Characterisation of α3β1 and αvβ3 integrin N-oligosaccharides in metastatic melanoma WM9 and WM239 cell lines

It is well documented that glycan synthesis is altered in some pathological processes, including cancer. The most frequently observed alterations during tumourigenesis are extensive expression of β1,6-branched complex type N-glycans, the presence of poly-N-acetyllactosamine structures, and high sialylation of cell surface glycoproteins. This study investigated two integrins, α₃β₁ and α_vβ₃, whose expression is closely related to cancer progression. Their oligosaccharide structures in two metastatic melanoma cell lines (WM9, WM239) were analysed with the use of matrix-assisted laser desorption ionisation mass spectrometry. Both examined integrins possessed heavily sialylated and fucosylated glycans, with β1,6-branches and short polylactosamine chains. In WM9 cells, α₃β₁ integrin was more variously glycosylated than α_vβ₃; in WM239 cells the situation was the reverse. Functional studies (wound healing and ELISA integrin binding assays) revealed that the N-oligosaccharide component of the tested integrins influenced melanoma cell migration on vitronectin and α₃β₁ integrin binding to laminin-5. Additionally, more variously glycosylated integrins exerted a stronger influence on these parameters. To the best of our knowledge, this is the first report concerning structural characterisation of α_vβ₃ integrin glycans in melanoma or in any cancer cells.     

A new resorufin-based α-glucosidase assay for high-throughput screening

Mutations in α-glucosidase cause accumulation of glycogen in lysosomes, resulting in Pompe disease, a lysosomal storage disorder. Small molecule chaperones that bind to enzyme proteins and correct the misfolding and mistrafficking of mutant proteins have emerged as a new therapeutic approach for the lysosomal storage disorders. In addition, α-glucosidase is a therapeutic target for type II diabetes, and α-glucosidase inhibitors have been used in the clinic as alternative treatments for this disease. We have developed a new fluorogenic substrate for the α-glucosidase enzyme assay, resorufin α-d-glucopyranoside. The enzyme reaction product of this new substrate emits at a peak of 590 nm, reducing the interference from fluorescent compounds seen with the existing fluorogenic substrate, 4-methylumbelliferyl-α-d-glucopyranoside. Also, the enzyme kinetic assay can be carried out continuously without the addition of stop solution due to the lower pK_a of the product of this substrate. Therefore, this new fluorogenic substrate is a useful tool for the α-glucosidase enzyme assay and will facilitate compound screening for the development of new therapies for Pompe disease.     

Km values of influenza virus neuraminidases for a new fluorogenic substrate, 4-methylumbelliferone N-acetyl neuraminic acid ketoside.

A fluorogenic substrate of neuraminidase, 4-methylumbelliferone N-acetylneuraminic acid ketoside (MUN), was synthesized. K_m values were obtained for Clostridium perfringens neuraminidase and strains of A- and B-type influenza neuraminidase from chicken red blood cell influenza virus eluates.     

The cDNA sequence of three hemocyanin subunits from the garden snail Helix lucorum

Hemocyanins are blue copper containing respiratory proteins residing in the hemolymph of many molluscs and arthropods. They can have different molecular masses and quaternary structures. Moreover, several molluscan hemocyanins are isolated with one, two or three isoforms occurring as decameric, didecameric, multidecameric or tubule aggregates. We could recently isolate three different hemocyanin isopolypeptides from the hemolymph of the garden snail Helix lucorum (HlH). These three structural subunits were named α_D-HlH, α_N-HlH and β-HlH. We have cloned and sequenced their cDNA which is the first result ever reported for three isoforms of a molluscan hemocyanin. Whereas the complete gene sequence of α_D-HlH and β-HlH was obtained, including the 5′ and 3′ UTR, 180 bp of the 5′ end and around 900 bp at the 3′ end are missing for the third subunit. The subunits α_D-HlH and β-HlH comprise a signal sequence of 19 amino acids plus a polypeptide of 3409 and 3414 amino acids, respectively. We could determine 3031 residues of the α_N-HLH subunit. Sequence comparison with other molluscan hemocyanins shows that α_D-HlH is more related to Aplysia californicum hemocyanin than to each of its own isopolypeptides. The structural subunits comprise 8 different functional units (FUs: a, b, c, d, e, f, g, h) and each functional unit possesses a highly conserved copper-A and copper-B site for reversible oxygen binding. Potential N-glycosylation sites are present in all three structural subunits. We confirmed that all three different isoforms are effectively produced and secreted in the hemolymph of H. lucorum by analyzing a tryptic digest of the purified native hemocyanin by MALDI-TOF and LC-FTICR mass spectrometry.     

A membrane-associated creatine kinase (EC 2.7.3.2) identified as an acidic species of the non-receptor,peripheral v-proteins in Torpedo acetylcholine receptor membranes

Human liver mRNA isolated from subjects phenotyped as homozygous PiMM or PiZZ α₁-antitrypsin, was translated in a reticulocyte cell-free system, and α₁-antitrypsin identified by immunoprecipitation. In the presence of dog pancreas membranes the translated α₁-antitrypsin appeared as a larger product. Treatment with endo-β-N-glucosaminidase yielded a protein smaller than the reticulocyte translated product, presumably due to removal of the N-terminal signal sequence by membranes and sugar residues by endo-β-N-glucosaminidase. Quantitation of α₁-antitrypsin translated from PiMM and PiZZ livers suggests that both mRNA species were present at the same cellular concentration, and that processing to the core glycosylation stage proceeded at identical rates.     

Large conductance Ca2+-activated K+ channel (BKCa) activating properties of a series of novel N-arylbenzamides: Channel subunit dependent effects

Large conductance calcium activated potassium channels (BK_Ca) are fundamental in the control of cellular excitability. Thus, compounds that activate BK_Ca channels could provide potential therapies in the treatment of pathologies of the cardiovascular and central nervous system. A series of novel N-arylbenzamide compounds, and the reference compound NS1619, were evaluated for BK_Ca channel opener properties in Human Embryonic Kidney (HEK293) cells expressing the human BK_Ca channel α-subunit alone or α + β1-subunit complex.Channel activity was determined using a non-radioactive Rb⁺ efflux assay to construct concentration effect curves for each compound. All N-arylbenzamide compounds and NS1619 evoked significant (p <0.05) concentration related increases in Rb⁺ efflux both in cells expressing α-subunit alone or α + β1-subunits. Co-expression of the β1-subunit modified the Rb⁺ efflux responses, relative to that obtained in cells expressing the α-subunit alone, for most of the N-arylbenzamide compounds, in contrast to NS1619. The EC₄₀ values of NS1619, BKMe1 and BKOEt1 were not significantly affected by the co-expression of the BK_Ca channel α + β1-subunits. In contrast, 5 other N-arylbenzamides (BKPr2, BKPr3, BKPr4, BKH1 and BKVV) showed a significant (p <0.05) 2- to 10-fold increase in EC₄₀ values when tested on the BK_Ca α + β1-subunit expressing cells compared to BK_Ca α-subunit expressing cells. Further, the E_max values for BKPr4, BKVV and BKH1 were lower in the BK_Ca channel α + β1-subunit expressing cells.In conclusion, the N-arylbenzamides studied, like NS1619, were able to activate BK_Ca channels formed of the α-subunit only. The co-expression of the β1-subunit, however, modified the ability of certain compounds to active the channel leading to differentiated pharmacodynamic profiles.     

Purification and properties of a β-N-acetylaminoglucohydrolase from malted barley

β-N-Acetylaminoglucohydrolase (β-2-acetylamino-2-deoxy-D-glucoside acetylaminodeoxyglucohydrolase, EC 3.2.1.30) was extracted from malted barley and purified. The partially purified preparation was free from α-and β-glucosidase, α- and β-galactosidase, α-mannosidase and β-mannosidase. This preparation was free from α-mannosidase only after affinity chromatography with p-amino-N-acetyl-β-D-glucosaminidine coupled to Sepharose. The enzyme was active between pH 3 and 6.5 and had a pH optimum at pH 5. A MW of 92000 was obtained by sodium dodecyl sulfate-acrylamide gel electrophoresis and a sedimentation coefficient of 4.65 was obtained from sedimentation velocity experiments. β-N-Acetylaminoglucohydrolase had a K_m of 2.5 × 10^?4 M using the p-nitrophenyl N-acetyl β-D-glucosaminidine as the substrate.     

Retarded protein folding of the human Z-type α1-antitrypsin variant is suppressed by Cpr2p

The human Z-type α₁-antitrypsin variant has a strong tendency to accumulate folding intermediates due to extremely slow protein folding within the endoplasmic reticulum (ER) of hepatocytes. Human α₁-antitrypsin has 17 peptidyl-prolyl bonds per molecule; thus, the effect of peptidyl-prolyl isomerases on Z-type α₁-antitrypsin protein folding was analyzed in this study. The protein level of Cpr2p, a yeast ER peptidyl-prolyl isomerase, increased more than two-fold in Z-type α₁-antitrypsin-expressing yeast cells compared to that in wild-type α₁-antitrypsin-expressing cells. When CPR2 was deleted from the yeast genome, the cytotoxicity of Z-type α₁-antitrypsin increased significantly. The interaction between Z-type α₁-antitrypsin and Cpr2p was confirmed by co-immunoprecipitation. In vitro folding assays showed that Cpr2p facilitated Z-type α₁-antitrypsin folding into the native state. Furthermore, Cpr2p overexpression significantly increased the extracellular secretion of Z-type α₁-antitrypsin. Our results indicate that ER peptidyl-prolyl isomerases may rescue Z-type α₁-antitrypsin molecules from retarded folding and eventually relieve clinical symptoms caused by this pathological α₁-antitrypsin.     

Glycosyltransferase activities of Ehrlich ascites tumor cells: Detection,isolation, and characterization using oligosaccharide-Synsorb beads

Detergent extracts of Ehrlich tumor cell membranes exhibit a host of glycosyltransferase activities which have been investigated using oligosaccharides immobilized to Synsorb beads as acceptors. Glycosidase digestions in combination with methylation analysis of the insoluble products have demonstrated the presence of an α(1,3)-galactosyltransferase and a β(1,3)-N-acetylglucosaminyltransferase, enzymes that utilize N-acetyllactosamine as their acceptor substrate. The two enzymes are presumably involved in the biosynthesis of α-d-galactosyl-terminated poly-N-acetyllactosamine glycans that occur on the surface of Ehrlich cells. In addition, a β-galactosyltransferase acting on N-acetylglucosamine and a separate β-N-acetylglucosaminyltransferase that is capable of incorporating GlcNAc into the trisaccharide β-d-GlcNAc(1,3)-β-d-Gal(1,4)-β-d-Glc-Synsorb have been identified. The Ehrlich cell α- and β-galactosyltransferases have been separated by chromatography on β-GlcNAc-Synsorb beads. In the presence of MnCl₂ and UDP the β-galactosyltransferase is specifically adsorbed to the monosaccharide column whereas the α-galactosyltransferase passes through unretarded.     

A proposed mechanism for the thermal denaturation of a recombinant Bacillus halmapalus α-amylase—the effect of calcium ions

The thermal stability of a recombinant α-amylase from Bacillus halmapalus α-amylase (BHA) has been investigated using circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). This α-amylase is homologous to other Bacillus α-amylases where crystallographic studies have identified the existence of three calcium binding sites in the structure. Denaturation of BHA is irreversible with a T_m of approximately 89 °C and DSC thermograms can be described using a one-step irreversible model. A 5 °C increase in T_m in the presence of 10-fold excess CaCl₂ was observed. However, a concomitant increase in the tendency to aggregate was also observed. The presence of 30–40-fold excess calcium chelator (ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis[β-aminoethyl ether] N,N,N′,N′-tetraacetic acid (EGTA)) results in a large destabilization of BHA, corresponding to about 40 °C lower T_m as determined by both CD and DSC. Ten-fold excess EGTA reveals complex DSC thermograms corresponding to both reversible and irreversible transitions, which probably originate from different populations of BHA/calcium complexes. Combined interpretation of these observations and structural information on homologous α-amylases forms the basis for a suggested mechanism underlying the inactivation mechanism of BHA. The mechanism includes irreversible thermal denaturation of different BHA/calcium complexes and the calcium binding equilibria. Furthermore, the model accounts for a temperature-induced reversible structural change associated with calcium binding.     

Role of alphaPhe-291 residue in the phosphate-binding subdomain of catalytic sites of Escherichia coli ATP synthase

The role of αPhe-291 residue in phosphate binding by Escherichia coli F₁F₀-ATP synthase was examined. X-ray structures of bovine mitochondrial enzyme suggest that this residue resides in close proximity to the conserved βR246 residue. Herein, we show that mutations αF291D and αF291E in E. coli reduce the ATPase activity of F₁F₀ membranes by 350-fold. Yet, significant oxidative phosphorylation activity is retained. In contrast to wild-type, ATPase activities of mutants were not inhibited by MgADP-azide, MgADP-fluoroaluminate, or MgADP-fluoroscandium. Whereas, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) inhibited wild-type ATPase essentially completely, ATPase in mutants was inhibited maximally by ∼75%, although reaction still occurred at residue βTyr-297, proximal to αPhe-291 in the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts in βE (empty) catalytic sites, as shown previously by X-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild-type but not in mutants. In addition, our data suggest that the interaction of αPhe-291 with phosphate during ATP hydrolysis or synthesis may be distinct.     

Synthesis and evaluation of novel photoreactive α-amino acid analog carrying acidic and cleavable functions

A novel photoreactive α-amino acid bearing an acidic residue and a cleavable diazirine was developed. To mimic common acidic α-amino acids, the residue was designed to be N-acylsulfonamide that possesses an acidic proton and is able to dissociate under the physiological conditions. The inhibition assay of its biotin-tagged derivative with glutamyl endopeptidase from Staphylococcus aureus V8 strain revealed a Ki_app value of 162 μM, which is slightly higher than the K_m value of a common substrate. Upon UV irradiation, this derivative specifically photolabeled glutamyl endopeptidase, l-glutamate dehydrogenase, glutamic oxalacetic transaminase, and l-glutamine synthetase, all the enzymes exhibit high affinity toward acidic α-amino acids. In addition, N-acylsulfonamide group functioned as a cleavable linker in mild basic solution after a brief N-alkylation. Either the multifunctional nature or the simple structure of this acidic α-amino acid surrogate would be useful as versatile photoreactive building block.     

Mass spectrometry and 13C nuclear magnetic resonance spectroscopy of compounds modeling the glycopeptide linkage of glycoproteins

The properties of several compounds useful as models for three-dimensional conformational studies and the investigation of the chemical degradation of glycopeptide linkages both of the N- and O-glycosidic type are described. Using the method of differential chemical shift in H₂O and D₂O as solvents, the carbon NMR spectrum of N-acetylglucosaminylasparagine, 1-N-acetyl-β-d-glucopyranosylamine, and 1-N-acetyl-2-acetamido-β-d-glucopyranosylamine has been assigned. Electron impact mass spectra of the peracetylated derivatives of the latter two compounds show a peak apparently unique to glycopyranosylamides at $\text{m}\text{e}\text{= 269}$, no analog of which is observed in the mass spectra of other peracetylated sugars. As models of the α-O-glycosidic linkage, fully assigned carbon NMR spectra of α-methyl-N-acetylgalactosamine (GalNAc), α-methyl-3-O-methyl GalNAc,and -GlcNAc as well as the disaccharide Glc-β-1 → 3 GalNAc are reported. Because certain anomalies in the chemical shifts and ¹J_CH observed in the disaccharide and in O-glycosylated glycoproteins are not observed in the simple model compounds, they may result from conformational interactions in the glycopeptides.     

Comparative genomics identifies new alpha class genes within the avian glutathione S-transferase gene cluster

Glutathione S-transferases (GSTs: EC2.5.1.18) are a superfamily of multifunctional dimeric enzymes that catalyze the conjugation of glutathione (GSH) to electrophilic chemicals. In most animals and in humans, GSTs are the principal enzymes responsible for detoxifying the mycotoxin aflatoxin B₁ (AFB₁) and GST dysfunction is a known risk factor for susceptibility towards AFB₁. Turkeys are one of the most susceptible animals known to AFB₁, which is a common contaminant of poultry feeds. The extreme susceptibility of turkeys is associated with hepatic GSTs unable to detoxify the highly reactive and electrophilic metabolite exo-AFB₁-8,9-epoxide (AFBO). In this study, comparative genomic approaches were used to amplify and identify the α-class tGST genes (tGSTA1.1, tGSTA1.2, tGSTA1.3, tGSTA2, tGSTA3 and tGSTA4) from turkey liver. The conserved GST domains and four α-class signature motifs in turkey GSTs (with the exception of tGSTA1.1 which lacked one motif) confirm the presence of hepatic α-class GSTs in the turkey. Four signature motifs and conserved residues found in α-class tGSTs are (1) xMExxxWLLAAAGVE, (2) YGKDxKERAxIDMYVxG, (3) PVxEKVLKxHGxxxL and (4) PxIKKFLXPGSxxKPxxx. A BAC clone containing the α-class GST gene cluster was isolated and sequenced. The turkey α-class GTS genes genetically map to chromosome MGA2 with synteny between turkey and human α-class GSTs and flanking genes. This study identifies the α-class tGST gene cluster and genetic markers (SNPs, single nucleotide polymorphisms) that can be used to further examine AFB₁ susceptibility and resistance in turkeys. Functional characterization of heterologously expressed proteins from these genes is currently underway.     

Origin of apparent negative cooperativity of F1-ATPase

In order to get insight into the origin of apparent negative cooperativity observed for F₁-ATPase, we compared ATPase activity and ATPMg binding of mutant subcomplexes of thermophilic F₁-ATPase, α_(W463F)3β_(Y341W)3γ and α_{(K175A/T176A/W463F)3}β_(Y341W)3γ. For α_(W463F)3β_(Y341W)3γ, apparent K_m's of ATPase kinetics (4.0 and 233 μM) did not agree with apparent K_m's deduced from fluorescence quenching of the introduced tryptophan residue (on the order of nM, 0.016 and 13 μM). On the other hand, in case of α_{(K175A/T176A/W463F)3}β_(Y341W)3γ, which lacks noncatalytic nucleotide binding sites, the apparent K_m of ATPase activity (10 μM) roughly agreed with the highest K_m of fluorescence measurements (27 μM). The results indicate that in case of α_(W463F)3β_(Y341W)3γ, the activating effect of ATP binding to noncatalytic sites dominates overall ATPase kinetics and the highest apparent K_m of ATPase activity does not represent the ATP binding to a catalytic site. In case of α_{(K175A/T176A/W463F)3}β_(Y341W)3γ, the K_m of ATPase activity reflects the ATP binding to a catalytic site due to the lack of noncatalytic sites. The Eadie-Hofstee plot of ATPase reaction by α_{(K175A/T176A/W463F)3}β_(Y341W)3γ was rather linear compared with that of α_(W463F)3β_(Y341W)3γ, if not perfectly straight, indicating that the apparent negative cooperativity observed for wild-type F₁-ATPase is due to the ATP binding to catalytic sites and noncatalytic sites. Thus, the frequently observed K_m's of 100-300 μM and 1-30 μM range for wild-type F₁-ATPase correspond to ATP binding to a noncatalytic site and catalytic site, respectively.     

Detection of esterase activity by chromogenic and fluorogenic probe based on an O-nitrobenzoxadiazole (O-NBD) unit

We designed a conjugated molecule bearing an O-nitrobenzoxadiazole (O-NBD) unit and an acetylated trimethyl lock as a chromogenic and fluorogenic probe for the detection of esterase activity. The designed molecule was briefly synthesized from a commercially available compound in two steps. Several experiments revealed that the conjugated molecule serves as a sensitive chromogenic and fluorogenic probe for the detection of porcine liver esterase activity. Mechanistic studies indicated that an intramolecular O- to N-NBD migration is involved in the chromogenic/fluorogenic phenomena. The results here would be helpful for designing other O-NBD-based chromogenic/fluorogenic probes in future.     

稻麦轮作系统冬小麦农田耕作措施对氧化亚氮排放的影响

2008—2011年,采用静态箱-气相色谱法对长江下游稻麦轮作系统冬小麦农田N2O排放进行了为期3a的田间原位观测,研究不同耕作措施(免耕、旋耕和翻耕)对冬小麦生长季N2O排放的影响。结果表明:不同耕作措施下冬小麦农田N2O排放高峰出现在施用基肥后的1个月内以及施用孕穗肥后的4月中旬至小麦成熟期,其余时间N2O排放通量均较小。年度和耕作措施对冬小麦农田N2O季节排放总量均有极显著影响(P<0.01),不同处理N2O季节排放总量表现为免耕>翻耕>旋耕,2008—2011年3年平均分别为2.50 kg/hm2、2.05 kg/hm2和1.66 kg/hm2,免耕比翻耕增加N2O排放22.0%(P<0.05),旋耕比翻耕减排19.0%(P<0.05)。冬小麦生长期内施用孕穗肥后1个月内N2O排放通量与农田土壤充水孔隙率(WFPS)及10 cm地温呈显著(P<0.05)或极显著(P<0.01)正相关,2009—2010年施用基肥后1个月内N2O排放通量与WFPS呈显著负相关(P<0.05)。结果说明旋耕是减少长江下游稻麦轮作系统冬小麦农田N2O排放的最佳耕作措施。     

Synthesis and characterization of cobalt oxide nanoparticles by thermal treatment process

The simple preparation of Co₃O₄ nanoparticles from a solid organometallic molecular precursor N-N′-bis(salicylaldehyde)-1,2-phenylenediimino cobalt(II); Co(salophen) has been achieved via two simple steps: firstly, the Co(salophen) precursor was precipitated from the reaction of cobalt(II) acetate and N-N′-bis(salicylaldehyde)-1,2-phenylenediimino; H₂salophen; in propanol under nitrogen condition; then, cubic phase Co₃O₄ nanoparticles with the size of mostly 30-50 nm could be produced by thermal treatment of the Co(salophen) in air at 773 K for 5 h. The as-synthesized products were characterized by powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), and scanning electronic microscopy (SEM). These results confirm that the resulting oxide was pure single-crystalline Co₃O₄ nanoparticles. The optical property test indicates that the absorption peak of the nanoparticles shifts towards short wavelength, and the blue shift phenomenon might be ascribed to the quantum effect. The hysteresis loops of the obtained samples reveal the ferromagnetic behaviors the enhanced coercivity (H_c) and decreased saturation magnetization (M_s) in contrast to their respective bulk materials.     

Backbone modifications in peptidic inhibitors of flaviviral proteases

The NS2B-NS3 protease is a promising target for the development of drugs against dengue virus (DENV), West Nile virus (WNV) and related flaviviruses. We report the systematic variation of the peptide backbone of the two lead compounds Bz-Arg-Lys-d-Phg-NH₂ and Bz-Arg-Lys-d-Phg(OBn)-NH₂. While inhibitory activity against WNV protease was generally decreased, the inhibitory potency against DENV protease could be conserved and increased in several peptidomimetics, particularly in those containing a (NMe)arginine fragment or an N-terminal α-keto amide. Methylation at the α-position of the C-terminal phenylglycine led to a 6-fold higher potency against DENV protease. Peptidomimetics with modified backbone showed increased resistance against hydrolytic attack by trypsin and α-chymotrypsin.     

Specific spectrophotometric assays for cathepsin B1.

Cathepsin B₁ from bovine spleen was partially purified by acetone fractionation and by chromatography on Sephadex G-150 and DEAE Sephadex A-50. The enzyme was shown to catalyze the hydrolysis of p-nitrophenyl benzyloxycarbonylglycinate and p-nitrophenyl α-N-benzyloxycarbonyl-l-lysinate. Under the assay conditions, cathepsin B₁ is the major enzyme present in bovine spleen homogenates hydrolyzing these substrates. The kinetic parameters for the hydrolysis of p-nitrophenyl benzyloxycarbonylglycinate and p-nitrophenyl α-N-benzyloxycarbonyl-l-lysinate were measured and compared with those obtained for other cathepsin B₁ substrates. These results form the basis of an improved spectrophotometric assay for this enzyme in which the liberation of p-nitrophenol from either the N-benzyloxycarbonyl glycine or lysine p-nitrophenyl ester is monitored continuously at 326 nm.