The pentose phosphate pathway in the endoplasmic reticulum

Approximately the same levels of six of the seven enzymes catalyzing reactions of the pentose phosphate pathway are in the cisternae of washed microsomes from rat heart, spleen, lung, and brain. Renal and hepatic microsomes also have detectable levels of these enzymes except ribulose-5-phosphate epimerase and ribose-5-phosphate isomerase. Their location in the cisternae is indicated by their latencies, i.e. requirement for disruption of the membrane for activity. In addition, transketolase, transaldolase, and glucose-6-phosphatase, a known cisternal enzyme, are inactivated by chymotrypsin and subtilisin only in disrupted hepatic microsomes under conditions in which NADPH-cytochrome c reductase, an enzyme on the external surface, is inactivated equally in intact and disrupted microsomes. The failure to detect the epimerase and isomerase in hepatic microsomes is due to inhibition of their assays by ketopentose-5-phosphatase. Xylulose 5-phosphate is hydrolyzed faster than ribulose 5-phosphate. A mild heat treatment destroys hepatic xylulose-5-phosphatase and glucose-6-phosphatase without affecting acid phosphatase. These results plus the established wide distribution of glucose dehydrogenase, the microsomal glucose-6-phosphate dehydrogenase, and its localization to the lumen of the endoplasmic reticulum suggest that most mammalian cells have two sets of enzymes of the pentose phosphate pathway: one is cytoplasmic and the other is in the endoplasmic reticulum. The activity of the microsomal pentose phosphate pathway is estimated to be about 1.5% that of the cytoplasmic pathway.     

Effects of lipids on cyclic-nucleotide phosphodiesterases

Several naturally-occurring lipids but not n-propanol, guanidine-HCl or a variety of synthetic detergents stimulate the 3′,5′-cyclic AMP-phosphodiesterase activities of a supernatant fraction of brain at 1.25 × 10^?7 M cAMP. The time courses of the reaction are linear in the presence and absence of lipid. On the other hand, lipid has different effects on various phosphodiesterase activities in fractions obtained after gel filtration of the crude extract. It stimulates the phosphodiesterase activities measured at 1.25 × 10^?7 M and 10^?4 M 3′,5′-cyclic-AMP and 1.25 × 10^?7 M 3′,5′-cyclic GMP in two of the fractions partially retained in the gel. However, lipid has little effect on the enzymatic hydrolysis of low concentrations of cAMP or cGMP and markedly inhibits the hydrolysis of high concentrations of cAMP by the fraction excluded from the gel.     

Intracellular vesicle movement, cAMP and myosin II in Dictyostelium

Dictyostelium amoebae were analyzed before and after rapid addition of 10(-6) M cAMP for cellular motility, dynamic shape changes, and intracellular particle movement. Before cAMP addition, amoebae moved in a persistent anterior fashion and were elongate with F-actin localized predominantly in the anterior pseudopod. Intracellular particles moved rapidly and anteriorly. Within seconds after 10(-6) M cAMP addition, cells stopped translocating, pseudopod formation ceased, intracellular particle movement was depressed, and F-actin was lost from the pseudopod and concomitantly relocalized in the cell cortex. After 10 seconds, expansion zones reappeared but were small and no longer anteriorly localized. Vesicle movement partially rebounded but was no longer anteriorly directed. The myosin II null mutant HS2215 exhibited both depressed cellular translocation and vesicle movement. The addition of cAMP to HS2215 cells did not result in any detectable change in the random, depressed movement of particles. The results with HS2215 suggest that myosin II is essential for (1) rapid cellular translocation, (2) cellular polarity, (3) rapid particle movement, (4) anteriorly directed particle movement, and (5) the cAMP response. Electron micrographs suggest that at least half of the particles examined in this study contain in turn smaller membrane bound vesicles or multilamellar membrane bodies. The possible role of these vesicles is discussed.     

Characterization and expression analysis of P5CS (Δ1-pyrroline-5-carboxylate synthase) gene in two distinct populations of the Atlantic Forest native species Eugenia uniflora L.

Molecular Biology Reports - Eugenia uniflora is an Atlantic Forest native species, occurring in contrasting edaphoclimatic environments. The identification of genes involved in response to abiotic...     

Cannabidiol inhibits lung cancer cell invasion and metastasis via intercellular adhesion molecule-1

Cannabinoids inhibit cancer cell invasion via increasing tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). This study investigates the role of intercellular adhesion molecule-1 (ICAM-1) within this action. In the lung cancer cell lines A549, H358, and H460, cannabidiol (CBD; 0.001-3 μM) elicited concentration-dependent ICAM-1 up-regulation compared to vehicle via cannabinoid receptors, transient receptor potential vanilloid 1, and p42/44 mitogen-activated protein kinase. Up-regulation of ICAM-1 mRNA by CBD in A549 was 4-fold at 3 μM, with significant effects already evident at 0.01 μM. ICAM-1 induction became significant after 2 h, whereas significant TIMP-1 mRNA increases were observed only after 48 h. Inhibition of ICAM-1 by antibody or siRNA approaches reversed the anti-invasive and TIMP-1-upregulating action of CBD and the likewise ICAM-1-inducing cannabinoids Δ(9)-tetrahydrocannabinol and R(+)-methanandamide when compared to isotype or nonsilencing siRNA controls. ICAM-1-dependent anti-invasive cannabinoid effects were confirmed in primary tumor cells from a lung cancer patient. In athymic nude mice, CBD elicited a 2.6- and 3.0-fold increase of ICAM-1 and TIMP-1 protein in A549 xenografts, as compared to vehicle-treated animals, and an antimetastatic effect that was fully reversed by a neutralizing antibody against ICAM-1 [% metastatic lung nodules vs. isotype control (100%): 47.7% for CBD + isotype antibody and 106.6% for CBD + ICAM-1 antibody]. Overall, our data indicate that cannabinoids induce ICAM-1, thereby conferring TIMP-1 induction and subsequent decreased cancer cell invasiveness.     

S-Palmitoylation and S-Oleoylation of Rabbit and Pig Sarcolipin

Sarcolipin (SLN) is a regulatory peptide present in sarcoplasmic reticulum (SR) from skeletal muscle of animals. We find that native rabbit SLN is modified by a fatty acid anchor on Cys-9 with a palmitic acid in about 60% and, surprisingly, an oleic acid in the remaining 40%. SLN used for co-crystallization with SERCA1a (Winther, A. M., Bublitz, M., Karlsen, J. L., Moller, J. V., Hansen, J. B., Nissen, P., and Buch-Pedersen, M. J. (2013) Nature 495, 265–2691; Ref. 1) is also palmitoylated/oleoylated, but is not visible in crystal structures, probably due to disorder. Treatment with 1 m hydroxylamine for 1 h removes the fatty acids from a majority of the SLN pool. This treatment did not modify the SERCA1a affinity for Ca²⁺ but increased the Ca²⁺-dependent ATPase activity of SR membranes indicating that the S-acylation of SLN or of other proteins is required for this effect on SERCA1a. Pig SLN is also fully palmitoylated/oleoylated on its Cys-9 residue, but in a reverse ratio of about 40/60. An alignment of 67 SLN sequences from the protein databases shows that 19 of them contain a cysteine and the rest a phenylalanine at position 9. Based on a cladogram, we postulate that the mutation from phenylalanine to cysteine in some species is the result of an evolutionary convergence. We suggest that, besides phosphorylation, S-acylation/deacylation also regulates SLN activity.     

The topology of phosphogluconate dehydrogenases in rat liver microsomes

Rat liver microsomes are known to contain a 6-phosphogluconate dehydrogenase which differs from the 6-phosphogluconate dehydrogenase in the soluble fraction. Microsomes which were washed once bind the soluble phosphogluconate dehydrogenase more tightly than they do glucose-6-phosphate dehydrogenase. Microsomes washed three times in 0.15 M Tris-HCl, pH 8.0, contain only the microsomal 6-phosphogluconate dehydrogenase. Two observations show that this dehydrogenase is located in the cisternae. First, this dehydrogenase is inactive in intact, three times washed microsomes. Second, proteolytic inactivation of 6-phosphogluconate dehydrogenase like that of the cisternal enzyme glucose-6-phosphatase requires disruption of the membrane. Under the conditions used, detergent did not affect the proteolytic inactivation of NADPH-cytochrome c reductase, an enzyme located on the external surface. The excellent correspondence between the activations of hexose phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in microsomes at various stages of disruption of the microsomal membrane produced by detergent supports the earlier contention that these two dehydrogenases are reducing NADP in the same region of the microsomes. A similar experiment which shows an exact correspondence between the activations of 6-phosphogluconate dehydrogenase and mannose-6-phosphatase with increasing concentrations of detergent indicates that the activation of the dehydrogenase can be explained solely by the penetration of the substrates to the active dehydrogenase within the microsomes and strongly suggests that the dehydrogenase is catalytically active in the cisternae.