Enhanced sugar chain detection by oriented immobilization of Fc-fused lectins

ABSTRACT

We report a novel scaffold for clustering and oriented immobilization of human IgG1 Fc-fused lectins on biosensors without chemical modifications. This approach uses a bio-nanocapsule (BNC) displaying a tandem form of IgG Fc-binding Z domains derived from Staphylococcus aureus protein A (ZZ-BNC). Incorporating ZZ-BNC effectively increased both the sensitivity and sugar chain-binding capacity compared with the condition without ZZ-BNC.     

Trace Elements in the Hair of Hemodialysis Patients

Trace element disturbance is often observed in hemodialysis patients. While trace element concentrations have been reported in blood samples from hemodialysis patients, they have not been well investigated in scalp hair. In the present study, 22 trace elemental concentrations were measured by inductively coupled plasma-atomic emission spectrometry in the scalp hair of 80 male hemodialysis patients and compared with those of 100 healthy male subjects. In hemodialysis patients, the concentrations of beryllium, arsenic, magnesium, chromium, manganese, iron, selenium, molybdenum, iodine, vanadium, and cobalt were significantly higher than those in healthy subjects, while lead, mercury, copper, germanium, and bromine were significantly lower than those in the former group. No significant differences were observed for lithium, aluminum, cadmium, zinc, boron, or nickel. There were significant positive correlations between the duration of hemodialysis and the magnesium and manganese concentrations. There was a significant negative correlation between cadmium concentration and the duration of hemodialysis. There were significant positive correlations between dialysis efficacy (Kt/V) and magnesium, manganese, zinc, and selenium concentrations. In conclusion, trace element concentrations of the scalp hair are different between hemodialysis patients and healthy subjects. Essential trace elements, such as magnesium, manganese, zinc, and selenium, may be affected by the duration of hemodialysis and Kt/V.     

Interaction of hexa-His tag with acidic amino acids results in facilitated refolding of halophilic nucleoside diphosphate kinase

We have previously reported that amino-terminal extension sequence containing hexa-His facilitated refolding and assembly of hexameric nucleoside diphosphate kinase from extremely halophilic archaeon Halobacterium salinarum (NDK). In this study, we made various mutations in both the tag sequence and within NDK molecule. SerNDK, in which hexa-His was replaced with hexa-Ser, showed no facilitated folding. In addition, HisD58GD63G, in which both Asp58 and Asp63 in NDK were replaced with Gly, also showed no refolding enhancement. These results suggest that hexa-His in His-tag interact cooperatively with either Asp58 or Asp63 or both. Furthermore, G114D mutant, which formed a dimer in low salt solution, was strongly stabilized by His-tag to form a stable hexamer.     

The effect of Ndrg2 expression on astroglial activation

N-myc downstream-regulated gene 2 (Ndrg2) is a differentiation- and stress-associated molecule predominantly expressed in astrocytes in the central nervous system (CNS). To study the expression and possible role of Ndrg2 in quiescent and activated astrocytes, mice were administrated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropypridine (MPTP), a Parkinson disease (PD)-related neurotoxin which causes both neurodegeneration and glial activation. Immunohistological analysis revealed that Ndrg2 was highly expressed in both types of astrocytes, but less so in astrocytes during the early process of activation. Ndrg2 was also expressed in astrocyte-like cells, but not in neurons, in human brains from PD and Cortico-basal degeneration (CBD) patients. In cultured astrocytes, gene silencing of Ndrg2 significantly enhanced the numbers of 5-bromo-2′-deoxy-uridine (BrdU)-incorporated and proliferating cell nuclear antigen (PCNA)-positive cells, and reduced the length of cell processes and the amount of F-actin. In contrast, adenovirus-mediated overexpression of Ndrg2 significantly reduced the numbers of BrdU-incorporated and PCNA-positive cells, and enhanced the amount of F-actin. Fractionation and immunocytochemical analysis further revealed that Ndrg2 was located in different cellular fractions including the cytosol and cell surface membranes. These results suggest that Ndrg2 may regulate astroglial activation through the suppression of cell proliferation and stabilization of cell morphology.     

Salt-dependent thermo-reversible α-amylase: cloning and characterization of halophilic α-amylase from moderately halophilic bacterium, <Emphasis Type="Italic">Kocuria varians</Emphasis>

A moderately halophilic bacterium, Kocuria varians, was found to produce active α-amylase (K. varians α-amylase (KVA)). We have observed at least six different forms of α-amylase secreted by this bacterium into the culture medium. Characterization of these KVA forms and cloning of the corresponding gene revealed that KVA comprises pre-pro-precursor form of α-amylase catalytic domain followed by the tandem repeats, which show high similarity to each other and to the starch binding domain (SBD) of other α-amylases. The observed six forms were most likely derived by various processing of the protein product. Recombinant KVA protein was successfully expressed in Escherichia coli as a fusion protein and was purified with affinity chromatography after cleavage from fusion partner. The highly acidic amino acid composition of KVA and the highly negative electrostatic potential surface map of the modeled structure strongly suggested its halophilic nature. Indeed, KVA showed distinct salt- and time-dependent thermal reversibility: when α-amylase was heat denatured at 85°C for 3 min in the presence of 2 M NaCl, the activity was recovered upon incubation on ice (50% recovery after 15 min incubation). Conversely, KVA denatured in 0.1 M NaCl was not refolded at all, even after prolonged incubation. KVA activity was inhibited by proteinaceous α-amylase inhibitor from Streptomyces nitrosporeus, which had been implicated to inhibit only animal α-amylases. KVA with putative SBD regions was found to digest raw starch.     

Cytoglobin expression of rectal subepithelial myofibroblasts: significant alterations of cytoglobin+ stromal cells in long-standing ulcerative colitis

Cytoglobin/stellate cell activation-associated protein (Cygb/STAP), a hemoprotein, functions as part of an O2 reservoir with protective effects against oxidative stress in hepatic stellate cells. Heterogeneous expression of the neural cell adhesion molecule (NCAM)+ and/or α-smooth muscle actin (αSMA)+ has been noted in subepithelial myofibroblasts and interstitial cells of the same lineage in the colorectum. We have demonstrated that early genomic instability of both epithelial and stromal cells in ulcerative colitis (UC) is important for colorectal tumorigenesis, as well as for mucosal remodeling. To further clarify possible roles of stromal cells in mucosal remodeling and tumor development in UC, we here focused on Cygb expression of subepithelial myofibroblasts and interstitial cells, as well as αSMA and HSP47. Noncancerous mucosa of resected rectae from UC patients with or without colorectal neoplasia (14 and 20 cases, respectively) and of sporadic rectal cancer cases (16) was analyzed immunohistochemically, as well as by immuno-fluorescence and electron microscopy. The results, heterogeneous phenotypes of Cygb+, αSMA+ and HSP47+ subepithelial myofibroblasts and interstitial cells, corresponding to rectal stellate cells, were demonstrated. A decrease of Cygb+ subepithelial myofibroblasts and an increase of αSMA+ interstitial cells were significant in UC, as compared to normal rectal mucosa. Furthermore, a decrease of Cygb+ subepithelial myofibroblasts, correlating with αSMA+ and HSP47+ cells, was significant in long-standing UC with neoplasia. In conclusion, there are heterogeneous phenotypes of Cygb+, αSMA+ and HSP47+ subepithelial myofibroblasts and interstitial cells in the rectal mucosa. Mucosal remodeling with alterations of Cygb+ and/or αSMA+/HSP47+ stromal cells might have some relation to UC-associated tumorigenesis.     

Suppressors of DnaA(ATP) imposed overinitiation in Escherichia coli

Chromosome replication in Escherichia coli is limited by the supply of DnaA associated with ATP. Cells deficient in RIDA (Regulatory Inactivation of DnaA) due to a deletion of the hda gene accumulate suppressor mutations (hsm) to counteract the overinitiation caused by an elevated DnaA(ATP) level. Eight spontaneous hda suppressor mutations were identified by whole-genome sequencing, and three of these were analysed further. Two mutations (hsm-2 and hsm-4) mapped in the dnaA gene and led to a reduced ability to initiate replication from oriC. One mutation (hsm-1) mapped to the seqA promoter and increased the SeqA protein level in the cell. hsm-1 cells had prolonged origin sequestration, reduced DnaA protein level and reduced DnaA-Reactivating Sequence (DARS)-mediated rejuvenation of DnaA(ADP) to DnaA(ATP) , all of which could contribute to the suppression of RIDA deficiency. Despite of these defects hsm-1 cells were quite similar to wild type with respect to cell cycle parameters. We speculate that since SeqA binding sites might overlap with DnaA binding sites spread throughout the chromosome, excess SeqA could interfere with DnaA titration and thereby increase free DnaA level. Thus, in spite of reduction in total DnaA, the amount of DnaA molecules available for initiation may not be reduced.     

Fluorometric determination of 2-oxoadipic acid, a common metabolite of tryptophan and lysine, by high-performance liquid chromatography with pre-chemical derivatization

2-Oxoadipic acid, a key metabolite of tryptophan and lysine, reacted with 1,2-diamino-4,5-methylenebenzene in an acidic solution to produce a fluorescent derivative. The reaction product was separated using a Tosoh ODS-80Ts column with 20 mmol/L of KH?PO?-K?HPO? buffer (pH 7.0) containing 26% methanol at a flow rate 0.8 mL/min. The excitation wavelength of detection was 367 nm, and the emission wavelength was 446 nm. The limit of quantification was 1 pmol per injection, sufficiently sensitive for the determination of 2-oxoadipic acid in human and experimental animal urine.     

Amelioration of D-galactosamine-induced acute liver injury in rats by dietary supplementation with betaine derived from sugar beet molasses

The effects of betaine supplementation on D-galactosamine-induced liver injury were examined in terms of hepatic and serum enzyme activities and of the levels of glutathione and betaine-derived intermediates. The rats induced with liver injury showed marked increases in serum enzyme activity, but those receiving dietary supplementation of 1% betaine showed enzyme activity levels similar to a control group without liver injury. Administration of betaine also increased both hepatic and serum glutathione levels, even following D-galactosamine injection. The activity of glutathione-related enzymes was markedly decreased following injection of D-galactosamine, but remained comparable to that of the control group in rats receiving 1% betaine. The concentrations of hepatic S-adenosyl methionine and cysteine showed similar trends to that observed for hepatic glutathione levels. These results indicate that 1% betaine has a hepatoprotective effect by increasing hepatic and serum glutathione levels along with glutathione-related enzyme activities in rats.     

Arginine increases the solubility of alkyl gallates through interaction with the aromatic ring

We have recently proposed the application of solubilizing effects of arginine to poorly soluble aromatic compounds for drug discovery research. In this study, we compared the solubilizing effects of arginine with those of other amino acids, salts and a surfactant using alkyl gallates as model drug substances of low aqueous solubility. The solubilizing effects of arginine on the alkyl gallates were distinct compared with those of other amino acids and salts; the effects were even greater than those achieved using a strongly chaotropic guanidinium ion. Transfer free energy of the alkyl gallates from water to arginine solution depended weakly on their dissolution free energy in water, which is in contrast to sodium dodecyl sulphate that showed strong dependence. The present results suggest that arginine solubilizes alkyl gallates through interaction with the aromatic moiety and sodium dodecyl sulphate does so by interacting with alkyl groups.