Eeva Sievi

Proteolytic Function of GPI-Anchored Plasma Membrane Protease Yps1p in the Yeast Vacuole and Golgi

Yps1p is a member of the GPI‐anchored aspartic proteases which reside at the plasma membrane of Saccharomyces cerevisiae. Here we show that in Δerg6 cells, where a late biosynthetic step of the membrane lipid ergosterol is blocked, part of Yps1p was targeted to the vacuole. There it overtook proteolytic functions of the Pep4p protease, resulting in processing of pro‐CPY to CPY in cells lacking the PEP4 gene. Yps1p was enriched in membrane microdomains, as it could be isolated in detergent‐insoluble complexes from both normal and Δerg6 cells. Vacuolar Yps1 caused degradation of a mammalian sialyltransferase ectodomain fusion protein (ST6Ne), which was directed from the Golgi to the vacuole in both normal and Δerg6 cells. Unexpectedly, ST6Ne was degraded also when arrested in the Golgi in a temperature‐sensitive sec7–1 mutant. Newly synthesized Yps1p, in transit to the plasma membrane, was also involved in the Golgi‐associated degradation. These data show that GPI‐anchored proteases, whose biological roles are unknown, may reside and function in different subcellular locations.

Exposure to high ambient temperature increases absorption and plasma concentrations of transdermal nicotine

Absorption and plasma concentrations of transdermally delivered drugs may be increased during heat exposure. We studied the effects of short‐term heat exposure in a sauna bath on the pharmacokinetics of transdermal nicotine, 25 mg/16 hr, in 12 healthy smokers in an open, randomized crossover study that consisted of a control session and a sauna bathing session. In the sauna session the subjects stayed seated in a sauna bath (mean temperature 82° C (180° F); mean relative humidity 28%) for three 10‐minute periods separated by two 5‐minute breaks. Sauna bathing significantly (p < 0.01 versus control) increased peak plasma concentration, area under the plasma concentration‐time curve from 0 to 1 hour, the amount of nicotine absorbed, and the mean plasma nicotine concentrations during heat exposure. No significant difference in nicotine area under the plasma concentration‐time curve from 0 to 3 hours was observed. In addition, the combined effects of transdermal nicotine and sauna bathing on hemodynamics, some psychomotor skills, and subjective symptoms were evaluated. We concluded that absorption and plasma concentrations of transdermally delivered nicotine may be increased during exposure to high ambient temperature, probably because of enhanced skin blood flow. However, no adverse symptoms were recorded.

Nicotine stereoisomers and cotinine stimulate prostaglandin E2 but inhibit thromboxane B2 and leukotriene E4 synthesis in whole blood

The effects of (-)-nicotine (0.0005-500 microM), (+)-nicotine (0.0005-50 microM) and (-)-cotinine (0.0005-500 microM) on arachidonic acid metabolism were investigated in Ca2+ ionophore A23187 (calcimycin)-stimulated human whole blood in vitro. (-)-Nicotine and (-)-cotinine stimulated prostaglandin E2 but inhibited thromboxane B2 synthesis, as has been observed previously in A23187-stimulated polymorphonuclear leukocytes and platelet-rich plasma [Saareks, V., Riutta, A., Mucha, I., Alanko, J., Vapaatalo, H., 1993. Nicotine and cotinine modulate eicosanoid production in human leukocytes and platelet rich plasma. Eur. J. Pharmacol., 248, 345-349.]. (+)-Nicotine also stimulated prostaglandin E2 but inhibited thromboxane B2 synthesis. High concentrations of (-)-nicotine and (-)-cotinine and even nanomolar concentrations of (+)-nicotine inhibited leukotriene E4 synthesis. These results indicate that (-)-nicotine and (-)-cotinine stimulate cyclooxygenase but inhibit thromboxane synthase and 5-lipoxygenase in whole blood in vitro. (+)-Nicotine is capable of affecting in the same direction as well.

Inhibitory effects of mesoionic 3‐aryl substituted oxatriazole‐5‐imine derivatives on vascular smooth muscle cell mitogenesis and proliferation in vitro

1 The effects of oxatriazole‐type (GEA 3162 and GEA 5624) nitric oxide (NO) donors on mitogenesis and proliferation were studied in vascular smooth muscle cell (VSMC) culture. The effects of the GEA‐compounds were compared with well‐known NO‐donors 3‐morpholinosydnonimine (SIN‐1) and S‐nitroso‐N‐acetylpenicillamine (SNAP). 2 All NO‐donors released NO and increased the production of cyclic GMP concentration‐dependently. The production of cyclic GMP was inhibited by the guanylate cyclase inhibitor, ODQ (1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one). 3 The NO‐donors inhibited basal and serum‐induced DNA synthesis concentration‐dependently. The GEA‐compounds were needed in concentrations 10 times lower than SIN‐1 and SNAP. GEA 3162, SIN‐1 and SNAP were also able to inhibit serum‐induced cell proliferation. GEA 5624 was ineffective. The antimitogenic effect of NO‐donors was not reduced by inhibiting the guanylate cyclase. 4 These results suggest that NO inhibits serum‐induced DNA synthesis and proliferation of VSMC by a cyclic GMP‐independent mechanism. The oxatriazole‐type NO‐donor GEA 3162 was found to be a more potent inhibitor of mitogenesis and cell proliferation than SIN‐1 and SNAP.

Antioxidative Properties of Lactobacillus GG Measured as Prostacyclin and Nitric Oxide Production in Endothelial Cell Culture

There is previous evidence on antioxidative activity of lactobacilli. In the present study, we found increased formation of prostacyclin and cGMP (a second messenger of nitric oxide) in endothelial cell incubation with Lactobacillus GG. This indicates the antioxidative property of Lactobacillus GG.

Interrelationships between salt and fish oil in stroke-prone spontaneously hypertensive rat.

The cardiovascular effects of a partially purified extract of fish oil, enriched in the n-3 series fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), were studied in stroke-prone spontaneously hypertensive rats (SHR-SP) fed with high- and low-sodium diets during 5 weeks. Addition of salt to the low-salt control diet at a level commonly found in human food items (6% NaCl of the dry weight of the diet) produced a remarkable rise in blood pressure, an increase in left ventricular weight-to-body weight ratio (LVH-index) and an increase in kidney weight-to-body weight ratio (RH-index). Fish oil (20% of the dry weight of the diet) did not significantly influence the blood pressure or LVH-index or RH-index during the low-salt control diet. However, fish oil completely prevented the remarkable rise in blood pressure and clearly antagonized the rise of both LVH- and RH-indices, induced by the high-salt diet. The fish oil supplementation increased the levels of the polyunsaturated fatty acids of the n-3 series and decreased those of the n-6 series in plasma and kidney, irrespective of the salt content of the diet. Fish oil lowered serum thromboxane B2 concentration by approximately 75%. During the high-salt diet, fish oil markedly decreased water intake and urine volume, and increased urinary sodium concentration by about 60%. Our findings show that, in addition to an antihypertensive effect, fish oil also decreases LVH and RH. These effects appear to be due to an improved ability to excrete sodium and could be explained by the observed changes in the fatty acid composition and metabolism.

Catechol estrogens as inhibitors of leukotriene synthesis

Estrogens have a beneficial effect on atherosclerosis and osteoporosis after menopause, but their exact mechanism of action is still unknown. The aim of the present study was to investigate the effects of estradiol and its metabolites catechol estrogens on arachidonic acid metabolism in vitro. Estradiol had no effect on arachidonic acid metabolism up to 33 microM in A23187-stimulated human whole blood. All catechol estrogens (2-hydroxyestradiol, 2-hydroxyestrone, 4-hydroxyestradiol and 4-hydroxyestrone) had similar kinds of actions on arachidonic acid metabolism, being over ten times more potent inhibitors of leukotriene synthesis (IC50 values 0.044-0.16 microM) than thromboxane (IC50 values 0.99-2.1 microM) and prostaglandin E2 synthesis (IC50 values 0.84-5.5 microM). It is suggested that some of the protective actions of estrogens--e.g., on atherosclerosis and osteoporosis--may be related to the inhibition of leukotriene synthesis by catechol estrogens.

Validation of the Hsp150 Polypeptide Carrier and HSP150 Promoter in Expression of Rat α2,3-Sialyltransferase in Yeasts

Heterologous glycoproteins usually do not fold properly in yeast cells and fail to leave the endoplasmic reticulum. Here we show that the Hsp150Δ polypeptide carrier promoted proper folding and secretion of the catalytic ectodomain of rat α2,3‐sialyltransferase (ST3Ne) in Pichia pastoris.The efficiency of the Hsp150Δ carrier in P. pastoris and Saccharomyces cerevisiae was at least as high as that of the MFα carrier. Most of Hsp150Δ‐ST3Ne and MFα‐ST3Ne remained noncovalently attached to the cell wall via the ST3Ne portion. The strength of the HSP150promoter was found to be comparable to that of the GAL1 promoter.

Glycan engineering of proteins with whole living yeast cells expressing rat liver α2,3-sialytransferase in the porous cell wall

The N‐glycans of recombinant proteins produced via the secretory pathway of cultured mammalian cells are often undersialylated, and insect cells lack sialytransferases. Undersialylated glycoproteins are rapidly cleared from the circulation, compromising the effect of pharmaceuticals. We show that incubation with living Saccharomyces cerevisiae cells expressing the catalytic ectodomain of rat liver α2,3‐sialyltransferase (ST3Ne) in the porous cell wall resulted in sialylation of glycoproteins. The K m values of the yeast enzyme for several substrates were similar to those of recombinant ST3Ne from insect cells and of authentic ST3N. The yeast strain provides an inexpensive self‐perpetuating source of ST3N activity for glycan engineering of recombinant proteins.

Antihypertensive Drugs Reduce Noradrenaline-induced Hypertrophy of Cultured Myocardial Cells

The cellular mechanisms of cardiac hypertrophy are still largely unknown. In‐vivo studies have demonstrated that antihypertensive drugs can regress hypertrophy independently of reductions in blood pressure. The antihypertrophic effects of metoprolol, propranolol, felodipine, verapamil and captopril were studied in neonatal cardiac myocyte culture. Prazosin was used as a positive control. Hypertrophy was defined as an increase in protein content measured by [3H]leucine incorporation.