Major challenges of glycomics are to characterize a glycome and identify functional glycans as ligands for glycan-binding proteins (GBPs). To address these issues we have developed a general strategy termed shotgun glycomics. We focus on glycosphingolipids (GSLs), a challenging class of glycoconjugates recognized by toxins, antibodies, and GBPs. We derivatized GSLs extracted from cells with a heterobifunctional fluorescent tag suitable for covalent immobilization. Fluorescent GSLs were separated by multidimensional chromatography, quantified, and coupled to glass slides to create GSL shotgun microarrays. The microarrays were interrogated with cholera toxin, antibodies, and sera from patients with Lyme disease to identify biologically relevant GSLs that were subsequently characterized by mass spectrometry. Shotgun glycomics incorporating GSLs and potentially glycoprotein-derived glycans provides an approach to accessing the complex glycomes of animal cells and offers a strategy for focusing structural analyses on functionally significant glycans.

Xenotropic murine leukemia-related virus (XMRV) was identified in association with human prostate cancer and chronic fatigue syndrome. To examine the infection potential, kinetics, and tissue distribution of XMRV in an animal model, we inoculated five macaques with XMRV intravenously. XMRV established a persistent, chronic disseminated infection, with low transient viremia and provirus in blood lymphocytes during acute infection. Although undetectable in blood after about a month, XMRV viremia was reactivated at 9 months, confirming the chronicity of the infection. Furthermore, XMRV Gag was detected in tissues throughout, with wide dissemination throughout the period of monitoring. Surprisingly, XMRV infection showed organ-specific cell tropism, infecting CD4 T cells in lymphoid organs including the gastrointestinal lamina propria, alveolar macrophages in lung, and epithelial/interstitial cells in other organs, including the reproductive tract. Of note, in spite of the intravenous inoculation, extensive XMRV replication was noted in prostate during acute but not chronic infection even though infected cells were still detectable by fluorescence in situ hybridization (FISH) in prostate at 5 and 9 months postinfection. Marked lymphocyte activation occurred immediately postinfection, but antigen-specific cellular responses were undetectable. Antibody responses were elicited and boosted upon reexposure, but titers decreased rapidly, suggesting low antigen stimulation over time. Our findings establish a nonhuman primate model to study XMRV replication/dissemination, transmission, pathogenesis, immune responses, and potential future therapies.Xenotropic murine leukemia-related virus (XMRV) is a novel gammaretrovirus, initially identified in human prostate cancer using a Virochip DNA microarray (43) in men with a low-activity variant of RNASEL, an enzyme involved in innate immunity via type I interferons (14). Although related to murine leukemia virus (MLV) and probably acquired by zoonotic infection, human tissue-derived XMRV clearly segregates from other gammaretroviruses, genotypically arguing against the hypothesis that such human infection is acquired via repeated zoonotic transmission (43). The association of XMRV with prostate cancer has since been confirmed by other laboratories, albeit with a potentially different cellular tropism (34). In addition, association with RNASEL deficiency has been variable (1, 7, 15, 34), suggesting that low levels of RNASEL may not be a requirement for productive infection or viral propagation in humans. Nevertheless, the association of RNASEL mutations and prostate cancer has been reinforced by the recent discovery that a prostate cell line, 22Rv1, was derived from a patient with a low-activity RNASEL genotype (15). RNASEL dysfunction has also been associated with another disease, chronic fatigue syndrome (CFS) (8,20,21,38,42), which prompted an investigation into a potential association of XMRV with CFS. In a geographically restricted cohort, up to 67% of CFS patients were found ...

Biochemical studies of Chlamydomonas flagellar axonemes revealed that radial spoke protein (RSP) 3 is an A-kinase anchoring protein (AKAP). To determine the physiological role of PKA anchoring in the axoneme, an RSP3 mutant, pf14, was transformed with an RSP3 gene containing a mutation in the PKA-binding domain. Analysis of several independent transformants revealed that the transformed cells exhibit an unusual phenotype: a fraction of the cells swim normally; the remainder of the cells twitch feebly or are paralyzed. The abnormal/paralyzed motility is not due to an obvious deficiency of radial spoke assembly, and the phenotype cosegregates with the mutant RSP3. We postulated that paralysis was due to failure in targeting and regulation of axonemal cAMP-dependent protein kinase (PKA). To test this, reactivation experiments of demembranated cells were performed in the absence or presence of PKA inhibitors. Importantly, motility in reactivated cell models mimicked the live cell phenotype with nearly equal fractions of motile and paralyzed cells. PKA inhibitors resulted in a twofold increase in the number of motile cells, rescuing paralysis. These results confirm that flagellar RSP3 is an AKAP and reveal that a mutation in the PKA binding domain results in unregulated axonemal PKA activity and inhibition of normal motility. INTRODUCTIONEukaryotic cilia and flagella are highly conserved organelles that are required for diverse and vital motile and sensory functions (Afzelius, 2004;Snell et al., 2004;Pan et al., 2005;Quarmby and Parker, 2005). Motile cilia and flagella are capable of complex, carefully coordinated movements that are important for embryonic development, fertilization, and maintenance of epithelial tracts (Silflow and Lefebvre, 2001;El Zein et al., 2003;McGrath and Brueckner, 2003). Ciliary and flagellar movement is mediated by the axoneme, a highly ordered microtubule-based structure, composed of hundreds of conserved proteins (Avidor-Reiss et al., 2004;Li et al., 2004;Pazour et al., 2005). Within the axoneme, exact spatial and temporal regulation of dynein-driven microtubule sliding is required for the production of precisely formed bends (Satir, 1968;Summers and Gibbons, 1971;Shingyoji et al., 1977;Brokaw, 1991). However, the mechanisms that regulate dynein and modulate the size and shape of the axonemal bend, parameters referred to as waveform, are poorly understood. Experimental studies of Chlamydomonas and other organisms have shown that several key structural components of the axoneme, including the inner dynein arms, central pair apparatus, and radial spokes, are important in the control of axonemal waveform, in a process involving protein phosphorylation (Brokaw et al., 1982;Brokaw and Kamiya, 1987;Porter and Sale, 2000;Kamiya, 2002;Padma et al., 2003;Smith and Yang, 2004;White et al., 2005). Here, we focus on the role of the radial spokes and an associated axonemal cAMP-dependent protein kinase (PKA), which operates to regulate dynein activity and axonemal motility.Previous in vitro studies o...

Familiarity with the advantages and challenges of the various methods used for HbA1c testing is essential when establishing therapeutic management and goals based on HbA1c results, especially in populations with a high prevalence of Hb variants.

Peptide hormones are potent signaling molecules that coordinate animal physiology, behavior, and development. A key step in activation of these peptide signals is their proteolytic processing from propeptide precursors by a family of proteases, the subtilisin-like proprotein convertases (PCs). Here, we report the functional dissection of amontillado (amon), which encodes the Drosophila homolog of the mammalian PC2 protein, using cell-type specific inactivation and rescue experiments, and we show that amon is required in the islet-like adipokinetic hormone (AKH)–producing cells that regulate sugar homeostasis. In Drosophila, AKH acts analogously to vertebrate glucagon to increase circulating sugar levels from energy stores, while insulin-like peptides (DILPs) act to decrease sugar levels. amon mutant larvae have significantly reduced hemolymph sugar levels, and thus phenocopy larvae where the AKH–producing cells in the corpora cardiaca have been ablated. Reduction of amon expression in these cells via cell-specific RNA inactivation also results in larvae with reduced sugar levels while expression of amon in AKH cells in an amon mutant background rescues hypoglycemia. Hypoglycemia in larvae resulting from amon RNA inactivation in the AKH cells can be rescued by global expression of the akh gene. Finally, mass spectrometric profiling shows that the production of mature AKH is inhibited in amon mutants. Our data indicate that amon function in the AKH cells is necessary to maintain normal sugar homeostasis, that amon functions upstream of akh, and that loss of mature AKH is correlated with loss of amon activity. These observations indicate that the AKH propeptide is a proteolytic target of the amon proprotein convertase and provide evidence for a conserved role of PC2 in processing metabolic peptide hormones.