Glycosylation pattern of humanized IgG-like bispecific antibody produced by recombinant CHO cells

The glycosylation pattern of a humanized anti-EGFR×anti-CD3 bispecific single-chain diabody with an Fc portion (hEx3-scDb-Fc) produced by recombinant Chinese hamster ovary cells was evaluated and compared with those of a recombinant humanized anti-IL-8 antibody (IgG1) and human serum IgG. N-Linked oligosaccharide structures were estimated by two-dimensional high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. No sialylation was observed with purified hEx3-scDb-Fc and the anti-IL-8 antibody. From the analysis of neutral oligosaccharides, approximately more than 90% of the N-linked oligosaccharides of hEx3-scDb-Fc and the anti-IL-8 antibody were alpha-1,6-fucosylated. The galactosylated biantennary oligosaccharides comprise over 40% of the total N-linked oligosaccharides in both hEx3-scDb-Fc and the anti-IL-8 antibody. The fully galactosylated biantennary oligosaccharides from hEx3-scDb-Fc and the anti-IL-8 antibody accounted for only 10% of the N-linked; however, more than 20% of the N-linked oligosaccharides were fully galactosylated biantennary oligosaccharides in human serum IgG. The glycosylation pattern of hEx3-scDb-Fc was quite similar to that of the anti-IL-8 antibody.     

Molecular cloning and characterization of the α-glucosidase II from Bombyx mori and Spodoptera frugiperda

The α-glucosidase II (GII) is a heterodimer of α- and β-subunits and important for N-glycosylation processing and quality control of nascent glycoproteins. Although high concentration of α-glucosidase inhibitors from mulberry leaves accumulate in silkworms (Bombyx mori) by feeding, silkworm does not show any toxic symptom against these inhibitors and N-glycosylation of recombinant proteins is not affected. We, therefore, hypothesized that silkworm GII is not sensitive to the α-glucosidase inhibitors from mulberry leaves. However, the genes for B. mori GII subunits have not yet been identified, and the protein has not been characterized. Therefore, we isolated the B. mori GII α- and β-subunit genes and the GII α-subunit gene of Spodoptera frugiperda, which does not feed on mulberry leaves. We used a baculovirus expression system to produce the recombinant GII subunits and identified their enzyme characteristics. The recombinant GII α-subunits of B. mori and S. frugiperda hydrolyzed p-nitrophenyl α-d-glucopyranoside (pNP-αGlc) but were inactive toward N-glycan. Although the B. mori GII β-subunit was not required for the hydrolysis of pNP-αGlc, a B. mori GII complex of the α- and β-subunits was required for N-glycan cleavage. As hypothesized, the B. mori GII α-subunit protein was less sensitive to α-glucosidase inhibitors than was the S. frugiperda GII α-subunit protein. Our observations suggest that the low sensitivity of GII contributes to the ability of B. mori to evade the toxic effect of α-glucosidase inhibitors from mulberry leaves.     

Denatured Mammalian Protein Mixtures Exhibit Unusually High Solubility in Nucleic Acid-Free Pure Water

Preventing protein aggregation is a major goal of biotechnology. Since protein aggregates are mainly comprised of unfolded proteins, protecting against denaturation is likely to assist solubility in an aqueous medium. Contrary to this concept, we found denatured total cellular protein mixture from mammalian cell kept high solubility in pure water when the mixture was nucleic acids free. The lysates were prepared from total cellular protein pellet extracted by using guanidinium thiocyanate-phenol-chloroform mixture of TRIzol, denatured and reduced total protein mixtures remained soluble after extensive dialysis against pure water. The total cell protein lysates contained fully disordered proteins that readily formed large aggregates upon contact with nucleic acids or salts. These findings suggested that the highly flexible mixtures of disordered proteins, which have fully ionized side chains, are protected against aggregation. Interestingly, this unusual solubility is characteristic of protein mixtures from higher eukaryotes, whereas most prokaryotic protein mixtures were aggregated under identical conditions. This unusual solubility of unfolded protein mixtures could have implications for the study of intrinsically disordered proteins in a variety of cells.     

Oxidative DNA damage is a preliminary step during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

The aim of this study was to investigate oxidative DNA damage during 4-nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinogenesis. For this purpose, male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution through their drinking water for 4, 12, and 20 weeks. Ten animals were used as negative control. The alkaline Comet assay modified with lesion-specific enzymes was used to detect single and double strand breaks, labile sites (SBs), and oxidised purines and pyrimidines. Although no histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure, oxidative DNA damage was detected in the ‘normal’ oral epithelium. In pre-neoplastic lesions and squamous cell carcinomas induced after 12 and 20 weeks following carcinogen exposure, respectively, oxidative DNA damage was also increased (P < 0.05) when compared to negative control. In conclusion, our results suggest that oxidative DNA damage is an early event during multistep carcinogenesis assay induced by 4NQO. This kind of approach should be considered to persons with high risk of oral cancer, such as in smokers or alcohol consumers.     

Non-synaptic transformation of gustatory receptor potential by stimulation of the parasympathetic fiber of the frog glossopharyngeal nerve

When the glossopharyngeal nerve (GP) in the frog was strongly stimulated electrically, slow potentials were elicited from the tongue surface and taste cells in the fungiform papillae. Injection of atropine completely blocked these slow potentials. The present and previous data indicate that the slow potentials induced in the tongue surface and taste cells are due to a liquid junction potential between saliva secreted from the lingual glands due to parasympathetic fiber activity and an adapting solution on the tongue surface. Intracellularly recorded depolarizing receptor potentials in taste cells induced by 0.5 M NaCl and 3 mM acetic acid were enhanced by depolarizing slow potentials induced by GP nerve stimulation, but were depressed by the hyperpolarizing slow potentials. On average, the receptor potential of taste cells for 0.5 M NaCl was increased by 25% by the GP nerve-induced slow potential, but the receptor potential of taste cells for 3 mM acetic acid was decreased by 1% by the slow potential. These transformations of receptor potentials in frog taste cells were not due to a synaptic event initiated between taste cells and the efferent nerve fiber, but due to a non-synaptic event, a lingual junction potential generated in the dorsal lingual epithelium by GP nerve stimulation.     

Plant cultured cells expressing human beta1,4-galactosyltransferase secrete glycoproteins with galactose-extended N-linked glycans

Previously, we generated transgenic tobacco BY2 suspension-cultured cells (GT6 cells) that produced human beta1,4-galactosyltransferase. In this study, we analyze the N-glycan structures of glycoproteins secreted from GT6 cells to the spent medium. The N-glycans were liberated by hydrazinolysis, and the resulting oligosaccharides were labeled with 2-aminopyridine (PA). The pyridylaminated glycans were purified by reversed-phase and size-fractionation HPLC. The structures of the PA sugar chains were identified by the combined use of 2D PA sugar chain mapping, MS/MS analysis, and exoglycosidase digestion. The distribution of proposed N-glycan structures of GT6-secreted glycoproteins (GalGNM5 [26.8%], GalGNM4 [18.4%], GalGNM3 [19.6%], and GalGNM3X [35.2%]) is different from that found in intracellular glycoproteins (M7A [9.3%], M7B [15.9%], M6B [19.5%], M5 [1.4%], M3X [6.6%], GalGNM5 [35.5%], and GalGNM3 [11.8%]). In vitro, sialic acid was transferred to sugar chains of extracellular glycoproteins from the GT6 spent medium. The results suggest that sugar chains of extracellular glycoproteins from the GT6 spent medium are candidates for substrates of sialic acid transfer.     

Newly identified repeat sequences, derived from human chromosome 21qter, are also localized in the subtelomeric region of particular chromosomes and 2q13, and are conserved in the chimpanzee genome

Subtelomeric regions have been a target of structural and functional studies of human chromosomes. Markers having a defined structure are especially useful to such studies. Here, we report 93 bp tandem repeat sequences found in the subtelomeric region of human chromosome 21q. They were also detected in the telomeric region of several other chromosomes. Interestingly, the repeat was also found in the 2q13 region which is known to be a position of chromosomal fusion, a major difference between the human and chimpanzee karyotypes. To the best of our knowledge, this repetitive sequence is a new member of human subtelomeric interspersed repeats.     

A first high-density map of 981 biallelic markers on human chromosome 14

As the largest set of sequence variants, single-nucleotide polymorphisms (SNPs) constitute powerful assets for mapping genes and mutations related to common diseases and for pharmacogenetic studies. A major goal in human genetics is to establish a high-density map of the genome containing several hundred thousand SNPs. Here we assayed 3.7 Mb (154,397 bp in 24 alleles) of chromosome 14 expressed sequence tags (ESTs) and sequence-tagged sites, for sequence variation in DNA samples from 12 African individuals. We identified and mapped 480 biallelic markers (459 SNPs and 21 small insertions and deletions), equally distributed between EST and non-EST classes. Extensive research in public databases also yielded 604 chromosome 14 SNPs (dbSNPs), 520 of which could be mapped and 19 of which are common between CNG (i.e., identified at the Centre National de Génotypage) and dbSNP polymorphisms. We present a dense map of SNP variation of human chromosome 14 based on 981 nonredundant biallelic markers present among 1345 radiation hybrid mapped sequence objects. Next, bioinformatic tools allowed 945 significant sequence alignments to chromosome 14 contigs, giving the precise chromosome sequence position for 70% of the mapped sequences and SNPs. In addition, these tools also permitted the identification and mapping of 273 SNPs in 159 known genes. The availability of this SNP map will permit a wide range of genetic studies on a complete chromosome. The recognition of 45 genes with multiple SNPs, by allowing the construction of haplotypes, should facilitate pharmacogenetic studies in the corresponding regions.     

Time-series metagenomic analysis reveals robustness of soil microbiome against chemical disturbance

Soil microbial communities have great potential for bioremediation of recalcitrant aromatic compounds. However, it is unclear which taxa and genes in the communities, and how they contribute to the bioremediation in the polluted soils. To get clues about this fundamental question here, time-course (up to 24 weeks) metagenomic analysis of microbial community in a closed soil microcosm artificially polluted with four aromatic compounds, including phenanthrene, was conducted to investigate the changes in the community structures and gene pools. The pollution led to drastic changes in the community structures and the gene sets for pollutant degradation. Complete degradation of phenanthrene was strongly suggested to occur by the syntrophic metabolism by Mycobacterium and the most proliferating genus, Burkholderia. The community structure at Week 24 (∼12 weeks after disappearance of the pollutants) returned to the structure similar to that before pollution. Our time-course metagenomic analysis of phage genes strongly suggested the involvement of the ‘kill-the-winner’ phenomenon (i.e. phage predation of Burkholderia cells) for the returning of the microbial community structure. The pollution resulted in a decrease in taxonomic diversity and a drastic increase in diversity of gene pools in the communities, showing the functional redundancy and robustness of the communities against chemical disturbance.