. The DM Area treatment basically involved detecting oestrous females in an arena closely surrounded on two sides by boars and mating these females in this arena. This mating system was designed to improve the physical and sexual environments of the pigs at mating. In contrast, the conventional treatment involved conducting oestrus detection and mating in the boar's accommodation pen. The study was conducted over an 18-month period at a commercial farm that housed 2400 breeding female pigs.

In order to control for the effects of the stockperson, an analysis was conducted on the reproductive performance of female pigs in which one stockperson assisted the matings in both treatments over a 12-month period. Gilts mated in the DM Area treatment had a higher (P<0.05) total litter size and a higher (P<0.01) litter size born alive than gilts mated in the conventional treatment (10.31 vs. 8.96 and 9.50 vs. 8.29, respectively). Although gilts in the DM Area treatment had a higher farrowing rate (93.2 vs. 87.9%), this difference was not significant (P>0.05). There were no significant (P>0.05) differences found between the reproductive performance of sows in the two treatments; however this comparison was confounded by sows in the DM Area treatment having a lower number of matings per oestrus than those in the other treatment. Observations on the sexual behaviour of pigs at 145 matings indicated that the boars in the DM Area treatment displayed a higher (P<0.05) number of bouts of courting behaviour directed towards the female than boars in the conventional treatment (8.3 and 6.4, respectively). These very limited observations on sexual behaviour suggest that changes in the courting behaviour of bears may be associated with changes in litter size. While there was some indication from the results of progesterone analysis of blood samples taken from unmated gilts that there may have been some differences between treatments in the sexual age of gilts at matings, these differences are unlikely to explain the differences in litter size between treatments. Further research is required to identify the component (s) of the DM Area treatment that are responsible for the improvement in litter size in gilts and to further examine the effects of the two treatments on the reproductive performance of sows.