Sialic acids are widely expressed as terminal carbohydrates on glycoconjugates of eukaryotic cells. Sialylation is crucial for a variety of cellular functions, such as cell adhesion or signal recognition, and regulates the biological stability of glycoproteins. The key enzyme of sialic acid biosynthesis is the bifunctional UDP-N-acetylglucosamine-2-epimerase͞N-acetylmannosamine kinase (UDP-GlcNAc 2-epimerase), which catalyzes the first two steps of sialic acid biosynthesis in the cytosol. We report that inactivation of the UDP-GlcNAc 2-epimerase by gene targeting causes early embryonic lethality in mice, thereby emphasizing the fundamental role of this bifunctional enzyme and sialylation during development. The need of UDP-GlcNAc 2-epimerase for a defined sialylation process is exemplified with the polysialylation of the neural cell adhesion molecule in embryonic stem cells.

Muscle wasting (cachexia) is an incurable complication associated with chronic infection and cancers that leads to an overall poor prognosis for recovery. Tumor necrosis factor-␣ (TNF␣) is a key inflammatory cytokine associated with cachexia. TNF␣ inhibits myogenic differentiation and skeletal muscle regeneration through downstream effectors of the p53 cell death pathway including PW1/Peg3, bax, and caspases. We report that p53 is required for the TNF␣-mediated inhibition of myogenesis in vitro and contributes to muscle wasting in response to tumor load in vivo. We further demonstrate that PW1 and p53 participate in a positive feedback regulatory loop in vitro. Consistent with this observation, we find that the number of PW1-expressing stem cells in skeletal muscle declines significantly in p53 nullizygous mice. Furthermore, gene transfer of a dominant-negative form of PW1 into muscle tissue in vivo blocks myofiber atrophy in response to tumor load. Taken together, these results show a novel role for p53 in mediating muscle stem cell behavior and muscle atrophy, and point to new targets for the therapeutic treatment of muscle wasting.

N-Acetylglucosamine is produced by the endogenous degradation of glycoconjugates and by the degradation of dietary glycoconjugates by glycosidases. It enters the pathways of aminosugar metabolism by the action of N-acetylglucosamine kinase. In this study we report the isolation and characterization of a cDNA clone encoding the murine enzyme. An open reading frame of 1029 base pairs encodes 343 amino acids with a predicted molecular mass of 37.3 kDa. The deduced amino-acid sequence contains matches of the sequences of eight peptides derived from tryptic cleavage of rat N-acetylglucosamine kinase. The recombinant murine enzyme was functionally expressed in Escherichia coli BL21 cells, where it displays N-acetylglucosamine kinase activity as well as N-acetylmannosamine kinase activity.The complete cDNA sequence of human N-acetylglucosamine kinase was derived from the nucleotide sequences of several expressed sequence tags. An open reading frame of 1032 base pairs encodes 344 amino acids and a protein with a predicted molecular mass of 37.4 kDa. Similarities between human and murine N-acetylglucosamine kinase were 86.6% on the nucleotide level and 91.6% on the amino-acid level. Amino-acid sequences of murine and human N-acetylglucosamine kinase show sequence similarities to other sugar kinases, and all five sequence motifs necessary for the binding of ATP by sugar kinases are present.Tissue distribution of murine N-acetylglucosamine kinase revealed an ubiquitous occurrence of the enzyme and a very high expression in testis. The size of the murine mRNA was 1.35 kb in all tissues investigated, with the exception of testis, where it was 1.45 kb mRNA of the murine enzyme was continuously expressed during mouse development. mRNA of the human enzyme was expressed in all investigated human tissues, as well as in cancer cell lines. In both the tissues and the cancer cell lines, the human mRNA was 1.35 kb in size.

The chemokine stromal-derived factor-1a (SDF-1a, CXCL12) and its receptor CXCR4 are implicated as key mediators of hematopoietic stem cell retention, cancer metastasis, and HIV infection. Their role in myocardial infarction (MI) is not as well defined. The noninvasive in vivo quantitation of CXCR4 expression is central to understanding its importance in these diverse processes as well in the cardiac response to injury. Methods: Recombinant SDF-1a was radiolabeled under aprotic conditions and purified by gel-filtration chromatography (GFC) using high-specific-activity 99m Tc-S-acetylmercaptoacetyltriserine-Nhydroxysuccinimide ([ 99m Tc-MAS 3 ]-NHS) prepared by solid-phase preloading. Radiotracer stability and transmetallation under harsh conditions were quantified by GFC. Affinity, specificity, and maximum number of binding sites (B max ) were quantified, with adenoviralexpressed CXCR4 on nonexpressing cells and endogenous receptor on rat neonatal cardiomyocytes, using a high-throughput live-cell-binding assay. Blood half-life, biodistribution, and clearance of intravenously injected [ 99m Tc-MAS 3 ]-SDF-1a were quantified in Sprague-Dawley rats before and after experimentally induced MI. Results: [ 99m Tc-MAS 3 ]-SDF-1a could be prepared in 2 h total with a specific activity of 8.0 · 10 7 MBq/mmol (2,166 Ci/mmol) and a radiochemical purity greater than 98%. Degradation of the radiotracer after boiling for 5 min, with and without 1 mM dithiothreitol, and transmetallation in 100% serum at 37°C for 4 h were negligible. [ 99m Tc-MAS 3 ]-SDF-1a exhibits high specificity for CXCR4 on the surface of living rat neonatal cardiomyocytes, with an affinity of 2.7 6 0.9 nM and a B max of 4.8 · 10 4 binding sites per cell. After intravenous injection, 99m Tc-labeled SDF-1a displays a blood half-life of 25.8 6 4.6 min, rapid renal clearance with only 26.2 6 6.1 percentage injected dose remaining in the carcass at 2 h, consistently low uptake in most organs (,0.1 percentage injected dose per gram), and no evidence of blood-brain barrier penetration. After MI was induced, CXCR4 expression levels in the myocardium increased more than 5-fold, as quantified using [ 99m Tc-MAS 3 ]-SDF-1a and confirmed using confocal immunofluorescence. Conclusion: We describe a 99m Tclabeled SDF-1a radiotracer that can be used as a sensitive and specific probe for CXCR4 expression in vivo and demonstrate that this radiotracer is able to quantify changes in CXCR4 expression under different physiologic and pathologic states. Taken together, CXCR4 levels should now be quantifiable in vivo in a variety of animal model systems of human diseases.

Chemokines are small secreted proteins with chemoattractant properties that play a key role in inflammation, metastasis, and embryonic development. We previously demonstrated a non-chemotactic role for one such chemokine pair, stromal cell-derived factor-1α (SDF-1α) and its G-protein coupled receptor (GPCR), CXCR4. SDF-1/CXCR4 are expressed on cardiac myocytes and have direct consequences on cardiac myocyte physiology by inhibiting contractility in response to the non-selective β-adrenergic receptor (βAR) agonist, isoproterenol. Due to the importance of β-adrenergic signaling in heart failure pathophysiology, we investigated the underlying mechanism involved in CXCR4 modulation of βAR signaling. Our studies demonstrate activation of CXCR4 by SDF-1 leads to a decrease in βAR-induced PKA activity as assessed by cAMP accumulation and PKA-dependent phosphorylation of phospholamban (PLB), an inhibitor of SERCA2a. We determined CXCR4 regulation of βAR downstream targets is β2AR dependent. We demonstrated a physical interaction between CXCR4 and β2AR as determined by co-immunoprecipitation, confocal microscopy and BRET techniques. The CXCR4-β2AR interaction leads to G-protein signal modulation and suggests the interaction is a novel mechanism for regulating cardiac myocyte contractility. Chemokines are physiologically and developmentally relevant to myocardial biology and represent a novel receptor class of cardiac modulators. The CXCR4-β2AR complex could represent a hitherto unknown target for therapeutic intervention.