Previously, we developed an alpha 2-6-sialic acid (Sia)-specific lectin (SRC) starting from an R-type galactose-specific lectin C-terminal domain. However, it showed relatively low affinity because ...of its monovalency. Here, we engineered a tandem repeat construct (SRC2) showing substantial affinity for alpha 2,6-sialylated N-glycans (in the order of 10 super(-) super(6)M in K sub(d)), almost comparable to a natural alpha 2-6Sia-specific lectin from Sambucus sieboldiana (SSA). Notably, its binding to branched N-glycans was found to be more selective than SSA. Nevertheless, SRC2 showed no apparent hemagglutinating activity, while it exerted strong erythrocyte-binding activity. This unique feature will help flow cytometry analysis, where usual lectins including SSA agglutinate cells. Some other biochemical properties investigated for SRC2, e.g., high productivity in bacteria and easy release of captured glycoproteins with lactose have demonstrated versatility of this mutant protein as a powerful tool for sialoglycomics.
Abstract The dopamine transporter has been shown to be the most relevant target site for the specificity of 1-methyl-4-phenylpyridinium ion (MPP+ ), a neurotoxin for dopaminergic neurons. In ...contrast, the mechanisms underlying the selective toxicity of manganese and rotenone, potentially toxic agents implicated in dopaminergic neuronal cell death, remain unknown. The aim of this study was to determine the cellular mechanisms of manganese or rotenone uptake in dopaminergic cells via the dopamine transporter. PC12 cells overexpressing the dopamine transporter, which were exposed to 10 μM MPP+ , showed extensive DNA fragmentation, a biochemical hallmark of apoptosis, whereas wild-type PC12 cells or vector-transfected PC12 cells, which were exposed to 5 mM MPP+ , did not show DNA fragmentation. In contrast, manganese and rotenone induced DNA fragmentation at slightly lower concentrations in PC12 cells overexpressing the dopamine transporter compared to control cells. Dopamine transporter inhibitors, such as mazindol, nomifensine, or GBR12909, inhibited MPP+ -induced DNA fragmentation but did not affect manganese- and rotenone-induced DNA fragmentation in PC12 cells overexpressing the dopamine transporter. Finally, manganese accumulated to similar levels in PC12 cells overexpressing the dopamine transporter and control PC12 cells following incubation with manganese chloride. These results suggested that the dopamine transporter dose not confer cytotoxicity to manganese and rotenone.
Previously, we developed an α2-6-sialic acid (Sia)-specific lectin (SRC) starting from an R-type galactose-specific lectin C-terminal domain. However, it showed relatively low affinity because of its ...monovalency. Here, we engineered a tandem repeat construct (SRC2) showing substantial affinity for α2,6-sialylated
N-glycans (in the order of 10
−6
M in
K
d), almost comparable to a natural α2-6Sia-specific lectin from
Sambucus sieboldiana (SSA). Notably, its binding to branched
N-glycans was found to be more selective than SSA. Nevertheless, SRC2 showed no apparent hemagglutinating activity, while it exerted strong erythrocyte-binding activity. This unique feature will help flow cytometry analysis, where usual lectins including SSA agglutinate cells. Some other biochemical properties investigated for SRC2, e.g., high productivity in bacteria and easy release of captured glycoproteins with lactose have demonstrated versatility of this mutant protein as a powerful tool for sialoglycomics.
Ingestion of the mushroom
Boletus venenatus causes a gastrointestinal syndrome, such as diarrhea. A family of isolectins (
B. venenatus lectins, BVLs) was isolated as the toxic principles from the ...mushroom. BVL ingestion resulted in fetal toxicity to mice and also caused diarrhea.
Ingestion of the toxic mushroom
Boletus venenatus causes a severe gastrointestinal syndrome, such as nausea, repetitive vomiting, diarrhea, and stomachache. A family of isolectins (
B. venenatus lectins, BVLs) was isolated as the toxic principles from the mushroom by successive 80% ammonium sulfate-precipitation, Super Q anion-exchange chromatography, and TSK-gel G3000SW gel filtration. Although BVLs showed a single band on SDS–PAGE, they were further divided into eight isolectins (BVL-1 to -8) by BioAssist Q anion-exchange chromatography. All the isolectins showed lectin activity and had very similar molecular weights as detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. Among them, BVL-1 and -3 were further characterized with their complete amino acid sequences of 99 amino acids determined and found to be identical to each other. In the hemagglutination inhibition assay, both proteins failed to bind to any mono- or oligo-saccharides tested and showed the same sugar-binding specificity to glycoproteins. Among the glycoproteins examined, asialo-fetuin was the strongest inhibitor. The sugar-binding specificity of each isolectin was also analyzed by using frontal affinity chromatography and surface plasmon resonance analysis, indicating that they recognized
N-linked sugar chains, especially Galβ1
→
4GlcNAcβ1
→
4Manβ1
→
4GlcNAcβ1
→
4GlcNAc (Type II) residues in
N-linked sugar chains. BVLs ingestion resulted in fatal toxicity in mice upon intraperitoneal administration and caused diarrhea upon oral administration in rats.
The transcription factor Pax6 is essential for the development of the central nervous system and it exerts its multiple functions by regulating the expression of downstream target molecules. To ...screen for genes downstream of Pax6, we performed comprehensive transcriptome profiling analyses in the early hindbrain of Pax6 homozygous mutant and wild-type rats using microarrays.
Comparison of quadruplicate microarray experiments using two computational methods allowed us to identify differentially expressed genes that have relatively small fold changes or low expression levels. Gene ontology analyses of the differentially expressed molecules demonstrated that Pax6 is involved in various signal transduction pathways where it regulates the expression of many receptors, signaling molecules, transporters and transcription factors. The up- or down-regulation of these genes was further confirmed by quantitative RT-PCR. In situ staining of Fabp7, Dbx1, Unc5h1 and Cyp26b1 mRNAs showed that expression of these transcripts not only overlapped with that of Pax6 in the hindbrain of wild-type and Pax6 heterozygous mutants, but also was clearly reduced in the hindbrain of the Pax6 homozygous mutant. In addition, the Pax6 homozygous mutant hindbrain showed that Cyp26b1 expression was lacked in the dorsal and ventrolateral regions of rhombomeres 5 and 6, and that the size of rhombomere 5 expanded rostrocaudally.
These results indicate that Unc5h1 and Cyp26b1 are novel candidates for target genes transactivated by Pax6. Furthermore, our results suggest the interesting possibility that Pax6 regulates anterior-posterior patterning of the hindbrain via activation of Cyp26b1, an enzyme that metabolizes retinoic acid.
This study aimed to investigate the association of the KLOTHO gene single nucleotide polymorphisms (SNPs), G-395A and C1818T, with various laboratory data in 219 Japanese hemodialysis (HD) patients.
...The genotyping of G-395A in the promoter region and C1818T in exon 4 was performed using polymerase chain reaction with confronting two-pair primers (PCR-CTPP) assay.
In HD patients, the allele frequencies of G-395A were 0.847 for the G allele and 0.153 for the A allele and those of the C1818T were 0.829 for the C allele and 0.171 for the T allele. There were no significant differences in allele frequencies of G-395A and those of the C1818T between HD patients and healthy subjects. Multivariate analysis adjusted for age and duration on HD demonstrated that uric acid was significantly high in A allele carriers of G-395A compared with GG genotype in all and female patients. Low-density lipoprotein cholesterol was significantly low in T allele carriers of C1818T compared with CC genotype in all and male patients.
KLOTHO gene SNPs G-395A and C1818T are associated with low-density lipoprotein cholesterol and uric acid in HD patients.
Neocarzinostatin (NCS) is the first discovered anti-tumor antibiotic having an enediyne-containing chromophore and an apoprotein with a 1: 1 complex. An artificial gene library for NCS apoprotein ...(apo-NCS) production in Escherichia coli was designed and constructed on a phage-display vector, pJuFo. The recombinant phages expressing pre-apo-NCS protein were enriched with a mouse anti-apo-NCS monoclonal antibody, 1C7D4. The apo-NCS gene (encsA) for E. coli was successfully cloned, and then re-cloned into the pRSET A vector. After the his-tagged apo-NCS protein had been purified and cleaved with enterokinase, the binding properties of the recombinant protein as to ethidium bromide (EtBr) were studied by monitoring of total fluorescence intensity and fluorescence polarization with a BEACON 2000 system and GraphPad Prism software. A dissociation constant of 4.4 ± 0.3 μM was obtained for recombinant apo-NCS in the fluorescence polarization study. This suggests that fluorescence polarization monitoring with EtBr as a chromophore mimic may be a simplified method for the characterization of recombinant apo-NCS binding to the NCS chromophore. When Phe78 on apo-NCS was substituted with Trp78 by site-directed mutagenesis using a two stage megaprimer poly-merase chain reaction, the association of the apo-NCS mutant and EtBr observed on fluorescence polarization analysis was of the same degree as in the case of the wild type, although the calculated maximum change (δITmax) in total fluorescence intensity decreased from 113.9 to 31.3. It was suggested that an environmental change of the bound EtBr molecule on F78W might have dramatically occurred as compared with in the case of wild type apo-NCS. This combination of monitoring of fluorescence polarization and total fluorescence intensity will be applicable for determination and prediction of the ligand state bound or associated with the target protein. The histone-specific proteolytic activity was also re-investigated using this recombinant apo-NCS preparation, and calf thymus histone HI, H2A, H2B, H3, and H4. The recombinant apo-NCS does not act as a histone protease because a noticeable difference was not observed between the incubation mixtures with and without apo-NCS under our experimental conditions.
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