Galactose 6-O-Sulfotransferases Are Not Required for the Generation of Siglec-F Ligands in Leukocytes or Lung Tissue

Eosinophil accumulation is a characteristic feature of the immune response to parasitic worms and allergens. The cell surface carbohydrate-binding receptor Siglec-F is highly expressed on eosinophils and negatively regulates their accumulation during inflammation. Although endogenous ligands for Siglec-F have yet to be biochemically defined, binding studies using glycan arrays have implicated galactose 6-O-sulfate (Gal6S) as a partial recognition determinant for this receptor. Only two sulfotransferases are known to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase 1 (C6ST-1). Here we use mice deficient in both KSGal6ST and C6ST-1 to determine whether these sulfotransferases are required for the generation of endogenous Siglec-F ligands. First, we characterize ligand expression on leukocyte populations and find that ligands are predominantly expressed on cell types also expressing Siglec-F, namely eosinophils, neutrophils, and alveolar macrophages. We also detect Siglec-F ligand activity in bronchoalveolar lavage fluid fractions containing polymeric secreted mucins, including MUC5B. Consistent with these observations, ligands in the lung increase dramatically during infection with the parasitic nematode, Nippostrongylus brasiliensis, which is known to induce eosinophil accumulation and mucus production. Surprisingly, Gal6S is undetectable in sialylated glycans from eosinophils and BAL fluid analyzed by mass spectrometry. Furthermore, none of the ligands we describe are diminished in mice lacking KSGal6ST and C6ST-1, indicating that neither of the known galactose 6-O-sulfotransferases is required for ligand synthesis. These results establish that ligands for Siglec-F are present on several cell types that are relevant during allergic lung inflammation and argue against the widely held view that Gal6S is critical for glycan recognition by this receptor.     

MemBrain: improving the accuracy of predicting transmembrane helices

Prediction of transmembrane helices (TMH) in alpha helical membrane proteins provides valuable information about the protein topology when the high resolution structures are not available. Many predictors have been developed based on either amino acid hydrophobicity scale or pure statistical approaches. While these predictors perform reasonably well in identifying the number of TMHs in a protein, they are generally inaccurate in predicting the ends of TMHs, or TMHs of unusual length. To improve the accuracy of TMH detection, we developed a machine-learning based predictor, MemBrain, which integrates a number of modern bioinformatics approaches including sequence representation by multiple sequence alignment matrix, the optimized evidence-theoretic K-nearest neighbor prediction algorithm, fusion of multiple prediction window sizes, and classification by dynamic threshold. MemBrain demonstrates an overall improvement of about 20% in prediction accuracy, particularly, in predicting the ends of TMHs and TMHs that are shorter than 15 residues. It also has the capability to detect N-terminal signal peptides. The MemBrain predictor is a useful sequence-based analysis tool for functional and structural characterization of helical membrane proteins; it is freely available at http://chou.med.harvard.edu/bioinf/MemBrain/.     

EGF upregulates Na+/H+ exchanger NHE1 by post-translational regulation that is important for cervical cancer cell invasiveness

Na+/H+ exchanger 1 (NHE1) is involved in cell migration but little is known about the signal pathways that regulate NHE1 activity and that are associated with tumor cell invasiveness. This study is to investigate the mechanisms by which epidermal growth factor (EGF) regulates NHE1 expression to promote cervical cancer cell invasiveness and the clinical significance in early-stage cervical cancer. NHE1 protein was scanty in normal or noncancerous cervical tissues of all surgical specimens examined (n = 92). Tumor tissues clearly expressed NHE1 protein with different amounts. The differential expression level of NHE1 is associated with the clinical outcome. NHE1 protein was also differentially expressed between normal cervical epithelial cells and two cervical cancer cell lines. Cervical cancer cells benefit some enhanced cellular functions from NHE1 abundance, such as cell volume regulation, migration, and invasion. Interestingly, NHE1 colocalized with EGF in cervical cancer tissues. Studies in cell culture systems indicated that EGF-stimulated NHE1 abundance in a time-dependent manner by post-translational regulation. This implies a likely autocrine or paracrine EGF stimulation of NHE1 production in vivo. In addition, the phosphoinositide 3-kinase pathway is the dominant signal controlling EGF-stimulated NHE1 abundance. Pharmacological inhibition of NHE1 activity markedly inhibited the basal and EGF-stimulated cervical cancer cell migration. Image studies and immunoprecipitaion experiments suggest that EGF-induced NHE1 translocation to the leading-edge lamellipodia, where NHE1 interacted with actin-associated protein Ezrin, thereby remodeling cytoskeleton and stimulating cervical cancer cell migration. In conclusion, EGF upregulates NHE1 by post-translational regulation that is important for cervical cancer cell invasiveness.     

Gender Difference in the Interaction Effects of Diabetes and Hypertension on Stroke among the Elderly in the Shih-Pai Study,Taiwan

Aims

To investigate the interaction effects of diabetes and hypertension on stroke, and also investigate the independent and interaction effects of parental history and environmental factors on diabetes and hypertension in a cross-sectional elderly population.

Methods

The Shih-Pai Community Medical Service Program was a community-based, fixed cohort study conducted between June 1999 and November 2002. Socio-demographic and clinical data of subjects aged 65 years and older were collected by well-trained interviewers during home visits. Interaction effects were analyzed using Rothman’s synergy index (SI).

Results

In total, 4,124 subjects were included in the study, with 2,284 males and 1,840 females. The synergistic interaction of diabetes and hypertension on stroke was statistically significant in women (SI = 3.16, 95% CI: 1.35–7.39). The synergistic interaction of parental diabetes and being overweight on diabetes was only statistically significant in men, and not in women (SI = 3.30, 95% CI: 1.00–10.83 in men, and SI = 1.15, 95% CI: 0.30–4.39 in women).

Conclusions

A synergistic interaction was found for diabetes and hypertension in both sexes when parental history and being overweight were combined. Furthermore, combining diabetes and hypertension in elderly women was significant in terms of the risk of stroke. Strategies to control risk factors in individuals at additional high risk are urgently needed.     

Angiotensin-converting enzyme inhibitor captopril attenuates ventilator-induced lung injury in rats.

We hypothesized that lung inflammation and parenchymal apoptosis in ventilator-induced lung injury (VILI) are related to ANG II and assessed the ability of the angiotensin-converting enzyme inhibitor captopril to attenuate VILI in rats. Adult male Sprague-Dawley rats were randomized to receive two ventilation strategies for 2 h: 1) tidal volume of 40 ml/kg, respiratory rate of 25 breaths/min, and inspiratory O2 fraction of 0.21 [high-volume, 0 positive end-expiratory pressure (HVZP) group] and 2) injection of captopril (100 mg/kg ip) 30 min before HVZP ventilation (HVZP+CAP group). Another group, which did not receive ventilation, served as the control. Mean arterial pressure was significantly lower in the HVZP+CAP group than in the HVZP group at 2 h of ventilation. Total protein levels were significantly higher in bronchoalveolar lavage fluid (BALF) recovered from HVZP-ventilated rats than from controls. BALF macrophage inflammatory protein-2 and lung ANG II were significantly higher in the HVZP group than in the control and HVZP+CAP groups. Lung ANG II levels correlated positively with BALF protein and macrophage inflammatory protein-2. The number of apoptotic airway and alveolar wall cells was significantly higher in the HVZP and HVZP+CAP groups than in the control group and significantly lower in the HVZP+CAP group than in the HVZP group. These results suggest that the efficiency of captopril to attenuate VILI is related to reduction of inflammatory cytokines and inhibition of apoptosis and indicate that VILI is partly mediated by the local angiotensin system.     

Hydrophilic aromatic residue and in silico structure for carbohydrate binding module

Carbohydrate binding modules (CBMs) are found in polysaccharide-targeting enzymes and increase catalytic efficiency. Because only a relatively small number of CBM structures have been solved, computational modeling represents an alternative approach in conjunction with experimental assessment of CBM functionality and ligand-binding properties. An accurate target-template sequence alignment is the crucial step during homology modeling. However, low sequence identities between target/template sequences can be a major bottleneck. We therefore incorporated the predicted hydrophilic aromatic residues (HARs) and secondary structure elements into our feature-incorporated alignment (FIA) algorithm to increase CBM alignment accuracy. An alignment performance comparison for FIA and six others was made, and the greatest average sequence identities and similarities were achieved by FIA. In addition, structure models were built for 817 representative CBMs. Our models possessed the smallest average surface-potential z scores. Besides, a large true positive value for liagnd-binding aromatic residue prediction was obtained by HAR identification. Finally, the pre-simulated CBM structures have been deposited in the Database of Simulated CBM structures (DS-CBMs). The web service is publicly available at http://dscbm.life.nthu.edu.tw/ and http://dscbm.cs.ntou.edu.tw/.     

Ketoconazole-evoked [Ca2+]i rises and non-Ca2+-triggered cell death in rabbit corneal epithelial cells (SIRC)

The effect of ketoconazole on cytosolic free Ca2+ concentrations ([Ca2+]i) and proliferation has not been explored in corneal cells. This study examined whether ketoconazole alters Ca2+ levels and causes cell death in SIRC rabbit corneal epithelial cells. [Ca2+]i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Ketoconazole at concentrations of 5 microM and above increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. The ketoconazole-induced Ca2+ influx was insensitive to L-type Ca2+ channel blockers and protein kinase C modulators. In Ca2+-free medium, after pretreatment with 50 microM ketoconazole, thapsigargin-(1 microM)-induced [Ca2+]i rises were abolished; conversely, thapsigargin pretreatment nearly abolished ketoconazole-induced [Ca2+]i rises. Inhibition of phospholipase C with 2 microM U73122 did not change ketoconazole-induced [Ca2+]i rises. At concentrations between 5 and 100 microM, ketoconazole killed cells in a concentration-dependent manner. The cytotoxic effect of 50 microM ketoconazole was not reversed by prechelating cytosolic Ca2+ with BAPTA. In summary, in corneal cells, ketoconazole-induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum and Ca2+ influx from unknown pathways. Furthermore, the cytotoxicity induced by ketoconazole was not caused via a preceding [Ca2+]i rise.     

Baicalein induces proliferation inhibition in B16F10 melanoma cells by generating reactive oxygen species via 12-lipoxygenase

In a previous study, we demonstrated that baicalein induces hydroxyl radical formation in human platelets but the mechanisms are unclear. Herein, we show, using an electron spin resonance technique, that baicalein also induces hydroxyl radical formation in B16F10 melanoma cells in a dose-dependent manner. Baicalein produced superoxide anions in the presence of an iron chelator and superoxide dismutase (SOD) inhibitor. We suggest that superoxide anions produced by baicalein were promptly converted to hydroxyl radicals through SOD and the Fenton reaction in B16F10 melanoma cells. According to Western blotting results, the 12-LOX protein was expressed in B16F10 melanoma cells, but baicalein had no effect on 12-LOX expression. Decreases in 12-LOX protein expression and hydroxyl radical signals occurred in a 12-LOX small interfering RNA knockdown protein group compared with the baicalein control. In the MTT assay, we also found that baicalein caused a reduction in cellular viability, which was reversed by the addition of ROS scavengers. On the basis of these data, we conclude that ROS formation catalyzed by 12-LOX is one possible mechanism of growth inhibition by baicalein in B16F10 melanoma cells.     

Phagocytosis of apoptotic cells is regulated by a UNC-73/TRIO-MIG-2/RhoG signaling module and armadillo repeats of CED-12/ELMO

BACKGROUND: Phagocytosis of cells undergoing apoptosis is essential during development, cellular turnover, and wound healing. Failure to promptly clear apoptotic cells has been linked to autoimmune disorders. C. elegans CED-12 and mammalian ELMO are evolutionarily conserved scaffolding proteins that play a critical role in engulfment from worm to human. ELMO functions together with Dock180 (a guanine nucleotide exchange factor for Rac) to mediate Rac-dependent cytoskeletal reorganization during engulfment and cell migration. However, the components upstream of ELMO and Dock180 during engulfment remain elusive. RESULTS: Here, we define a conserved signaling module involving the small GTPase RhoG and its exchange factor TRIO, which functions upstream of ELMO/Dock180/Rac during engulfment. Complementary studies in C. elegans show that MIG-2 (which we identify as the homolog of mammalian RhoG) and UNC-73 (the TRIO homolog) also regulate corpse clearance in vivo, upstream of CED-12. At the molecular level, we identify a novel set of evolutionarily conserved Armadillo (ARM) repeats within CED-12/ELMO that mediate an interaction with activated MIG-2/RhoG; this, in turn, promotes Dock180-mediated Rac activation and cytoskeletal reorganization. CONCLUSIONS: The combination of in vitro and in vivo studies presented here identify two evolutionarily conserved players in engulfment, TRIO/UNC73 and RhoG/MIG-2, and the TRIO --> RhoG signaling module is linked by ELMO/CED-12 to Dock180-dependent Rac activation during engulfment. This work also identifies ARM repeats within CED-12/ELMO and their role in linking RhoG and Rac, two GTPases that function in tandem during engulfment.     

Overexpression, one-step purification, and biochemical characterization of a recombinant gamma-glutamyltranspeptidase from Bacillus licheniformis

A truncated gene from Bacillus lichenifromis ATCC 27811 encoding a recombinant γ-glutamyltranspeptidase (BLrGGT) was cloned into pQE-30 to generate pQE-BLGGT, and the overexpressed enzyme was purified from the crude extract of IPTG-induced E. coli M15 (pQE-BLGGT) to homogeneity by nickel-chelate chromatography. This protocol yielded over 25 mg of purified BLrGGT per liter of growth culture under optimum conditions. The molecular masses of the subunits of the purified enzyme were determined to be 41 and 22 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature for the recombinant enzyme were 6–8 and 40 °C, respectively. The chloride salt of metal ions Mg²⁺, K⁺, and Na⁺ can activate BLrGGT, whereas that of Pb²⁺ dramatically inhibited it. The substrate specificity study showed that l-γ-glutamyl-p-nitroanilide (l-γ-Glu-p-NA) is a preference for the enzyme. Steady-state kinetic study revealed that BLrGGT has a k _cat of 105 s⁻¹ and a K _m of 21 μM when using l-γ-Glu-p-NA as the substrate. With this overexpression and purification system, BLrGGT can now be obtained in quantities necessary for structural characterization and synthesis of commercially important γ-glutamyl compounds.