Rab2 requires PKC iota/lambda to recruit beta-COP for vesicle formation

The small GTPase Rab2 initiates the recruitment of soluble components necessary for protein sorting and recycling from pre-Golgi intermediates. Our previous studies showed that Rab2 required protein kinase C (PKC) or a PKC-like protein to recruit β-COP to membrane (Tisdale EJ, Jackson M. Rab2 protein enhances coatomer recruitment to pre-Golgi intermediates. J Biol Chem 1998;273: 17269–17277). We investigated the role of PKC in Rab2 function by first determining the active isoform that associates with membranes used in our assay. Western blot analysis detected three isoforms: PKCα, Γ and ι/Λ. A quantitative binding assay was used to measure recruitment of these kinases when incubated with Rab2. Only PKCι/Λ translocated to membrane in a dose-dependent manner. Microsomes treated with anti-PKCι/Λ lost the ability to bind β-COP, suggesting that Rab2 requires PKCι/Λ for β-COP recruitment. The recruitment of β-COP to membranes is not regulated by PKCι/Λ kinase activity. However, PKCι/Λ kinase activity was necessary for Rab2-mediated vesicle budding. We found that the addition of either a kinase-deficient PKCι/Λ mutant or atypical PKC pseudosubstrate peptide to the binding assay drastically reduced vesicle formation. These data suggest that Rab2 causes translocation of PKCι/Λ to v ¯esicular t ¯ubular c ¯lusters (VTCs), which promotes the recruitment of COPI to generate retrograde-transport vesicles.     

Cloning and functional analysis of the rhesus macaque ABCG2 gene. Forced expression confers an SP phenotype among hematopoietic stem cell progeny in vivo

Hematopoietic cells can be highly enriched for repopulating ability based upon the efflux of the fluorescent Hoechst 33342 dye by sorting for SP (side population) cells, a phenotype attributed to expression of ABCG2, a member of the ABC transporter superfamily. Intriguingly, murine studies suggest that forced ABCG2 expression prevents hematopoietic differentiation. We cloned the full-length rhesus ABCG2 and introduced it into a retroviral vector. ABCG2-transduced human peripheral blood progenitor cells (PBPCs) acquired the SP phenotype but showed significantly reduced growth compared with control. Two rhesus macaques received autologous PBPCs split for transduction with the ABCG2 or control vectors. Marking levels were similar between fractions with no discrepancy between bone marrow and peripheral blood marking. Analysis for the SP phenotype among bone marrow and mature blood populations confirmed ABCG2 expression at levels predicted by vector copy number long term, demonstrating no block to differentiation in the large animal. In vitro studies showed selective protection against mitoxantrone among ABCG2-transduced rhesus PBPCs. Our results confirm the existence of rhesus ABCG2, establish its importance in conferring the SP phenotype, suggest no detrimental effect of its overexpression upon differentiation in vivo, and imply a potential role for its overexpression as an in vivo selection strategy for gene therapy applications.     

Downregulation of ubiquitin-dependent proteolysis by eicosapentaenoic acid in acute starvation.

A number of acute wasting conditions are associated with an upregulation of the ubiquitin-proteasome system in skeletal muscle. Eicosapentaenoic acid (EPA) is effective in attenuating the increased protein catabolism in muscle in cancer cachexia, possibly due to inhibition of 15-hydroxyeicosatetraenoic acid (15-HETE) formation. To determine if a similar pathway is involved in other catabolic conditions, the effect of EPA on muscle protein degradation and activation of the ubiquitin-proteasome pathway has been determined during acute fasting in mice. When compared with a vehicle control group (olive oil) there was a significant decrease in proteolysis of the soleus muscles of mice treated with EPA after starvation for 24 h, together with an attenuation of the proteasome "chymotryptic-like" enzyme activity and the induction of the expression of the 20S proteasome alpha-subunits, the 19S regulator and p42, an ATPase subunit of the 19S regulator in gastrocnemius muscle, and the ubiquitin-conjugating enzyme E2(14k). The effect was not shown with the related (n-3) fatty acid docosahexaenoic acid (DHA) or with linoleic acid. However, 2,3,5-trimethyl-6-(3-pyridylmethyl)1,4-benzoquinone (CV-6504), an inhibitor of 5-, 12- and 15-lipoxygenases also attenuated muscle protein catabolism, proteasome "chymotryptic-like" enzyme activity and expression of proteasome 20S alpha-subunits in soleus muscles from acute fasted mice. These results suggest that protein catabolism in starvation and cancer cachexia is mediated through a common pathway, which is inhibited by EPA and is likely to involve a lipoxygenase metabolite as a signal transducer.     

Glyceraldehyde-3-phosphate dehydrogenase is required for vesicular transport in the early secretory pathway

Protein transport in the early secretory pathway requires Rab2 GTPase. This protein promotes the recruitment of soluble components that participate in protein sorting and recycling from pre-Golgi intermediates (vesicular tubular clusters (VTCs)). We previously reported that a constitutively activated form of Rab2 (Q65L) as well as Rab2 wild type promoted vesicle formation from VTCs. These vesicles contained Rab2, beta-COP, p53/gp58, and protein kinase Ciota/lambda but lacked anterograde-directed cargo. To identify other candidate Rab2 effectors, the polypeptide composition of the vesicles was further analyzed. We found that vesicles released in response to Rab2 also contained the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). To study the relationship of this enzyme to Rab2 function, we performed a quantitative binding assay to measure recruitment of GAPDH to membrane when incubated with Rab2. Rab2-treated microsomes showed a 5-10-fold increase in the level of membrane-associated GAPDH. We generated an affinity-purified anti-GAPDH polyclonal to study the biochemical role of GAPDH in the early secretory pathway. The antibody arrests transport of a reporter molecule in an assay that reconstitutes ER to Golgi traffic. Furthermore, the affinity-purified antibody blocked the ability of Rab2 to recruit GAPDH to membrane. However, the antibody did not interfere with Rab2 stimulated vesicle release. These data suggest that GAPDH is required for ER to Golgi transport. We propose that membranes incubated with anti-GAPDH and Rab2 form "dead end" vesicles that are unable to transport and fuse with the acceptor compartment.     

Development of a sensitive chemiluminescent neuraminidase assay for the determination of influenza virus susceptibility to zanamivir

Determination of the sensitivity of influenza viruses to neuraminidase (NA) inhibitors is presently based on assays of NA function because, unlike available cell culture methods, the results of such assays are predictive of susceptibility in vivo. At present the most widely used substrate in assays of NA function is the fluorogenic reagent 2'-O-(4-methylumbelliferyl)-N-acetylneuraminic acid (MUN). A rapid assay with improved sensitivity is required because a proportion of clinical isolates has insufficient NA to be detectable in the current fluorogenic assay, and because some mutations associated with resistance to NA inhibitors reduce the activity of the enzyme. A chemiluminescence-based assay of NA activity has been developed that uses a 1,2-dioxetane derivative of sialic acid (NA-STAR) as the substrate. When compared with the fluorogenic assay, use of the NA-STAR substrate results in a 67-fold reduction in the limit of detection of the NA assay, from 200 pM (11 fmol) NA to 3 pM (0.16 fmol) NA. A panel of isolates from phase 2 clinical studies of zanamivir, which were undetectable in the fluorogenic assay, was tested for activity using the NA-STAR substrate. Of these 12 isolates with undetectable NA activity, 10 (83%) were found to have detectable NA activity using the NA-STAR substrate. A comparison of sensitivity to zanamivir of a panel of influenza A and B viruses using the two NA assay methods has been performed. IC(50) values for zanamivir using the NA-STAR were in the range 1.0-7.5 nM and those for the fluorogenic assay in the range 1. 0-5.7 nM (n = 6). The NA-STAR assay is a highly sensitive, rapid assay of influenza virus NA activity that is applicable to monitoring the susceptibility of influenza virus clinical isolates to NA inhibitors.     

Extensive labeling with [3H]ethanolamine of a hydrophilic protein of animal cells

Murine T-lymphomas and Thy-1- mutants were labeled overnight with [3H]ethanolamine to detect proteins which possess a glycophospholipid anchor. When labeled cells were treated with 10% trichloroacetic acid and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, both Thy-1 and a second intensely labeled protein (46 kDa) were observed. The presence of the radiolabeled 46-kDa protein in wild type and class E Thy-1 negative cells (cells in which Thy-1 is synthesized but cannot be labeled with [3H]ethanolamine) suggested incorporation into a distinct moiety. Labeling of the 46-kDa protein with [3H]ethanolamine is rapidly inhibited by cycloheximide. Further characterization of the 46-kDa protein by subcellular fractionation and Triton X-114 partitioning indicated that the protein is located in the cytosol. The protein is basic and does not bind to either concanavalin A or wheat germ agglutinin. Labeling of a 46-kDa protein has also been demonstrated in Chinese hamster ovary, COS, rat myeloma, cloned human T-lymphocytes, and HeLa cells. Pronase digestion of the [3H]ethanolamine-labeled 46-kDa protein of wild type lymphoma cells generated a nonbasic and polar labeled fragment which is labile to strong acid and base ([3H]ethanolamine is liberated), insensitive to periodate oxidation and alkaline phosphatase, and does not bind to concanavalin A or wheat germ agglutinin. Judging from methylation studies, the labeled ethanolamine residue does not contain a free amino group. Based on these results, we report a novel post-translational modification of selected protein(s) by the covalent addition of [3H]ethanolamine.     

Hydroxyurea-Increased Fetal Hemoglobin Is Associated with Less Organ Damage and Longer Survival in Adults with Sickle Cell Anemia

Background

Adults with sickle cell anemia (HbSS) are inconsistently treated with hydroxyurea.

Objectives

We retrospectively evaluated the effects of elevating fetal hemoglobin with hydroxyurea on organ damage and survival in patients enrolled in our screening study between 2001 and 2010.

Methods

An electronic medical record facilitated development of a database for comparison of study parameters based on hydroxyurea exposure and dose. This study is registered with ClinicalTrials.gov, number NCT00011648.

Results

Three hundred eighty-three adults with homozygous sickle cell disease were analyzed with 59 deaths during study follow-up. Cox regression analysis revealed deceased subjects had more hepatic dysfunction (elevated alkaline phosphatase, Hazard Ratio = 1.005, 95% CI 1.003–1.006, p<0.0.0001), kidney dysfunction (elevated creatinine, Hazard Ratio = 1.13, 95% CI 1.00–1.27, p = 0.043), and cardiopulmonary dysfunction (elevated tricuspid jet velocity on echocardiogram, Hazard Ratio = 2.22, 1.23–4.02, p = 0.0082). Sixty-six percent of subjects were treated with hydroxyurea, although only 66% of those received a dose within the recommended therapeutic range. Hydroxyurea use was associated with improved survival (Hazard Ratio = 0.58, 95% CI 0.34–0.97, p = 0.040). This effect was most pronounced in those taking the recommended dose of 15–35 mg/kg/day (Hazard Ratio 0.36, 95% CI 0.17–0.73, p = 0.0050). Hydroxyurea use was not associated with changes in organ function over time. Further, subjects with higher fetal hemoglobin responses to hydroxyurea were more likely to survive (p = 0.0004). While alkaline phosphatase was lowest in patients with the best fetal hemoglobin response (95.4 versus 123.6, p = 0.0065 and 96.1 versus 113.6U/L, p = 0.041 at first and last visits, respectively), other markers of organ damage were not consistently improved over time in patients with the highest fetal hemoglobin levels.

Conclusions

Our data suggest that adults should be treated with the maximum tolerated hydroxyurea dose, ideally before organ damage occurs. Prospective studies are indicated to validate these findings.     

What's in a name; Genetic structure in Solanum section Petota studied using population-genetic tools

Background  

The taxonomy and systematic relationships among species of Solanum section Petota are complicated and the section seems overclassified. Many of the presumed (sub)species from South America are very similar and they are able to exchange genetic material. We applied a population genetic approach to evaluate support for subgroups within this material, using AFLP data. Our approach is based on the following assumptions: (i) accessions that may exchange genetic material can be analyzed as if they are part of one gene pool, and (ii) genetic differentiation among species is expected to be higher than within species.     

Synergizing metabolic flux analysis and nucleotide sugar metabolism to understand the control of glycosylation of recombinant protein in CHO cells

Background

The photorespiratory nitrogen cycle in C₃ plants involves an extensive diversion of carbon and nitrogen away from the direct pathways of assimilation. The liberated ammonia is re-assimilated, but up to 25% of the carbon may be released into the atmosphere as CO₂. Because of the loss of CO₂ and high energy costs, there has been considerable interest in attempts to decrease the flux through the cycle in C₃ plants. Transgenic tobacco plants were generated that contained the genes gcl and hyi from E. coli encoding glyoxylate carboligase (EC 4.1.1.47) and hydroxypyruvate isomerase (EC 5.3.1.22) respectively, targeted to the peroxisomes. It was presumed that the two enzymes could work together and compete with the aminotransferases that convert glyoxylate to glycine, thus avoiding ammonia production in the photorespiratory nitrogen cycle.

Results

When grown in ambient air, but not in elevated CO₂, the transgenic tobacco lines had a distinctive phenotype of necrotic lesions on the leaves. Three of the six lines chosen for a detailed study contained single copies of the gcl gene, two contained single copies of both the gcl and hyi genes and one line contained multiple copies of both gcl and hyi genes. The gcl protein was detected in the five transgenic lines containing single copies of the gcl gene but hyi protein was not detected in any of the transgenic lines. The content of soluble amino acids including glycine and serine, was generally increased in the transgenic lines growing in air, when compared to the wild type. The content of soluble sugars, glucose, fructose and sucrose in the shoot was decreased in transgenic lines growing in air, consistent with decreased carbon assimilation.

Conclusions

Tobacco plants have been generated that produce bacterial glyoxylate carboligase but not hydroxypyruvate isomerase. The transgenic plants exhibit a stress response when exposed to air, suggesting that some glyoxylate is diverted away from conversion to glycine in a deleterious short-circuit of the photorespiratory nitrogen cycle. This diversion in metabolism gave rise to increased concentrations of amino acids, in particular glutamine and asparagine in the leaves and a decrease of soluble sugars.