Lovastatin selectively inhibits ras activation of the 12-O-tetradecanoylphorbol-13-acetate response element in mammalian cells.

To evaluate ras-mediated signal transduction, an alkaline phosphatase gene (SEAP) was placed under the control of the ras-inducible phorbol ester response element (TRE) in murine fibroblasts (TRE-SEAP cells). The Kirsten ras gene was placed under the control of the glucocorticoid-inducible mouse mammary tumor virus promoter and introduced into the TRE-SEAP cells. Dexamethasone increased ras expression in the TRE-SEAP cells carrying the Kirsten ras gene and stimulated SEAP activity 25-fold. Lavostatin blocked dexamethasone induction of SEAP activity (50% inhibitory concentration, 0.5 microM) but did not affect phorbol ester-induced SEAP activity in the same cells. Lovastatin also did not block forskolin induction of SEAP activity in cells expressing SEAP under the control of the cyclic AMP response element.     

Transcapillary escape rate of albumin in humans during exercise-induced hypervolemia

Haskell, Andrew, Ethan R. Nadel, Nina S. Stachenfeld, KeiNagashima, and Gary W. Mack. Transcapillary escape rate of albuminin humans during exercise-induced hypervolemia. J. Appl. Physiol. 83(2): 407-413, 1997.To test thehypotheses that plasma volume (PV) expansion 24 h after intenseexercise is associated with reduced transcapillary escape rate ofalbumin (TER_alb) and that localchanges in transcapillary forces in the previously active tissues favorretention of protein in the vascular space, we measured PV,TER_alb, plasma colloid osmoticpressure (COP_p), interstitialfluid hydrostatic pressure (Pi), and colloid osmotic pressure in legmuscle and skin and capillary filtration coefficient (CFC) in the armand leg in seven men and women before and 24 h after intense uprightcycle ergometer exercise. Exercise expanded PV by 6.4% at 24 h (43.9 ± 0.8 to 46.8 ± 1.2 ml/kg, P < 0.05) and decreased total protein concentration (6.5 ± 0.1 to6.3 ± 0.1 g/dl, P < 0.05) andCOP_p (26.1 ± 0.8 to 24.3 ± 0.9 mmHg, P < 0.05), although plasmaalbumin concentration was unchanged. TER_alb tended to decline (8.4 ± 0.5 to 6.5 ± 0.7%/h, P = 0.11) and was correlated with the increase in PV(r = 0.69,P < 0.05). CFC increased in the leg(3.2 ± 0.2 to 4.3 ± 0.5 µl · 100 g¹ · min¹ · mmHg¹,P < 0.05), and Pi showed a trend toincrease in the leg muscle (2.8 ± 0.7 to 3.8 ± 0.3 mmHg, P = 0.08). These datademonstrate that TER_alb isassociated with PV regulation and that local transcapillary forcesin the leg muscle may favor retention of albumin in the vascular spaceafter exercise.

     

An antibody that blocks insulin-like growth factor (IGF) binding to the type II IGF receptor is neither an agonist nor an inhibitor of IGF-stimulated biologic responses in L6 myoblasts

To better define the biologic function of the type II insulin-like growth factor (IGF) receptor, we raised a blocking antiserum in a rabbit by immunizing with highly purified rat type II IGF receptor. On immunoblots of crude type II receptor preparations, only bands corresponding to the type II IGF receptor were seen with IgG 3637, indicating that the antiserum was specific for the type II receptor. Competitive binding and chemical cross-linking experiments showed that IgG 3637 blocked binding of 125I-IGF-II to the rat type II IGF receptor, but did not block binding of 125I-IGF-I to the type I IGF receptor, nor did IgG 3637 block binding of 125I-insulin to the insulin receptor. In addition, IgG 3637 did not inhibit the binding of 125I-IGF-II to partially purified 150- and 40-kDa IGF carrier proteins from adult and fetal rat serum. L6 myoblasts have both type I and type II IGF receptors. IGF-I was more potent than IGF-II in stimulating N-methyl-alpha-[14C]aminoisobutyric acid uptake, 2-[3H]deoxyglucose uptake, and [3H]leucine incorporation into cellular proteins. IgG 3637 did not stimulate either 2-[3H]deoxyglucose uptake, N-methyl-alpha-[14C]aminoisobutyric acid uptake, or [3H]leucine incorporation into protein when tested alone. Furthermore, IgG 3637 at concentrations sufficient to block type II receptors under conditions of the uptake and incorporation experiments did not cause a shift to the right of the dose-response curve for stimulation of these biologic functions by IGF-II. We conclude that the type II IGF receptor does not mediate IGF stimulation of N-methyl-alpha-[14C]aminoisobutyric acid and 2-[3H]deoxyglucose uptake and protein synthesis in L6 myoblasts; presumably, the type I receptor mediates these biologic responses. The anti-type II receptor antibody inhibited IGF-II degradation in the media by greater than 90%, suggesting that the major degradative pathway for IGF-II in L6 myoblasts utilizes the type II IGF receptor.     

Ley/H: an endothelial-selective, cytokine-inducible, angiogenic mediator

Endothelial cells (ECs) are key participants in angiogenic processes that characterize tumor growth, wound repair, and inflammatory diseases, such as human rheumatoid arthritis (RA). We and others have shown that EC molecules, such as soluble E-selectin, mediate angiogenesis. Here we describe an EC molecule, Lewisy-6/H-5-2 glycoconjugate (Ley/H), that shares some structural features with the soluble E-selectin ligand, sialyl Lewisx (sialyl Lex). One of the main previously recognized functions of Lewisy is as a blood group glycoconjugate. Here we show that Ley/H is rapidly cytokine inducible, up-regulated in RA synovial tissue, where it is cell-bound, and up-regulated in the soluble form in angiogenic RA compared with nonangiogenic osteoarthritic joint fluid. Soluble Ley/H also has a novel function, for it is a potent angiogenic mediator in both in vitro and in vivo bioassays. These results suggest a novel paradigm of soluble blood group Ags as mediators of angiogenic responses and suggest new targets for therapy of diseases, such as RA, that are characterized by persistent neovascularization.     

A peptide-doxorubicin 'prodrug' activated by prostate-specific antigen selectively kills prostate tumor cells positive for prostate-specific antigen in vivo

We covalently linked doxorubicin with a peptide that is hydrolyzable by prostate-specific antigen. In the presence of prostate tumor cells secreting prostate-specific antigen, the peptide moiety of this conjugate, L-377,202, was hydrolyzed, resulting in the release of leucine-doxorubicin and doxorubicin, which are both very cytotoxic to cancer cells. However, L-377,202 was much less cytotoxic than conventional doxorubicin to cells in culture that do not secrete prostate-specific antigen. L-377,202 was approximately 15 times more effective than was conventional doxorubicin at inhibiting the growth of human prostate cancer tumors in nude mice when both drugs were used at their maximally tolerated doses. Nude mice inoculated with human prostate tumor cells secreting prostate-specific antigen showed considerable reductions in tumor burden with minimal total body weight loss when treated with L-377, 202. This improvement in therapeutic index correlated with the selective localization of leucine-doxorubicin and free doxorubicin in tissues secreting prostate-specific antigen after exposure to L-377,202.     

The effect of delayed feeding on the post-feeding behaviour of sows

Previous research into oral persistence in pigs has shown that the relationship between motivation and performance of stereotypy is not a simple one, and that non-specific motivational factors such as arousal may be important. For chronically food-restricted sows the time before food arrives is characterised by increasing excitement and arousal which may be carried over into the post-feeding period facilitating the performance of persistent oral behaviour. To investigate this possibility, arousal was manipulated by delaying feeding. Five and 15 min delays were used and sows experienced delayed feeding 1 day in every 4. Principal components analysis (PCA) was used to group the behaviour data into two factors, ACTIVITY/CHEW and NOSE/ROOT/PEN and these scores were then analysed using ANOVA as well as the appropriate individual behaviours. The delay of feeding increased the rate of eating, presumably reflecting increased arousal (P<0.05). ACTIVITY/CHEW scores were highest in 5 min delay sets, and an interaction between day of set and delay length indicated that ACTIVITY/CHEW scores peaked on different days within a set (P<0.05). Time spent chewing was highest on the day of delay (P<0.05), while standing was highest in days 1 and 2 post-delay (P<0.05). The NOSE/ROOT/PEN scores were lower on the day of delay than on other days (P<0.05), and were lowest in the first delay set than in sets later in the trial (P<0.01). It appeared that increasing arousal in the pre-feeding period does affect activity and oral behaviours in the post-feeding period. However, some of the effects appeared in a more extended and diffuse manner, perhaps due to the general disturbance created by the experimental regime and the sows' experience of long-term food restriction.