Phosphorylation of the norepinephrine transporter at threonine 258 and serine 259 is linked to protein kinase C-mediated transporter internalization

Recently, we have demonstrated the phosphorylation- and lipid raft-mediated internalization of the native norepinephrine transporter (NET) following protein kinase C (PKC) activation (Jayanthi, L. D., Samuvel, D. J., and Ramamoorthy, S. (2004) J. Biol. Chem. 279, 19315-19326). Here we tested an hypothesis that PKC-mediated phosphorylation of NET is required for transporter internalization. Phosphoamino acid analysis of 32P-labeled native NETs from rat placental trophoblasts and heterologously expressed wild type human NET (WT-hNET) from human placental trophoblast cells revealed that the phorbol ester (beta-PMA)-induced phosphorylation of NET occurs on serine and threonine residues. Beta-PMA treatment inhibited NE transport, reduced plasma membrane hNET levels, and stimulated hNET phosphorylation in human placental trophoblast cells expressing the WT-hNET. Substance P-mediated activation of the G alpha(q)-coupled human neurokinin 1 (hNK-1) receptor coexpressed with the WT-hNET produced effects similar to beta-PMA via PKC stimulation. In striking contrast, an hNET double mutant harboring T258A and S259A failed to show NE uptake inhibition and plasma membrane redistribution by beta-PMA or SP. Most interestingly, the plasma membrane insertion of the WT-hNET and hNET double mutant were not affected by beta-PMA. Although the WT-hNET showed increased endocytosis and redistribution from caveolin-rich plasma membrane domains following beta-PMA treatment, the hNET double mutant was completely resistant to these PKC-mediated effects. In addition, the PKC-induced phosphorylation of hNET double mutant was significantly reduced. In the absence of T258A and S259A mutations, alanine substitution of all other potential phosphosites within the hNET did not block PKC-induced phosphorylation and down-regulation. These results suggest that Thr-258 and Ser-259 serve as a PKC-specific phospho-acceptor site and that phosphorylation of this motif is linked to PKC-induced NET internalization.     

POM-POM2/cellulose synthase interacting1 is essential for the functional association of cellulose synthase and microtubules in Arabidopsis

In plants, regulation of cellulose synthesis is fundamental for morphogenesis and plant growth. Cellulose is synthesized at the plasma membrane, and the orientation of synthesis is guided by cortical microtubules; however, the guiding mechanism is currently unknown. We show that the conditional root elongation pom2 mutants are impaired in cell elongation, fertility, and microtubule-related functions. Map-based cloning of the POM-POM2 locus revealed that it is allelic to CELLULOSE SYNTHASE INTERACTING1 (CSI1). Fluorescently tagged POM2/CSI1s associated with both plasma membrane-located cellulose synthases (CESAs) and post-Golgi CESA-containing compartments. Interestingly, while CESA insertions coincided with cortical microtubules in the pom2/csi1 mutants, the microtubule-defined movement of the CESAs was significantly reduced in the mutant. We propose that POM2/CSI1 provides a scaffold between the CESAs and cortical microtubules that guide cellulose synthesis.     

Rapid genomic-scale analysis of Escherichia coli O104:H4 by using high-resolution alternative methods to next-generation sequencing

Two technologies, involving DNA microarray and optical mapping, were used to quickly assess gene content and genomic architecture of recent emergent Escherichia coli O104:H4 and related strains. In real-time outbreak investigations, these technologies can provide congruent perspectives on strain, serotype, and pathotype relationships. Our data demonstrated clear discrimination between clinically, temporally, and geographically distinct O104:H4 isolates and rapid characterization of strain differences.     

Characterization of the metabolic flux and apoptotic effects of O-hydroxyl- and N-acyl-modified N-acetylmannosamine analogs in Jurkat cells

The supplementation of the sialic acid biosynthetic pathway with exogenously supplied N-acetylmannosamine (ManNAc) analogs has many potential biomedical and biotechnological applications. In this work, we explore the structure-activity relationship of Man-NAc analogs on cell viability and metabolic flux into the sialic acid biosynthetic pathway to gain a better understanding of the fundamental biology underlying "glycosylation engineering" technology. A panel of ManNAc analogs bearing various modifications on the hydroxyl groups as well as substitutions at the N-acyl position was investigated. Increasing the carbon chain length of ester derivatives attached to the hydroxyl groups increased the metabolic efficiency of sialic acid production, whereas similar modification to the N-acyl group decreased efficiency. In both cases, increases in chain length decreased cell viability; DNA ladder formation, Annexin V-FITC two-dimensional flow cytometry assays, caspase-3 activation, and down-regulation of sialoglycoconjugate-processing enzymes established that the observed growth inhibition and toxicity resulted from apoptosis. Two of the panel of 12 analogs tested, specifically Ac(4)ManNLev and Ac(4) ManNHomoLev, were highly toxic. Interestingly, both of these analogs maintained a ketone functionality in the same position relative to the core monosaccharide structure, and both also inhibited flux through the sialic acid pathway (the remainder of the less toxic analogs either increased or had no measurable impact on flux). These results provide fundamental insights into the role of sialic acid metabolism in apoptosis by demonstrating that ManNAc analogs can modulate apoptosis both indirectly via hydroxylgroup effects and directly through N-acyl-group effects.     

Aluminum concentrations in tissues of rats: effect of soft drink packaging

Aluminum is a commonly occurring trace element for which no nutritional requirements have been set. Some non-conclusive evidence exists suggesting a need of aluminum for growth, reproduction or health of man and animals. There is concern that exposure or consumption of aluminum may be toxic to humans and animals. The objective of the current study was to compare tissue levels of aluminum of rats fed soft drinks packaged in aluminum cans, glass bottles or distilled water. Thirty male weanling rats (Sprague-Dawley) were divided into three treatment groups of 10 rats each. All rats were fed rodent chow ad libitum throughout the study. Three different fluids, i.e. distilled water, diet soft drinks from aluminum cans and diet soft drinks from glass bottles, were fed for a period of 3 weeks. Aluminum contents of tissues were measured by atomic absorption spectrophotometry. Canned soft drink fed rats had significantly higher blood, liver and bone aluminum concentration than rats that were given glass bottled soft drink. There was a 69% higher bone aluminum concentration and 16% lower femur weight in rats fed aluminum canned soft drinks when compared with rats fed with distilled water.     

On the intracellular localization of the ecdysone 20-monooxygenase in Musca domestica. L

The cytochrome P-450-dependent 20-monooxygenation of ecdysone is catalyzed both by mitochondria and microsomes isolated from Musca domestica (L.) larvae; however, about 50% of the activity is associated with mitochondria, and 37% is associated with microsomes. Pretreatment of larvae with ecdysone results in an increase in Vmax and a decrease in Km values in mitochondria but not in microsomes. Phenobarbital, a known cytochrome P-450 inducer, increases the cytochrome P-450 levels in microsomes without affecting the 20-monooxygenase activity, but both the cytochrome P-450 levels and monooxygenase activity are depressed in mitochondria from phenobarbital-pretreated larvae. The ecdysone 20-monooxygenase activity is equally distributed between mitochondria and microsomes in adult insects. Pretreatment of the insects with ecdysone does not significantly modify the 20-monooxygenase activity of either mitochondrial or microsomal fractions, but the cytochrome P-450 levels are reduced in mitochondria. Phenobarbital also depresses the mitochondrial cytochrome P-450 levels while markedly increasing the microsomal cytochrome P-450 levels. However, no significant changes in ecdysone 20-monooxygenase activity are produced by phenobarbital pretreatment. The effects of ecdysone on adult cytochrome P-450 are mostly evidenced in mitochondria isolated from females, whereas in males the changes are not statistically significant. It is concluded that the mitochondrial ecdysone 20-monooxygenase is under regulatory control by ecdysone in the larval stage, which suggests that only the mitochondrial activity has a physiological role during insect development in M. domestica. In adults, both the mitochondrial and microsomal ecdysone 20-monooxygenase activities are not responsive to ecdysone, which, coupled to their high Km values, indicates that the reaction may not be of physiological importance in adult insects and that the mitochondrial cytochrome P-450 species being depressed by ecdysone in females are possibly not involved in ecdysone metabolism.     

Metabolic installation of thiols into sialic acid modulates adhesion and stem cell biology

Metabolic 'oligosaccharide engineering' methods based on N-acetyl-D-mannosamine (ManNAc) analogs allow the glycocalyx of living cells to be remodeled. Herein we report the analog Ac(5)ManNTGc (1) that enables thiols to be expressed in surface sialic acids. By locating this versatile functional group on the outer periphery of normally nonadhesive human Jurkat cells, we obtained spontaneous cell-cell clustering and attachment to complementary maleimide-derivatized substrates. When analyzed in human embryoid body-derived (hEBD) stem cells, Ac(5)ManNTGc induced beta-catenin expression and altered cell morphology, consistent with neuronal differentiation. Notably, these effects were modulated by the growth substrate of the cells, with a stronger response observed on a gold surface than on glass. Together, these results establish sugar analogs as small-molecule tools for tissue engineering by providing a method for attaching cells to scaffolds via their surface carbohydrates as well as offering a means to influence stem cell fates.