Glycoprotein Biosynthesis in Chlamydomonas II. No Evidence for Lipid Intermediates in Protein Galactosylation

A crude membrane fraction from Chlamydomonas reinhardii transferredradioactivity from UDP-[¹⁴C]galactose to endogenous lipids.Most of the radioactivity was detected in digalactosyl diacylglycerolmoieties in which only the distal galactose residue (-linkedto monogalactosyl diacylglycerol) was labeled. A second compoundwas identified as monogalactosyl diacylglycerol labeled in thegalactose moiety ß-linked to glycerol. In additionto these two galactolipid species, trace amounts of radioactiveglucose were detected in the aqueous phase after mild acid treatmentof the total lipid fraction. This demonstrates the presenceof a 4-epimerase and provides indirect evidence for the presenceof a small amount of polyprenyl-monophosphate-glucose whichwas presumably not detectable per se because of the bulk ofneutral galactolipids. The failure to detect polyprenyl-phosphate-galactoseor mild acid labile galactose at any time during the incubationsuggests that galactosylation of Chlamydomonas proteins mightoccur without the involvement of lipid intermediates. (Received May 10, 1982; Accepted August 21, 1982)     

Glycoprotein Biosynthesis in Chlamydomonas: I. IN VITRO INCORPORATION OF GALACTOSE FROM UDP-[C]GALACTOSE INTO MEMBRANE-BOUND PROTEIN

A crude membrane fraction from Chlamydomonas reinhardii was found to catalyze d-galactose transfer from UDP-galactose to endogenous proteins. Highest incorporation rates were achieved by incubation at 25 degrees C and pH 7.5 in the presence of 10 millimolar Fe(2+). Hydrolytic studies on the labeled polymer revealed that radioactivity was attached to protein via an alkali-stable and acid-labile linkage. Identification of galactose as the only labeled sugar in the acid hydrolysate and results of a tentative estimation of the molecular weight of the charged alkaline degradation product indicate that monomeric galactose units are transferred to form an O-glycosidic bond with peptidyl hydroxyproline. No indications were found for a similar linkage to serine which, in contrast to the hydroxyproline-O-glycoside linkage, is acid-stable but is cleaved by beta-elimination. Chromatography of the sodium dodecyl sulfate-solubilized polymer on Sepharose-6B demonstrated that galactosyl residues are mainly associated with proteins which are of considerably higher molecular weight than are the majority of sodium dodecyl sulfate-denatured membrane proteins in this fraction.     

Mechanisms of suicidal erythrocyte death.

Erythrocyte injury such as osmotic shock, oxidative stress or energy depletion stimulates the formation of prostaglandin E2 through activation of cyclooxygenase which in turn activates a Ca2+ permeable cation channel. Increasing cytosolic Ca2+ concentrations activate Ca2+ sensitive K+ channels leading to hyperpolarization, subsequent loss of KCl and (further) cell shrinkage. Ca2+ further stimulates a scramblase shifting phosphatidylserine from the inner to the outer cell membrane. The scramblase is sensitized for the effects of Ca2+ by ceramide which is formed by a sphingomyelinase following several stressors including osmotic shock. The sphingomyelinase is activated by platelet activating factor PAF which is released by activation of phospholipase A2. Phosphatidylserine at the erythrocyte surface is recognised by macrophages which engulf and degrade the affected cells. Moreover, phosphatidylserine exposing erythrocytes may adhere to the vascular wall and thus interfere with microcirculation. Erythrocyte shrinkage and phosphatidylserine exposure ('eryptosis') mimic features of apoptosis in nucleated cells which however, involves several mechanisms lacking in erythrocytes. In kidney medulla, exposure time is usually too short to induce eryptosis despite high osmolarity. Beyond that high Cl- concentrations inhibit the cation channel and high urea concentrations the sphingomyelinase. Eryptosis is inhibited by erythropoietin which thus extends the life span of circulating erythrocytes. Several conditions trigger premature eryptosis thus favouring the development of anemia. On the other hand, eryptosis may be a mechanism of defective erythrocytes to escape hemolysis. Beyond their significance for erythrocyte survival and death the mechanisms involved in 'eryptosis' may similarly contribute to apoptosis of nucleated cells.     

PI3 kinase dependent stimulation of gastric acid secretion by dexamethasone.

Excessive gastric acid secretion plays an important role in the pathogenesis of peptic ulcers. Dexamethasone, a widely used drug, is known to stimulate gastric acid secretion and increase the incidence of peptic ulcers. However little is known about the mechanism of the dexamethasone's effect on parietal cells. The present study was performed to investigate the contribution of the phosphatidylinositol-3-kinase (PI3 kinase) to dexamethasone induced stimulation of gastric acid secretion. In vivo pretreatment with dexamethasone injections (150 microg/100g for 3 days) or in vitro exposure to (10 microM for > 20 minutes) significantly increased acid secretion in isolated gastric glands approximately 2-3 fold. The dexamethasone induced stimulation of gastric acid secretion was concentration dependent and significantly blunted by the H+/K2+ ATPase inhibitor omeprazole (200 microM), the PI3 kinase inhibitor Wortmannin (500 nM), the protein kinase inhibitor staurosporine (2.5 microM) and the Cl(-) channel blocker NPPB (100 microM); but not by the H(2) antagonist cimetidine (100 microM). In conclusion, it was observed that dexamethasone's effect on proton extrusion requires the activity of a PI3 kinase pathway, an apical Cl(-) channel and the H2+/K2+ ATPase.     

Blocking NF-κB and Akt by Hsp90 inhibition sensitizes Smac mimetic compound 3-induced extrinsic apoptosis pathway and results in synergistic cancer cell death

NF-κB and Akt are two main cell survival pathways that attenuate the anticancer efficacy of therapeutics. Our previous studies demonstrated that the Smac mimetic compound 3 (SMC3) specifically suppresses c-IAP1 and induces TNF-α autocrine to kill cancer cells. However, SMC3 also induces a cell survival signal through NF-κB activation. In this report, we further found that SMC3 potently activates Akt, which inhibits SMC3-induced cancer cell death. Strikingly, concurrent blocking NF-κB and Akt resulted in a significantly potentiated cytotoxicity. Because heat shock protein 90 (Hsp90) plays an important role in maintaining the integrity of both the NF-κB and Akt pathways in cancer cells, we examined if suppression of Hsp90 is able to potentiate SMC3-induced cancer cell death. The results show that targeting Hsp90 does not interfere with SMC3-induced c-IAP1 degradation and TNF-α autocrine, the key processes for SMC3-induced cancer cell apoptosis. However, Hsp90 inhibitors effectively blocked SMC3-induced NF-κB activation through degradation of RIP1 and IKKβ, two key components of the NF-κB activation pathway, and reduced both the constitutive and SMC3-induced Akt activity through degradation of the Akt protein. Consistently, with the co-treatment of SMC3 and Hsp90 inhibitors, apoptosis was markedly sensitized and a synergistic cytotoxicity was observed. The results suggest that concurrent targeting c-IAP1 and Hsp90 by combination of SMC3 and Hsp90 inhibitors is an effective approach for improving the anticancer value of SMC3.     

The rat thymus--a site of atrial natriuretic peptide synthesis

The atrium of the heart has been demonstrated to represent the major site of synthesis of atrial natriuretic peptide (ANP), a potent natriuretic, diuretic and vasoactive hormone. Our recent studies revealed ANP-like material outside the heart, namely, in lymphoid follicles of the intestine and in the thymus, and now we report data demonstrating the thymus as a site of synthesis for ANP. The experimental evidence is as follows: firstly, the immunoreactive material detected corresponds chromatographically with the precursor of ANP. Secondly, the thymus contains mRNA for ANP. Thirdly, immunohistochemistry locates ANP-like material to cortical thymocytes with particularly dense staining in the subcapsular areas of the thymus. Interestingly, both ANP-like material and the mRNA coding for ANP were expressed to a larger extent in newborn rats as compared to adult animals, suggesting that ANP may be involved in the development and/or function of T-cells.