Functional UDP-xylose Transport across the Endoplasmic Reticulum/Golgi Membrane in a Chinese Hamster Ovary Cell Mutant Defective in UDP-xylose Synthase

In mammals, xylose is found as the first sugar residue of the tetrasaccharide GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser, initiating the formation of the glycosaminoglycans heparin/heparan sulfate and chondroitin/dermatan sulfate. It is also found in the trisaccharide Xylα1-3Xylα1-3Glcβ1-O-Ser on epidermal growth factor repeats of proteins, such as Notch. UDP-xylose synthase (UXS), which catalyzes the formation of the UDP-xylose substrate for the different xylosyltransferases through decarboxylation of UDP-glucuronic acid, resides in the endoplasmic reticulum and/or Golgi lumen. Since xylosylation takes place in these organelles, no obvious requirement exists for membrane transport of UDP-xylose. However, UDP-xylose transport across isolated Golgi membranes has been documented, and we recently succeeded with the cloning of a human UDP-xylose transporter (SLC25B4). Here we provide new evidence for a functional role of UDP-xylose transport by characterization of a new Chinese hamster ovary cell mutant, designated pgsI-208, that lacks UXS activity. The mutant fails to initiate glycosaminoglycan synthesis and is not capable of xylosylating Notch. Complementation was achieved by expression of a cytoplasmic variant of UXS, which proves the existence of a functional Golgi UDP-xylose transporter. A ∼200 fold increase of UDP-glucuronic acid occurred in pgsI-208 cells, demonstrating a lack of UDP-xylose-mediated control of the cytoplasmically localized UDP-glucose dehydrogenase in the mutant. The data presented in this study suggest the bidirectional transport of UDP-xylose across endoplasmic reticulum/Golgi membranes and its role in controlling homeostasis of UDP-glucuronic acid and UDP-xylose production.Xylose is only known to occur in two different mammalian glycans. First, xylose is the starting sugar residue of the common tetrasaccharide, GlcAβ1,3Galβ1,3Galβ1,4Xylβ1-O-Ser, attached to proteoglycan core proteins to initiate the biosynthesis of glycosaminoglycans (GAGs)² (1). Second, xylose is found in the trisaccharide Xylα1,3Xylα1,3Glcβ1-O-Ser in epidermal growth factor (EGF)-like repeats of proteins, such as blood coagulation factors VII and IX (2) and Notch (3) (Fig. 1). Two variants of O-xylosyltransferases (XylT1 and XylT2) are responsible for the initiation of glycosaminoglycan biosynthesis, which differ in terms of acceptor specificity and tissue distribution (4-7), and two different enzymatic activities have been identified that catalyze xylosylation of O-glucose residues added to EGF repeats (8-10). On Notch, O-glucose occurs on EGF repeats in a similar fashion as O-fucose, which modifications have been shown to influence ligand-mediated Notch signaling (11-16). Recently, rumi, the gene encoding the Notch O-glucosyltransferase in Drosophila, has been identified, and inactivation of the gene was found to cause a temperature-sensitive Notch phenotype (17). Although this finding clearly demonstrated that O-glucosylation is essential for Notch signaling, the importance of xylosylation for Notch functions remains ambiguous.Open in a separate windowFIGURE 1.UDP-xylose metabolism in mammalian cells. A, UDP-Xyl is synthesized in two steps from UDP-Glc by the enzymes UGDH, forming UDP-GlcA, and UXS, also referred to as UDP-glucuronic acid decarboxylase. UGDH is inhibited by the product of the second enzyme, UDP-Xyl (42). B, in mammals, UDP-Xyl is synthesized within the lumen of the ER/Golgi, where it is substrate for different xylosyltransferases incorporating xylose in the glycosaminoglycan core (XylT1 and XylT2) or in O-glucose-linked glycans. The nucleotide sugar transporter SLC35D1 (52) has been shown to transport UDP-GlcA over the ER membrane and SLC35B4 (29) to transport UDP-Xyl over the Golgi membrane. The function of this latter transporter is unclear.Several different Chinese hamster ovary (CHO) cell lines with defects in GAG biosynthesis have been isolated by screening for reduced incorporation of sulfate (18) and reduced binding of fibroblast growth factor 2 (FGF-2) (19, 20) and by direct selection with FGF-2 conjugated to the plant cytotoxin saporin (21). Isolated cells (called pgs, for proteoglycan synthesis mutants) (21) exhibited defects in various stages of GAG biosynthesis, ranging from the initiating xylosyltransferase to specific sulfation reactions (18, 19, 21-25). Mutants that affect overall GAG biosynthesis were shown to have a defect in the assembly of the common core tetrasaccharide. Interestingly, these latter mutants could be separated into clones in which GAG biosynthesis can be restored by the external addition of xylosides as artificial primers and those that cannot (18). The two mutants belonging to the first group are pgsA-745 and pgsB-761. Although pgs-745 is defective in XylT2 (4-6, 18), pgsB-761 exhibits a defect in galactosyltransferase I (B4GalT7), the enzyme that catalyzes the first step in the elongation of the xylosylated protein (25 (see Fig. 1B). Restoration of GAG biosynthesis in the latter mutant presumably occurs through a second β1-4-galactosyltransferase, able to act on xylosides when provided at high concentration but not on the endogenous protein-linked xylose.Here we describe the isolation of a third CHO cell line (pgsI-208) with the xyloside-correctable phenotype. The mutant is deficient in UDP-xylose synthase (UXS), also known as UDP-glucuronic acid decarboxylase. This enzyme catalyzes the synthesis of UDP-Xyl, the common donor substrate for the different xylosyltransferases, by decarboxylation of UDP-glucuronic acid. Importantly, UXS in the animal cell is localized in the lumen of the ER and/or Golgi (26-28), superseding at first sight the need for the Golgi UDP-xylose transporter, which has been recently cloned and characterized (29). Using this cell variant, experiments were designed that establish the functional significance of UDP-Xyl transport with respect to UDP-glucuronic acid production and xylosylation.     

Caspase-independent apoptosis in Friend’s erythroleukemia cells: role of mitochondrial ATP synthesis impairment in relocation of apoptosis-inducing factor and endonuclease G

Mitochondria have emerged as the central components of both caspase-dependent and independent apoptosis signalling pathways through release of different apoptogenic proteins. We previously documented that parental and differentiated Friend’s erythroleukemia cells were induced to apoptosis by oligomycin and H₂O₂ exposure, showing that the energy impairment occurring in both cases as a consequence of a severe mitochondrial F₀F₁ATPsynthase inactivation was a common early feature. Here we provide evidence for AIF and Endo G mitochondrio-nuclear relocation in both cases, as a component of caspase-independent apoptosis pathways. No detectable change in mitochondrial transmembrane potential and no variation in mitochondrial levels of Bcl-2 and Bax are observed. These results point to the osmotic rupture of the mitochondrial outer membrane as occurring in response to cell exposure to the two energy-impairing treatments under conditions preserving the mitochondrial inner membrane. A critical role of the mitochondrial F₀F₁ATP synthase inhibition in this process is also suggested.     

Development and Validation of a HPV-32 Specific PCR Assay

Background

The present work aims at determining HCV genotypes in patients with chronic HCV infection, in Gaza strip, Palestine. The most common risk factors for HCV transmission were also evaluated in conjunction with the genotyping data.

Results

The study shows that there are only two major genotypes of HCV in Gaza Strip: Genotype 1 (subtypes 1a and 1b) collectively contribute to 28.3% of the cases, and genotype 4 (subtypes 4a and 4c/d) collectively contribute to 64.1% of the cases. Mixed infection with the two genotypes was also present among 7.6% of the cases. In this study a statistically significant relationship was established between the distribution of these genotypes and the patients' living place, traveling history, history of blood transfusion and history of surgical operations.

Conclusion

The present study is the first to link HCV genotyping in Gaza strip with its possible roots of transmission. Traveling to endemic countries, especially Egypt; blood transfusion and surgical operations are major roots of HCV infection in Gaza strip. The results indicate that iatrogenic and nosocomial procedures may be responsible for the majority of HCV infections in Gaza strip.     

Lossless filter for multiple repeats with bounded edit distance

Background  

Identifying local similarity between two or more sequences, or identifying repeats occurring at least twice in a sequence, is an essential part in the analysis of biological sequences and of their phylogenetic relationship. Finding such fragments while allowing for a certain number of insertions, deletions, and substitutions, is however known to be a computationally expensive task, and consequently exact methods can usually not be applied in practice.     

Potential Contribution of Adipose Tissue to Elevated Serum Cystatin C in Human Obesity

Cystatin C, an endogenous inhibitor of cathepsin proteases has emerged as a biomarker of cardiovascular risk and reduced renal function. Epidemiological studies indicate that serum cystatin C increased in human obesity. Here, we evaluated the contribution of adipose tissue to this elevation, based on our previous observation that cystatin C is produced by in vitro differentiated human adipocytes. We measured serum cystatin C in 237 nonobese (age: 51 ± 0.8 years; BMI: 22.8 ± 0.11 kg/m²) and 248 obese subjects (age: 50 ± 0.8 years; BMI: 34.7 ± 0.29 kg/m²). Creatinine‐based estimated glomerular filtration rate (eGFR) was calculated to account for renal status. Cystatin C gene expression and secretion were determined on surgical adipose tissue biopsies in a distinct group of subjects. Serum cystatin C is elevated in obese subjects of both genders, independently of reduced eGFR. Cystatin C mRNA is expressed in subcutaneous and omental adipose tissue, at twice higher levels in nonadipose than in adipose cells. Gene expression and cystatin C release by adipose tissue explants increase two‐ to threefold in obesity. These data confirm elevation of serum cystatin C in human obesity and strongly argue for a contribution of increased production of cystatin C by enlarged adipose tissue. Because cystatin C has the potential to affect adipose tissue and vascular homeostasis through local and/or systemic inhibition of cathepsins, this study adds a new factor to the list of adipose tissue secreted bioactive molecules implicated in obesity and obesity‐linked complications.     

Impact of the Method of G6PD Deficiency Assessment on Genetic Association Studies of Malaria Susceptibility

Background

Clinical association studies have yielded varied results regarding the impact of glucose-6-phosphate dehydrogenase (G6PD) deficiency upon susceptibility to malaria. Analyses have been complicated by varied methods used to diagnose G6PD deficiency.

Methodology/Prinicipal Findings

We compared the association between uncomplicated malaria incidence and G6PD deficiency in a cohort of 601 Ugandan children using two different diagnostic methods, enzyme activity and G6PD genotype (G202A, the predominant East African allele). Although roughly the same percentage of males were identified as deficient using enzyme activity (12%) and genotype (14%), nearly 30% of males who were enzymatically deficient were wild-type at G202A. The number of deficient females was three-fold higher with assessment by genotype (21%) compared to enzyme activity (7%). Heterozygous females accounted for the majority (46/54) of children with a mutant genotype but normal enzyme activity. G6PD deficiency, as determined by G6PD enzyme activity, conferred a 52% (relative risk [RR] 0.48, 95% CI 0.31–0.75) reduced risk of uncomplicated malaria in females. In contrast, when G6PD deficiency was defined based on genotype, the protective association for females was no longer seen (RR = 0.99, 95% CI 0.70–1.39). Notably, restricting the analysis to those females who were both genotypically and enzymatically deficient, the association of deficiency and protection from uncomplicated malaria was again demonstrated in females, but not in males (RR = 0.57, 95% CI 0.37–0.88 for females).

Conclusions/Significance

This study underscores the impact that the method of identifying G6PD deficient individuals has upon association studies of G6PD deficiency and uncomplicated malaria. We found that G6PD-deficient females were significantly protected against uncomplicated malaria, but this protection was only seen when G6PD deficiency is described using enzyme activity. These observations may help to explain the discrepancy in some published association studies involving G6PD deficiency and uncomplicated malaria.     

RNA Editing Genes Associated with Extreme Old Age in Humans and with Lifespan in C. elegans

Background

The strong familiality of living to extreme ages suggests that human longevity is genetically regulated. The majority of genes found thus far to be associated with longevity primarily function in lipoprotein metabolism and insulin/IGF-1 signaling. There are likely many more genetic modifiers of human longevity that remain to be discovered.

Methodology/Principal Findings

Here, we first show that 18 single nucleotide polymorphisms (SNPs) in the RNA editing genes ADARB1 and ADARB2 are associated with extreme old age in a U.S. based study of centenarians, the New England Centenarian Study. We describe replications of these findings in three independently conducted centenarian studies with different genetic backgrounds (Italian, Ashkenazi Jewish and Japanese) that collectively support an association of ADARB1 and ADARB2 with longevity. Some SNPs in ADARB2 replicate consistently in the four populations and suggest a strong effect that is independent of the different genetic backgrounds and environments. To evaluate the functional association of these genes with lifespan, we demonstrate that inactivation of their orthologues adr-1 and adr-2 in C. elegans reduces median survival by 50%. We further demonstrate that inactivation of the argonaute gene, rde-1, a critical regulator of RNA interference, completely restores lifespan to normal levels in the context of adr-1 and adr-2 loss of function.

Conclusions/Significance

Our results suggest that RNA editors may be an important regulator of aging in humans and that, when evaluated in C. elegans, this pathway may interact with the RNA interference machinery to regulate lifespan.     

Bcl-2 is an integral component of mitotic chromosomes

We previously demonstrated that phospho-Thr56 Bcl-2 colocalizes with Ki-67 and nucleolin in nuclear structures in prophase cells and is detected on mitotic chromosomes in later mitotic phases. To gain insight into the fine localization of Bcl-2 on mitotic chromosomes, we further investigated Bcl-2 localization by immunostaining of Bcl-2 with known components of metaphase chromosomes and electron microscopic immunocytochemistry. Immunofluorescence analysis on HeLa mitotic cells together with chromatin immunoprecipitation assays showed that Bcl-2 is associated with the condensed chromatin. Co-immunostaining experiments performed on mitotic chromosome spreads demonstrated that Bcl-2 is not localized on the longitudinal axis of chromatids with the condensin complex, but partially colocalizes with histone H3 on some regions of the mitotic chromosome. Finally, most of the Bcl-2 staining overlaps with Ki-67 staining at the chromosome periphery. Bcl-2 localization at the periphery and over the mitotic chromosome was confirmed by immunoelectron microscopy on mitotic cells.Our results indicate that Bcl-2 is an integral component of the mitotic chromosome.