High level expression of the IL-1 receptor related T1 receptor in insect cells.

Baculovirus-dependent expression of membrane-associated and secreted, heavily modified proteins in insect cells often results in low yields. To optimize expression of T1, a heavily glycosylated receptor related to Interleukin-1 receptor type I, deletion mutants comprising either the heavily glycosylated extracellular domain or the isolated transmembrane and cytoplasmic portions of the T1 receptor were expressed from recombinant baculoviruses. As shown here, the use of a Baculovirus-derived leader sequence (GP67) in combination with an insect cell line with increased secretory capacity resulted in yields of several mg per liter (10(9) cells) of culture of both purified secretory T1 glycoprotein and membrane-associated T1 receptor.

Bone matrix deposition of T1, a homologue of interleukin 1 receptors.

The mouse T1 glycoprotein is a secreted molecule of the immunoglobulin superfamily with significant homology to interleukin 1 receptors. It is expressed during bone development, and the extracellular diffusible gene product is found associated with newly formed bone but not cartilage matrix. During osteogenic differentiation of mandibular condyles of newborn mice in vitro, T1 gene expression is induced shortly after cultivation and is observed throughout the differentiation process. The temporal expression pattern of the gene is an mandibular condyles indicates that T1 expression is an early marker of osteogenic differentiation. This view is substantiated by the analysis of T1 gene regulation in continuous osteogenic cell lines. Both in differentiating osteoblast-like KM-1K cells derived from mandibular condyles and in MC3T3 cells, T1 gene activity is preferentially associated with early differentiation stages. In mandibular condyles, the secreted extracellular T1 protein is deposited into newly formed osteoid but not into cartilage matrix. This novel bone matrix protein may locally modulate the availability of its ligand.

New Lignan Glucosides from Stemmadenia minima*.

Reinvestigation of the polar fractions from the stem bark of STEMMADENIA MINIMA A. Gentry (Apocynaceae) afforded two known and four new lignan glucosides. Their structures were deduced by spectroscopic and chemical methods.

Modes of action and combination effects of polychlorinated biphenyls and gamma-hexachlorocyclohexane on the regulation of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The effect of polychlorinated biphenyls, gamma-hexachlorocyclohexane and the effect of a combination of these substances on the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase were investigated. As known from previous investigations polychlorinated biphenyls interfere with the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in rat liver via enzyme-lipid interaction and at the pretranslational level. In contrast to polychlorinated biphenyls, gamma-hexachlorocyclohexane did not alter the lipid status of the microsomal membrane. Thus the location of the 3-hydroxy-3-methylglutaryl coenzyme A reductase, and consequently the catalytic activity of the enzyme was not changed. As with polychlorinated biphenyls, gamma-hexachlorocyclohexane interacted with enzyme regulation at the pretranslational level. Northern dot hybridization experiments showed a decrease in the level of m-RNA coding for 3-hydroxy-3-methylglutaryl coenzyme A reductase. The effect of combination of gamma-hexachlorocyclohexane and polychlorinated biphenyls was not additive. The gamma-hexachlorocyclohexane effect appeared to play a more important role than that of the polychlorinated biphenyls. The results indicate that the combination effects are as important as the effects of the single compounds when making risk assessments for xenobiotics.

Effect of alterations in vitro and in vivo of the cholesterol content in rat liver microsomes on the activity of 3-hydroxy-3-methylglutaryl-CoA reductase.

3-Hydroxy-3-methylglutaryl-CoA reductase, the regulatory enzyme of cholesterol synthesis in liver, is bound to the microsomal fraction. Lipoprotein-bound cholesterol (from human serum) in vitro inhibits the microsomal bound 3-hydroxy-3-methylglutaryl-CoA reductase. The solubilized enzyme, however, is not inhibited. Immunotitration of the microsomal enzyme with a monospecific antibody reveals that the loss in enzyme activity caused by cholesterol corresponds well with the lowering of the equivalence points. In contrast, the equivalence points of the solubilized enzyme remain unchanged. This indicates that the inhibitory effect of cholesterol is restricted to the microsomal bound enzyme. In vivo different physiological conditions lead to relatively small changes in the cholesterol content in the microsomes while drastic changes in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase are observed. When microsomes from these rats are incubated with exogenous cholesterol, the activity of the enzyme is always found to be decreased to the same extent independent of the physiological condition of the animals. The findings suggest that 3-hydroxy-3-methylglutaryl-CoA reductase may be "masked" by a cholesterol-mediated modification of the microsomal membrane.

Effect of unsaturated fatty acids on the biosynthesis of glucose-repressed enzymes in yeast.

In anaerobically glucose-grown yeast isocitrate lyase (EC 4.1.3.1.), and malate dehydrogenase (EC 1.1.1.37.) are repressed by glucose. 24 h cultures still contain 0.3--0.4% glucose in the medium, which is enough to completely repress these activities. Aeration of these cells, in buffer containing acetate, initiates the formation of the three enzymes. Within 16 h, the specific activities of these enzymes increase about 140, 120 and 70-fold, respectively. Glucose-6-phosphate dehydrogenase activity was not altered. When the yeast was grown anaerobically, but with a supplement of an unsaturated fatty acid in the medium, synthesis of the three enzymes was much faster and the specific activities after 16 h of derepression were considerably higher. A relationship exists between the number of double bonds in the unsaturated fatty acid molecule and its capability to stimulate enzyme synthesis: linolenic acid is more effective than linoleic acid, which, in turn, is much more effective than oleic acid. Increasing periods of aeration with glucose of anaerobically grown cells prior to derepression results in an increasing stimulation of enzyme synthesis on subsequent derepression. Anaerobic incubation of yeast in the presence of an unsaturated fatty acid in advance to derepression also increased the velocity of enzyme formation. It is suggested that during the aeration period with glucose and during anaerobic incubation with an unsaturated fatty acid a more active protein synthesizing apparatus was formed.

In vivo effect of cholesterol feeding on the short term regulation of hepatic hydroxymethylglutaryl coenzyme A reductase during the diurnal cycle.

Light-dark-cycled rats were fed a 3% cholesterol-supplemented diet at the beginning of the dark phase. Cholesterol-fed and control animals were taken at intervals throughout the following 12 h and the microsomal and solubilized hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase was isolated. Immunotitrations of this microsomal and solubilized enzyme were performed with a monospecific antibody to 3-hydroxy-3-methylglutaryl coenzyme A reductase. In contrast to the specific activity of the enzyme, which differs extremely during the diurnal cycle, the immunotitrations obtained from cholesterol-fed and control animals, yielded in identical antisera equivalence points. On the other hand, when the enzyme was phosphorylated in vitro, the antisera equivalence points corresponded to the alterations of the specific activity. In contrast to the results published by Higgins and Rudney ((1973) Nature New Biol. 246, 60-61), our data prove that even the in vivo short term changes in enzyme activity are due to changes in the quantity of enzyme rather than to a modulation of the catalytic activity.

Effect of unsaturated fatty acids on sterol biosynthesis in yeast.

Lipid-depleted yeast, grown anaerobically, contains only very low amounts of sterols. The hydroxymethylglutaryl-CoA reductase activity, the regulatory enzyme of sterol synthesis in yeast, is also low. Aeration of such cells in a buffer containing a carbon source induces hydroxymethylglutaryl-CoA reductase activity and increases sterol synthesis. The velocity of the increase depends on the carbon source present during the aeration period. Glucose and sugars that are easily converted to glucose were found to be most effective. A supplement of unsaturated fatty acids during anaerobic growth causes a several-fold greater velocity of the enzyme induction and of sterol biosynthesis. Linolenic acid (30 microM) accelerated sterol biosynthesis about 7-fold. Activities of galactokinase and galactose-1-phosphate uridyltransferase, which are involved in the conversion of galactose to glucose, increased several-fold in the supplemented cells within 6 h of aeration, concomitantly with stimulation of sterol synthesis from galactose. It is suggested that the stimulation of enzyme induction and sterol biosynthesis in fatty acid supplemented cells is due to a completion of the protein-synthesizing apparatus during cell growth. A markedly enhanced capacity of these cells to incorporate leucine into acid-precipitable protein supports this assumption.

Sterol biosynthesis in yeast. 3-Hydorxy-3-methylglutaryl-Coenzyme A reductase as a regulatory enzyme.

Anaerobically and aerobically grown yeast contains 3-hydroxy-3-methylglutaryl-CoA reductase, which is located in the mitochondrial fraction of the cell. Anaerobically grown yeast has a low specific activity of 3-hydroxy-3-methylglutaryl-CoA reductase and a low sterol content. Aeration of this yeast in buffer, without growth, results in an increase in the specific activity of the enzyme, which is paralleled by an increase in the sterol content. This induction has an oscillatory profile with yeast grown anaerobically for 24 h and a linear pattern with cells grown anaerobically for 72 h. With the latter type of yeast, glucose is necessary for an induction, whereas with the other yeast an induction occurs with and without glucose. By an anaerobic incubation in buffer of the yeast grown anaerobically for 24 h, the oscillatory profile can be transformed into a linear one. The extent of induction of the reductase is strictly dependent on the concentration of glucose present. Sterols increase in whole cells, but they do not increase in the mitochondrial fraction. The induction of 3-hydroxy-3-methylglutaryl-CoA reductase is strongly inhibited by cycloheximide, but is not affected by chloramphenicol. The induction of the enzyme is closely connected with the glucose metabolism of the cell; fructose, mannose, and ethanol can also induce the reductase.