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Toxoplasmosis is a worldwide parasitosis that is usually asymptomatic; cell-mediated immunity, particularly T cells, is a crucial mediator of the immune response against this parasite. Membrane protein expression has been studied for a long time in T lymphocytes, providing vital information to determine functional checkpoints. However, less is known about the role of post-translational modifications in T cell function. Glycosylation plays essential roles during maturation and function; particularly, sialic acid modulation is determinant for accurate T cell regulation of processes like adhesion, cell-cell communication, and apoptosis induction. Despite its importance, the role of T cell sialylation during infection remains unclear. Herein, we aimed to evaluate whether different membrane sialylation motifs are modified in T cells during acute Toxoplasma gondii infection using different lectins. To this end, BALB/c Foxp3EGFP mice were infected with T. gondii, and on days 3, 7, and 10 post-infection, splenocytes were obtained to analyze conventional (Foxp3-) CD4+ and CD8+ populations by flow cytometry. Among the different lectins used for analysis, only Sambucus nigra lectin, which detects sialic acid a2,6 linkages, revealed two distinctive populations (SNBright and SN-/Dim) after infection. Further characterization of CD4+ and CD8+ SN-/Dim lymphocytes showed that these are highly activated cells, with a TEf/EM or TCM phenotype that produce high IFN-γ levels, a previously undescribed cell state. This work demonstrates that glycan membrane analysis in T cells reveals previously overlooked functional states by evaluating only protein expression. [ABSTRACT FROM AUTHOR]

TC25G alloy was heat treated at 950 °C/3 h, AC + 580 °C/6 h, AC and the bimodal structure with primary α phase + β transition structure was obtained. The creep properties of the alloy were tested in 550–600 °C/150–250 MPa. The results show that the precipitations of silicide and α2 phase is accompanied by the creep process. α2 phase plays a dispersion strengthening role in both the primary and steady-state creep stages. However, in the accelerated creep stage, the mechanism of α2 phase and dislocation changes from cutting mechanism to bypassing mechanism, and the strengthening effect is weakened. Silicide inhibits grain boundary slip mainly in the primary creep stage, and inhibits dislocation slip in the steady-state and accelerated creep stages. At 550 °C, n = 1.6 and Q = 280–371 kJ/mol (150–250 MPa) indicate that the creep of the alloy is a self-diffusion process, and the creep deformation is mainly controlled by dislocation slip. At 570–600 °C, n = 3.2 indicates that the dislocation climb controls the creep deformation. Meanwhile, compared with Q = 274 kJ/mol at low stress (150 MPa), Q = 365 kJ/mol at the high stress (200–250 MPa) indicates that the second phase precipitation enhancement is enhanced. [ABSTRACT FROM AUTHOR]

Toxoplasmosis is a worldwide parasitosis that is usually asymptomatic; cell-mediated immunity, particularly T cells, is a crucial mediator of the immune response against this parasite. Membrane protein expression has been studied for a long time in T lymphocytes, providing vital information to determine functional checkpoints. However, less is known about the role of post-translational modifications in T cell function. Glycosylation plays essential roles during maturation and function; particularly, sialic acid modulation is determinant for accurate T cell regulation of processes like adhesion, cell-cell communication, and apoptosis induction. Despite its importance, the role of T cell sialylation during infection remains unclear. Herein, we aimed to evaluate whether different membrane sialylation motifs are modified in T cells during acute Toxoplasma gondii infection using different lectins. To this end, BALB/c Foxp3EGFP mice were infected with T. gondii, and on days 3, 7, and 10 post-infection, splenocytes were obtained to analyze conventional (Foxp3-) CD4+ and CD8+ populations by flow cytometry. Among the different lectins used for analysis, only Sambucus nigra lectin, which detects sialic acid α2,6 linkages, revealed two distinctive populations (SNBright and SN-/Dim) after infection. Further characterization of CD4+ and CD8+ SN-/Dim lymphocytes showed that these are highly activated cells, with a TEf/EM or TCM phenotype that produce high IFN-γ levels, a previously undescribed cell state. This work demonstrates that glycan membrane analysis in T cells reveals previously overlooked functional states by evaluating only protein expression.

The monocyte adhesion to endothelial cells is an early step in chronic inflammation. Interferon-γ (IFN-γ) is regarded as a master regulator of inflammation development. However, the significance and mechanisms of IFN-γ in the monocyte adhesion to endothelial cells remains largely unknown. IFN-γ up-regulates PD-L1 on various types of cells. Here, we performed flow cytometry to examine the contribution of IFN-γ-induced PD-L1 expression on monocyte adhesion to endothelial cells. Up-regulation of PD-L1 by IFN-γ enhanced the adhesion of monocytes to endothelial cells. By immunoprecipitation and lectin blot, PD-L1 in endothelial cells interacted with CD169/Siglec 1 in monocytes depending on the α2,3-sialylation of PD-L1. ST3Gal family (ST3β-galactoside α-2,3-sialyltransferase) was the major glycosyltransferase responsible for the α2,3-sialylation of membrane proteins. Down-regulation of ST3Gal4 by RNAinterference partially reduced the α2,3-sialylation of PD-L1 and the PD-L1-CD169 interaction. Finally, purified PD-L1 protein with α2,3-sialylation, but not PD-L1 protein without α2,3-sialylation, partially reduced IFN-γ-induced monocyte adhesion to endothelial cells. These findings provide evidence that the interaction between PD-L1 and CD169 promoted monocyte adhesion to endothelial cells and might elucidate a new mechanism of monocyte adhesion to endothelial cells. [ABSTRACT FROM AUTHOR]

Oxidative stress contributes to the pathogenesis of neurodegenerative diseases. With the aim to find reagents that reduce oxidative stress, a phage display library was screened for peptides mimicking α2,6-sialyllactose (6′-SL), which is known to beneficially influence neural functions. Using Sambucus nigra lectin, which specifically binds to 6′-SL, we screened a phage display library and found a peptide comprising identical sequences of 12 amino acids. Mimetic peptide, reverse peptide and scrambled peptide were tested for inhibition of 6′-SL binding to the lectin. Indeed, lectin binding to 6′-SL was inhibited by the most frequently identified mimetic peptide, but not by the reverse or scrambled peptides, showing that this peptide mimics 6′-SL. Functionally, mimetic peptide, but not the reverse or scrambled peptides, increased viability and expression of neural cell adhesion molecule L1 in SK-N-SH human neuroblastoma cells, and promoted survival and neurite outgrowth of cultured mouse cerebellar granule neurons challenged by H2O2-induced oxidative stress. The combined results indicate that the 6′-SL mimetic peptide promotes neuronal survival and neuritogenesis, thus raising hopes for the treatment of neurodegenerative diseases. This study was approved by the Medical Ethics Committee of Shantou University Medical College, China (approval No. SUMC 2014-004) on February 20, 2014.

ST3Gal IV is one of the principal sialyltransferases responsible for the biosynthesis of α2, 3-sialic acid to the termini N-glycans or O-glycans of glycoproteins and glycolipids. It has been reported that ST3Gal IV expression is associated with gastric carcinoma, pancreatic adenocarcinoma and breast cancer. While the expression and functions of ST3Gal IV in cervical cancer are still poorly understood. In this study, we found that ST3Gal IV was downregulated in human cervical cancer tissues compared to normal cervix tissues, and ST3Gal IV expression was negatively associated with the pathological grade of cervical cancer. ST3Gal IV upregulation inhibited the growth and proliferation of cervical cancer HeLa and SiHa cells in vitro and in vivo. Furthermore, ST3Gal IV overexpression enhanced the expression of several Notch pathway components such as Jagged1, Notch1, Hes1 and Hey1, while cell cycle protein expression like Cyclin D1, Cyclin E1, CDK2 and CDK4 were decreased. These results indicate that expression of ST3Gal IV is reduced in cervical cancer and plays a negative role in cell proliferation via Notch/p21/CDKs signaling pathway. Thus, sialyltransferase ST3Gal IV might be a target for the diagnosis and therapy of cervical cancer.

ST3Gal IV is one of the principal sialyltransferases responsible for the biosynthesis of α2, 3-sialic acid to the termini N -glycans or O- glycans of glycoproteins and glycolipids. It has been reported that ST3Gal IV expression is associated with gastric carcinoma, pancreatic adenocarcinoma and breast cancer. While the expression and functions of ST3Gal IV in cervical cancer are still poorly understood. In this study, we found that ST3Gal IV was downregulated in human cervical cancer tissues compared to normal cervix tissues, and ST3Gal IV expression was negatively associated with the pathological grade of cervical cancer. ST3Gal IV upregulation inhibited the growth and proliferation of cervical cancer HeLa and SiHa cells in vitro and in vivo. Furthermore, ST3Gal IV overexpression enhanced the expression of several Notch pathway components such as Jagged1, Notch1, Hes1 and Hey1, while cell cycle protein expression like Cyclin D1, Cyclin E1, CDK2 and CDK4 were decreased. These results indicate that expression of ST3Gal IV is reduced in cervical cancer and plays a negative role in cell proliferation via Notch/p21/CDKs signaling pathway. Thus, sialyltransferase ST3Gal IV might be a target for the diagnosis and therapy of cervical cancer. [ABSTRACT FROM AUTHOR]

The present study investigated the effect of rutin on high glucose-induced actin, a2, smooth muscle, aorta (ACTA2) and p38 protein expression in diabetic nephropathy (DN). Human mesangial cells were divided into a control group, high glucose-induced mesangial cell group, high glucose + captopril group, and high glucose + rutin group (low, middle and high doses of rutin). Cell viability, adenosine 5'-triphosphate (ATP) content, cell cycle, and ACTA2 and p38 protein expression were examined using MTT assay, ATP assay kit, flow cytometry and immunofluorescence staining in cultured human mesangial cells, respectively. Cell viability, ATP content, and ACTA2 and p38 expression increased significantly in high glucose-induced mesangial cells (P<0.05). However, at concentrations of 0.2, 0.4 and 0.8 µmol/l rutin was able to inhibit high glucose-induced human mesangial cell viability, ATP content, and ACTA2 and p38 expression and improve the cell cycle progression of mesangial cells. In conclusion, ACTA2 and p38 proteins may have important roles in DN. Rutin may inhibit the expression of ACTA2 and p38 and may be utilized in the prevention and treatment of DN. [ABSTRACT FROM AUTHOR]

Background: Various adjuvants are being used with local anesthetics for prolongation of intraoperative and postoperative analgesia. Dexmedetomidine, the highly selective 2 adrenergic agonist is a new neuraxial adjuvant gaining popularity. Aim: The purpose of this study was to compare the onset, duration of sensory and motor block, hemodynamic effects, postoperative analgesia, and adverse effects of dexmedetomidine, clonidine, and fentanyl used intrathecally with hyperbaric 0.5% bupivacaine for spinal anesthesia. Settings and Design: The study was conducted in prospective, double blind manner. It included 120 American Society of Anesthesiology (ASA) class I and II patients undergoing lower limb surgery under spinal anesthesia after approval from hospital ethics committee with written and informed consent of patients. Materials and Methods: The patients were randomly allocated into four groups (30 patients each). Group BS received 12.5 mg hyperbaric bupivacaine with normal saline, group BF received 12.5 mg bupivacaine with 25 g fentanyl, group BC received 12.5 mg of bupivacaine supplemented 30 g clonidine, and group BD received 12.5 mg bupivacaine plus 5 g dexmedetomidine. The onset time to reach peak sensory and motor level, the regression time of sensory and motor block, hemodynamic changes, and side effects were recorded. Results: Patients in Group BD had significantly longer sensory and motor block times than patients in Groups BC, BF, and BS with Groups BC and BF having comparable duration of sensory and motor block. The mean time of two segment sensory block regression was 147 ± 21 min in Group BD, 117 ± 22 in Group BC, 119 ± 23 in Group BF, and 102 ± 17 in Group BS (P < 0.0001). The regression time of motor block to reach modified Bromage zero (0) was 275 ± 25, 199 ± 26, 196 ± 27, 161 ± 20 in Group BD, BC, BF, and BS, respectively (P < 0.0001). The onset times to reach T8 dermatome and modified Bromage 3 motor block were not significantly different between the groups. Dexmedetomidine group showed significantly less and delayed requirement of rescue analgesic. Conclusions: Intrathecal dexmedetomidine is associated with prolonged motor and sensory block, hemodynamic stability, and reduced demand of rescue analgesics in 24 h as compared to clonidine, fentanyl, or lone bupivacaine.

Cortical spreading depression (SD) is a transient propagating neuronal excitation followed by depression, which is generally accepted as the underlying cause of migraine. The inhibitory γ-aminobutyric acid type A (GABA A ) receptor activation not only reduces cortical SD frequency and propagation, but also relieves migraine headache. This study aims to determine the role of major α subtypes of GABA A receptor in mediating SD genesis and propagation using an efficient in vitro chick retinal model. We firstly demonstrated that abundant α2, and to a lesser extent, α5 of GABA A receptor expression in the chick retina, enabled the tissue useful for studying GABA A receptor pharmacology and SD. Marked suppression of SD by SL651498 and TPA023 was observed at 10 μmol L −1 and 50 μmol L −1 , respectively, suggesting a critical role of GABA A receptor α subtypes, in particular α2, in modulating retinal SD elicitation and propagation. The negative data on NS11394 at 3 μmol L −1 on SD and the little positive selectivity of TPA023 for α5 did not support that α5 subtype is involved in SD genesis and propagation. Our data provide strong evidence that α2, but not α5 is involved in the early stage of migraine, indicating that α2 subtype is a possible drug target related to migraine with aura. [ABSTRACT FROM AUTHOR]

We previously found that a lectin, Sambucus sieboldiana agglutinin (SSA), bound to α2,6-sialylated glycan epitopes on transferrin and inhibited anti-transferrin antibody binding to the antigen in ELISA (SSA inhibition). Here we report that SSA inhibition is applicable to immunohistochemistry, localizing α2,6-sialylated transferrin in the liver. Immunohistochemistry using anti-transferrin polyclonal antibody revealed that transferrin was detected in hepatocytes near interlobular veins. Addition of SSA lectin markedly attenuated the staining. Sialidase treatment of a liver section abolished SSA binding and concomitantly cancelled SSA inhibition, suggesting that SSA binding to glycan epitopes on the section was essential for the inhibition. To examine the importance of proximity between antigen epitopes and SSA-binding (glycosylation) sites, we prepared two anti-peptide antibodies against partial amino acid sequences of transferrin. One antibody (Tf-596Ab) is against a peptide sequence, Cys596-Ala614, which is proximal to N-glycosylation sites (Asn-432 and Asn- 630). The other (Tf-120Ab) is against a peptide sequence, Val120-Cys137, distal to the sites. The staining signals of Tf-596Ab were reduced by the addition of SSA, whereas those of Tf-120Ab were reduced only a little. This result suggests that proximity of the antigen epitope to SSA binding sites is critical for SSA inhibition in immunohistochemistry. [ABSTRACT FROM AUTHOR]

N-Glycans of human proteins possess both α2,6- and α2,3-linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3-linkage due to the absence of α2,6-sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)-producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC-Sambucus nigra (SNA) lectin that preferentially binds α2,6-linked SA. The presence of α2,6-linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2-fold compared to the control. For host cell engineering, the CHOZN® GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single-cell clones were derived from the enriched population and selected based on FITC-SNA staining and St6gal1 expression. Two clones ('ST6GAL1 OE Clone 31 and 32') were confirmed for the presence of α2,6-linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6-linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human-like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of 'bio-better' protein therapeutics and cell culture vaccine production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:334-346, 2015 [ABSTRACT FROM AUTHOR]

α2,6-Sialylated glycans are crucial molecular targets for cancer diagnosis and clinical research. In this work, a novel ultrasensitive electrochemical biosensor was fabricated based on a graphite oxide (GO), Prussian blue (PB), and PTC-NH2 (an ammonolysis product of 3,4,9,10-perylenetetracarboxylic dianhydride) nanocomposite for the selective detection of α2,6-sialylated glycans. To increase the sensitivity of the electrochemical biosensor, gold nanoparticles (GNPs) were immobilized on a GO-PB-PTC-NH2 modified glassy carbon electrode (GCE). Sambucus nigra agglutinins (SNAs), which specifically bind with α2,6-sialylated glycans, were covalently immobilized on GNPs for the sensitive detection of α2,6-sialylated glycans in serum. This proposed method can be applied to human serum, and it worked well over a broad linear range (0.1pgmL−1-500ngmL−1) with detection limits of 0.03pgmL−1. Moreover, recovery of the spiked samples ranged from 100.2% to 105.0%, suggesting that this excellent electrochemical biosensor can be used for the practical detection of α2,6-sialylated glycans. [ABSTRACT FROM AUTHOR]

Background: Various adjuvants are being used with local anesthetics for prolongation of intraoperative and postoperative analgesia. Dexmedetomidine, the highly selective α2 adrenergic agonist is a new neuraxial adjuvant gaining popularity. Aim: The purpose of this study was to compare the onset, duration of sensory and motor block, hemodynamic effects, postoperative analgesia, and adverse effects of dexmedetomidine, clonidine, and fentanyl used intrathecally with hyperbaric 0.5% bupivacaine for spinal anesthesia. Settings and Design: The study was conducted in prospective, double blind manner. It included 120 American Society of Anesthesiology (ASA) class I and II patients undergoing lower limb surgery under spinal anesthesia after approval from hospital ethics committee with written and informed consent of patients. Materials and Methods: The patients were randomly allocated into four groups (30 patients each). Group BS received 12.5 mg hyperbaric bupivacaine with normal saline, group BF received 12.5 mg bupivacaine with 25 µg fentanyl, group BC received 12.5 mg of bupivacaine supplemented 30 µg clonidine, and group BD received 12.5 mg bupivacaine plus 5 µg dexmedetomidine. The onset time to reach peak sensory and motor level, the regression time of sensory and motor block, hemodynamic changes, and side effects were recorded. Results: Patients in Group BD had significantly longer sensory and motor block times than patients in Groups BC, BF, and BS with Groups BC and BF having comparable duration of sensory and motor block. The mean time of two segment sensory block regression was 147 ± 21 min in Group BD, 117 ± 22 in Group BC, 119 ± 23 in Group BF, and 102 ± 17 in Group BS (P < 0.0001). The regression time of motor block to reach modified Bromage zero (0) was 275 ± 25, 199 ± 26, 196 ± 27, 161 ± 20 in Group BD, BC, BF, and BS, respectively (P < 0.0001). The onset times to reach T8 dermatome and modified Bromage 3 motor block were not significantly different between the groups. Dexmedetomidine group showed significantly less and delayed requirement of rescue analgesic. Conclusions: Intrathecal dexmedetomidine is associated with prolonged motor and sensory block, hemodynamic stability, and reduced demand of rescue analgesics in 24 h as compared to clonidine, fentanyl, or lone bupivacaine. [ABSTRACT FROM AUTHOR]

Abstract: Gangliosides are a family of sialylated glycosphingolipids enriched in the outer leaflet of neuronal membranes, in particular at synapses. Therefore, they have been hypothesized to play a functional role in synaptic transmission. We have measured in detail the electrophysiological parameters of synaptic transmission at the neuromuscular junction (NMJ) ex vivo of a GD3-synthase knockout mouse, expressing only the O- and a-series gangliosides, as well as of a GM2/GD2-synthase*GD3-synthase double-knockout (dKO) mouse, lacking all gangliosides except GM3. No major synaptic deficits were found in either null-mutant. However, some extra degree of rundown of acetylcholine release at high intensity use was present at the dKO NMJ and a temperature-specific increase in acetylcholine release at 35 °C was observed in GD3-synthase knockout NMJs, compared with wild-type. These results indicate that synaptic transmission at the NMJ is not crucially dependent on the particular presence of most ganglioside family members and remains largely intact in the sole presence of GM3 ganglioside. Rather, presynaptic gangliosides appear to play a modulating role in temperature- and use-dependent fine-tuning of transmitter output. [Copyright &y& Elsevier]

Abstract: We show that if X is an uncountable productive γ-set [F. Jordan, Productive local properties of function spaces, Topology Appl. 154 (2007) 870–883], then there is a countable such that is not Hurewicz. Along the way we answer a question of A. Miller by showing that an increasing countable union of γ-spaces is again a γ-space. We will also show that λ-spaces with the Hurewicz property are precisely those spaces for which every co-countable set is Hurewicz. [Copyright &y& Elsevier]

Abstract: Sialyltransferases are key enzymes for the biosynthesis of sialyl-glycoproteins and sialyl-lipids and the genes encoding sialyltransferases have been cloned from mammalian and bacterial source. In the mammalian sialyltransferase, existence of three conserved regions, named sialyl motifs, has been demonstrated. On the other hand, two short motifs, named D/E-D/E-G motif and HP motif, have been reported in the bacterial sialyltransferases very recently. From the results of multiple alignments among the sialyltransferases belonging to Glycosyltransferase family 80 and crystal structures of two reported sialyltransferases, it is clearly demonstrated that the third conserved-functional motif exists in the bacterial sialyltransferases that have been classified into Glycosyltransferase family 80 in this study. [Copyright &y& Elsevier]

Background: Cutaneous lymphocyte-associated antigen (CLA) is a surface glycoprotein expressed by skin-homing T cells. This carbohydrate moiety expressed on mucin-like surface glycoproteins, including P-selectin glycoprotein ligand 1 and CD43, confers binding activity to dermal endothelial E-selectin and is critical for T-cell recruitment to the skin. Vitamin A (retinoic acid [RA]) and the active form of vitamin D3 (1,25 dihydroxyvitamin D3 [1,25D(3)]) have been used to treat certain T cell–mediated inflammatory skin diseases, as well as cutaneous T-cell lymphomas; however, their effect on CLA expression has not been studied. Objective: We analyzed the effects of RA and 1,25D(3) on expression of CLA and other lymphocyte-homing receptors on human T cells. Methods: We cultured human T cells with 1,25D(3) and RA and analyzed the expression of CLA and other homing receptors. We also pretreated mice with either vitamin and then induced an antigen-dependent contact hypersensitivity response. Results: Both RA and 1,25D(3) downregulated expression of the CLA and, in parallel, functional E-selectin ligand. Whereas RA increased expression of the gut-homing receptor α4β7 and reduced L-selectin expression, 1,25D(3) had no effect on other homing receptors. In an in vivo assay treatment with RA or 1,25D(3) downregulated the skin infiltration of effector CD4+ T cells. Conclusion: These findings suggest that 1,25D(3) can selectively downregulate CLA expression without influencing lymphocyte migration patterns to other tissues. [Copyright &y& Elsevier]

Abstract: CD22/Siglec-2 is a B cell membrane-bound lectin that recognizes glycan ligands containing α2,6-linked sialic acid, and negatively regulates signaling through the B cell antigen receptor (BCR). Previous studies demonstrated that synthetic sialosides that bind to CD22 augment BCR signaling by inhibiting CD22-mediated BCR regulation. Here we demonstrate that, after antigen stimulation, CD22 forms a cap together with BCR, and translocates to lipid rafts. Both co-capping of CD22 with BCR and translocation of CD22 to lipid rafts were markedly blocked by a synthetic α2,6-linked sialic acid, Neu5Gcα2-6GalβSE. These results strongly suggest that synthetic glycan ligand excludes CD22 from BCR-containing lipid rafts. Because CD22-mediated signal regulation requires phosphorylation of CD22 by Lyn that localizes in lipid rafts and is activated by BCR, synthetic glycan ligand regulates localization of CD22 crucial for signal regulation. [Copyright &y& Elsevier]

Abstract: In Ramos cells, a human Burkitt''s lymphoma cell line, stimulation of the B cell antigen receptor with anti-IgM antibody (Ab) induces apoptosis as indicated by a decrease in cell viability and an increase in DNA fragmentation and cell surface exposure of phosphatidylserine. Furthermore, these changes are suppressed by incubating the cells in α1-acid glycoprotein (AGP)-coated tissue culture plates. Here, we found that, during Anti-IgM Ab-induced apoptosis in Ramos cells, caspase-3 is activated downstream of caspase-8 and the mitochondrial pathway is activated, as indicated by a loss of mitochondrial membrane potential, an increase in the release of cytochrome c to the cytoplasm, and enhanced Bax expression. Anti-IgM Ab-induced apoptosis of neuraminidase-treated Ramos cells was suppressed by incubating the cells on plates coated with AGP, which contains a high concentration of α2,6-linked sialic acid. The incubation on plates coated with AGP also suppressed anti-IgM Ab-stimulated caspase-3 activity and increased the level of X-linked inhibitor of apoptosis protein (XIAP), but it did not affect caspase-8 activity, the mitochondrial membrane potential, cytochrome c release, or Bax expression. The results indicate that the interaction of Ramos cells with immobilized α2,6-linked sialic acid enhances XIAP expression, directly or indirectly suppressing caspase-3 activity and inhibiting anti-IgM Ab-induced apoptosis. [Copyright &y& Elsevier]

Abstract: The N-glycans in human immunoglobulin G were engineered through the bacterial glycosyltransferase-catalyzed galactosylation and sialylation with in situ regeneration of sugar-nucleotides. For the galactosylation and sialylation, β1,4-galactosyltransferase from H. pylori and α2,3-sialyltransferase from N. gonorrhoeae were expressed in Escherichia coli. For the regeneration of UDP-galactose and CMP-NeuAc, the required enzymes were also expressed in E. coli and their extracts were employed as biocatalysts. Using the catalytic system, we carried out the galactosylation of terminal GlcNAc residues with UDP-Gal regeneration and sialylation of terminal Gal residues with CMP-NeuAc regeneration, respectively. When the oligosaccharides in human IgG were analyzed by HPLC (NP and WAX) and MALDI-TOF, the galactosylation reaction showed 86.1% increase in terminal galactosylation of N-linked oligosaccharide in IgG, and the sialylation reaction gave 69.6% increase in terminal sialylation. To achieve a maximum sialylation of IgG, simultaneous galactosylation and sialylation reaction was conducted by incubating all of the biocatalysts required for the galactosylation, sialylation and sugar-nucleotide regenerations in one reactor, resulting in 80.2% increase in terminal sialylation. [Copyright &y& Elsevier]

The centrally acting α2-adrenoceptor agonists clonidine and dexmedetomidine have been used with success to provide haemodynamic stability for patients undergoing surgery. Particularly in the case of patients with overt or underlying cardiac disease the actions of α2-adrenoceptor agonists, which include maintenance of stable systemic blood pressure and low heart rate and a reduction in overall oxygen consumption, can be expected to reduce the risk of procedure-related cardiac events. This expectation has been corroborated in clinical trials with clonidine, dexmedetomidine and mivazerol and meta-analyses; additional large controlled trials would be instructive in establishing a robust estimate of the scale of the benefit. In addition, α2-adrenoceptor agonists used as premedication have been shown to substantially reduce anaesthetic requirements among surgical patients, and the use of these agents has been associated with a reduced risk of postoperative delirium, which may be expected to improve considerably the postoperative course for at-risk patients. Dexmedetomidine is the only α2-adrenoceptor agonist currently approved for use in the intensive care unit. A distinctive feature of dexmedetomidine in that setting is that in addition to haemodynamic stability it confers a distinctive and advantageous quality of sedation: patients are tranquil but responsive to requests from attending staff. This review examines the pharmacological principles underlying the use of α2-adrenoceptor agonists as adjuncts to surgery and clinical experience in that indication. [ABSTRACT FROM AUTHOR]

Abstract: The hemagglutinin (HA) of influenza viruses initiates infection by binding to sialic acid on the cell surface via α2,6 (human) or α2,3 (avian) linkage. The influenza neuraminidase (NA) can cleave both α2,3- and α2,6-linked sialic acids, but all influenza NAs have a marked preference for the non-human α2,3 linkage. Recent H3N2 influenza viruses have lost the ability to agglutinate chicken red blood cells. To determine if changes in HA specificity or affinity correlate with NA specificity or activity, we examined red cell binding and elution of a series of H3N2 viruses. We found that the NA activity of many influenza viruses does not release binding by their HA. In some egg-adapted strains, lack of elution correlates with low levels of viral NA activity, and these elute rapidly when bacterial NA is added. However, a Fujian-like virus, A/Oklahoma/323/03, does not elute by its own NA or with Vibrio cholerae sialidase, and it binds to red cells pre-treated with V. cholerae sialidase. It elutes after addition of the broad specificity Micromonospora viridifaciens sialidase. Human glycophorin inhibits A/Oklahoma/323/03 hemagglutination 6-fold better than fetuin. We conclude that specific forms of sialic acid are used as receptor by recent human H3N2 influenza viruses, perhaps involving branched α2,6 sialic acid or α2,8 sialic acid structures on O-linked carbohydrates. The virus itself has no O-linked glycans, so even though the NA is not able to cleave receptors on cells, the viruses will not self-aggregate. It will be important to monitor efficacy of neuraminidase inhibitors in case there are NA-resistant receptors in the human respiratory tract that allow the viruses to be less dependent on NA activity. [Copyright &y& Elsevier]

Abstract: The N-linked oligosaccharides on human choriogonadotropin (hCG) and follitropin (hFSH) α-subunit loop 2 (α2) have a dominant influence on hormone efficacy. hCG analogs lacking this oligosaccharide retain approximately 40% the efficacy of the fully glycosylated hormone in cyclic AMP accumulation assays. Previous efforts to reduce efficacy further have involved removing the other N-linked oligosaccharides. We found that some intersubunit disulfide crosslinks reduced the efficacies of hCG analogs lacking only the α2 oligosaccharide. The least active analog was an hCG/hFSH chimera containing hFSH residues 95–108 in place of hCG residues 101–114 and a disulfide bond between α-subunit residue 37 and β-subunit residue 33. While it bound lutropin receptors 2- to 3-fold better than hCG and follitropin receptors 10–30% as well as hFSH, it had less than 10% and 5% the efficacies of either hormone. This suggests that complete deglycosylation will not be required to produce glycoprotein hormone analogs that have low efficacies. [Copyright &y& Elsevier]

Affinities and efficacies of chemically diverse ligands—some of them used as clinical agents—were examined, employing [3H]RX821,002 and [35S]GTPγS binding assays, respectively, at human (h) cloned, hα2A, hα2B and hα2C adrenoceptors (AR) expressed in Chinese hamster ovary (CHO) cells. As compared to noradrenaline (NA, efficacy defined as 100%), the majority of the 13 agonists tested generally behaved as partial agonists. Amongst 18 antagonists, pKB and pKi values, which were highly correlated for each α2-AR subtype, failed to reveal any strikingly selective agents. Inverse agonist properties were not detected for any antagonist, consistent with a lack of constitutive activity suggested by the monophasic inhibition of [35S]GTPγS binding by GTPγS. These data should facilitate interpretation of experimental and clinical actions of adrenergic agonists. Moreover, they emphasize the continuing need for α2-AR subtype-selective antagonists in order to define further the roles and therapeutic relevance of hα2A-, hα2B-, and hα2C-AR. [Copyright &y& Elsevier]

Glycoprotein-S-sulfotransferase (GP3ST) is a key enzyme in downregulating the expression of Galαl,3Gaiβ1,4GlcNAc-R (the α-Gal epitope), via enzymatic competition with an αl,3 galactosyltransferase (αl,3GT), such as α2,6 sialyltransferase (α2,6ST). In this study, we report the dominance of GP3ST over αl,3GT using transfected pig endothelial cell (PEC) lines. The introduction of the GP3ST gene into PEC suppresses its antigenicity with respect to normal human pooled serum (NHS), including the α-Gal epitope and the Hanganutziu-Deicher (H-D) antigen, and, in addition, reduces the susceptibility to NHS in complement-mediated cell lysis. Western and lectin blot analyses of the products of parental PEC and its transfectants indicated that proteins smaller than 66 kDa have a diminished reactivity with NHS and the IB4 lectin. The levels of the a-Gal epitope in neutral glycosphingolipids were also decreased in the GP3ST transfectants as detected in thin layer chromatography by immunostaining. These data indicate that GP3ST is very effective in reducing xenoepitope levels. [ABSTRACT FROM AUTHOR]

For the resection of an esophagus carcinoma a mortality rate of 2 to 30% was described. It is still unclear whether an abdominothoracic or transhiatal intervention is superior regarding the outcome. To investigate the prognostic value of fibrinolytic markers, plasmin-α2-antiplasmin (PAP) and D-dimer (DD) values were determined daily in the early postoperative period for 11 days. In addition, the course of PAP and DD concentrations was compared with the method of esophagectomy. Of the 28 patients enclosed in the study, 5 died between day 10 and day 34 owing to adult respiratory distress syndrome and septicemia. The PAP and DD concentrations increased in survivors after surgery until day 5 and day 7, respectively. The concentrations were twofold and 10-fold higher than the upper reference level. In contrast, four of five nonsurvivors showed an inadequate increase in PAP concentrations within the reference range, whereas the course of DD was inconspicuous. The sensitivity and specificity of PAP and DD in respect to a fatal outcome was calculated by receiver operating characteristic analysis based on all results: sensitivity 76% (PAP-cut off value 760 μg/L) and 49% (DD 6 mg/L), specificity 77% and 72%, respectively. The biochemical markers showed no significant differences between the abdominothoracic and transhiatal esophagectomy. In the abdominothoracic intervention, lower PAP and higher DD concentrations were observed. The results showed that the PAP concentrations could detect a fatal outcome within the first 5 days after surgery. [ABSTRACT FROM AUTHOR]

Previously, we demonstrated that β1,4galactosyltransferase (gal-T1) reversibly segregates from α2,6sialyltransferase (ST6Gal) to swollen vesicles after monensin treatment of the cells. To further explore this phenomenon, we investigated the response to monensin of various Golgi proteins. Within 30 min of monensin treatment, gal-T1 moved from the Golgi apparatus, as defined by localization of giantin, to swollen vesicles whereas ST6Gal, α2,3(N)sialyltransferase, mannosidase II, and N-acetylgalactosaminyltransferase 2 remained associated with the Golgi apparatus. Stably transfected CHO cells exhibited a similar phenomenon of monensin-induced displacement of recombinant gal-T1 to swollen vesicles while recombinant ST6Gal remained colocalized with endogenously expressed giantin. Gal-T1 and the cation-insensitive mannose 6-phosphate receptor colocalized in swollen vesicles as observed at both light and electron microscopic levels. When monensin was replaced by chloroquine, gal-T1 remained arrested in swollen vesicles. Brefeldin A treatment known to cause relocation of Golgi-associated gal-T1 to the endoplasmic reticulum had no effect on gal-T1 trapped in swollen vesicles. This evidence suggests that monensin blocks gal-T1 trafficking in post-Golgi structures and argues against swelling of gal-T1-containing trans Golgi cisternae as previously assumed. [ABSTRACT FROM AUTHOR]

The case of a 70-year-old patient with an epithelial mesothelioma is presented. The patient suffered from dyspnea due to a right-sided recurrent hemorrhagic effusion. Cytological analysis of the effusion revealed marked lymphocytosis and tumor cells, some of them multinucleated with prominent nucleoli. Open lung biopsy revealed nodular thickening of the diaphragmatic, visceral, and parietal pleura; histological examination of biopsies detected intravascular growth of tumor cells. Immunocytochemical characterization of cultured tumor cells and the biopsy specimens showed positive staining to vimentin and cytokeratin, but negative reaction with antibodies against epithelial membrane associated antigen, leukocyte common antigen, van Willebrand factor, and neurofilament. Hence, the tumor was classified as malignant mesothelioma. In vitro, interferon-α2 produced a dose-dependent inhibition of proliferation in the cultured mesothelioma cells. Two weeks after initiation of interferon-α2 treatment the patient improved and the pleural effusion vanished. [ABSTRACT FROM AUTHOR]

Agonists at the α 2 adrenergic receptor produce sedation, increase focus, provide analgesia, and induce centrally mediated hypotension and bradycardia, yet neither their dynamic interactions with adrenergic receptors nor their modulation of neuronal circuit activity is completely understood. Photoaffinity ligands of α 2 adrenergic agonists have the potential both to capture discrete moments of ligand-receptor interactions and to prolong naturalistic drug effects in discrete regions of tissue in vivo. We present here the synthesis and characterization of a novel α 2 adrenergic agonist photolabel based on the imidazole medetomidine called azi-medetomidine . Azi-medetomidine shares protein association characteristics with its parent compound in experimental model systems and by molecular dynamics simulation of interactions with the α 2A adrenergic receptor. Azi-medetomidine acts as an agonist at α 2A adrenergic receptors, and produces hypnosis in Xenopus laevis tadpoles. Azi-medetomidine competes with the α 2 agonist clonidine at α 2A adrenergic receptors, which is potentiated by photolabeling, and azi-medetomidine labels moieties on the α 2A adrenergic receptor as determined by mass spectrometry in a manner consistent with a simulated model. This novel α 2 adrenergic agonist photolabel can serve as a powerful tool for in vitro and in vivo investigations of adrenergic signaling.

Epithelial ovarian cancer (EOC) has the highest mortality rate among gynecologic cancers due to advanced stage presentation, peritoneal dissemination, and refractory ascites at diagnosis. We investigated the role of α2,3-sialyltransferase type I (ST3GalI) by analyzing human ovarian cancer datasets and human EOC tissue arrays. We found that high expression of ST3GalI was associated with advanced stage EOC. Transwell migration and cell invasion assays showed that high ST3GalI expression enhanced migration of EOC cells. We also observed that there was a linear relation between ST3GalI expression and epidermal growth factor receptor (EGFR) signaling in EOC patients, and that high ST3GalI expression blocked the effect of EGFR inhibitors. Co-Immunoprecipitation experiments demonstrated that ST3GalI and EGFR were present in the same protein complex. Inhibition of ST3GalI using a competitive inhibitor, Soyasaponin I (SsaI), inhibited tumor cell migration and dissemination in the in vivo mouse model with transplanted MOSEC cells. Further, SsaI synergistically enhanced the anti-tumor effects of EGFR inhibitor on EOC cells. Our study demonstrates that ST3GalI regulates ovarian cancer cell migration and peritoneal dissemination via EGFR signaling. This suggests α2,3-linked sialylation inhibitors in combination with EGFR inhibitors could be effective agents for the treatment of EOC.

As a subset of glycosyltransferases, the family of sialyltransferases catalyze transfer of sialic acid (Sia) residues to terminal non-reducing positions on oligosaccharide chains of glycoproteins and glycolipids, utilizing CMP-Neu5Ac as the activated sugar nucleotide donor. In the four known sialyltransferase families (ST3Gal, ST6Gal, ST6GalNAc and ST8Sia), the ST8Sia family catalyzes synthesis of α2, 8-linked sialic/polysialic acid (polySia) chains according to their acceptor specificity. We have determined the 3D structural models of the ST8Sia family members, designated ST8Sia I (1), II(2), IV(4), V(5), and VI(6) using the Phyre2 server. Accuracy of these predicted models are based on the ST8Sia III crystal structure as the calculated template. The common structural features of these models are: (1) Their parallel templates and disulfide bonds are buried within the enzymes and are predominately surrounded by helices; (2) The anti-parallel β-sheets are located at the N-terminal region of the enzymes; (3) The mono-sialytransferases (mono-STs), ST8Sia I and ST8Sia VI, contain only a single pair of disulfide bonds, and there are no anti-parallel β-sheets in ST8Sia VI; (4) The Nterminal region of all of the mono-STs are located some distant away from their core structure; (5) These conformational features show that the 3D structures of the mono-STs are less compact than the two polySTs, ST8Sia II and ST8Sia IV, and the oligo-ST, ST8Sia III. These structural features relate to the catalytic specificity of the monoSTs; (6) In contrast, the more compact structural features of ST8Sia II, ST8Sia IV and ST8Sia III relate to their ability to catalyze the processive synthesis of oligo- (ST8Sia III) and polySia chains (ST8Sia II & ST8Sia IV); (7) Although ST8Sia II, III and IV have similar conformations in their corresponding polysialyltransferase domain (PSTD) and polybasic region (PBR) motifs, the structure of ST8Sia III is less compact than ST8Sia II and ST8Sia IV, and the amino acid components of the several three-residue-loops in the two motifs of ST8Sia III are different from that in ST8Sia II and ST8Sia IV. This is likely the structural basis for why ST8Sia III is an oligoST and not able to polysialylate and; (8) In contrast, essentially all amino acids within the threeresidue- loops in the PSTD of ST8Sia II and ST8Sia IV are highly conserved, and many amino acids in the loops and the helices of these two motifs are critical for NCAM polysialylation, as determined by mutational analysis and confirmed by our recent NMR results. In summary, these new findings provide further insights into the molecular mechanisms underlying polyST-NCAM recognition, polySTpolySia/ oligoSia interactions, and polysialylation of NCAM., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

ST6Gal-I sialyltransferase adds α2,6-linked sialic acids to the terminal ends of glycan chains of glycoproteins and glycolipids. ST6Gal-I is reportedly upregulated in many cancers, including hepatocellular carcinoma, ovarian cancer and breast cancer. However, the expression and function of ST6Gal-I in prostate cancer (PCa) and the mechanism underlying this function remain largely unknown. In this study, we observed that ST6Gal-I expression was upregulated in human PCa tissues compared to non-malignant prostate tissues. High ST6Gal-I expression was positively correlated with Gleason scores, seminal vesicle involvement and poor survival in patients with PCa. ST6Gal-I knockdown in aggressive prostate cancer PC-3 and DU145 cells significantly inhibited the proliferation, growth, migration and invasion capabilities of these cells. ST6Gal-I knockdown decreased the levels of several PI3K/Akt/GSK-3β/ β-catenin pathway components, such as p-PI3K, (Ser473)p-Akt, (Ser9)p-GSK-3β and β-catenin. Furthermore, targeting this pathway with a PI3K inhibitor or Akt RNA interference decreased p-Akt, p-GSK-3β and β-catenin expression, resulting in decreased PC-3 and DU145 proliferation, migration and invasion. Taken together, these results indicate that ST6Gal-I plays a critical role in cell proliferation and invasion via the PI3K/Akt/GSK-3β/β-catenin signaling pathway during PCa progression and that it might be a promising target for PCa prognosis determination and therapy.

Antibacterial peptides (ABPs) with cancer-selective toxicity have received much more attention as alternative chemotherapeutic agents in recent years. However, the basis of their anticancer activity remains unclear. The modification of cell surface glycosylation is a characteristic of cancer cells. The present study investigated the effect of glycosylation, in particular sialic acid, on the anticancer activity of ABPs. We showed that aurein 1.2, buforin IIb and BMAP-28m exhibited selective cytotoxicity toward MX-1 and MCF-7 breast cancer cells. The binding activity, cytotoxicity and apoptotic activity of ABPs were enhanced by the presence of O-, N-glycoproteins, gangliosides and sialic acid on the surface of breast cancer cells. Among N-, O-glycoproteins and ganglioside, O-glycoproteins almost had the strongest effect on the binding and cytotoxicity of the three peptides. Further, up-regulation of hST6Gal1 in CHO-K1 cells enhanced the susceptibility of cells to these peptides. Finally, the growth of MX-1 xenograft tumors in mice was significantly suppressed by buforin IIb treatment, which was associated with induction of apoptosis and inhibition of vascularization. These data demonstrate that the three peptides bind to breast cancer cells via an interaction with surface O-, N-glycoproteins and gangliosides. Sialic acids act as key glycan binding sites for cationic ABP binding to glycoproteins and gangliosides. Therefore, glycosylation in breast cancer cells plays an important role in the anticancer activity of ABPs, which may partly explain their cancer-selective toxicity. Anticancer ABPs with cancer-selective cytotoxicity will be promising candidates for anticancer therapy in the future., (Copyright © 2013 Elsevier Inc. All rights reserved.)